Auditory cortex shapes sound responses in the inferior colliculus — TEI-tagged view

BlackwellpersName, LesickopersName, RaopersName, De BiasipersName, GeffenpersName (2020date). “Auditory cortex shapes sound responses in the inferior colliculus.” eLifetitle·j.

Object of study — the descending feedbackterm projection from auditory cortexterm to inferior colliculusterm, and what it does to sound responses in the structure below it

Research question — whether selectively driving or silencing cortico-collicular projections — and separately, changing cortical inhibition — alters spontaneous and sound-evoked activity in the inferior colliculusterm

Contribution — an asymmetry: activating the projection changes collicular responses, silencing it changes nothing, and interneuron-driven changes in cortex do not reach the colliculus at all

Full text — tagged

Abstract The extensive feedback from the auditory cortexterm (AC) to the inferior colliculusterm (IC) supports critical aspects of auditory behavior but has not been extensively characterized. Previous studies demonstrated that activity in IC is altered by focal electrical stimulation and pharmacological inactivation of AC, but these methods lack the ability to selectively manipulate projection neurons. We measured the effects of selective optogenetic modulationterm·var of cortico-collicular feedbackterm projections on IC sound responses in mice. Activation of feedback increased spontaneous activityterm and decreased stimulus selectivity in IC, whereas suppression had no effect. To further understand how microcircuits in AC may control collicular activity, we optogenetically modulated the activity of different cortical neuronal subtypes, specifically parvalbumin-positive (PV) and somatostatin-positive (SST) inhibitory interneurons. We found that modulating the activity of either type of interneuron did not affect IC sound-evoked activity. Combined, our results identify that activation of excitatory projections, but not inhibition-driven changes in cortical activity, affects collicular sound responses.

Research organism: Mouse eLife digest How do we hear the world around us? Hearing begins when hair cells in the inner ear translate incoming sound waves into electrical signals. These signals travel via the auditory nerve and the brainstem to the midbrain, where an area called the inferior colliculusterm processes them. The inferior colliculusterm then passes the signals on to another area deep within the brain, the thalamus, which processes the signals further before it too passes them on to an area of the brain’s outer layer called the auditory cortexterm.

At each stage of the auditory pathway, the signals undergo more complex processing than at the previous stage. Researchers have tended to think of this pathway as a one-way route from the ear to the brain. But in reality, feedback occurs at various points along the pathway, enabling areas that do higher processing to shape the responses of areas earlier in the pathway. This feedback is particularly prevalent in the auditory system, where one such strong feedback route is from the auditory cortexterm to the inferior colliculusterm. This reverse connection helps animals learn new behavioral responses to sounds, for example, to run away from a loud noise.

By manipulating the activity of this pathway in mice using a technique called optogenetics, BlackwellpersName et al. provide further clues to how the auditory pathway works. Optogenetics involves introducing light-sensitive ion channels into neurons, and then using light to activate or inhibit those neurons on demand. BlackwellpersName et al. show that activating the feedback pathway from the auditory cortexterm to the inferior colliculusterm in awake mice changes how the inferior colliculusterm responds to sounds. By contrast, inhibiting the pathway has no effect on inferior colliculusterm responses. This suggests that the feedback pathway is not active all the time, but instead influences inferior colliculusterm activity only during specific behavior, for example, perhaps when we are listening for a specific sound like the ringing of a phone.

Understanding how the brain processes sound is important for understanding how we communicate and why we appreciate music. It could also help in treating hearing loss. Stimulating the inferior colliculusterm using a device implanted in the brainstem can improve hearing in people with certain types of deafness. Strengthening or weakening the feedback pathway from the auditory cortexterm to the inferior colliculusterm could make these implants more effective. In the future, it may even be possible that stimulating the pathway directly could restore hearing without any implant being required.

Introduction Information processing is typically studied as a set of computations ascending along a hierarchy of sensory areas, often overlooking the role of descending feedbackterm between these nuclei. In the auditory system, the auditory cortexterm (AC) sends extensive feedback to nuclei earlier in the auditory pathway, including the auditory thalamus ( Alitto and Usrey, 2003 ; Rouiller and Durif, 2004 ; Winer et al., 2001 ) and the inferior colliculusterm (IC) ( Bajo and Moore, 2005 ; Bajo et al., 2007 ; Coomes et al., 2005 ; Doucet et al., 2003 ; Saldaña et al., 1996 ; Williamson and Polley, 2019 ). Previous studies have demonstrated the importance of cortical feedback to IC in auditory behaviors ( Bajo et al., 2010 ; Xiong et al., 2015 ), however, the mechanisms by which information processing is shaped via the descending feedbackterm pathway remain poorly characterized. In this study, we selectively modulated activity in AC, targeting either excitatory projections or inhibitory interneurons, to quantify how AC shapes spontaneous activityterm and sound-evoked responses in IC.

Previous studies demonstrated that neuronal responses to sounds in IC are altered by focal electrical stimulation and inactivation of AC. Cortical stimulation shifted tuning properties of IC neurons toward those of the stimulated neurons in frequency ( Jen et al., 1998 ; Jen and Zhou, 2003 ; Ma and Suga, 2001a ; Yan et al., 2005 ; Yan and Suga, 1998 ; Zhou and Jen, 2007 ), amplitude ( Jen and Zhou, 2003 ; Yan et al., 2005 ; Zhou and Jen, 2007 ), azimuth ( Zhou and Jen, 2007 ; Zhou and Jen, 2005 ), and duration ( Ma and Suga, 2001b ). Stimulation of AC had mixed effects on sound-evoked responses in IC, increasing and decreasing responses in different subpopulations of neurons ( Jen et al., 1998 ; Zhou and Jen, 2005 ). Consistent with this effect, different patterns of direct cortico-collicular activation enhanced or suppressed white noise-induced responses in IC ( Vila et al., 2019 ). AC inactivation studies, on the other hand, found less consistent effects on IC responses. Whereas one study found that inactivation of AC caused a shift in best frequencyterm in IC neurons ( Zhang et al., 1997 ), several other studies showed that inactivation of AC had no effect on frequency selectivityterm in IC ( Jen et al., 1998 ), but rather modulated sound-evoked and spontaneous activityterm ( Gao and Suga, 1998 ; Popelár et al., 2003 ; Popelář et al., 2016 ). Cortico-collicular feedbackterm is critical to auditory learning, specifically learning to adapt to a unilateral earplug during sound localization ( Bajo et al., 2010 ). Pairing electrical leg stimulation with a tone induced a shift in best frequencyterm of IC neurons, while presentation of a tone alone was insufficient ( Gao and Suga, 1998 ; Gao and Suga, 2000 ). Furthermore, cortico-collicular feedbackterm was necessary to induce running in response to a loud noise ( Xiong et al., 2015 ).

In AC, modulation of sound responses is not a monophasic process, but instead is shaped by interactions between excitatory and inhibitory cortical neurons. Modulating activity of different inhibitory interneuron subtypes in AC narrowed frequency tuning and attenuated tone-evoked responses of excitatory neurons, while suppression had the opposite effect ( Aizenberg et al., 2015 ; Hamilton et al., 2013 ; Phillips and Hasenstaub, 2016 ; Seybold et al., 2015 ). Electrical stimulation of AC, cooling or pharmacological inactivation affected the amplitude of sound-evoked responses and shifted the best frequencyterm of neurons in IC, but it remains unknown how specific these effects are to direct feedback, and whether the effects of intra-cortical inhibition propagate to the IC.

The goal of the present study is to examine the role of cortico-collicular projections in shaping sound responses in IC. IC receives glutamatergic ( Feliciano and Potashner, 1995 ) inputs from neurons originating predominantly in layer 5term of AC ( Bajo and Moore, 2005 ; Bajo et al., 2007 ; Coomes et al., 2005 ; Doucet et al., 2003 ; Saldaña et al., 1996 ; Winer et al., 1998 ). We used viral transfection methods to selectively drive excitatory or inhibitory opsin expression in AC-IC projections. We then recorded neuronal activity in IC and tested how activation or suppression of AC-IC projections affected spontaneous activityterm and sound-evoked responses in IC. To better understand whether and how intra-cortical network interactions propagated to IC, we manipulated the activity of the two most common inhibitory neuronal subtypes in AC, Parvalbumin-(PV) and Somatostatin-(SST) positive interneurons ( Rudy et al., 2011 ).

Results Activating direct cortico-collicular feedbackterm modulates activity in the inferior colliculusterm Our first goal was to characterize the effects of activating the direct cortico-collicular projections on tone-evoked responses in IC. A combination of cortical anterograde and collicular retrograde viral transfections was used in order to achieve specific viral transfection of cortico-collicular neurons. We delivered either an excitatory opsin, ChannelRhodopsin2 (ChR2term) or an inhibitory opsin, ArchaerhodopsinT (ArchTterm), bilaterally, specifically to the neurons in the auditory cortexterm which project to the inferior colliculusterm ( Figure 1A ). To achieve such specificity, we injected a retrograde virus that encoded Cre recombinase (Retro2 AAV.Cre) in IC ( Figure 1B ). This retrograde viral construct ensured that neurons projecting to IC expressed Cre recombinase a few weeks later. At the same time, we injected a virus that encoded ChR2term or ArchTterm in reversed fashion under the FLEX cassette in AC (AAV.Flex.ChR2term, AAV.Flex.ArchTterm) ( Figure 1C ). This strategy ensured that only neurons expressing Cre recombinase in the auditory cortexterm would express ChR2term or ArchTterm in AC. Therefore, opsin was expressed exclusively in AC-IC projecting neurons, which terminate across all regions of IC, including central nucleus (CNICterm) and dorsal cortex of IC (DCICterm), from which we recorded ( Figure 1D ). Shining light over AC of these mice would therefore directly activate or suppress only these cortico-collicular feedbackterm projections to IC.

Figure 1. Opsin expression in corticocollicular feedbackterm.

( A ) Experimental design: To yield selective labeling of auditory cortico-collicular projections, a retroAAV-Cre-GFP construct is injected into the IC and AAV-FLEX-opsin-tdTomato construct is injected into the AC bilaterally. Recordings are made in multiple bilateral locations of the IC (left) of awake mice. Cannulas are implanted bilaterally in the auditory cortexterm, and a green/blue laser is used to silence/activate the cortico-collicular pathway (right). ( B ) Expression of the retro AAV-Cre-GFP construct in the IC. Scale bar: 1000 μm. ( C ) Selective expression of the AAV-FLEX-opsin-tdTomato construct in cell bodies in layer 5term (L5) and 6 (L6) of the AC that project to the IC. Fiber and terminal labeling is also present in the medial geniculate body, a known target of auditory cortico-collicular cells. Scale bar: 2000 μm. ( D ) Expression of the AAV-FLEX-opsin-tdTomato construct in fibers and terminals at multiple rostro-cadual levels of the IC. Expression, as measured by fluorescence intensity (bottom), is strongest in medial portions of the central nucleus of the IC and lateral portions of the external nucleus of the IC. Scale bar: left, 1000 μm; middle and right, 500 μm.

First, we measured and quantified neuronal spiking in IC in response to stimulation or suppression of cortico-collicular projections using ChR2term or ArchTterm. To activate this projection, we shone blue laser over AC, recorded neuronal activity in IC, and quantified the effects of manipulating feedback in the absence of sound ( Figure 2A,C,D ). We measured the spiking activity in IC as we varied the duration of laser manipulation (1 ms, 5 ms, 25 ms, 250 ms). As exected, activation of cortico-collicular neurons resulted, on average, in an increase in firing rate of IC neurons. This effect persisted at the 5 ms, 25 ms, and 250 ms laser duration ( Figure 2C ; 1 ms : p=5.3e-4, ON = 5.6 ± 0.6 Hz, OFF = 4.9 ± 0.5 Hz; 5 ms : p=0.0027, ON = 6.9 ± 0.7 Hz, OFF = 4.8 ± 0.5 Hz; 250 ms : p=0.005, ON = 5.6 ± 0.6 Hz, OFF = 4.8 ± 0.5 Hz). Whereas the direction of the effect was consistent with our prior expectations, the magnitude of the effect was unexpectedly small. This suggests that AC targets a small subpopulation of IC neurons and that the effect of activation does not spread far within IC.

Figure 2. Effects of activating and inactivating cortico-collicular feedbackterm activity on firing rate of neurons in IC in the absence of sounds and in response to sound clicks.

( A ) Example IC unit response to two laser pulse durationsterm·var, activation (ChR2term) (blue), 25 ms (left) and 250 ms (right). Top: raster plot of spike times. Bottom: average firing rate. Blue bar indicates the timing of the laser. ( B ) Example unit response to clicks with (blue) and without laser (activation, ChR2term). Axes same as A. We presented 6 clicks at 10 Hz every second, starting at 0 s. ( C ) Change in IC unit activity in response to different laser durations with cortico-collicular acitvation using ChR2term (1 ms, 5 ms, 25 ms, 250 ms). Top panels : Histogram of changes in unit activity between laser ON and laser OFF conditions, + indicates median, ∨ indicates mean. These distributions are not normally distributed (Kolmogorov-Smirnov testterm; 1 ms : p=3.07e-15, mean = 1.1 stdevs, median = 0.21 stdevs; 5 ms : p=4.4e-20, mean = 1.7 stdevs, median = 0.19 stdevs; 25 ms : p=5.5e-17, mean = 1.6 stdevs, median = −0.021 stdevs; 250 ms : p=3.4e-12, mean = 0.58 stdevs, median = 0.101 stdevs). Bottom panels : Firing rate of IC units in laser ON versus laser OFF conditions (Statistics for the difference across the neuronal population: 1 ms : p=5.3e-4, ON = 5.6 ± 0.6 Hz, OFF = 4.9 ± 0.5 Hz; 5 ms : p=0.0027, ON = 6.9 ± 0.7 Hz, OFF = 4.8 ± 0.5 Hz; 250 ms : p=0.005, ON = 5.6 ± 0.6 Hz, OFF = 4.8 ± 0.5 Hz)). ( D ) Change in IC unit activity in response to different laser durations with cortico-collicular inactivation using ChR2term (1 ms, 5 ms, 25 ms, 250 ms). Top panels: Histogram of changes in unit activity between laser ON and laser OFF conditions, + indicates median, ∨ indicates mean. These distributions are normally distributed (Kolmogorov-Smirnov testterm). Bottom panels: Firing rate of units in laser ON versus laser OFF conditions (Statistics for the difference across the neuronal population: 2 5 ms : p=0.039, ON = 9.5 ± 0.7 Hz, OFF = 9.5 ± 0.7 Hz; 250 ms : p=0.02, ON = 9.5 ± 0.8 Hz, OFF = 9.5 ± 0.8 Hz). ( E ) IC population responses to clicks exhibit a decrease in click response with cortico-cllicular activation using ChR2term (left; p=0.001, click ON = 10.4 ± 1.2 Hz, click OFF = 10.7 ± 1.1 Hz) and an increase in spontaneous activityterm (bottom; p=0.0031, spont ON = 5 ± 0.87 Hz, spont OFF = 4.3 ± 0.92 Hz). Left panel: Click-evoked firing rate of IC units on laser ON versus laser OFF conditions. Center panel: box plot of change in click-evoked firing rate On box plots, red line indicates median, white ◆ indicates mean. Right panel: Change in click response versus change in spontaneous activityterm due to laser activation. Bottom panel: Spontaneous activityterm on laser ON versus laser OFF trials. ( F ) IC population responses to clicks exhibit no change in activity with cortico-collicular suppression using ArchTterm. On box plots, red line indicates median, white ◆ indicates mean. Axes as in E.

We tested the effect of suppressing AC-IC neurons on firing responses in IC and, surprisingly, we detected little difference in firing in IC neurons ( Figure 2D ; 2 5 ms : p=0.039, ON = 9.5 ± 0.7 Hz, OFF = 9.5 ± 0.7 Hz; 250 ms : p=0.02, ON = 9.5 ± 0.8 Hz, OFF = 9.5 ± 0.8 Hz). This result suggests that AC-IC inputs at rest do not contribute to IC responses, but rather modulate collicular activity only when activated.

Next, our goal was to characterize modulation of sound-evoked responses in IC by cortical feedback. We first tested the effects of feedback modulation on acoustic click responses. We chose clicks as the initial stimulus because they drive fast responses in both AC and IC. Laser stimulation began 250 ms prior to click train onset to allow for the response to the laser to come to a steady state, and continued activation throughout the clicks ( Figure 2B ). Activating the feedback using ChR2term had a weak suppressive effect on the firing rate of IC neurons in response to clicks. Consistent with previous experiments, we observed an overall increase in spontaneous activityterm ( Figure 2E , bottom; p=0.0031, spont ON = 5 ± 0.87 Hz, spont OFF = 4.3 ± 0.92 Hz). Activating feedback caused a small decrease in click-evoked response ( Figure 2E , left; p=0.001, click ON = 10.4 ± 1.2 Hz, click OFF = 10.7 ± 1.1 Hz).

By contrast, suppressing cortico-collicular feedbackterm using ArchTterm had no effect on IC click responses ( Figure 2F ). Because attenuating the input does not affect the activity in IC during clicks, this result further suggests that the strength of the baseline signal from AC to IC, even in the presence of cortical activity, is not sufficient to modulate IC responses.

We also tested whether the effects of cortico-collicular modulation differed across tone frequency range. We presented a stimulus that consisted of tones of 50 frequencies ranging from 3 to 70 kHz. To modulate the feedback from AC, we tested three different laser onsetsterm·var (−100 ms, −20 ms, +8 ms) relative to tone onset to isolate the effect of timing on affecting IC responses. These delays were chosen for the following reasons: −100 ms delay would allow for the laser effect on cortical activity to come to a steady state, allowing to quantify the effect throughout the tone pip; +8 ms is set up to mimic the time scale of cortical response to a tone, effectively amplifying the onset of the cortical response; −20 ms delay is an intermediate value.

We found that activating cortico-collicular feedbackterm using ChR2term increased spontaneous activityterm of IC neurons ( Figure 3A , left; −100 ms: p=0.022, ON = 4.3 ± 0.47 Hz; OFF = 3.6 ± 0.38; −20 ms: p=8.6e-6, ON = 5.6 ± 0.63, OFF = 3.7 ± 0.47). However, overall the feedback decreased tone-evoked response magnitudeterm in IC, which we defined as the difference between spontaneous and tone-evoked response, at all laser onsetsterm·var ( Figure 3A , right; −100 ms: p=3.2e-5, ON = 14.2 ± 0.98 Hz; OFF = 15.5 ± 1.04 Hz; −20 ms: p=1.4e-6, ON = 11.8 ± 1.08 Hz, OFF = 13.9 ± 1 Hz; +8 ms: p=0.0034, ON = 13.03 ± 1.03, OFF = 13.7 ± 1.03). This suggests that the broad activation of feedback upregulates the baseline activity of IC neurons, but decreases tone-evoked response (Figure 6C, −100 ms: p=0.018, ON = 19.01 ± 1.2 Hz, OFF = 18.5 ± 1.2 Hz; +8 ms: p=0.007, ON = 17.3 ± 1.2 Hz; OFF = 16.9 ± 1.2 Hz). By contrast, suppressing the feedback using ArchTterm had no effect on either spontaneous activityterm or tone-evoked response magnitudeterm ( Figure 3B ), suggesting that at baseline AC does not provide strong modulation of IC activity, as removing it does not affect sound-evoked effects in IC.

Figure 3. Effects of modulating feedback activity on spontaneous and tone-evoked response magnitudeterm in IC.

Left panels: neuronal activity (spontaneous, left or tone-evoked, right) on laser ON versus laser OFF trials. Right panels: Box plots of neuronal activity (spontaneous, left or tone-evoked, right) for laser ON and laser OFF trials. Magenta circles indicate single units. On box plots, red line indicates median, white ◆ indicates mean. ( A ) Activating feedback using ChR2term increased IC spontaneous activityterm (left; −100 ms : p=0.022, ON = 4.3 ± 0.47 Hz; OFF = 3.6 ± 0.38; −20 ms : p=8.6e-6, ON = 5.6 ± 0.63, OFF = 3.7 ± 0.47) and decreased tone-evoked response magnitudeterm (right; −100 ms : p=3.2e-5, ON = 14.2 ± 0.98 Hz; OFF = 15.5 ± 1.04 Hz; −20 ms : p=1.4e-6, ON = 11.8 ± 1.08 Hz, OFF = 13.9 ± 1 Hz; +8 ms : p=0.0034, ON = 13.03 ± 1.03, OFF = 13.7 ± 1.03). ( B ) Suppressing feedback using ChR2term had no effect on IC spontaneous activityterm or tone-evoked response magnitudeterm.

Figure 3—figure supplement 1. Effects of activating cortico-collicular feedbackterm on noise correlationsterm·def in IC.

( A ) TOP: Distribution of noise correlationsterm·def of IC tone response for laser OFF (gray) condition (mean = 0.069) and laser ON (blue, activation with ChR2term) condition (mean = 0.057) BOTTOM: Distribution of change in correlation between laser ON and laser OFF conditions (mean = −0.017). ( B ) TOP: Distribution of noise correlationsterm·def of spontaneous activityterm for laser OFF (gray) condition (mean = 0.021) and laser ON (blue) condition (mean = 0.012) BOTTOM: Distribution of change in correlation between laser ON and laser OFF conditions (mean = −0.008).

Figure 3—figure supplement 2. Effects of modulating cortico-collicular feedbackterm in AC on activity in AC in two example units.

( A ) Example unit with increased spontaneous and tone-evoked activity in AC with activation of cortico-collicular feedbackterm using ChR2term in AC averaged across all frequencies. ( B ) Example unit with decreased spontaneous and tone-evoked activity in AC with suppression of cortico-collicular feedbackterm using ArchTterm in AC averaged across all frequencies.

We then examined the effect of feedback on frequency tuning of IC units. Activation of feedback using ChR2term decreased frequency selectivityterm in the subsets of IC units that exhibited a decrease in tone-evoked response magnitudeterm or increase in spontaneous activityterm ( Figure 4A ), but not in units that exhibited an increase in tone-evoked response magnitudeterm or decrease in spontaneous activityterm ( Figure 4B , mag decrease: −20 ms, p=0.00031, sparse ON = 0.49 ± 0.027, sparse OFF = 0.55 ± 0.025; +8 ms, p=0.00029, sparse ON = 0.46 ± 0.027, sparse OFF = 0.55 ± 0.024; spont increase: −100 ms, p=0.011, sparse ON = 0.49 ± 0.032, sparse OFF = 0.56 ± 0.031; −20 ms, p=0.00018, sparse ON = 0.46 ± 0.034, sparse OFF = 0.56 ± 0.029; +8 ms, p=2.2e-6, sparse ON = 0.41 ± 0.032, sparse OFF = 0.55 ± 0.029).

Figure 4. Effect of modulating direct cortico-collicular feedbackterm at different latencies relative to tone onset on frequency selectivityterm in IC.

On box plots, red line indicates median, white ◆ indicates mean. ( A ) Left panels: Sparsenessterm for IC units on laser ON (activation with ChR2term) versus laser OFF trials. Right panels: Average sparsenessterm for laser ON and laser OFF trials. Activating feedback using ChR2term decreased frequency selectivityterm in a subset of IC units (mag decrease: −20 ms, p=0.00031, sparse ON = 0.49 ± 0.027, sparse OFF = 0.55 ± 0.025; +8 ms, p=0.00029, sparse ON = 0.46 ± 0.027, sparse OFF = 0.55 ± 0.024; spont increase: −100 ms, p=0.011, sparse ON = 0.49 ± 0.032, sparse OFF = 0.56 ± 0.031; −20 ms, p=0.00018, sparse ON = 0.46 ± 0.034, sparse OFF = 0.56 ± 0.029; +8 ms, p=2.2e-6, sparse ON = 0.41 ± 0.032, sparse OFF = 0.55 ± 0.029). ( B ) Left panels: Sparsenessterm for laser ON (suppression with ArchTterm) versus laser OFF trials. Right panels: Average sparsenessterm for laser ON and laser off trials. Suppressing feedback using ArchTterm has little effect on frequency selectivityterm in IC (mag decrease: −100 ms, p=0.011, sparse ON = 0.53 ± 0.021, sparse OFF = 0.51 ± 0.021; mag increase: −100 ms, p=0.019, sparse ON = 0.48 ± 0.021, sparse OFF = 0.49 ± 0.021; spont increase: −100 ms, p=0.043, sparse ON = 0.46 ± 0.029, sparse OFF = 0.45 ± 0.029).

Interestingly, these changes were observed almost exclusively in cells located in DCICterm rather than CNICterm ( Figure 5 ). Specifically, activation of feedback increased spontaneous activityterm ( Figure 5A , left; −100 ms : p=0.0204, ON = 3.5 ± 0.5 Hz, OFF = 2.5 ± 0.3 Hz; −20 ms : p=2.8e-7, ON = 5.2 ± 0.7 Hz, OFF = 2.3 ± 0.3 Hz) and decreased tone-evoked response magnitudeterm ( Figure 5A , right, −100 ms : p=3.3e-6, ON = 14.8 ± 1.4 Hz, OFF = 16.5 ± 1.5 Hz; −20 ms : p=6.3e-7, ON = 11.8 ± 1.6 Hz, OFF = 14.7 ± 1.4 Hz) in cells located in DCICterm, but not in CNICterm ( Figure 5B ). Similarly, activation of feedback decreased frequency selectivityterm ( Figure 5C ; mag decrease: −20 ms, p=6.3e-4, sparse ON = 0.46 ± 0.03, sparse OFF = 0.53 ± 0.03; +8 ms, p=4.7e-5, sparse ON = 0.41 ± 0.03, sparse OFF = 0.53 ± 0.03; spont increase: −100 ms, p=0.0044, sparse ON = 0.43 ± 0.04, sparse OFF = 0.52 ± 0.04; −20 ms, p=0.00013, sparse ON = 0.39 ± 0.04, sparse OFF = 0.53 ± 0.04; +8 ms, p=5.2e-6, sparse ON = 0.33 ± 0.03, OFF = 0.53 ± 0.04) in cells located in DCICterm, but not in CNICterm ( Figure 5D ). This is consistent with the enhanced density of AC projections in DCICterm as compared to CNICterm ( Figure 1D ). To verify that these results were not due to a select few high firing rates of units in DCICterm, we repeated the analysis comparing only units with spontaneous activityterm in the laser OFF condition less than 40 Hz. With these matched firing rates we still observed the same statistically significant effects (DCICterm – mag decrease −100 ms: p=3.3e-6, ON = 14.8 Hz, OFF = 16.6 Hz; −20 ms: p=6.3e-7, ON = 12.1 Hz, OFF = 12.6 Hz; spont increase −100 ms: p=0.02 ON=3.5 Hz, OFF = 2.5 Hz; −20 ms: p=2.8e-7, ON = 5.1 Hz, OFF = 4.9 Hz; CNICterm – mag decrease +8 ms: p=0.0039, ON = 11.7 Hz, OFF = 12.6 Hz).

Figure 5. Effects of activating cortico-collicular feedbackterm with ChR2term on responses in dorsal and central IC ( A,C ) and DCICterm ( B,D ) recording sitesterm·var.

On box plots, red line indicates median, white ◆ indicates mean. ( A ) Increase in spontaneous activityterm (left; −100 ms : p=0.0204, ON = 3.5 ± 0.5 Hz, OFF = 2.5 ± 0.3 Hz; −20 ms : p=2.8e-7, ON = 5.2 ± 0.7 Hz, OFF = 2.3 ± 0.3 Hz) and decrease in tone-evoked response magnitudeterm (right, −100 ms : p=3.3e-6, ON = 14.8 ± 1.4 Hz, OFF = 16.5 ± 1.5 Hz; −20 ms : p=6.3e-7, ON = 11.8 ± 1.6 Hz, OFF = 14.7 ± 1.4 Hz) for cells in DCICterm with activation of cortico-collicular feedbackterm using ChR2term. Left panels: neuronal activity (spontaneous, left or tone-evoked, right) on laser ON versus laser off trials. Right panels: Average neuronal activity (spontaneous, left or tone-evoked, right) for laser on and laser off trials. ( B ) Little change in spontaneous activityterm or tone-evoked response magnitudeterm (right; +8 ms: p=0.0027, ON = 13 ± 1.2 Hz, OFF = 13.6 ± 1.2 Hz) for cells in CNICterm with activation of cortico-collicular feedbackterm using ChR2term. Panels as in A. ( C ) Activating feedback with ChR2term decreases frequency selectivityterm in a subset of units (mag decrease: −20 ms, p=6.3e-4, sparse ON = 0.46 ± 0.03, sparse OFF = 0.53 ± 0.03; +8 ms, p=4.7e-5, sparse ON = 0.41 ± 0.03, sparse OFF = 0.53 ± 0.03; spont increase: −100 ms, p=0.0044, sparse ON = 0.43 ± 0.04, sparse OFF = 0.52 ± 0.04; −20 ms, p=0.00013, sparse ON = 0.39 ± 0.04, sparse OFF = 0.53 ± 0.04; +8 ms, p=5.2e-6, sparse ON = 0.33 ± 0.03, OFF = 0.53 ± 0.04) for cells in DCICterm. Left panels: Sparsenessterm for laser ON versus laser OFF trials. Right panels: Average sparsenessterm for laser on and laser off trials. ( D ) Activating feedback has no effect on frequency selectivityterm of cells in CNICterm. Panels as in C.

To elucidate changes in the shape of the frequency response functions in IC with activation of cortico-cortical feedback beyond the general changes in selectivity we observed with changes in sparsenessterm, we fitted linear regressionsterm·stat to light off versus light on firing rates. This analysis can reveal whether changes in frequency selectivityterm are linear/subtractive, multiplicative/divisive, or some combination of these transformations. For example, a slope of 1 and a positive y-intercept would represent a linear increase in frequency response (equal increase in response across all frequencies). Over the population of IC neurons with decreased tone-evoked response magnitudeterm and/or increased spontaneous activityterm, the median slopes and y-intercepts of ranked linear fits to frequency responses are less than 1 and above zero, respectively ( Figure 6A ). These results, in combination with the decreased tone-evoked activity observed ( Figure 6A ; Figure 6B ; Figure 6C , −100 ms: p=0.018, ON = 19.01 ± 1.2 Hz, OFF = 18.5 ± 1.2 Hz; +8 ms: p=0.007, ON = 17.3 ± 1.2 Hz; OFF = 16.9 ± 1.2 Hz), indicate that the decrease in frequency selectivityterm was due to a decrease in response to tones at preferred frequencies, not non-preferred frequencies. This result suggests that the suppressive effect of the cortico-collicular feedbackterm is preferential for higher firing responses. Suppressing feedback resulted in very small (<0.04%) change in sparsenessterm, therefore it did not affect frequency selectivityterm in IC ( Figure 4B ).

Figure 6. Effects of activating direct cortico-collicular feedbackterm on frequency tuning properties of IC neurons.

( A ) Example IC unit response timecourse (top) and frequency response function (bottom) for laser ON (blue) and laser off (gray) conditions (activation using ChR2term). Bottom insets represent ranked linear fits for example unit. ( B ) Slope coefficients (top, mag decrease: −100 ms , median = 0.88; −20 ms , median = 0.92; +8 ms , median = 0.903; spont increase: −100 ms , median = 0.99; −20 ms , median = 1; +8 ms , median = 0.94) and y-intercepts (bottom, mag decrease: −100 ms , median = 0.0086; −20 ms , median = 0.014; +8 ms , median = 0.022; spont increase: −100 ms , median = 0.014; −20 ms , median = 0.025; +8 ms , median = 0.037) of ranked linear fits of the frequency response function of IC neurons , red horizontal lines indicate median, red vertical lines indicate interquartile range. ( C ) Left panels: Tone-evoked response for laser ON versus laser off trials. Magenta circles indicate single units. Right panels: Average tone-evoked response for laser ON and laser OFF trials. On box plots, red line indicates median, white ◆ indicates mean. Activating feedback slightly decreased tone-evoked response, averaged across 7 most preferred frequencies ( −100 ms: p=0.018, ON = 19.01 ± 1.2 Hz, OFF = 18.5 ± 1.2 Hz; +8 ms: p=0.007, ON = 17.3 ± 1.2 Hz; OFF = 16.9 ± 1.2 Hz) ( D ) Best frequencies for laser off versus laser ON trials. Activating feedback had little effect on best frequencyterm (spont increase: −100 ms , p=0.022).

Correlations in trial-to-trial responsiveness of neurons can affect the information encoded by a neuronal population ( Abbott and Dayan, 1999 ; Averbeck et al., 2006 ; Nirenberg and Latham, 2003 ; Shadlen and Newsome, 1998 ; Zohary et al., 1994 ). To test whether activation of cortico-collicular feedbackterm affected correlations in trial-to-trial variability of response strength between units recorded simultaneously we measured noise correlationsterm·def. We found that activating cortico-collicular feedbackterm using ChR2term had no effect on noise correlationsterm·def during spontaneous activityterm or tone response ( Figure 3—figure supplement 1 ).

To better understand the effect of modulation of cortical activity on spectro-temporal receptive fieldterm properties of IC neurons, we presented a continuous signal comprised of dynamic random chordsterm (DRCs) sampled from a uniform distribution of loudness values per frequency bin. The unbiased nature of this stimulus allowed us to estimate the spectro-temporal receptive fieldterm of neurons (STRF), which quantifies the dynamics of sound waveform in time and frequency that lead to a neuronal response. During the DRC stimulus, we turned on the laser every other second for 250 ms to activate cortico-collicular projections with ChR2term. We found that a subset of IC units reduced their mean DRC-evoked firing rates (N = 56), whereas another subset of IC units increased their mean DRC-evoked firing rate when we activated cortico-collicular feedbackterm (N = 47). We next separately computed the receptive fields for each neuron when laser was off and when laser was on and compared the STRFs for activation with ChR2term ( Figure 7A) . To quantify those changes, we identified the positive (activation) and negative (suppressive) regions in the STRFs and compared them for laser ON and laser OFF conditions. In the subset of neurons whose firing rate increased with laser STRFs changed: only 42% of positive lobes, and 63% of negative lobes persisted with the laser ( Figure 7B , left). Of those lobes that persisted, for positive lobes, there was on average a decrease in temporal width (p=0.00098, ON = 0.0303 ± 0.0018 s, OFF = 0.037 ± 0.0023 s), frequency selectivityterm (p=0.00042, ON = 6.7 ± 1.1 Hz, OFF = 9.6 ± 1.8 Hz) and STRF size (p=0.00036, ON = 46.05 ± 6.9 pixels, OFF = 77.5 ± 12.04 pixels), whereas for negative lobes, we did not detect any changes ( Figure 7C , left). For IC units whose firing rate was decreased, there was a much smaller change in the lobes with cortico-collicular activation, with 78% and 79% of positive and negative lobes persisting, respectively ( Figure 7B , right). For both positive lobes and negative lobes, the only difference was an increase in the temporal width when laser activated cortico-collicular feedbackterm ( Figure 7C , right; Positive lobes: p=0.026, ON = 0.034 ± 0.0025 s, OFF = 0.032 ± 0.0025 s; Negative lobes: p=0.02, ON = 0.037 ± 0.0028 s, OFF = 0.032 ± 0.0024 s). The decrease in STRF size in units with increased DRC-evoked response and decrease in sparsenessterm across the population is consistent with the interpretation that the effect of the cortico-collicular feedbackterm leads to a decrease in responsiveness to tones that evoke the greatest responses (in the center of the receptive field) and an increase to stimuli that evoke weaker activity. In other words, neuronal firing increases overall, but selective responses to specific frequency bands decrease. Because the majority of the units were recorded as multi-units, it is possible that this change in selectivity is due to the change in firing rate of differentially tuned single units.

Figure 7. Effects of activating direct cortico-collicular feedbackterm using ChR2term on STRF properties of IC units.

( A ) Example STRF with and without activation of feedback for IC units that increase firing rate (left) and decrease firing rate (right) in response to cortico-collicular feedbackterm activation using ChR2term. ( B ) Number of positive and negative lobes that persisted with laser (stable). ( C ) Changes in STRF parameters of stable positive and negative lobes. Left panels: STRF parameter for laser OFF versus laser ON trials. Center panels: Average positive lobe STRF parameter for laser ON and laser off trials. Right panels: Average negative lobe STRF parameter for laser on and laser off trials. On box plots, red line indicates median, white ◆ indicates mean. For units with an increase in firing rate we observed a decrease in temporal width (p=0.00098, ON = 0.0303 ± 0.0018 s, OFF = 0.037 ± 0.0023 s), frequency width (p=0.00042, ON = 6.7 ± 1.1 Hz, OFF = 9.6 ± 1.8 Hz), and overall size (p=0.00036, ON = 46.05 ± 6.9 pixels, OFF = 77.5 ± 12.04 pixels) of positive lobes. For units with a decrease in firing rate we observed only a small increase in temporal width for both positive (p=0.026, ON = 0.034 ± 0.0025 s, OFF = 0.032 ± 0.0025 s) and negative (p=0.02, ON = 0.037 ± 0.0028 s, OFF = 0.032 ± 0.0024 s) lobes.

Modulating inhibitory neuronal activity in AC does not affect collicular sound responses Auditory responses in AC are shaped by interactions between excitatory and inhibitory neurons ( Wood et al., 2017 ). To determine how modulating frequency selectivityterm in AC might affect tone-evoked responses in IC in a frequency-selective fashion, we perturbed the excitatory-inhibitory interactions by modulating two different classes of inhibitory interneuron known to contribute to sound responses in AC: PV and SST inhibitory interneurons ( Figure 8A ). We found that modulating PV interneuron activity in AC had no significant effects on spontaneous and tone-evoked activity or frequency selectivityterm in IC ( Figure 8B–E ) despite modulating frequency selectivityterm, spontaneous activityterm, and tone-evoked response magnitudeterm in AC. Specifically, in AC, activating PVs decreased spontaneous activityterm and tone-evoked response magnitudeterm ( Figure 8F ; spontaneous activityterm: p=2.2e-9, ON = 0.88 ± 0.16 Hz, OFF = 2.7 ± 0.34 Hz; tone-evoked response magnitudeterm: p=8.02e-7, ON = 6.9 ± 1.06 Hz, OFF = 11.5 ± 1.3 Hz) and increased frequency selectivityterm ( Figure 8G ; p=3.3e-12, ON = 0.59 ± 0.022, OFF = 0.42 ± 0.02), while suppressing PVs increased spontaneous activityterm ( Figure 8I ; p=5.2e-5, ON = 4.08 ± 0.54 Hz, OFF = 3.06 ± 0.55 Hz) and decreased frequency selectivityterm ( Figure 8J ; p=3.8e-5, ON = 0.34 ± 0.027, OFF = 0.41 ± 0.031). This suggests that the changes in cortical activity driven by manipulation of PV activity does not propagate to the inferior colliculusterm.

Figure 8. Effects of modulating PV interneurons in AC on tone-evoked responses in IC and AC.

On box plots, red line indicates median, white ◆ indicates mean. ( A ) Stain for PV (center) and opsin-GFP (left). Scale bar: 50 um. ( B ) Activating PVs had no effect on spontaneous activityterm or tone-evoked response magnitudeterm in IC. Magenta circles indicate single units. ( C ) Activating PVs had no effect on frequency selectivityterm in IC. ( D ) Suppressing PVs had no effect on spontaneous activityterm or tone-evoked response magnitudeterm in IC. Magenta circles indicate single units. ( E ) Suppressing PVs had weak effects on frequency selectivityterm in IC (mag decrease: p=0.0068, ON = 0.57 ± 0.092, OFF = 0.5 ± 0.087). ( F ) Activating PVs decreased spontaneous activityterm (p=2.2e-9, ON = 0.88 ± 0.16 Hz, OFF = 2.7 ± 0.34 Hz) and tone-evoked response magnitudeterm (p=8.02e-7, ON = 6.9 ± 1.06 Hz, OFF = 11.5 ± 1.3 Hz) in AC. ( G ) Activating PVs increased frequency selectivityterm in putative excitatory units in AC (p=3.3e-12, ON = 0.59 ± 0.022, OFF = 0.42 ± 0.02). ( H ) Activating PVs affected putative excitatory units across all layers of AC. ( I ) Suppressing PVs increased spontaneous activityterm (p=5.2e-5, ON = 4.08 ± 0.54 Hz, OFF = 3.06 ± 0.55 Hz) but did not affect tone-evoked response magnitudeterm in AC. ( J ) Suppressing PVs decreased frequency selectivityterm in AC (p=3.8e-5, ON = 0.34 ± 0.027, OFF = 0.41 ± 0.031). ( K ) Suppressing PVs affected putative excitatory units across all layers of AC. B,D,F,I Left panels: neuronal activity (spontaneous, left or tone-evoked, right) on laser ON versus laser OFF trials. Right panels: Average neuronal activity (spontaneous, left or tone-evoked, right) for laser ON and laser OFF trials. C,E,G,J Left panels: Sparsenessterm for laser ON versus laser off trials. Right panels: Average sparsenessterm for laser on and laser off trials. ( H,K ) Change in spontaneous activityterm (laser ON trials – laser off trials), left panels: for units at each tetrode; right panels: separated into supragranular: tetrodes 1–3; granular: tetrodes 5,6; infragranular: tetrodes 7–10.

Figure 8—figure supplement 1. Effects of activating PV interneurons in AC on spontaneous activityterm in IC (top) and AC (bottom) in both anesthetized and awake (black edges) preparations.

TOP: Activation of PV interneurons in AC had no effect on spontaneous activityterm in IC for either awake (N = 57 units, ON = 7.4 ± 1.4 Hz, OFF = 7.2 ± 1.3 Hz) or anesthetized (N = 138, ON = 10.5 ± 0.92 Hz, OFF = 10.6 ± 0.9 Hz) recordings. BOTTOM: Activation of PV interneurons in AC decreased spontaneous activityterm in AC for both awake (N = 14, p=8.5e-4, ON = 0.93 ± 0.24 Hz, OFF = 3.3 ± 0.87 Hz) and anesthetized (N = 53, p=2.2e-7, ON = 0.87 ± 0.19, OFF = 2.6 ± 0.36) recordings.

Different interneuron classes may function in distinct networks, so we also tested the effects of modulating SST interneurons in AC. Modulating SST interneurons had no effect on tone-evoked activity ( Figure 9B,D , right) or frequency selectivityterm ( Figure 9C,E ), but suppressing SST interneurons increased spontaneous activityterm in IC ( Figure 9D , left; p=0.029, ON = 6.9 ± 0.66 Hz, OFF = 6.7 ± 0.63 Hz), a change that we also observed with activation of the direct feedback projections ( Figure 2A , left). Similar to PV interneurons, activating SST interneurons decreased spontaneous activityterm and tone-evoked response magnitudeterm in AC ( Figure 9F ; spontaneous activityterm: p=1.4e-12, ON = 0.97 ± 0.25 Hz, OFF = 2.9 ± 0.37 Hz; tone-evoked response magnitudeterm: p=1.3e-15, ON = 3.3 ± 0.59 Hz, OFF = 11.4 ± 1.2 Hz) and increased frequency selectivityterm ( Figure 9G ; p=1.6e-13, ON = 0.69 ± 0.024, OFF = 0.47 ± 0.019). In AC, suppressing SSTs reduced spontaneous activityterm, but had no significant effect on tone-evoked response magnitudeterm or frequency selectivityterm ( Figure 9I,J ; p=8.1e-4, ON = 3.6 ± 0.41 Hz, OFF = 2.2 ± 0.38 Hz). This lack of effect was not due to the relatively small effect of light penetrating to the deep layers. In fact, to confirm that modulating PV and SST activity in AC affected activity of units in L5/6 where the feedback projections we computed changes in spontaneous activityterm at each tetrode which spanned the entire auditory cortexterm. We found that activity was modulated across the layers ( Figures 8 – 9 H,K). Whereas modulating PVs did not have an effect on IC activity, SST suppression resulted in an increase in spontaneous, but not tone-evoked activity in IC, which suggests that inhibitory modulation of sound responses in AC does not propagate to IC.

Figure 9. Effects of modulating SST interneurons in AC on tone-evoked responses in IC and AC.

On box plots, red line indicates median, white ◆ indicates mean. ( A ) Stain for SST (center) and opsin-GFP (left). Scale bar: 50 um. ( B ) Activating SSTs had no effect on spontaneous activityterm or tone-evoked response magnitudeterm in IC. Magenta circles indicate single units. ( C ) Activating SSTs had no effect on frequency selectivityterm in IC. ( D ) Suppressing SSTs increased spontaneous activityterm (p=0.029, ON = 6.9 ± 0.66 Hz, OFF = 6.7 ± 0.63 Hz) but did not affect tone-evoked response magnitudeterm in IC. Magenta circles indicate single units. ( E ) Suppressing SSTs had no effect on frequency selectivityterm in IC. ( F ) Activating SSTs decreased spontaneous activityterm (p=1.4e-12, ON = 0.97 ± 0.25 Hz, OFF = 2.9 ± 0.37 Hz) and tone-evoked response magnitudeterm (p=1.3e-15, ON = 3.3 ± 0.59 Hz, OFF = 11.4 ± 1.2 Hz) in AC. ( G ) Activating SSTs increased frequency selectivityterm in putative excitatory units in AC (p=1.6e-13, ON = 0.69 ± 0.024, OFF = 0.47 ± 0.019). ( H ) Activating SSTs affected putative excitatory units across all layers of AC. ( I ) Suppressing SSTs increased spontaneous activityterm (p=8.1e-4, ON = 3.6 ± 0.41 Hz, OFF = 2.2 ± 0.38 Hz) but did not affect tone-evoked response magnitudeterm in AC. ( J ) Suppressing SSTs had no effect on frequency selectivityterm in AC. ( K ) Suppressing SSTs affected putative excitatory units across all layers of AC. B,D,F,I Left panels: neuronal activity (spontaneous, left or tone-evoked, right) on laser on versus laser off trials. Right panels: Average neuronal activity (spontaneous, left or tone-evoked, right) for laser on and laser off trials. C,E,G,J Left panels: Sparsenessterm for laser on versus laser off trials. Right panels: Average sparsenessterm for laser on and laser off trials. ( H,K ) Change in spontaneous activityterm (laser on trials – laser off trials), left panels: for units at each tetrode; right panels: separated into supragranular: tetrodes 1–3; granular: tetrodes 5,6; infragranular: tetrodes 7–10.

Discussion Auditory cortexterm sends extensive projections to IC ( Bajo and Moore, 2005 ; Bajo et al., 2007 ; Coomes et al., 2005 ; Doucet et al., 2003 ; Saldaña et al., 1996 ; Winer et al., 1998 ). Our results demonstrate that activation of this cortico-collicular feedbackterm modulates sound responses in IC by upregulating spontaneous IC activity, decreasing frequency selectivityterm and reducing spectro-temporal receptive fieldterm size in IC ( Figures 2 , 3 , 4 ). This effect was localized to DCICterm, corresponding to the sites with largest density of AC projections ( Figure 5 ). Interestingly, suppressing cortico-collicular feedbackterm had little effect on IC activity ( Figures 2 , 3 ), which suggests that at baseline and during passive tone presentation the feedback does not affect activity of IC neurons. We also found that SST, but not PV inhibitory interneurons modulated IC spontaneous activityterm, suggesting that the effects of modulation of cortical activity by PVs does not back-propagate to IC, whereas SST-driven modulation affects spontaneous activityterm, but not tone-evoked responses ( Figures 8 , 9 ). Overall our findings imply that direct cortico-collicular feedbackterm can modulate responses to simple (pure tones) and more complex (DRCs) auditory stimuli by reducing, rather than increasing, sound selectivity. This modulation occurs independently of the activity of cortical inhibitory interneurons.

Whereas optogenetic manipulation allows for temporally precise control and cell-type specificity, the technique lacks the spatial specificity of electrical stimulation. Since both AC and cortico-collicular projections are tonotopically organized ( Barnstedt et al., 2015 ; Lim and Anderson, 2007 ; Markovitz et al., 2013 ; Straka et al., 2015 ), spatially specific stimulation can more accurately mimic frequency-specific responses by activating specific regions in the tonotopic map. Previous studies found that electrical stimulation of AC caused best frequencies in IC to shift towards the best frequencies of the stimulated site in AC ( Gao and Suga, 1998 ; Gao and Suga, 2000 ; Ma and Suga, 2001a ; Yan et al., 2005 ; Yan and Suga, 1998 ; Zhou and Jen, 2007 ). These results suggest that cortico-collicular feedbackterm may be important for increasing the representation of behaviorally relevant stimuli in IC. However, when activating direct feedback, we did not observe consistent changes in best frequencyterm of IC neurons ( Figure 6D ). This may be due to activation of feedback projections across cortex, and therefore across the tonotopic map, in this paradigm.

Previous studies found no effect of activating projection terminals on responses in the CNICterm, only shell regions of IC ( Xiong et al., 2015 ), which are the predominant targets of cortico-collicular feedbackterm. In our study we were able to record from a larger neuronal population, revealing a subset of neurons in central IC that were modulated by feedback. This is consistent with another study which observed enhanced white noise-induced activity in a small subset of neurons in the CNICterm with activation of direct cortico-collicular feedbackterm ( Vila et al., 2019 ). The effects in central IC may be due to direct cortico-collicular feedbackterm, intra-collicular circuits ( Malmierca et al., 1995 ; Miller et al., 2005 ; Saldaña and Merchań, 1992 ; Sturm et al., 2014 ), or a combination of these mechanisms.

Previous inactivation studies have shown mixed effects on sound-evoked responses in IC, but consistently demonstrated no effect on frequency selectivityterm, agreeing with our results. Specifically, suppression of AC increased or decreased IC sound responses in distinct subsets of cells ( Popelár et al., 2003 ; Popelář et al., 2016 ) while suppression of direct cortico-collicular feedbackterm terminals in IC decreased sound-evoked responses ( Xiong et al., 2015 ). It is possible that we saw little effect from suppression of cortico-collicular projections because inactivation through the auditory cortexterm suppressed only a subset of feedback projections to IC. Combined, these studies suggest that at baseline the cortex provides only weak modulation of IC activity.

Understanding how AC modulates sound responses in central IC may provide insight into how AC drives changes in auditory behavior. Previously, we found that behavioral frequency discrimination acuity changed after differential auditory fear conditioning and these changes were driven by the auditory cortexterm ( Aizenberg and GeffenpersName, 2013 ; Aizenberg et al., 2015 ). The behavioral task used to test frequency discrimination acuity was a modified pre-pulse inhibition (PPI) of the acoustic startle reflex task. Although AC can modulate this behavior, PPI is still observed after decerebration ( Li and Frost, 2000 ) and the underlying circuit is believed to be subcortical ( Fendt et al., 2001 ). The IC is a critical structure in the PPI circuit ( Fendt et al., 2001 ; Leitner and Cohen, 1985 ; Li et al., 1998 ), with the central nucleus of IC receiving ascending auditory projections and the external nucleus of IC acting as the output station ( Fendt et al., 2001 ; Li and Yue, 2002 ). The broad frequency tuning in the shell regions of the IC ( Barnstedt et al., 2015 ; Syka et al., 2000 ) made the central nucleus of IC, which has sharper tuning and a tonotopic organization ( Barnstedt et al., 2015 ; Ehret et al., 2003 ; Malmierca et al., 2008 ; Syka et al., 2000 ), a good candidate for how AC may drive changes in frequency discrimination acuity. Limited evidence for a descending pathway from AC to the pedunculopontine tegmental nucleus ( Schofield and Motts, 2009 ) suggests another alternative pathway by which AC modulates frequency discrimination.

We activated cortico-collicular neurons by shining light in the cortex, thus initiating spikes at the level of the cell body as would occur in response to a sound. An important consideration with this method, however, is that these neurons do not only send collaterals to IC, but also to the auditory thalamus ( Asokan et al., 2018 ; Williamson and Polley, 2019 ), amygdala ( Asokan et al., 2018 ), and striatum ( Asokan et al., 2018 ). There is also evidence of descending projections to IC from thalamus ( Winer et al., 2002 ) and amygdala ( Marsh et al., 2002 ). When interpreting our results, it is important to consider that the effects we observe from activating cortico-collicular feedbackterm may also be due, at least in part, to these secondary pathways. One caveat is that we used a square pulse for optogenetic manipulations, whereas a more complex stimulation pattern potentially can achieve stronger activation ( Vila et al., 2019 ).

One caveat of our study is that our experiments were performed in passively listening mice. It is possible that the lack of effect we observe with suppression of cortico-collicular feedbackterm is due to the lack of task engagement. Previous studies have found that inactivation of this pathway affects innate responses to sound ( Xiong et al., 2015 ) and auditory learning ( Bajo et al., 2010 ). Furthermore, studies have found that task engagement modulates neuronal responses to sounds ( Downer et al., 2017 ; Fritz et al., 2003 ; Kuchibhotla et al., 2017 ; Lakatos et al., 2013 ; McGinley et al., 2015 ). Thus, we would expect suppression of this pathway to affect sound processing in IC during a behavioral task. Future studies should explore how activity changes with an animal actively engaged in a behavioral task.

We observed little effect of modulating AC inhibitory interneurons on activity in IC despite the changes observed in AC ( Figures 8 and 9 ), which suggests there may be another neuron subtype that plays a modulatory role during cortico-collicular plasticity in driving feedback modulation. One possibility that was not explored here are cholinergic inputs from the nucleus basalis (NB). NB, a cholinergic nucleus in the basal forebrain, has been implicated as a key structure in learning-induced auditory plasticity. Studies have found that pairing NB stimulation with presentation of a tone has been sufficient to induce receptive field plasticity in AC ( Kilgard and Merzenich, 1998 ; Kilgard et al., 2001 ; Weinberger, 2003 ) and that blocking muscarinic receptors in AC impairs this plasticity ( Miasnikov et al., 2008 ). Of greater interest is the receptive field plasticity also observed in IC. Acetylcholine applied to AC increased spontaneous and tone-evoked activity in both AC and IC ( Ji et al., 2001 ). Furthermore, pairing AC stimulation with nucleus basalis stimulation ( Ma and Suga, 2003 ) or pairing tone presentation with nucleus basalis stimulation ( Zhang et al., 2005 ) shifted best frequencies in both AC and IC and application of atropine in AC inhibited this IC plasticity ( Zhang et al., 2005 ). It is plausible that NB cholinergic inputs may be responsible for driving responses of IC neurons rather than PV or SST inhibitory interneurons. However, SSTs in AC are depolarized by application of cholinergic agonist ( Kuchibhotla et al., 2017 ). While the lack of effect we observe with interneuron inhibition might suggest that SST and PV interneurons are not involved in this circuit, we must also consider that direct activation of feedback is not exactly equivalent to disinhibition. Direct activation causes synchronized activation of cells, while disinhibition allows for asynchronous activation. This difference may explain the different results. Furthermore, the lack of effect we observe with inhibitory interneuron activation is consistent with our results from suppressing cortico-collicular feedbackterm. Thus, while another neuronal subtype may be responsible for driving feedback activity in a behavioral context, inhibitory interneurons may still play a role in shaping responses of feedback once active.

Materials and methods Key resources table.

Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background ( Mus musculus ) Pvalb-Cre mice Jackson Laboratories B6;129P2-Pvalbtm1(cre)Arbr/J RRID: IMSR_JAX:008069 Strain, strain background ( Mus musculus ) Sst-Cre mice Jackson Laboratories Ssttm2.1(cre)Zjh/J RRID: IMSR_JAX:013044 Strain, strain background ( Mus musculus ) Wild type Jackson Laboratories C57BL/6J RRID: IMSR_JAX:000664 Strain, strain background ( Mus musculus ) Cdh23 mice Jackson Laboratories Cdh23tm2.1Kjn/J RRID: IMSR_JAX:018399 Recombinant DNA reagent AAV9-CAG-FLEX-ChR2-tdTomato Penn VectorCore Addgene 18917 Recombinant DNA reagent AAV9-CAG-FLEX-ArchT-GFP UNC Vector Core Addgene 99039 Recombinant DNA reagent AAV9-CAG-FLEX-ArchT-tdTomato UNC Vector Core Addgene 28305 Recombinant DNA reagent RetroAAV2 hSyn Cre-GFP In house Vector generated and maintained in the di BiasipersName lab Software, algorithm Matlab Mathworks Mathworks.com RRID: SCR_001622 Software, algorithm ImageJ NIH RRID: SCR_003070 Animals All experiments were performed in adult male and female mice (supplier: Jackson Laboratories; age, 12–15 wk; weight, 22–32 g; wild-type C57BL/6J RRID: IMSR_JAX:000664 ; Pvalb-Cre mice, strain: B6;129P2-Pvalbtm1(cre)Arbr/J RRID: IMSR_JAX:008069 ; Sst-Cre mice, strain: Ssttm2.1(cre)Zjh/J RRID: IMSR_JAX:013044 ; Cdh23 mice, strain: Cdh23tm2.1Kjn/J RRID: IMSR_JAX:018399 , or Pvalb-Cre x Cdh23 or Sst-Cre x Cdh23 crosses). Mice were housed at 28°C on a 12 hr light–dark cycle with water and food provided ad libitum, less than five animals per cage. In Pvalb-Cr e mice Cre recombinase (Cre) was expressed in parvalbumin-positive interneuronsterm, and in Sst-Cre , Cre was expressed in somatostatin-positive interneuronsterm. All animal work was conducted according to the guidelines of University of Pennsylvanian IACUC (protocol number 803266) and the AALAC Guide on Animal Research. Anesthesia by isoflurane and ketamine and euthanasia by CO2 were used. All means were taken to minimize the pain or discomfort of the animals during and following the experiments. All experiments were performed during the animals' dark cycle. Original spike data and code are available on Dryad ( http://doi.org/10.5061/dryad.1t61c80 ).

Viral vectors Modified AAVs encoding ArchTterm (AAV9-CAG-FLEX-ArchT-GFP or AAV9-CAG-FLEX-ArchT-tdTomato; UNC Vector Core) or ChR2term (AAV9-CAG-FLEX-ChR2-tdTomato; Penn Vector Core) were used for selective suppression or excitation, respectively. Retrograde AAV virus encoding Cre (retro AAV-hSyn-Cre-GFP) was custom made in our laboratory. Briefly, RetroAAV2 hSyn Cre-GFP was packaged using the Helper-Free system (Agilent) and the retrograde trafficking plasmid, Retro2, which bears capsid mutations in serotype 2.

Surgery and virus injection At least 21 days prior to electrophysiological recordings, mice were anesthetized with isoflurane to a surgical plane. The head was secured in a stereotactic holder. The mouse was subjected to a small craniotomy (2 × 2 mm) over AC under aseptic conditions. Viral particles were injected (750 nl) bilaterally using a syringe pump (Pump 11 Elite, Harvard Apparatus) targeted to AC (coordinates relative to bregma: −2.6 mm anterior,±4.3 mm lateral, +1 mm ventral). Fiber-optic cannulas (Thorlabs, Ø200 μm Core, 0.22 NA) were implanted bilaterally over the injection site at depth of 0.5 mm from the skull surface. For a subset of mice, to target direct feedback, the mouse was also subjected to a craniotomy over IC (1 × 4 mm). Retro AAV viral construct was injected (3 × 200 nl) via glass syringe (30–50 um diameter) using a syringe pump (Pump 11 Elite, Harvard Apparatus) bilaterally in IC. Craniotomies were covered with a removable silicon plug. A small headpost was secured to the skull with dental cement (C and B Metabond) and acrylic (Lang Dental). For postoperative analgesia, slow release Buprenex (0.1 mg/kg) and Bupivicane (2 mg/kg) were injected subcutaneously. An antibiotic (5 mg/kg Baytril) was injected subcutaneously daily (for 4 days) at the surgical site during recovery. Virus spread was confirmed in all mice postmortem by visualization of the fluorescent protein expression in fixed brain tissue, and its colocalization with PV or SST or, for feedback cohort, expression in AC layer 5term/6, following immuno-histochemical processing with the appropriate antibody.

Confocal imaging To confirm the location of viral injection sites, tissue sections containing the IC or AC were cut at 40 μm using a cryostat. Sections were mounted onto glass slides and imaged using Zeiss LSM 800 confocal microscope. 20X images were taken throughout the entire IC or AC, and tiles were stitched together to form a composite image using Zen software. Masks were drawn around the edge of the tissue to hide the embedding medium using Photoshop.

Acoustic stimuli Stimuli were delivered via a magnetic speaker (Tucker-Davis Technologies) directed toward the mouse’s head. Speakers were calibrated prior to the experiments to ± 3 dB over frequencies between 3 and 70 kHz by placing a microphone (Brüel and Kjaer) in the location of the ear contralateral to the recorded AC hemisphere, recording speaker output and filtering stimuli to compensate for acoustic aberrations ( Carruthers et al., 2013 ). Free-standing speaker was used to approximate the conditions under which the mouse typically experiences sounds in awake state.

Clicks To obtain a quick assessment of auditory onset responses we used click trains. Click trains were composed of six 50 ms clicks at 70 dB sound pressure level relative to 20 microPascals (SPL) with a 50 ms ISI. Click trains were repeated 120 times with a 450 ms ISI between trains. Alternating click trains were also paired with 1 s laser stimulation beginning 250 ms prior to click train onset.

Frequency tuning stimuli To measure tuning for direct feedback cohorts, a train of 50 pure tones of frequencies spaced logarithmically between 3 and 70 kHz, at 70 dB SPL in pseudo-random order was presented 20 times. Each tone was 50 ms duration (5 ms cosine squared ramp up and down) with an inter-stimulus interval (ISI) of 450 ms. Alternating tones were paired with continuous 250 ms laser pulse at either −100 ms, −20 ms, or +8 ms onset relative to tone onset.

For Sst-Cre and Pvalb-Cre cohorts, a train of pure tones of 35 frequencies spaced logarithmically between 3 and 70 kHz and 8 uniformly spaced intensities from 0 to 70 dB SPL were presented 10 times in a pseudo-random order. Alternating tones were paired with continuous 250 ms laser pulse at −100 ms relative to tone onset.

Dynamic Random Chordsterm (DRCs) To measure spectro-temporal receptive fieldsterm (STRFs) we constructed DRCs from 20 ms chords (with 1 ms ramp) of 50 frequencies spaced logarithmically between 5 and 40 kHz with average intensity of 50 dB SPL and 20 dB SPL standard deviation selected from a uniform distribution for each chord. Total duration was 40 min with a 250 ms continuous laser pulse presented every 1 s.

Electrophysiological recordings All recordings were carried out inside a double-walled acoustic isolation booth (Industrial Acoustics). Mice were placed in the recording chamber, and a headpost was secured to a custom base, immobilizing the head. Activity of neurons in AC were recorded via a custom silicon multi-channel probe (Neuronexus), lowered in the area targeting AC via a stereotactic instrument following a craniotomy at a 35-degree angle. The electrode tips were arranged in a vertical fashion that permits recording the activity of neurons across the depth of the auditory cortexterm and the inferior colliculusterm. Activity of neurons in IC were recorded via the same custom probes, lowered in the area targeting IC via a stereotactic instrument following either a craniotomy ( Sst-Cre and Pvalb-Cre cohorts) or removal of the silicon plug, vertically. Recording sitesterm·var in IC were identified based on anatomical markers and stereotaxic coordinates as dorsal cortex (<0.5 mm of midline) or central nucleus of IC (center of central nucleus of IC ~1 mm lateral of midline). Recordings were made in multiple locations across IC. Electro-physiological data from 32 channels were filtered between 600 and 6000 Hz (spike responses), digitized at 32 kHz and stored for offline analysis (Neuralynx). Spikes belonging to single neurons and multi-units were detected using commercial software (Plexon). Original spike data and code are available on Dryad ( http://doi.org/10.5061/dryad.1t61c80 ). We examined the experimental groups described in Table 1 (note: we do not distinguish here between Sst-Cre / Pvalb Cre and Sst-Cre x Cdh23 and Pvalb-Cre x Cdh23 ). Initial experiments were performed under anesthesia to control for stability or recordings. For cohorts with awake and anesthetized recordings data were analyzed separately, but we observed no difference in our results so data were combined ( Figure 8—figure supplement 1 ). We used power analysis for effect size of 25% and expected variance in firing rate (50%) to determine the minimum total number of units for each experimental group at N = 44. All groups included a greater number of units (single- and multi-units were combined in analysis). The number of units in measuring tone-evoked responses are specified in Table 1 . Mice that did not show effect of laser activation or suppression in auditory cortexterm were excluded. We recorded from AC in Cdh23+ChR2term mice and Cdh23+ArchTterm mice to verify an effect in AC ( Figure 3—figure supplement 2 ) in all mice used in our analysis.

Table 1. Number of recorded units and mice used in our analyses for each experimental group and condition.

Group Recording area Single units Multi units Awake total mice Awake total units Anesthetized total mice Anesthetized total units Total units Cdh23-ChR2 IC 4 187 5 Male 191 - - 191 Cdh23-ArchT IC 24 181 4 Male 205 - - 205 PV-ChR2 IC 25 198 4 Male 85 12 Male 138 223 AC 3 64 14 53 67 PV-ArchT IC 6 40 4 Male 46 - - 46 AC 3 56 59 - 59 SST-ChR2 IC 15 90 7 Male 76 8 Male 29 105 AC 9 90 49 50 99 SST-ArchT IC 16 161 2 Male 2 Female 140 4 Male 2 Female 37 177 AC 4 155 123 36 159 Photostimulation of neuronal activity Neurons were stimulated by application of continuous light pulse delivered from either blue (473 nm, BL473T3-150, used for ChR2term stimulation) or green DPSS laser (532 nm, GL532T3-300, Slocs lasers, used for ArchTterm stimulation) through implanted cannulas. Laser power measured through cannulas was 3 mW. Timing of the light pulse was controlled with microsecond precision via a custom control shutter system, synchronized to the acoustic stimulus delivery.

Neural response analysis Unit selection Units were selected based on pure-tone responsiveness. For each unit we identified the 7 frequencies that elicited the highest response and averaged activity across these trials (and the highest 3 amplitudes for stimulus with multiple amplitudes). Units with tone-evoked activity (75 ms window after tone onset) less than two standard deviations above the spontaneous activityterm (50 ms window prior to tone onset) in no laser condition were excluded from the analysis. Both single units and high quality multi-units (multi-units with <1% of spikes with <1 ms inter-spike-interval) were used.

Spontaneous activityterm and tone-evoked response magnitudeterm Feedback cohort: Spontaneous activityterm was the average firing rate in a 20 ms window (to allow for equivalent comparison between −100 ms and −20 ms laser onsetterm·var conditions) prior to tone onset of top 7 preferred frequencies.

Sst-Cre/Pvalb-Cre cohorts: Spontaneous activityterm was the average firing rate in a 50 ms window prior to tone onset of top 7 preferred frequencies and 3 highest amplitudes.

All mice: Tone-evoked response magnitudeterm was calculated as the difference between the average tone-evoked response in a 75 ms window after tone onset and the spontaneous activityterm.

Sparsenessterm. To examine frequency selectivityterm of neurons, sparsenessterm of frequency tuning was computed as: S p a r s e n e s s = 1 − ( ∑ i = 1 i = n F R i n ) 2 ∑ i = 1 i = n F R i 2 n where FR i is tone-evoked firing rate response to tone at frequency i , and n is number of frequencies used ( Weliky et al., 2003 ). Subgroups of neurons used in sparsenessterm analyses were separated based on changes in spontaneous activityterm or tone-evoked response magnitudeterm in response to cortico-collicular activation or suppression. Units were selected if > 1 standard deviation change in spontaneous activityterm or tone-evoked response magnitudeterm based on the -100 ms laser onsetterm·var trials.

Linear fits across frequencies Linear fits were calculated using linear regression (fitlm.m; MATLAB)term·stat over 50 data points, one for each of the 50 frequencies tested ( Natan et al., 2017a ). The 50 data points were separately calculated as the mean FR over all repeats of each frequency.

Best Frequencyterm Best frequencyterm was defined as the frequency that elicited the maximum response.

STRF Analysis To calculate the STRF we separated the stimulus into 1 s chunks, concatenating the 250 ms laser ON chunks and the 250 ms laser OFF chunks immediately preceding laser onsetterm·var. These data were then used to calculate the average spectrogram preceding a spike. To allow for finer temporal resolution of the STRFs we upsampled the DRCs using nearest neighbor interpolation. Subsequently we averaged the STRF across the eight stimulus files. To determine the significance of the lobes, the z -score of pixelsterm·stat was computed relative to the baseline values from an STRF generated with scrambled spike trains, using Stat4ci toolbox ( Chauvin et al., 2005 ; Natan et al., 2017b ). We ran this significance test 100 times and any pixel identified as significant more than 90 times was considered significant. Lobes were matched between laser ON and laser OFF trials by comparing the overlap of the lobes, requiring a 50% overlap of the smallest lobe size to be a match. From the STRF, the peak time, temporal width, peak frequency, and frequency width of the positive and negative lobes were measured ( Schneider and Woolley, 2010 ; Shechter and Depireux, 2007 ; Woolley et al., 2006 ).

Noise correlationterm Noise correlationsterm·def were calculated as the pairwise Pearson correlation coefficientterm·stat between the spike counts of units recorded simultaneously. For spontaneous activityterm spikes were counted in a window 20 ms before tone onset and for tone response spikes were counted in a window 75 ms after tone onset.

Statistical analyses Significant differences and P values were calculated using paired Wilcoxon sign-rank testterm (unless noted otherwise) with standard MATLAB routine. For the laser alone data, to compare distributions to standard normal distribution data were normalized by mean and standard deviation and then significant differences and P values were calculated by Kolmogorov-Smirnoff testterm with standard MATLAB routine. Mean ± standard error of the meanterm·stat was reported unless stated otherwise. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

Acknowledgements This work was supported by National Institutes of Health (Grant numbers NIH R03DC013660, NIH R01DC014779, NIH R01DC015527, NIH R01NS113241), Klingenstein Award in Neuroscience, Human Frontier in Science Foundation Young Investigator Award and the Pennsylvania Lions Club Hearing Research Fellowship to MNG. MNG is the recipient of the Burroughs Wellcome Award at the Scientific Interface. JMB was supported by NIH T32MH017168. We thank the members of the GeffenpersName laboratory and the Hearing Research Center at the University of Pennsylvania, including Dr. Steve Eliades and Dr. Yale Cohen for comments on an earlier version of the manuscript. We also thank Sarah Kwon for assistance with confocal imaging.

Funding Statement The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information Maria N GeffenpersName, Email: mgeffen@pennmedicine.upenn.edu.

Barbara G Shinn-Cunningham, Carnegie Mellon University, United States.

Catherine Emily Carr, University of Maryland, United States.

Funding Information This paper was supported by the following grants: National Institute on Deafness and Other Communication Disorders R03DC013660 to Maria N GeffenpersName.

National Institute on Deafness and Other Communication Disorders NIH R01DC014779 to Maria N GeffenpersName.

National Institute on Deafness and Other Communication Disorders NIH R01DC015527 to Maria N GeffenpersName.

Human Frontier Science Program Young Investigator Award to Maria N GeffenpersName.

Pennsylvania Lions Club Hearing Research Fellowship to Maria N GeffenpersName.

Klingenstein Award in Neurosciences to Maria N GeffenpersName.

National Institute of Mental Health 1F32MH120890 to Alexandria MH LesickopersName.

National Institutes of Health R01NS113241 to Maria N GeffenpersName.

Additional information Competing interests No competing interests declared.

Author contributions Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing.

Formal analysis, Validation, Investigation, Visualization, Methodology.

Investigation, Visualization, Methodology.

Resources, Visualization.

Ethics Animal experimentation: All animal work was conducted according to the guidelines of University of Pennsylvanian IACUC (protocol number 803266) and the AALAC Guide on Animal Research. Anesthesia by isoflurane and ketamine and euthanasia by CO2 were used. All means were taken to minimize the pain or discomfort of the animals during and following the experiments. All experiments were performed during the animals' dark cycle.

Additional files Transparent reporting form elife-51890-transrepform.docx (248.5KB, docx) Data availability Original spike data and code are available on Dryad ( http://doi.org/10.5061/dryad.1t61c80 ).

The following dataset was generated: BlackwellpersName JM, LesickopersName A, RaopersName W, De BiasipersName M, GeffenpersName MN. 2019. Data from: The role of feedback from the auditory cortexterm in shaping responses to sounds in the inferior colliculusterm. Dryad Digital Repository.

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Nature. 1994;370:140–143. doi: 10.1038/370140a0. [ DOI ] [ PubMed ] [ Google Scholar ] eLife. doi: 10.7554/eLife.51890.sa1 Decision letter Editor: Catherine Emily Carr 1 Reviewed by: Sarah Woolley , Heather Read 1 University of Maryland, United States Roles Catherine Emily Carr : Reviewing Editor Sarah Woolley : Reviewer Heather Read : Reviewer In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary: Sensory processing is typically studied along an ascending hierarchy of brain areas, often ignoring feedback between these areas. The auditory cortexterm sends an extensive feedback to the inferior colliculusterm, and the authors have found that activation of cortico-collicular feedbackterm reduces sound selectivity in the inferior colliculusterm by increasing neuronal firing overall, and by attenuating responses in preferred frequency bands. Interestingly, this modulation occurs independently of the activity of cortical inhibitory interneurons. Suppression of this feedback also had no effect on sound-evoked responses in the inferior colliculusterm, suggesting that during passive sound presentation feedback does not shape activity in the inferior colliculusterm.

Decision letter after peer review: [Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.] Thank you for submitting your work entitled "The role of feedback from the auditory cortexterm in shaping responses to sounds in the inferior colliculusterm" for consideration by eLife . Your article has been reviewed by three peer reviewers, including Catherine Emily Carr as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Sarah Woolley (Reviewer #3).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife .

The major concerns of the reviewers were similar, and focused on controls that we think you could carry out with more extensive analyses of your existing data set. We recognize major strengths of the study, which are that your results provide an interesting and detailed picture of how descending input affects IC activity, and align with previous findings.

Our consensus decision is therefore that you should carry out the controls, measurements and analyses needed to address the issues highlighted below. We think this would take too long for a major revision; however, we encourage you to resubmit a manuscript that addresses the points below.

Reviewer #1: This paper uses optogenetic modulationterm·var of cortico-collicular feedbackterm projections to examine sound responses in mouse IC. In addition to activation of this feedback, the paper included experiments to modulate parvalbumin-positive and somatostatin positive inhibitory interneurons. Their main conclusion was that activation of excitatory projections, but not inhibition-driven increases in cortical activity, affected collicular sound responses.

The findings are interesting and surprising. Previous studies had found that electrical stimulation of AC caused best frequencies in IC to shift towards the best frequencies of the stimulated site in AC, and the authors suggest that this may be because the optogenetic stimulation activated whole frequency map, rather than the differently selective electrical stimulation used earlier.

Major concerns: The results are reported as a population that lumps single and multiunit responses, although clear that there are multiple cell types, and IC divisions. The authors observe wide variety of responses to activation of cortico-collicular inputs, shown in the figures. I am less concerned about lumping single and multi-unit responses (although a comparison of the two would be nice), and more concerned about differentiating between the dorsal and central divisions of the IC, since the cortical projection is more prominent in the dorsal regions. The authors state they were able to record from a subset of neurons in central IC, but further support for where they recorded would be useful.

Reviewer #2: This study examined the effects of several optogenetic manipulations of activity in the auditory cortexterm (AC) on various measures of neuronal functionality in the inferior colliculusterm (IC).

One major issue with the study is that the stated goal was to examine the physiological role of projections from the AC to the IC. The first approach taken was then to activate or suppress cortico-collicular projections optogenetically. In order to infer from this what role these projections might play, it is important that these manipulations affect inputs to the IC in a physiologically plausible way. A number of steps would need to be taken in order to determine if this is the case. Firstly, it is not clear how sparse the expression of the retrograde virus is in the AC. Figure 1B suggests that the opsins could be expressed very sparsely, but there is no quantification. Secondly, optogenetic activation provides highly synchronised activation, the magnitude of which may or may not be within physiological ranges. If the manipulations lead to changes in cortico-collicular activity that are not within these ranges, it is not clear what conclusion can be drawn regarding the role of these inputs generally. The second part of the study that approaches the same question by manipulating the activity of inhibitory subpopulations in the cortex would seem to be a better approach. Effectively disinhibiting the cortex should have the effect of asynchronously activating excitatory input to the IC, but in this case little to no effect was observed in the IC.

A second major issue is related to the statistical analyses employed. The first section of the paper claims that activating direct cortico-collicular feedbackterm modulates activity in the IC. The initial experimental approach used was to optogenetically activate or suppress IC-projecting neurons in the AC and measure change in spontaneous activityterm in the inferior colliculusterm. It would then make sense to conduct a statistical analysis that addresses the question of whether the spontaneous rate of neuronal units in the IC is different in the laser ON conditions vs the laser OFF conditions. Instead, the authors have chosen to use a Kolmogorov-Smirnoff testterm to ask when the distribution of change in firing rate was normally distributed. However, it is not possible to conclude from this that the cortical activation actually has caused a significant change. Instead, it would be appropriate to conduct a dependant-samples t-test for laser ON vs laser OFF firing rates (or indeed a Wilcoxon sign-rank testterm if these data are not normally distributed, as is applied elsewhere in the manuscript). Looking at the data presented in Figure 1E suggests that this would be unlikely to find any significant changes. If anything, the central tendency of the distribution of changes is in the opposite direction to that concluded (and expected) by the authors: in Figure 1E, peaks in the distribution appear to be slightly below zero. The mean and median values appear to be skewed away from this peak by a very small number of units showing high deviations in firing rate.

Reviewer #3: This study consists of two major groups of experiments investigating the effects of descending inputs from the auditory cortexterm (AC) to the inferior colliculusterm (IC) on spontaneous and sound-evoked activity in IC neurons. First, viral vectors are used to specifically infect AC neurons that project to IC with opsins that excite or suppress AC-IC neurons, and the effects of exciting or suppressing these neurons on IC spontaneous and sound-evoked activity are measured. Second, interneuron-class-specific manipulations in AC are used to test whether inhibitory activity in AC impacts IC spontaneous and sound-evoked activity. The goals of the study are largely exploratory/descriptive, rather than hypothesis-driven. With this in mind, the results are informative despite many negative results. Overall, these experiments show that IC activity is influenced by AC-IC input in very specific ways; spontaneous firing in IC units increases with AC-IC input and the linear mapping of IC responses to specific stimulus frequencies is disrupted by AC-IC input. Important methodological and interpretation-related concerns need to be addressed, however.

1) The effects of optogenetic excitation and suppression of AC neurons projecting to IC on IC responses were small to none (Figures 1-3). These results prompt a few methodological concerns.

A) Is it possible that the viral infection methods used to specifically label AC-IC neurons resulted in low rates of infection? This would limit the effects that AC manipulation could have on activity in the IC.

B) Is it possible that the light stimulation did not reach layer 5term with enough power to effectively manipulate AC-IC neurons? This is addressed for the PV and SOM interneuron manipulations, but not for the AC-IC projection neurons.

C) It isn't clear from the Results that the recorded IC neurons were in the spatial location of AC-IC termination.

D) Based on Figures 1, AC stimulation significantly affected a small number of IC units, resulting in small or no average differences between the light on and light off conditions. Were effects of AC manipulation on frequency tuning analyzed separately for these units?

E) The use of response magnitude (difference between evoked and spontaneous rates) to measure effects of AC manipulation on tone evoked responses could explain why tone-evoked responses appeared to decrease with AC stimulation because spontaneous activityterm increased. Were firing rates evoked by the same frequencies with laser on and laser off compared for each neuron? This would avoid conflation with the laser on-laser off differences in spontaneous activityterm.

2) The results of the experiments using tones and clicks and those of experiments using the dynamic random chordterm (DRC) stimulus are difficult to reconcile. Figure 4 shows what appear to be large effects of AC stimulation on IC receptive fields (STRFs). Given this, the explanation for the disappearance of significant positive and negative lobes with laser stimulation should be pursued. For example, if the interpretation given at the end of subsection “Activating direct cortico-collicular feedbackterm modulates activity in the inferior colliculusterm” should be tested by going back to the tone-evoked responses and measuring effects of AC stimulation on responses to tones outside of the 7 frequencies that evoked the largest responses from a unit.

3) So much work clearly went into the PV and SOM interneuron manipulations and recordings in Ac and IC. The results of those experiments and interpretation of the results should be further explained. For example, why were the effects of interneuron manipulation observed in AC but not IC units?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.] Thank you for resubmitting your work entitled "The role of feedback from the auditory cortexterm in shaping responses to sounds in the inferior colliculusterm." for further consideration by eLife . Your revised article has been evaluated by Barbara Shinn-Cunningham (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: Summary The manuscript had been previously reviewed at eLife , and rejected because the reviewers felt that the controls, measurements and analyses needed to address the issues raised by reviewers would take too long for a major revision; however, we encouraged resubmission. The new manuscript has been reviewed, and we felt that the study and topic area merit publication in eLife .

Nevertheless, several important caveats to the approach and analyses remain, which could be addressed in the Materials and methods and/or Discussion. We will attach the relevant reviews in addition to the summary here, and suggest that you acknowledge and address the following points in your revised submission. Other thoughtful suggestions follow, and we include the reviews with suggestions. The other reviewers had no major changes.

Major changes: 1) That there are potential limitations of using rectangular laser pulses as described previously (Vila et al., 2019) 2) That in spite of the cell-specific expression there hypothetically could be mixed suppressive and excitatory effects that are lost in multi-unit spike rate and spectro-temporal receptive field (STRF)term·def analyses used here.

3) There are also concerns about normality in spike rate distributions. With respect to the spike rate effects in the new Figure 5, panel A (dorsal IC, -100 ms laser condition), if the spike rate distributions are not normal then the bar graphs could be replaced with box-plots emphasizing that there are indeed spike-rate outliers. Note that the appropriate statistical comparisons should be performed.

Here are the full reviews that include suggestions for revision. The main points are summarized above.

Reviewer #1: 1) In their revision, the authors have sidestepped one of my major concerns, namely: "Secondly, optogenetic activation provides highly synchronised activation, the magnitude of which may or may not be within physiological ranges. If the manipulations lead to changes in cortico-collicular activity that are not within these ranges, it is not clear what conclusion can be drawn regarding the role of these inputs generally." At the very least, the authors should explicitly acknowledge the limitations of using rectangular laser pulses in this context and the associated limitations to the generality of their conclusions. For example, a recent study addressing the same questions has shown that these effects in the IC strongly depend on patterns of laser activation in the AC (Vila et al., 2019).

2) A new important issue has appeared in the revised manuscript, which merits careful consideration. The new Figure 5 is used to support the conclusion that "…units recorded from dorsal recording sitesterm·var are significantly affected by activation of feedback, but centrally located units are not". However, the firing rates of some dorsal units are twice as high as the central ones'. Compare the scales in panels A and B. Could the effect in, for example, -100 ms tone-evoked "dorsal" be due to those high-firing-rate units being suppressed by the laser? One can clearly see that those data points are below the diagonal. It would be crucial to reanalyse the data presented in panel A, where the firing rates are up to 100 Hz, by using only those units whose firing rates are comparable to those in panel B, i.e., less than 40 Hz. Otherwise, the conclusion might reflect the effect of the laser on the small number of relatively rare units with high firing rates, disproportionately suppressed by the laser. As those cells are relatively rare (about 5%?), they might have been missed in the central IC (71 units; versus 120 units in the dorsal IC). Without this analysis, I am not convinced by the authors' conclusion.

3) The authors have indicated in the revision letter which data points corresponded to single units (red dots). This information is very useful. Yet they chose not to indicate it in the revised manuscript. Why? I think it must be indicated.

4) In general, my concern with multi-unit data is that it is possible, given the bidirectional effects of the laser stimulation, that some nearby neurons are modulated positively and other neurons are modulated negatively. If these neurons are recorded as a multiunit, then the total effect will be a weighted sum of the individual effects. This will bias not only the determination of the effects on the firing rates, but may also induce artefacts in the calculation of STRFs, unless all individual neurons contributing to the multiunit have the same selectivity. However, since they were not recoded as single units, their individual selectivity is unknown. You can see, for example, that the receptive field in Figure 7A 'laser on" differs significantly from that in the "laser off" condition. These are units that increase their firing rate with the laser on. Which means there are more spikes; yet the STRF almost vanishes. This illustrates what I have said above: what the authors interpret as a change in selectivity could be due to a change in the firing rates of differentially tuned single units, all contributing to the vector sum during STRF calculation, without any change in individual neurons' selectivity.

5) The authors persist in using bars and standard errors for data that are so obviously non-normally distributed.

Reviewer #2: The authors have thoroughly responded to reviewers' critiques with new analyses, revised figures and additional information in the Materials and methods, Results and Discussion. I am satisfied that this paper will contribute importantly to our understanding of how the auditory system's descending projections shape sound coding.

Reviewer #3: Clearly cortical feedback to collicular structures including superior and inferior colliculi plays a pivotal role in shaping neural pathway responses and behavior and yet little is known about auditory circuits mediating this process. Prior studies have dumped pharmacological agents on cortex or electrically stimulated cortex to explore the sound processing of these feedback circuits. However, these manipulations are not cell-type specific. This is a rigorous and well-executed study demonstrating for the first time cell-types involved in cortical feedback shaping of sound evoked responses in central and dorsal IC. I have only very minor comments and suggestions to improve the write up as outlined below.

Title, "Role of feedback from auditory cortexterm in shaping responses to sounds in inferior colliculusterm" Title.

1) Shorten title?, e.g. "Shaping inferior colliculusterm responses to sound through cortical feedback" Introduction section.

1) Please open with a paragraph explaining why we care about cortico-collicular feedbackterm? You end paragraph two with some discussion of why we may care. Maybe round this out and move up to the top paragraph of Introduction.

2) Introduction paragraph three, add a captivating introduction sentence to the paragraph, e.g., "Cortical output modulations of collicular activity is not a monolithic process but instead is shaped by interactions between excitatory and inhibitory cortical neurons." 3) Introduction paragraph two, you have a long sentence "Whereas inactivation studies, on the other hand, found less consistent effects on IC responses.…) Please simplify. Perhaps, "Prior studies find pharmacological inactivation of AC alters tone-evoked responses of IC neurons (Zhang, 1997) whereas others do not (Jen, 1998)." Is the Jen, 1997 study using pharma to inactivate AC?

Terminology throughout manuscript 1) Non-specialist audiences will not get what the difference is between the dorsal cortex of IC and central nucleus. In parts of the manuscript you refer to central nucleus simply as "IC" whereas others it is "central IC". Please lay this out clearly and refer to all your recordings that are verified as being in Central IC as being in CIC being consistent with your abbreviation for dorsal cortex as DCICterm.

Materials and methods.

1) In Materials and methods, clearly explain your rationale for doing these initial experiments under anesthesia e.g., " To obtain precise responses across large numbers of IC neurons, these experiments were carried out under anesthesia. Though cortical spike rate responses are reduced under anesthesia, the general frequency response organization remains the same. " 2) “Acoustic stimuli” subsection, please give your rationale for delivering sound from a free field speaker. Perhaps, one rationale is to more closely approximate conditions animals may experience in awake conditions? Note the caveats. If you are calibrating next to the contralateral ear in free field and then using a filter to compensate for sound reflections -presumably you have a means of insuring that the set-up (stereotax unit, electrode holders, amplifiers etc are in fixed spatial positions relative to the ear and you have distinct filters for left and right ear configurations). Using ear-tubes may be a better approach as the mice have relatively uniform ear canals and calibrating can be more precise.

3) “Acoustic stimuli” section, provide a rationale for including click train responses in your sound battery. Click train responses have been used in the past for ABR and penetrating electrode studies but these sounds are fraught with spectral splatter that makes interpretation of the response relatively difficult. Perhaps, your rationale was to get a quick assessment of auditory onset responses?

“Neural Response Analysis” subsection, you define FRi as "tone-evoked response" here and above (describe that you're assessing firing rate). Can you please explain that Fri is a "tone-evoked firing rate response"? Also please explain the rationale for computing sparsenessterm and the rationale for your 20 ms time scale for doing so. For example, there are prior studies that have compared cortical and collicular time-scales for sparse coding and find that central IC has an order of magnitude shorter integration times (~10ms) than cortex and that on those time scales encoding in IC is indeed sparse (Chen, Read, Escabi, J Neurosci 2012; https://www.ncbi.nlm.nih.gov/pubmed/22723685).

Results section.

1) Results section, first paragraph. The authors are commended for carrying out these important, rigorous and difficult studies with cell specific modulation and e-phys but they should recognize that most of your audience is less informed about theses dual viral techniques. Please do a better job of setting up this paragraph so as not to lose the general audience. Following the first introduction sentence please help orient the non-expert with something like, "A combination of local and retrograde viral transfections were made in order to achieve cell and circuit specific viral transfections of cortico-collicular neurons".

2) In general, most paragraphs in the Results section do a great job of introducing the rationale for experiments and analyses. Thank you!

3) Discussion paragraph three, here you refer to central IC as "central IC" but many other places throughout the manuscript you are ambiguous e.g. Discussion paragraph one, you say things like "…little effect on IC activity" is this central and dorsal IC? Also you say "effects of modulation of cortical activity by PVs does not backpropagate to IC". Do you DCICterm here?

eLife. 2020 Jan 31;9:e51890. doi: 10.7554/eLife.51890.sa2 Author response Collection date 2020.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.] Reviewer #1: […] Major concerns: The results are reported as a population that lumps single and multiunit responses, although clear that there are multiple cell types, and IC divisions. The authors observe wide variety of responses to activation of cortico-collicular inputs, shown in the figures. I am less concerned about lumping single and multi-unit responses (although a comparison of the two would be nice), and more concerned about differentiating between the dorsal and central divisions of the IC, since the cortical projection is more prominent in the dorsal regions. The authors state they were able to record from a subset of neurons in central IC, but further support for where they recorded would be useful.

This is an important concern, and a very good suggestion. During the experiment, we targeted two recordings in the central nucleus of IC and two recordings in the dorsal cortex of IC (closer to midline). Previously, we combined these recordings together in the analysis. In the revision, we separated the units based on whether the recording siteterm·var was more central or more dorsal. Indeed, we find that units recorded from dorsal recording sitesterm·var are significantly affected by activation of feedback, but centrally located units are not.

We have included these results in Figure 5 and the Results: “Interestingly, these changes are observed almost exclusively in cells located in DCICterm rather than CNICterm (Figure 5). Specifically, activation of feedback increased spontaneous activityterm (Figure 5A, left; -100 ms: p = 0.0204, ON = 3.5 ± 0.5 Hz, OFF = 2.5 ± 0.3 Hz; -20 ms: p = 2.8e-7, ON = 5.2 ± 0.7 Hz, OFF = 2.3 ± 0.3 Hz) and decreased tone-evoked response magnitudeterm (Figure 5A, right, -100 ms: p = 3.3e-6, ON = 14.8 ± 1.4 Hz, OFF = 16.5 ± 1.5 Hz; -20 ms: p = 6.3e-7, ON = 11.8 ± 1.6 Hz, OFF = 14.7 ± 1.4 Hz) in cells located in DCICterm, but CNICterm (Figure 5B). Similarly, activation of feedback decreased frequency selectivityterm (Figure 5C; mag decrease: -20 ms, p = 6.3e-4, sparse ON = 0.46 ± 0.03, sparse OFF = 0.53 ± 0.03; +8 ms, p = 4.7e-5, sparse ON = 0.41 ± 0.03, sparse OFF = 0.53 ± 0.03; spont increase: -100 ms, p = 0.0044, sparse ON = 0.43 ± 0.04, sparse OFF = 0.52 ± 0.04; -20 ms, p = 0.00013, sparse ON = 0.39 ± 0.04, sparse OFF = 0.53 ± 0.04; +8 ms, p = 5.2e-6, sparse ON = 0.33 ± 0.03, OFF = 0.53 ± 0.04) in cells located in DCICterm, but not CNICterm (Figure 5D). This is consistent with the enhanced density of AC projections in dorsal as compared to central IC (Figure 1D).”

We also amended the Materials and methods to clarify identification of our recording sitesterm·var “Recording sitesterm·var in IC were identified based on anatomical markers and stereotaxic coordinates as dorsal cortex (<0.5 mm of midline) or central nucleus of IC (center of central nucleus of IC ~1 mm lateral of midline). Recordings were made in multiple locations across IC.”

Reviewer #2: […] One major issue with the study is that the stated goal was to examine the physiological role of projections from the AC to the IC. The first approach taken was then to activate or suppress cortico-collicular projections optogenetically. In order to infer from this what role these projections might play, it is important that these manipulations affect inputs to the IC in a physiologically plausible way. A number of steps would need to be taken in order to determine if this is the case. Firstly, it is not clear how sparse the expression of the retrograde virus is in the AC. Figure 1B suggests that the opsins could be expressed very sparsely, but there is no quantification.

We compared the strength of opsin expression in slices in multiple mice, and found that the transfection was as strong as was evident in previously published studies. We quantified the expression by counting the number of cell bodies that were expressing the fluorescent protein. We found that in layer 5term of the auditory cortexterm, we had ~5 cell bodies/100um x 100um ROI. This level of expression is similar to that observed by Williamson and Polley, 2019, Figure 1A, left. We now include a new figure (Figure 1) that demonstrates the spread of virus expression in IC, the transfected cells in AC, and the labelled projections within IC. Furthermore, we quantified the distribution of projections in IC, which shows that AC projects not just to the external shell, but also to the central nucleus.

Secondly, optogenetic activation provides highly synchronised activation, the magnitude of which may or may not be within physiological ranges. If the manipulations lead to changes in cortico-collicular activity that are not within these ranges, it is not clear what conclusion can be drawn regarding the role of these inputs generally. The second part of the study that approaches the same question by manipulating the activity of inhibitory subpopulations in the cortex would seem to be a better approach. Effectively disinhibiting the cortex should have the effect of asynchronously activating excitatory input to the IC, but in this case little to no effect was observed in the IC.

This is a great point, and we added in the Discussion: “While the lack of effect we observe with interneuron inhibition might suggest that SOM and PV interneurons are not involved in this circuit, we must also consider that direct activation of feedback is not exactly equivalent to disinhibition. Direct activation causes synchronized activation of cells, while disinhibition allows for asynchronous activation. This difference may explain the different results.”

A second major issue is related to the statistical analyses employed. The first section of the paper claims that activating direct cortico-collicular feedbackterm modulates activity in the IC. The initial experimental approach used was to optogenetically activate or suppress IC-projecting neurons in the AC and measure change in spontaneous activityterm in the inferior colliculusterm. It would then make sense to conduct a statistical analysis that addresses the question of whether the spontaneous rate of neuronal units in the IC is different in the laser ON conditions vs the laser OFF conditions. Instead, the authors have chosen to use a Kolmogorov-Smirnoff testterm to ask when the distribution of change in firing rate was normally distributed. However, it is not possible to conclude from this that the cortical activation actually has caused a significant change. Instead, it would be appropriate to conduct a dependant-samples t-test for laser ON vs laser OFF firing rates (or indeed a Wilcoxon sign-rank testterm if these data are not normally distributed, as is applied elsewhere in the manuscript). Looking at the data presented in Figure 1E suggests that this would be unlikely to find any significant changes. If anything, the central tendency of the distribution of changes is in the opposite direction to that concluded (and expected) by the authors: in Figure 1E, peaks in the distribution appear to be slightly below zero. The mean and median values appear to be skewed away from this peak by a very small number of units showing high deviations in firing rate.

To address this concern, we performed a paired Wilcoxon sign-rank testterm on the laser on vs laser off firing rate of each unit. We still observe significant increases in firing rate with ChR2term activation of AC-IC feedback. We have updated the Results and Figure 2 legend to reflect the new statistics as well as to include mean and s.e.m. of the changes in firing rate for each significant duration. We also updated Figure 2 to include scatter plots to show the changes in firing rate across the population in addition to the distributions.

Figure 2 C: Changes in spontaneous activityterm with activation of AC-IC projections ( 1 ms : p = 5.3e-4, ON = 5.6 ± 0.6 Hz, OFF = 4.9 ± 0.5 Hz; 5 ms : p = 0.0027, ON = 6.9 ± 0.7 Hz, OFF = 4.8 ± 0.5 Hz; 250 ms : p = 0.005, ON = 5.6 ± 0.6 Hz, OFF = 4.8 ± 0.5 Hz) Figure 2 D: Changes in spontaneous activityterm with suppression of AC-IC projections (2 5 ms : p = 0.039, ON = 9.5 ± 0.7 Hz, OFF = 9.5 ± 0.7 Hz; 250 ms : p = 0.02, ON = 9.5 ± 0.8 Hz, OFF = 9.5 ± 0.8 Hz) Reviewer #3: […] Overall, these experiments show that IC activity is influenced by AC-IC input in very specific ways; spontaneous firing in IC units increases with AC-IC input and the linear mapping of IC responses to specific stimulus frequencies is disrupted by AC-IC input. Important methodological and interpretation-related concerns need to be addressed, however.

1) The effects of optogenetic excitation and suppression of AC neurons projecting to IC on IC responses were small to none (Figures 1-3). These results prompt a few methodological concerns.

A) Is it possible that the viral infection methods used to specifically label AC-IC neurons resulted in low rates of infection? This would limit the effects that AC manipulation could have on activity in the IC.

Please see response to reviewer #2, question 1.

B) Is it possible that the light stimulation did not reach layer 5term with enough power to effectively manipulate AC-IC neurons? This is addressed for the PV and SOM interneuron manipulations, but not for the AC-IC projection neurons.

We observe effects of light stimulation across all layers in cortex (Figure 5 H,K; Figure 6 H,K). We use identical procedures for cannula implant and laser power for the AC-IC projection cohorts. Amended Materials and methods to … “We recorded from AC in our Cdh23+ChR2term mice and Cdh23+ArchTterm mice to verify an effect in AC (Figure 3—figure supplement 2) in all mice used in our analysis” … for clarification C) It isn't clear from the Results that the recorded IC neurons were in the spatial location of AC-IC termination.

Please see response to reviewer #1, question 1.

D) Based on Figures 1, AC stimulation significantly affected a small number of IC units, resulting in small or no average differences between the light on and light off conditions. Were effects of AC manipulation on frequency tuning analyzed separately for these units?

Yes. In Figures 3, 5, and 6, we separately analyze frequency selectivityterm for different subsets of neurons based on effect of light on condition. We state this in the Results section, for example “Activation of feedback decreased frequency selectivityterm in the subsets of units that also showed a decrease in tone-evoked response magnitudeterm or increase in spontaneous activityterm, but not in units that showed an increase in tone-evoked response magnitudeterm or decrease in spontaneous activityterm”, as well as in the Materials and methods section “Subgroups of neurons used in sparsenessterm analyses were separated based on changes in spontaneous activityterm or tone-evoked response magnitudeterm in response to cortico-collicular activation or suppression. Units were selected if > 1 standard deviation change based on the -100 ms laser onsetterm·var trials.”

E) The use of response magnitude (difference between evoked and spontaneous rates) to measure effects of AC manipulation on tone evoked responses could explain why tone-evoked responses appeared to decrease with AC stimulation because spontaneous activityterm increased. Were firing rates evoked by the same frequencies with laser on and laser off compared for each neuron? This would avoid conflation with the laser on-laser off differences in spontaneous activityterm.

We compared spontaneous activityterm, tone-evoked activity, and tone-evoked response magnitudeterm in response to the same frequency for each neuron (chosen as the best frequencyterm in the laser OFF condition), each under laser-ON and laser-OFF conditions. We find the same effects as when averaging across top 7 frequencies in each condition. We find an increase in spontaneous activityterm (left; -20 ms : p = 0.0094, ON = 6.5 ± 0.5, OFF = 4.1 ± 0.9) and decrease in tone-evoked response magnitudeterm (right; -100 ms : p = 8.2e-5, ON = 14.7 ± 1.1 Hz; OFF = 17.9 ± 1.3; -20 ms : p = 1.6e-6, ON = 12.9 ± 1.4, OFF = 17.6 ± 1.3; +8 ms : p = 0.013, ON = 15.3 ± 1.3, OFF = 16.6 ± 1.3), as in Figure 2A in manuscript, as well as a decrease in tone-evoked activity (middle; -100 ms : p = 2.8e-4, ON = 19.2 ± 1.3 Hz; OFF = 21.4 ± 1.4; -20 ms : p = 3.7e-4, ON = 19.4 ± 1.4, OFF = 21.7 ± 1.4; +8 ms : p = 9.8e-5, ON = 19.05 ± 1.4, OFF = 20.9 ± 1.4), as in Figure 3E in manuscript.

2) The results of the experiments using tones and clicks and those of experiments using the dynamic random chordterm (DRC) stimulus are difficult to reconcile. Figure 4 shows what appear to be large effects of AC stimulation on IC receptive fields (STRFs). Given this, the explanation for the disappearance of significant positive and negative lobes with laser stimulation should be pursued. For example, if the interpretation given at the end of subsection “Activating direct cortico-collicular feedbackterm modulates activity in the inferior colliculusterm” should be tested by going back to the tone-evoked responses and measuring effects of AC stimulation on responses to tones outside of the 7 frequencies that evoked the largest responses from a unit.

We address changes in activity outside of most preferred frequencies in Figure 6, which examines changes in the shape of the frequency response curve. We find that units decrease response at more preferred frequencies, suggested by the linear fit slopes being less than 1 and supported by Figure 6B, and increase at less preferred frequencies, suggested by linear fit y-intercepts being greater than 1 (further evidence provided Author response image 1 ).

Author response image 1. Response averaged across 7 less preferred frequencies (29-35th most preferred frequencies).

Activation of direct cortico-collicular feedbackterm increases tone-evoked activity (middle; +8 ms: p = 6.4e-4, ON = 7.1 ± 0.7, OFF = 5.7 ± 0.6) at less preferred frequencies.

For clarification of our interpretation of these results we added “Activation of feedback decreased frequency selectivityterm in the subsets of units that also showed a decrease in tone-evoked response magnitudeterm or increase in spontaneous activityterm, but not in units that showed an increase in tone-evoked response magnitudeterm or decrease in spontaneous activityterm”, as well as in the Materials and methods section “Subgroups of neurons used in sparsenessterm analyses were separated based on changes in spontaneous activityterm or tone-evoked response magnitudeterm in response to cortico-collicular activation or suppression. Units were selected if > 1 standard deviation change based on the -100 ms laser onsetterm·var trials.”

3) So much work clearly went into the PV and SOM interneuron manipulations and recordings in Ac and IC. The results of those experiments and interpretation of the results should be further explained. For example, why were the effects of interneuron manipulation observed in AC but not IC units?

To address our interpretation as to why we observe effects in AC, but not IC, with manipulation of interneurons in AC we added to the Discussion … “We observed little effect of modulating AC inhibitory interneurons on activity in IC despite the changes observed in AC (Figures 8, 9), which suggests there may be another neuron subtype that plays a modulatory role during cortico-collicular plasticity in driving feedback modulation. […] Thus, while another neuronal subtype may be responsible for driving feedback activity in a behavioral context, inhibitory interneurons may still play a role in shaping responses of feedback once active.”

[Editors’ note: what follows is the authors’ response to the second round of review.] Nevertheless, several important caveats to the approach and analyses remain, which could be addressed in the Materials and methods and/or Discussion. We will attach the relevant reviews in addition to the summary here, and suggest that you acknowledge and address the following points in your revised submission. Other thoughtful suggestions follow, and we include the reviews with suggestions. The other reviewers had no major changes.

Major changes: 1) That there are potential limitations of using rectangular laser pulses as described previously (Vila et al., 2019) Added to the Discussion: “One caveat to consider in interpreting these results is that we used a square pulse for optogenetic manipulations, whereas a more complex stimulation pattern potentially can achieve stronger activation (Vila et al., 2019).”

2) That in spite of the cell-specific expression there hypothetically could be mixed suppressive and excitatory effects that are lost in multi-unit spike rate and spectro-temporal receptive field (STRF)term·def analyses used here.

Added to the Results: “Because the majority of the units were recorded as multi-units, this change in selectivity may be due to the change in firing rate of differentially tuned single units.”

3) There are also concerns about normality in spike rate distributions. With respect to the spike rate effects in the new Figure 5, panel A (dorsal IC, -100 ms laser condition), if the spike rate distributions are not normal then the bar graphs could be replaced with box-plots emphasizing that there are indeed spike-rate outliers. Note that the appropriate statistical comparisons should be performed.

All bar plots changed to box plots to elaborate the distribution of the data. We used non-parametric statistical methods as described in “Statistical Analyses” where appropriate.

Here are the full reviews that include suggestions for revision. The main points are summarized above.

Reviewer #1: 1) In their revision, the authors have sidestepped one of my major concerns, namely: "Secondly, optogenetic activation provides highly synchronised activation, the magnitude of which may or may not be within physiological ranges. If the manipulations lead to changes in cortico-collicular activity that are not within these ranges, it is not clear what conclusion can be drawn regarding the role of these inputs generally." At the very least, the authors should explicitly acknowledge the limitations of using rectangular laser pulses in this context and the associated limitations to the generality of their conclusions. For example, a recent study addressing the same questions has shown that these effects in the IC strongly depend on patterns of laser activation in the AC (Vila et al., 2019).

Added to the Discussion: “One caveat to consider in interpreting these results is that we used a square pulse for optogenetic manipulations, whereas a more complex stimulation pattern potentially can achieve stronger activation (Vila et al., 2019).”

2) A new important issue has appeared in the revised manuscript, which merits careful consideration. The new Figure 5 is used to support the conclusion that "…units recorded from dorsal recording sitesterm·var are significantly affected by activation of feedback, but centrally located units are not". However, the firing rates of some dorsal units are twice as high as the central ones'. Compare the scales in panels A and B. Could the effect in, for example, -100 ms tone-evoked "dorsal" be due to those high-firing-rate units being suppressed by the laser? One can clearly see that those data points are below the diagonal. It would be crucial to reanalyse the data presented in panel A, where the firing rates are up to 100 Hz, by using only those units whose firing rates are comparable to those in panel B, i.e., less than 40 Hz. Otherwise, the conclusion might reflect the effect of the laser on the small number of relatively rare units with high firing rates, disproportionately suppressed by the laser. As those cells are relatively rare (about 5%?), they might have been missed in the central IC (71 units; versus 120 units in the dorsal IC). Without this analysis, I am not convinced by the authors' conclusion.

We added the following to the manuscript: “To verify these results were not due to a select few high firing units found in DCICterm, we repeated the analysis comparing only units with spontaneous activityterm in the laser OFF condition less than 40 Hz. With these matched firing rates we still see the same statistically significant effects (DCICterm – mag decrease -100 ms: p = 3.3e-6, ON = 14.8 Hz, OFF = 16.6 Hz; -20 ms: p = 6.3e-7, ON = 12.1 Hz, OFF = 12.6 Hz; spont increase -100 ms: p = 0.02 ON = 3.5 Hz, OFF = 2.5 Hz; -20 ms: p = 2.8e-7, ON = 5.1 Hz, OFF = 4.9 Hz; CNICterm – mag decrease +8 ms: p = 0.0039, ON = 11.7 Hz, OFF = 12.6 Hz).”

3) The authors have indicated in the revision letter which data points corresponded to single units (red dots). This information is very useful. Yet they chose not to indicate it in the revised manuscript. Why? I think it must be indicated.

We have added reference to the IC single units in our figures as in the revision letter.

4) In general, my concern with multi-unit data is that it is possible, given the bidirectional effects of the laser stimulation, that some nearby neurons are modulated positively and other neurons are modulated negatively. If these neurons are recorded as a multiunit, then the total effect will be a weighted sum of the individual effects. This will bias not only the determination of the effects on the firing rates, but may also induce artefacts in the calculation of STRFs, unless all individual neurons contributing to the multiunit have the same selectivity. However, since they were not recoded as single units, their individual selectivity is unknown. You can see, for example, that the receptive field in Figure 7A 'laser on" differs significantly from that in the "laser off" condition. These are units that increase their firing rate with the laser on. Which means there are more spikes; yet the STRF almost vanishes. This illustrates what I have said above: what the authors interpret as a change in selectivity could be due to a change in the firing rates of differentially tuned single units, all contributing to the vector sum during STRF calculation, without any change in individual neurons' selectivity.

Added to the Results: “Because the majority of the units were recorded as multi-units, this change in selectivity may be due to the change in firing rate of differentially tuned single units.”

5) The authors persist in using bars and standard errors for data that are so obviously non-normally distributed.

All bar plots changed to box plots to elaborate the distribution of the data.

Reviewer #3: Clearly cortical feedback to collicular structures including superior and inferior colliculi plays a pivotal role in shaping neural pathway responses and behavior and yet little is known about auditory circuits mediating this process. […] I have only very minor comments and suggestions to improve the write up as outlined below.

Title, "Role of feedback from auditory cortexterm in shaping responses to sounds in inferior colliculusterm" 1) Shorten title?, e.g. "Shaping inferior colliculusterm responses to sound through cortical feedback" Good point. We shortened the title to “Auditory cortexterm shapes sound responses in the inferior colliculusterm”.

Introduction section.

1) Please open with a paragraph explaining why we care about cortico-collicular feedbackterm? You end paragraph two with some discussion of why we may care. Maybe round this out and move up to the top paragraph of Introduction.

Amended end of first paragraph to: “Previous studies have demonstrated the importance of cortical feedback to IC in auditory behaviors (Bajo, Nodal, Moore, and King, 2010; Xiong et al., 2015), however, the mechanisms by which information processing is shaped via the descending feedbackterm pathway remain poorly characterized.”

2) Introduction paragraph three, add a captivating introduction sentence to the paragraph, e.g., "Cortical output modulations of collicular activity is not a monolithic process but instead is shaped by interactions between excitatory and inhibitory cortical neurons." Changed first sentence to: “In AC, modulation of sound responses is not a monolithic process, but instead is shaped by interactions between excitatory and inhibitory cortical neurons.”

3) Introduction paragraph two, you have a long sentence "Whereas inactivation studies, on the other hand, found less consistent effects on IC responses…) Please simplify. Perhaps, "Prior studies find pharmacological inactivation of AC alters tone-evoked responses of IC neurons (Zhang, 1997) whereas others do not (Jen, 1998)." Is the Jen, 1997 study using pharma to inactivate AC?

Changed to: “Whereas one study found inactivation of AC caused a shift in best frequencyterm in IC neurons (Zhang, Suga, and Yan, 1997), several other studies showed that inactivation of AC had no effect on frequency selectivityterm in IC (Jen et al., 1998), but rather modulated sound-evoked and spontaneous activityterm (Gao and Suga, 1998; J Popelář, Nwabueze-Ogbo, and Syka, 2003; Jiří Popelář et al., 2016)”

We still want to include a bit more information about the previous studies than would be in the suggested simplified sentence, but we reworded for clarity.

Terminology throughout manuscript 1) Non-specialist audiences will not get what the difference is between the dorsal cortex of IC and central nucleus. In parts of the manuscript you refer to central nucleus simply as "IC" whereas others it is "central IC". Please lay this out clearly and refer to all your recordings that are verified as being in Central IC as being in CIC being consistent with your abbreviation for dorsal cortex as DCICterm.

This has been corrected. Now all uses to IC refer to cases when we are not distinguishing between the different regions.

Materials and methods.

1) In Materials and methods, clearly explain your rationale for doing these initial experiments under anesthesia e.g., " To obtain precise responses across large numbers of IC neurons, these experiments were carried out under anesthesia. Though cortical spike rate responses are reduced under anesthesia, the general frequency response organization remains the same." To Materials and methods – Electrophsiological Recordings we added: “Initial experiments were performed under anesthesia to control for stability or recordings.”

2) “Acoustic stimuli” subsection, please give your rationale for delivering sound from a free field speaker. Perhaps, one rationale is to more closely approximate conditions animals may experience in awake conditions? Note the caveats. If you are calibrating next to the contralateral ear in free field and then using a filter to compensate for sound reflections -presumably you have a means of insuring that the set-up (stereotax unit, electrode holders, amplifiers etc are in fixed spatial positions relative to the ear and you have distinct filters for left and right ear configurations). Using ear-tubes may be a better approach as the mice have relatively uniform ear canals and calibrating can be more precise.

We have added to the stimulus section: “Free-standing speaker was used to approximate the conditions under which the mouse typically experiences sounds in awake state.”

We are very careful at calibrating the speaker. All the parts of the setup are at exact fixed positions relative to the speaker. We use the white noise approach for the calibration procedure. We record the signal at 3 different slightly offset positions of a high quality (B and K) calibration microphone.

3) “Acoustic stimuli” section, provide a rationale for including click train responses in your sound battery. Click train responses have been used in the past for ABR and penetrating electrode studies but these sounds are fraught with spectral splatter that makes interpretation of the response relatively difficult. Perhaps, your rationale was to get a quick assessment of auditory onset responses?

Added: “To obtain a quick assessment of auditory onset responses we used click trains.”

“Neural Response Analysis” subsection, you define FRi as "tone-evoked response" here and above (describe that you're assessing firing rate). Can you please explain that Fri is a "tone-evoked firing rate response"?

Amended to read “tone-evoked firing rate response”

Also please explain the rationale for computing sparsenessterm and the rationale for your 20 ms time scale for doing so. For example, there are prior studies that have compared cortical and collicular time-scales for sparse coding and find that central IC has an order of magnitude shorter integration times (~10ms) than cortex and that on those time scales encoding in IC is indeed sparse (Chen, Read, Escabi, J Neurosci 2012; https://www.ncbi.nlm.nih.gov/pubmed/22723685).

We are using a measure sparsenessterm to measure frequency selectivityterm within an individual neuron as it is less sensitive to the shape of the frequency response function than gaussian widths (Aizenberg et al., 2015). This is distinct from the “sparsenessterm” definition in the Chen et al. study.

Results section.

1) Results section, first paragraph. The authors are commended for carrying out these important, rigorous and difficult studies with cell specific modulation and e-phys but they should recognize that most of your audience is less informed about theses dual viral techniques. Please do a better job of setting up this paragraph so as not to lose the general audience. Following the first introduction sentence please help orient the non-expert with something like, "A combination of local and retrograde viral transfections were made in order to achieve cell and circuit specific viral transfections of cortico-collicular neurons".

Added the following sentence after the introductory sentence of this paragraph to help clarify technique: “A combination of cortical anterograde and collicular retrograde viral transfections in order to achieve specific viral transfection of cortico-collicular neurons.”

2) In general, most paragraphs in the Results section do a great job of introducing the rationale for experiments and analyses. Thank you!

Thank you for your kind comment.

3) Discussion paragraph three, here you refer to central IC as "central IC" but many other places throughout the manuscript you are ambiguous e.g. Discussion paragraph one, you say things like "…little effect on IC activity" is this central and dorsal IC? Also you say "effects of modulation of cortical activity by PVs does not backpropagate to IC". Do you DCICterm here?

When we use the abbreviation ‘IC’ we are not distinguishing between CNICterm and DCICterm. We only use the terms CNICterm and DCICterm when we are explicitly referring to the recordings where we distinguish between these two areas.

Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations BlackwellpersName JM, LesickopersName A, RaopersName W, De BiasipersName M, GeffenpersName MN. 2019. Data from: The role of feedback from the auditory cortexterm in shaping responses to sounds in the inferior colliculusterm. Dryad Digital Repository. [ DOI ] Supplementary Materials Transparent reporting form elife-51890-transrepform.docx (248.5KB, docx) Data Availability Statement Original spike data and code are available on Dryad ( http://doi.org/10.5061/dryad.1t61c80 ).

The following dataset was generated: BlackwellpersName JM, LesickopersName A, RaopersName W, De BiasipersName M, GeffenpersName MN. 2019. Data from: The role of feedback from the auditory cortexterm in shaping responses to sounds in the inferior colliculusterm. Dryad Digital Repository.