MurraypersName, Woloszynowska-FraserpersName, Ansel-BollepallipersName, ColepersName, FoggettipersName, CrouchpersName, RiedelpersName, WulffpersName (2015date). “Parvalbumin-positive interneurons of the prefrontal cortex support working memory and cognitive flexibility.” Scientific Reportstitle·j.
Object of study — whether one class of prefrontal interneuron accounts for all of the behavioural consequences of prefrontal dysfunction, or only some
Research question — which PFC-dependent behaviours require intact parvalbumin interneuron output, and which survive its loss
Contribution — shows that silencing PV interneurons reproduces the cognitiveterm deficits of a whole-region lesion but none of the social or psychostimulant ones, so a composite syndrome maps onto separable circuit elements
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | prefrontal parvalbumin interneuron control of working memory and cognitive flexibility — “Thus various modalities of PFC-dependent working memory critically depend on PVI integrity.” | |
| claim-type | mechanism — “By sampling for behavioral alterations that map onto distinct symptom categories in schizophrenia, we show that dysfunction of PVI signaling in the PFC specifically produces deficits in the cognitive domain, but does not give rise to PFC-dependent correlates of negative or positive symptoms.” | |
| method — evidence | — “To directly test how chronic impairment of PVI signaling affects PFC-dependent behavior, we selectively blocked output from PVIs in the mouse PFC by cell-type- and region-selective expression of tetanus toxin light chain (TeLC).” | |
| object of study — evidence | — “Although these disturbances all appear to involve PFC dysfunction, it is unclear whether specific circuit elements within the PFC contribute differentially to symptom complexity.” | |
| research question — evidence | — “To distinguish PFC-dependent behaviors which require intact PVI signaling from those that do not, we compared PVI-selective interference with non-cell-type-selective perturbations of the same circuitry in behavioral assays that were chosen to detect alterations that map onto symptom categories (positive, negative, cognitive) in schizophrenia.” | |
| contribution — evidence | — “By sampling for behavioral alterations that map onto distinct symptom categories in schizophrenia, we show that dysfunction of PVI signaling in the PFC specifically produces deficits in the cognitive domain, but does not give rise to PFC-dependent correlates of negative or positive symptoms.” | |
| transfer | from clinical psychiatry: the tripartite positive/negative/cognitive symptom division, imported as a way of choosing which rodent assays to run — “To distinguish PFC-dependent behaviors which require intact PVI signaling from those that do not, we compared PVI-selective interference with non-cell-type-selective perturbations of the same circuitry in behavioral assays that were chosen to detect alterations that map onto symptom categories (positive, negative, cognitive) in schizophrenia.” | transfers[0] |
| transfer | from molecular microbiology: tetanus toxin light chain, used as a genetically targetable silencer of transmitter release — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | transfers[1] |
| transfer | from pharmacology: amphetamine-induced locomotion as a behavioural proxy for subcortical dopamine tone — “It is thought that both positive symptoms and increased amphetamine sensitivity are due to enhanced subcortical dopaminergic tone, which in turn correlates with and might be secondary to PFC dysfunction in schizophrenia” | transfers[2] |
| mechanism | PV interneurons generate the fast network oscillations that synchronise prefrontal and coupled circuits, and that synchrony is what supports information processing during working memory — so blocking PV output removes it — “Generation of network oscillations in turn is thought to depend on the activity of fast-spiking putative PVIs” | mechanisms[0] |
| mechanism | Loss of glutamatergic prefrontal afferent control dysregulates ventral brainstem dopamine neurons, raising subcortical dopamine and hence amphetamine sensitivity after PFC lesion — “Consistent with this idea, lesions of the PFC in rodents also increase amphetamine sensitivity and subcortical dopamine presumably through dysregulation of dopaminergic neurons in the ventral brainstem after loss of glutamatergic PFC afferent control” | mechanisms[1] |
| mechanism | A division of labour among GABAergic interneuron sub-populations means silencing only the PV class removes one specific circuit function, producing a cognitive-only deficit profile rather than the full lesion profile — “In contrast, selective inactivation of one sub-population of GABAergic interneurons – PVIs – caused deficits only in the cognitive domain, suggesting that these interneurons may subserve specific circuit functions in the PFC.” | mechanisms[2] |
| mechanism | tetanus toxin light chain is expressed only where Cre flips its reading frame, and it stops release by cleaving VAMP2 — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | mechanisms[3] |
| mechanism | PV interneurons target the soma, proximal dendrites and axon initial segment of principal cells, which is why they can govern regional output — “In the isocortex PVIs mainly comprise basket and chandelier cells, which control network activity by targeting the soma and proximal dendrites or the axon initial segment of principal cells, respectively” | mechanisms[4] |
| mechanism | the proposed route from PV loss to cognitive deficit runs through network oscillations, which are thought to depend on fast-spiking interneurons — “At the systems level synchronous network activity within the PFC and across functionally coupled brain structures may aid information processing during working memory 56 , 57 . Generation of network oscillations in turn is thought to depend on the activity of fast-spiking putative PVIs” | mechanisms[5] |
| mechanism | the route from prefrontal damage to amphetamine hypersensitivity runs through loss of cortical control over subcortical dopamine — “It is thought that both positive symptoms and increased amphetamine sensitivity are due to enhanced subcortical dopaminergic tone, which in turn correlates with and might be secondary to PFC dysfunction in schizophrenia” | mechanisms[6] |
| mechanism | impaired PV signalling may be where many separate genetic and environmental risks converge — “Accordingly impaired signaling of PVIs may form a common endpoint for different genetic and environmental factors leading to PFC malfunction and schizophrenia-associated deficits” | mechanisms[7] |
| builds-on | the authors' own earlier study of hippocampal parvalbumin interneurons using the same AAV-FLEX-TeLC construct (cited as ref. 26) — “However, using the same AAV-FLEX-TeLC we have previously shown that transmission of hippocampal PVIs was fully abolished 10 days after virus injection despite residual VAMP2 immunoreactivity 26 .” | claim_relations[0] |
| extends | post-mortem findings of reduced GAD67 and parvalbumin in schizophrenia — “In particular reduced mRNA levels of the 67 kD isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD67) have been consistently reported 9 , 10 , 11 , 12 , 13 . These changes seem to dominantly affect a subclass of GABAergic interneurons, that expresses the calcium-binding protein PV, which itself shows reduced expression in schizophrenia” | claim_relations[1] |
| confirms | prior lesion work establishing PFC involvement in social behaviour and amphetamine sensitivity — “Compared with controls, PFC-Lesion animals showed significantly higher levels of locomotor activity ( Fig. 6F ), with increased peak activity ( p = 0.04; t = 1.97) and slower decay kinetics ( p = 0.005; t = 3.25), confirming the importance of the PFC for amphetamine sensitivity.” | claim_relations[2] |
| qualifies | the view that PV interneuron pathology explains the breadth of prefrontal dysfunction in schizophrenia — “In summary, impaired signaling of PVIs in the PFC, which is thought to form a core pathology in schizophrenia, may be causally involved in the development of cognitive symptoms. However, amphetamine-hypersensitivity and deficits in social-approach avoidance behavior as indicators of positive and negative symptoms, respectively, are not precipitated by PVI dysfunction. Based on these data we suggest that different aspects of the composite symptomatology of PFC dysfunction in schizophrenia can be allocated to specific neuronal elements.” | claim_relations[3] |
| supports | bicuculline studies in which total disinhibition of the PFC reproduced the full lesion-like profile — “Interestingly, complete disruption of GABA A -receptor-mediated inhibition in the PFC by bicuculline infusion has been shown to cause similar behavioral alterations as those we report here for PFC-lesion animals” | claim_relations[4] |
| distinguishes-from | somatostatin interneurons, silenced the same way with no working memory effect — “Such a “division of labor” scenario between different populations of interneurons is further supported by our finding that analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC of Sst-Cre mice 71 caused no change in working memory performance in the same task that was clearly impaired after inactivation of PVIs” | claim_relations[5] |
| sense | PFC dysfunction is used at two different grains here - a whole-region lesion and the loss of one interneuron class - and the paper's point is that they are NOT the same manipulation — “We found that PFC lesions produced behavioral alterations in all three symptom categories, whereas selective inactivation of PVIs caused deficits only in the cognitive domain.” | senses[0] |
| sense | working memory in this paper spans three tasks with different demands: repeat investigation, spontaneous alternation, and a 60s delayed match-to-place — “we used a delayed (60s) match-to-place test in a Y-maze configuration in the water maze, where the percentage of correct responses in match trials represents trial unique short-term memory” | senses[1] |
| sense | cognitive, negative and positive symptoms are used as ANIMAL CORRELATES of clinical categories, assigned by which behavioural assay was chosen — “To distinguish PFC-dependent behaviors which require intact PVI signaling from those that do not, we compared PVI-selective interference with non-cell-type-selective perturbations of the same circuitry in behavioral assays that were chosen to detect alterations that map onto symptom categories (positive, negative, cognitive) in schizophrenia.” | senses[2] |
| sense | mouse PFC here means a block spanning prelimbic, infralimbic, cingulate and medial orbital cortex, treated as integrating what three separate human regions do — “Additionally, TeLC expression was found in the cingulate and medial orbital cortices, demonstrating functional removal of PVIs across an area of mouse PFC thought to integrate functions of the dorsolateral, medial and orbital PFC in humans” | senses[3] |
| sense | inactivation means blocked transmitter release with the cell alive, not cell death - the contrast case in the same paper kills the tissue — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | senses[4] |
| control | the analysis carried out blind to the experimental group — held — rules out experimenter expectancy in a manual analysis — “Analysis was carried out blind to the experimental group.” | controls[0] |
| control | the manual re-investigation also performed by an experimenter blind to the experimental group — held — rules out the blinding being lost at the second pass - the re-check is the step most likely to be done informally, and it was blinded too — “Reinvestigation was analyzed manually by an experimenter blind to the experimental group.” | controls[1] |
| differs in | somatostatin interneurons, silenced the same way with no working memory effect — THE AXIS IS WHICH INTERNEURON POPULATION IS SILENCED, WITH THE MANIPULATION HELD IDENTICAL. 'analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC.' Same toxin, same region, different cell class and no working-memory effect - so the result is attributable to the population rather than to the method, which is exactly what a single-population study cannot establish. — “Such a “division of labor” scenario between different populations of interneurons is further supported by our finding that analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC of Sst-Cre mice 71 caused no change in working memory performance in the same task that was clearly impaired after inactivation of PVIs” — “analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC” | claim_relations[5] |
| audit | tetanus toxin light chain —causes→ VAMP2 — Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but TeLC stops transmitter release BY CLEAVING VAMP2; 'causes VAMP2' is loose. — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | concept_edges[0] |
| note | tetanus toxin light chain —causes→ VAMP2 — cleavage is the mechanism of the silencing, and the antibody against VAMP2 is also how coverage is checked — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | concept_edges[0] |
| edge | basket —is-a→ PVIs — “In the isocortex PVIs mainly comprise basket and chandelier cells, which control network activity by targeting the soma and proximal dendrites or the axon initial segment of principal cells, respectively” | concept_edges[1] |
| edge | chandelier cells —is-a→ PVIs — “In the isocortex PVIs mainly comprise basket and chandelier cells, which control network activity by targeting the soma and proximal dendrites or the axon initial segment of principal cells, respectively” | concept_edges[2] |
| note | PVIs —contrasted-with→ somatostatin — CRITERION: same toxin, same task, no working memory deficit - the cell-type specificity control — “Such a “division of labor” scenario between different populations of interneurons is further supported by our finding that analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC of Sst-Cre mice 71 caused no change in working memory performance in the same task that was clearly impaired after inactivation of PVIs” | concept_edges[4] |
| edge | PVIs —causes→ working memory — “Thus various modalities of PFC-dependent working memory critically depend on PVI integrity.” | concept_edges[5] |
| audit | PVIs —causes→ Behavioral flexibility — Audit 2026-09-28: DOUBTFUL - read against its span and kept, but PV-silenced mice were slower at reversal; the edge lacks its direction and names PVIs for their silencing. — “In contrast, during reversal, PFC-PV-TeLC and PFC-Lesion mice needed more time to learn the new platform location although this was only significant for PFC-PV-TeLC mice (PFC-PV-TeLC vs. PFC-PV-GFP, day effect F(3,84) = 24; p < 0.001; group effect F(4,84) = 8.8; p = 0.006; and PFC-Lesion vs. PFC-Saline, F(3,60) = 2.1; p = 0.16)” | concept_edges[6] |
| note | PVIs —causes→ Behavioral flexibility — the deficit is real but transient - PV silencing delays relearning where the lesion prevents it in a third of animals — “In contrast, during reversal, PFC-PV-TeLC and PFC-Lesion mice needed more time to learn the new platform location although this was only significant for PFC-PV-TeLC mice (PFC-PV-TeLC vs. PFC-PV-GFP, day effect F(3,84) = 24; p < 0.001; group effect F(4,84) = 8.8; p = 0.006; and PFC-Lesion vs. PFC-Saline, F(3,60) = 2.1; p = 0.16)” | concept_edges[6] |
| weight | PVIs —causes→ Behavioral flexibility — 0.9 — “In contrast, during reversal, PFC-PV-TeLC and PFC-Lesion mice needed more time to learn the new platform location although this was only significant for PFC-PV-TeLC mice (PFC-PV-TeLC vs. PFC-PV-GFP, day effect F(3,84) = 24; p < 0.001; group effect F(4,84) = 8.8; p = 0.006; and PFC-Lesion vs. PFC-Saline, F(3,60) = 2.1; p = 0.16)” | concept_edges[6] |
| audit | PFC —causes→ social preference — Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span writes 'social approach/avoidance behavior' and 'novelty preference', not 'social preference'. — “Accordingly social approach/avoidance behavior as well as social recognition memory/novelty preference depends on the PFC circuitry. PV-positive interneurons, however, do not seem to be a crucial component of this circuit.” | concept_edges[7] |
| note | PFC —causes→ social preference — SIGN: the region is required and the PV population is not - this is the dissociation, stated — “Accordingly social approach/avoidance behavior as well as social recognition memory/novelty preference depends on the PFC circuitry. PV-positive interneurons, however, do not seem to be a crucial component of this circuit.” | concept_edges[7] |
| audit | PFC —distinct-from→ spatial reference memory — Audit 2026-09-28: DOUBTFUL - read against its span and kept, but the span says spatial reference memory does NOT DEPEND on PFC; distinct-from paraphrases independence. — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length (PFC-PV-TeLC/PFC-PV-GFP, day effect: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effect: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memory per se does not depend on the PFC” | concept_edges[9] |
| note | PFC —distinct-from→ spatial reference memory — CRITERION: both perturbed groups acquired the platform normally, so the reversal deficit is not a learning deficit — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length (PFC-PV-TeLC/PFC-PV-GFP, day effect: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effect: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memory per se does not depend on the PFC” | concept_edges[9] |
| outcome (stated by the tagger, no quotation) | control — held | controls[0] |
| outcome (stated by the tagger, no quotation) | control — held | controls[1] |
| for which use | limitation — any direct comparison between the silencing and the lesion arms: THIS EXPERIMENTAL DESIGN PRECLUDES A DIRECT 2 x 2 STATISTICAL COMPARISON — “This experimental design precludes a direct 2 × 2 statistical comparison.” | limitations[0] |
| for which use | limitation — the cell-type specificity claim: TRANSGENE-EXPRESSION IN PV-NEGATIVE CELLS WAS SCARCE (4.3%) AND LIKELY INCLUDES PVIS CONTAINING PV LEVELS BELOW DETECTION THRESHOLD - the identity of those cells is inferred, not established — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | limitations[1] |
| for which use | limitation — the water-maze expression data: ONLY COHORT 1 WAS QUANTITATIVELY ASSESSED (N = 10/17) — “Only cohort 1 was quantitatively assessed (n = 10/17).” | limitations[2] |
| for which use | limitation — what 'PV-silenced' means in every result: BILATERAL INFUSIONS OF AAV-FLEX-TeLC RESULTED IN EXPRESSION OF TeLC IN OVER 75% OF PVIs, so a residual population stays active — “Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIs in prelimbic and infralimbic regions of the PFC” | limitations[3] |
| for which use | limitation — the completeness of the transmission block: THE RESIDUAL VAMP2 IMMUNOREACTIVITY IS LIKELY DUE TO LOW AFFINITY RECOGNITION OF VAMP2 CLEAVAGE PRODUCTS, with completeness argued from a previous hippocampal result rather than measured here — “The residual VAMP2 immunoreactivity is likely due to low affinity recognition of VAMP2 cleavage products by the antibody 31 . However, using the same AAV-FLEX-TeLC we have previously shown that transmission of hippocampal PVIs was fully abolished 10 days after virus injection despite residual VAMP2 immunoreactivity” | limitations[4] |
| duplicate of | limitation — limitations[1] — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | limitations[5] |
| for which use | limitation — the same scope as limitations[1], with the figure: 4.3% of transgene expression was in PV-negative cells, read by the authors as PV below detection rather than as off-target expression — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | limitations[5] |
| for which use | limitation — the water-maze results specifically: ONLY COHORT 1 WAS QUANTITATIVELY ASSESSED (n = 10/17). FOR COHORT 2 AND ALL PFC-PV-GFP ANIMALS CORRECT VIRAL EXPRESSION WAS CONFIRMED VISUALLY — “Water maze experiments were replicates of two cohorts. Only cohort 1 was quantitatively assessed (n = 10/17). For cohort 2 and all PFC-PV-GFP animals correct viral expression was confirmed visually.” | limitations[6] |
| for which use | limitation — the reversal deficit, which is a delay rather than a prevention: THIS DEFICIT WAS TRANSIENT IN PFC-PV-TeLC ANIMALS, WHERE ALL MICE EVENTUALLY ACQUIRED THE NEW LOCATION — “Analysis of reversal performance by subject revealed that this deficit was transient in PFC-PV-TeLC animals, where all mice eventually acquired the new location.” | limitations[7] |
| replication | untested | |
| limitation | The two manipulations (viral silencing and lesion) were run as separate sequential experiments, so the four groups cannot be compared in a single factorial analysis. — “This experimental design precludes a direct 2 × 2 statistical comparison.” | limitations[0] |
| limitation | A small fraction of transgene expression fell in PV-negative cells, whose identity is inferred rather than established. — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | limitations[1] |
| limitation | Viral expression was verified quantitatively in only one of the two water-maze cohorts. — “Only cohort 1 was quantitatively assessed (n = 10/17).” | limitations[2] |
| limitation | silencing covers over 75% of PV cells, not all of them, so a residual population remains active — “Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIs in prelimbic and infralimbic regions of the PFC” | limitations[3] |
| limitation | VAMP2 immunoreactivity was reduced but not abolished; completeness of the block is argued from a previous result in a different brain region — “The residual VAMP2 immunoreactivity is likely due to low affinity recognition of VAMP2 cleavage products by the antibody 31 . However, using the same AAV-FLEX-TeLC we have previously shown that transmission of hippocampal PVIs was fully abolished 10 days after virus injection despite residual VAMP2 immunoreactivity” | limitations[4] |
| limitation | 4.3% of transgene expression was in PV-negative cells, which the authors read as PV below detection rather than off-target — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | limitations[5] |
| limitation | in the water maze only one of two cohorts was quantitatively verified histologically; the rest was confirmed by eye — “Water maze experiments were replicates of two cohorts. Only cohort 1 was quantitatively assessed (n = 10/17). For cohort 2 and all PFC-PV-GFP animals correct viral expression was confirmed visually.” | limitations[6] |
| limitation | the reversal deficit after PV silencing is transient, so the manipulation delays rather than prevents relearning — “Analysis of reversal performance by subject revealed that this deficit was transient in PFC-PV-TeLC animals, where all mice eventually acquired the new location.” | limitations[7] |
finding · quantity · variable · definition · key-term — grounded in the full text| finding | Silencing PV interneurons in the prefrontal cortex produced deficits only in the cognitive domain, whereas non-selective PFC lesions disrupted behaviours mapping onto all three schizophrenia symptom categories. — “We found that PFC lesions produced behavioral alterations in all three symptom categories, whereas selective inactivation of PVIs caused deficits only in the cognitive domain.” | content_tags.findings[0] |
| finding | Both PV-silenced and lesioned mice showed increased repeat investigations. — “both PFC-PV-TeLC and PFC-Lesion animals showed increased levels of repeat investigations” | content_tags.findings[1] |
| finding | Spontaneous alternation in the Y-maze was reduced in both PV-silenced and lesioned mice relative to controls. — “( C ) In the same cohorts of mice spontaneous alternation in a Y-maze is reduced in PFC-PV-TeLC animals and in ( D ), PFC-Lesion animals compared to respective controls.” | content_tags.findings[2] |
| finding | In the delayed match-to-place task control mice improved significantly on match trials while PV-silenced mice did not. — “Whereas PFC-PV-GFP animals showed significant improvement in match trials, PFC-PV-TeLC animals did not ( Fig. 3E ).” | content_tags.findings[3] |
| finding | During initial water-maze training both PV-silenced and lesioned mice learned the platform location as well as their controls. — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length” | content_tags.findings[4] |
| finding | Whereas PV-silenced mice met the reversal criterion upon overtraining, more than 30% of lesioned mice failed to. — “However, whereas PFC-PV-TeLC mice met reversal criterion upon overtraining, more than 30% of PFC-Lesion mice failed to do so.” | content_tags.findings[5] |
| finding | Lesioned mice failed to discriminate a familiar from an unfamiliar conspecific. — “In contrast PFC-Lesion animals did not discriminate between familiar and unfamiliar mice” | content_tags.findings[6] |
| finding | Amphetamine produced hypersensitivity — higher peak locomotion and slower decay — in lesioned mice only. — “Both groups show enhanced locomotor activity after receiving amphetamine but PFC-Lesion animals show a hypersensitivity as indicated by increased peak locomotor activity (inset) and slower decay kinetics.” | content_tags.findings[7] |
| finding | PV interneurons do not seem to be required for the PFC's regulation of amphetamine sensitivity. — “However, PVIs do not seem to be required for this regulatory function of the PFC.” | content_tags.findings[8] |
| finding | Local field potential recordings showed no sign of enhanced synchronous activity after PV-cell silencing. — “However, local field potential (LFP) recordings from PFC showed no indication of enhanced synchronous activity in PFC-PV-TeLC mice.” | content_tags.findings[9] |
| finding | Applying the same tetanus-toxin silencing to somatostatin-expressing interneurons left working memory intact, showing the deficit is specific to the PV population. — “analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC of Sst-Cre mice 71 caused no change in working memory performance in the same task that was clearly impaired after inactivation of PVIs” | content_tags.findings[10] |
| finding | The viral strategy expressed the toxin in over 75% of PV interneurons in prelimbic and infralimbic cortex. — “Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIs in prelimbic and infralimbic regions of the PFC ( Fig. 1C,D,F ).” | content_tags.findings[11] |
| finding | VAMP2 immunoreactivity was strongly reduced in toxin-positive terminals compared with GFP-positive terminals, confirming the toxin cleaved its synaptic target. — “As expected, immunoreactivity for VAMP2 was strongly reduced in TeLC-positive terminals of PFC-PV-TeLC mice when compared with GFP-positive terminals of PFC-PV-GFP mice” | content_tags.findings[12] |
| finding | both PV silencing and whole-region lesion increased repeat investigations in the hole-board test — “both PFC-PV-TeLC and PFC-Lesion animals showed increased levels of repeat investigations (PFC-PV-TeLC vs. PFC-PV-GFP, p = 0.04; t = 1.97; PFC-Lesion vs. PFC-Saline, p = 0.04; t = 1.98)” | content_tags.findings[13] |
| finding | spontaneous alternation fell from about 70% to about 57% after PV silencing — “This deficit was confirmed in a spontaneous alternation task in the Y-maze, where alternation levels were high in PFC-PV-GFP and PFC-Saline animals (70.0 ± 3.0% and 74.3 ± 4.1% respectively), but significantly lower in PFC-PV-TeLC and PFC-Lesion animals (56.9 ± 2.7% and 60.0 ± 2.3% respectively)” | content_tags.findings[14] |
| finding | PV-silenced mice failed to improve in delayed match-to-place while controls did — “Whereas PFC-PV-GFP animals showed significant improvement in match trials, PFC-PV-TeLC animals did not” | content_tags.findings[15] |
| finding | reversal learning was slowed by both manipulations, significantly so for PV silencing — “In contrast, during reversal, PFC-PV-TeLC and PFC-Lesion mice needed more time to learn the new platform location although this was only significant for PFC-PV-TeLC mice (PFC-PV-TeLC vs. PFC-PV-GFP, day effect F(3,84) = 24; p < 0.001; group effect F(4,84) = 8.8; p = 0.006; and PFC-Lesion vs. PFC-Saline, F(3,60) = 2.1; p = 0.16)” | content_tags.findings[16] |
| finding | the reversal deficit differed in severity: PV-silenced mice reached criterion with overtraining, over 30% of lesioned mice never did — “However, whereas PFC-PV-TeLC mice met reversal criterion upon overtraining, more than 30% of PFC-Lesion mice failed to do so.” | content_tags.findings[17] |
| finding | lesioned mice showed strongly reduced interaction with a conspecific, while PV-silenced mice interacted like their controls — “During sociability testing PFC-Lesion animals showed strongly reduced interaction with the conspecific animal ( p = 0.003; t = 3.57) ( Fig. 6B ). However, PFC-PV-TeLC mice displayed similar levels of social interaction as PFC-PV-GFP control animals” | content_tags.findings[18] |
| finding | PV-silenced mice did not differ from controls in their response to amphetamine — “In contrast, PFC-PV-TeLC animals did not differ from controls in amphetamine response (peak activity, t < 1; activity decay, t < 1)” | content_tags.findings[19] |
| finding | the whole-region lesion produced deficits in all three symptom domains and PV silencing only in the cognitive one — “We found that PFC lesions produced behavioral alterations in all three symptom categories, whereas selective inactivation of PVIs caused deficits only in the cognitive domain.” | content_tags.findings[20] |
| finding | CONTROL NULL: local field potentials showed no enhanced synchrony, so the deficits are not seizure activity from lost inhibition — “In principle, loss of PVI-mediated inhibition could induce hyper-activity in pyramidal cells and induce ictal discharges in the PFC, which in turn could complicate the interpretation of behavioral data. However, local field potential (LFP) recordings from PFC showed no indication of enhanced synchronous activity in PFC-PV-TeLC mice. PFC-PV-TeLC and PFC-PV-GFP mice displayed similar absolute power at different spectral frequency bands” | content_tags.findings[21] |
| finding | CONTROL NULL: neither manipulation altered locomotion or anxiety-related behaviour at baseline — “Neither PFC-PV-TeLC nor PFC-Lesion mice showed obvious neurological deficits or alterations in locomotion and anxiety-related behavior” | content_tags.findings[22] |
| finding | CONTROL NULL: nose-poke counts were alike across groups, so the working memory deficit is not reduced exploration — “Although all groups of mice showed similar overall levels of nose pokes ( Fig. 2E,F ), indicating similar exploratory activity” | content_tags.findings[23] |
| finding | CONTROL NULL: initial platform acquisition was normal in both perturbed groups, indicating that spatial reference memory per se does not depend on the PFC — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length (PFC-PV-TeLC/PFC-PV-GFP, day effect: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effect: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memory per se does not depend on the PFC” | content_tags.findings[24] |
| finding | CONTROL NULL: silencing somatostatin interneurons the same way left working memory intact — “Such a “division of labor” scenario between different populations of interneurons is further supported by our finding that analogous tetanus toxin-based targeting of a different, somatostatin (Sst)-expressing, population of interneurons in the PFC of Sst-Cre mice 71 caused no change in working memory performance in the same task that was clearly impaired after inactivation of PVIs” | content_tags.findings[25] |
| finding | lesioned mice failed to tell a familiar conspecific from an unfamiliar one — “In contrast PFC-Lesion animals did not discriminate between familiar and unfamiliar mice” | content_tags.findings[26] |
| finding | social recognition and approach depend on PFC circuitry, but PV interneurons do not seem to be a crucial component of it — “Accordingly social approach/avoidance behavior as well as social recognition memory/novelty preference depends on the PFC circuitry. PV-positive interneurons, however, do not seem to be a crucial component of this circuit.” | content_tags.findings[27] |
| quantity | over 75% — proportion of prelimbic and infralimbic PV interneurons expressing the tetanus toxin transgene — “Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIs in prelimbic and infralimbic regions of the PFC ( Fig. 1C,D,F ).” | content_tags.quantities[0] |
| quantity | 4.3% — off-target transgene expression in PV-negative cells — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | content_tags.quantities[1] |
| quantity | more than 30% — share of lesioned mice that never reached the reversal-learning criterion, versus none of the PV-silenced mice — “However, whereas PFC-PV-TeLC mice met reversal criterion upon overtraining, more than 30% of PFC-Lesion mice failed to do so.” | content_tags.quantities[2] |
| quantity | over 75% — share of PV interneurons expressing the silencer in prelimbic and infralimbic cortex — “Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIs in prelimbic and infralimbic regions of the PFC” | content_tags.quantities[3] |
| quantity | 4.3% — transgene expression in PV-negative cells, read by the authors as PV below detection — “Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIs containing PV levels below detection threshold.” | content_tags.quantities[4] |
| quantity | 70.0% and 74.3% vs 56.9% and 60.0% — Y-maze alternation in the two control groups against the two perturbed groups — “This deficit was confirmed in a spontaneous alternation task in the Y-maze, where alternation levels were high in PFC-PV-GFP and PFC-Saline animals (70.0 ± 3.0% and 74.3 ± 4.1% respectively), but significantly lower in PFC-PV-TeLC and PFC-Lesion animals (56.9 ± 2.7% and 60.0 ± 2.3% respectively)” | content_tags.quantities[5] |
| quantity | more than 30% — lesioned mice that never reached reversal criterion, against none of the PV-silenced — “However, whereas PFC-PV-TeLC mice met reversal criterion upon overtraining, more than 30% of PFC-Lesion mice failed to do so.” | content_tags.quantities[6] |
| quantity | 9344 — PV-positive cells counted for the coverage estimate — “For analyses of GFP-TeLC-expessing PV-positive neurons (n = 9344 cells) sections were taken from at least 3 points along the rostro-caudal extent of the PFC and immunostained for GFP and PV.” | content_tags.quantities[7] |
| quantity | 60s — delay in the match-to-place test of trial-unique memory — “we used a delayed (60s) match-to-place test in a Y-maze configuration in the water maze, where the percentage of correct responses in match trials represents trial unique short-term memory” | content_tags.quantities[8] |
| variable | non-cell-type-specific PFC lesions (independent) — “In a second set of experiments we also made non-cell-type-specific PFC lesions, which have previously been used to investigate the behavioral role of the PFC, by injection of the fiber-sparing neurotoxin ibotenic acid” | content_tags.variables[1] |
| variable | percentage alternation (dependent) — “Percentage alternation was calculated as the number of total complete alternations, divided by the total number of arm entries minus two and expressed as a percentage.” | content_tags.variables[2] |
| variable | path length — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length (PFC-PV-TeLC/PFC-PV-GFP, day effect: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effect: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memory per se does not depend on the PFC” | content_tags.variables[6] |
| variable | spontaneous alternation — “This deficit was confirmed in a spontaneous alternation task in the Y-maze, where alternation levels were high in PFC-PV-GFP and PFC-Saline animals (70.0 ± 3.0% and 74.3 ± 4.1% respectively), but significantly lower in PFC-PV-TeLC and PFC-Lesion animals (56.9 ± 2.7% and 60.0 ± 2.3% respectively)” | content_tags.variables[7] |
| variable | repeat investigations — “both PFC-PV-TeLC and PFC-Lesion animals showed increased levels of repeat investigations (PFC-PV-TeLC vs. PFC-PV-GFP, p = 0.04; t = 1.97; PFC-Lesion vs. PFC-Saline, p = 0.04; t = 1.98)” | content_tags.variables[8] |
| variable | peak activity — “Compared with controls, PFC-Lesion animals showed significantly higher levels of locomotor activity ( Fig. 6F ), with increased peak activity ( p = 0.04; t = 1.97) and slower decay kinetics ( p = 0.005; t = 3.25), confirming the importance of the PFC for amphetamine sensitivity.” | content_tags.variables[9] |
| definition | working memory — “Working memory represents a short-lasting on-line memory buffer that holds information relevant to ongoing tasks” | content_tags.definitions[0] |
| definition | behavioral flexibility — “Behavioral flexibility, which permits the individual to appropriate behavioral strategies to a changing environment, is another core function of the PFC that is impaired in schizophrenia” | content_tags.definitions[1] |
| definition | tetanus toxin light chain (TeLC) — “Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | content_tags.definitions[2] |
| definition | complete alternation — “A complete alternation was defined as an animal entering each of the three arms in sequence without re-entering a previously explored arm.” | content_tags.definitions[3] |
| definition | perfect trial — “A perfect trial was defined as the animal leaving the start arm and immediately entering the goal arm.” | content_tags.definitions[4] |
| definition | discrimination index — “Time spent with stranger 2 was divided by time spent with stranger 1 to give a discrimination index, where 1 (dashed line in Fig. 6C,D ) indicates equal time spent with each stranger.” | content_tags.definitions[5] |
| definition | working memory — “Working memory represents a short-lasting on-line memory buffer that holds information relevant to ongoing tasks” | content_tags.definitions[6] |
| definition | Behavioral flexibility — “Behavioral flexibility, which permits the individual to appropriate behavioral strategies to a changing environment, is another core function of the PFC that is impaired in schizophrenia” | content_tags.definitions[7] |
| definition | tetanus toxin light chain — “As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIs 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2, a protein required for synaptic vesicle docking” | content_tags.definitions[8] |
| definition | match-to-place — “we used a delayed (60s) match-to-place test in a Y-maze configuration in the water maze, where the percentage of correct responses in match trials represents trial unique short-term memory” | content_tags.definitions[9] |
| definition | hole-board — “We implemented a hole-board-based working memory test by analyzing repeat investigations of previously explored holes.” | content_tags.definitions[10] |
| key-terms | PVIs, PFC, tetanus toxin light chain, ibotenic acid, working memory, reversal learning, social preference, amphetamine, schizophrenia, somatostatin, VAMP2, spontaneous alternation |
| concept | PFC — not defined in this paper's own words; prefrontal cortex; here prelimbic, infralimbic, cingulate and medial orbital — “Compared with controls, PFC-Lesion animals showed significantly higher levels of locomotor activity ( Fig. 6F ), with increased peak activity ( p = 0.04; t = 1.97) and slower decay kinetics ( p = 0.005; t = 3.25), confirming the importance of the PFC for amphetamine sensitivity.” | concepts[4] |
| concept | amphetamine — not defined in this paper's own words; psychostimulant whose locomotor effect indexes subcortical dopamine — “Compared with controls, PFC-Lesion animals showed significantly higher levels of locomotor activity ( Fig. 6F ), with increased peak activity ( p = 0.04; t = 1.97) and slower decay kinetics ( p = 0.005; t = 3.25), confirming the importance of the PFC for amphetamine sensitivity.” | concepts[16] |
| t-test t | as written: two-tailed paired or unpaired Student’s t-test — “Unless otherwise stated data were analysed with two-tailed paired or unpaired Student’s t-test as appropriate.” | content_tags.statistics[0] |
| ANOVA F | as written: two-way ANOVA with treatment as between-subject and trial/day as within-subject factors — “We also used two-way ANOVA with treatment as between-subject and trial/day as within-subject factors.” | content_tags.statistics[1] |
| chi-squared / Fisher's exact test | as written: Fisher’s exact or Chi-Square tests — “Contingency analysis applied Fisher’s exact or Chi-Square tests.” | content_tags.statistics[2] |
| significance level (alpha) alpha | as written: alpha greater than 5% — “The null hypothesis was rejected for alpha greater than 5%.” | content_tags.statistics[3] |
| spectral power analysis (Fast Fourier Transform) | as written: spectral power analysis conducted using Fast Fourier Transform — “Artefact free continuous recordings (120s) of LFPs were chosen and spectral power analysis conducted using Fast Fourier Transform in BrainVision Analyzer 2.0 software” | content_tags.statistics[4] |
| t | — “As expected, immunoreactivity for VAMP2 was strongly reduced in TeLC-positive terminals of PFC-PV-TeLC mice when compared with GFP-positive terminals of PFC-PV-GFP mice ( p < 0.0001; t = 22.2; n = 4 animals per group; Fig. 1G ).” | content_tags.statistics[5] |
| day effect | — “During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path length (PFC-PV-TeLC/PFC-PV-GFP, day effect: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effect: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memory per se does not depend on the PFC” | content_tags.statistics[6] |
| Fast Fourier Transform | — “Local field potentials (LFPs) from PFC were recorded while animals were freely moving in their holding cage. Low weight dummies were removed and replaced by a wireless data logger for continuous recording of 4 channels and on board memory (NeuroLogger, New Behavior, Zurich, Switzerland), set at a sampling rate of 200 Hz.” | content_tags.statistics[7] |
| claim type | mechanism — a second reading reaching the same value; recorded: mechanism — “Our results suggest that distinct aspects of the complex symptomatology of PFC dysfunction in schizophrenia can be attributed to specific prefrontal circuit elements.” | alternatives.claim_type |
| method | cell-type-selective synaptic silencing with Cre-dependent tetanus toxin light chain, compared against a titrated excitotoxic lesion of the same region, across a behavioural battery — a second reading DIFFERING from the recorded value; recorded: behavioural-experiment — “We reasoned that this approach would confirm the PFC-dependence of our behavioral tests and that qualitative comparison of the behavioral deficits between PV-cell-selective and non-cell-type-selective circuit lesions would allow us to dissociate general PFC functions from those that specifically involve PVIs.” | alternatives.method |
| phenomenon | the behavioural syndrome produced by prefrontal dysfunction, split by which circuit element is removed — a second reading DIFFERING from the recorded value; recorded: prefrontal parvalbumin interneuron control of working memory and cognitive flexibility — “We found that PFC lesions produced behavioral alterations in all three symptom categories, whereas selective inactivation of PVIs caused deficits only in the cognitive domain.” | alternatives.phenomenon |
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Parvalbumin-positive interneurons of the prefrontal cortex support working memoryterm and cognitiveterm flexibility Andrew J MurraypersName Andrew J MurraypersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 2 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA 1, 2, * , Marta U Woloszynowska-FraserpersName Marta U Woloszynowska-FraserpersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 1, * , Laura Ansel-BollepallipersName Laura Ansel-BollepallipersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 3 Institute of Physiology, Christian Albrechts University Kiel, 24098 Kiel, Germany 1, 3 , Katy L H ColepersName Katy L H ColepersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 1 , Angelica FoggettipersName Angelica FoggettipersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 3 Institute of Physiology, Christian Albrechts University Kiel, 24098 Kiel, Germany 1, 3 , Barry CrouchpersName Barry CrouchpersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 1 , Gernot RiedelpersName Gernot RiedelpersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 1, a , Peer WulffpersName Peer WulffpersName 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 3 Institute of Physiology, Christian Albrechts University Kiel, 24098 Kiel, Germany 1, 3, b 1 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 2 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA 3 Institute of Physiology, Christian Albrechts University Kiel, 24098 Kiel, Germany a Email: g.riedelpersName@abdn.ac.uk b Email: p.wulffpersName@physiologie.uni-kiel.de * These authors contributed equally to this work.
Received 2015 Jun 26; Accepted 2015 Oct 20; Collection date 2015.
Copyright © 2015, Macmillan Publishers Limited This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ PMCID: PMC4660359 PMID: 26608841 Abstract Dysfunction of parvalbumin (PV)-positive GABAergic interneurons (PVIsterm) within the prefrontal cortex (PFCterm) has been implicated in schizophreniaterm pathology. It is however unclear, how impaired signaling of these neurons may contribute to PFCterm dysfunction. To identify how PVIsterm contribute to PFC-dependent behaviors we inactivated PVIsterm in the PFCterm in mice using region- and cell-type-selective expression of tetanus toxin light chain (TeLC)term·def and compared the functional consequences of this manipulation with non-cell-type-selective perturbations of the same circuitry. By sampling for behavioral alterations that map onto distinct symptom categories in schizophreniaterm, we show that dysfunction of PVIterm signaling in the PFCterm specifically produces deficits in the cognitiveterm domain, but does not give rise to PFC-dependent correlates of negativeterm or positiveterm symptoms. Our results suggest that distinct aspects of the complex symptomatology of PFCterm dysfunction in schizophreniaterm can be attributed to specific prefrontal circuit elements.
Functional imaging studies have suggested that dysfunction of the prefrontal cortex (PFCterm) underlies a multitude of deficits associated with schizophreniaterm, comprising positiveterm (e.g. delusions and hallucinations), negativeterm (e.g. emotional and social dysfunction) and cognitiveterm (e.g. impairments in working memoryterm and cognitiveterm flexibility) symptoms 1 , 2 , 3 , 4 , 5 . Although these disturbances all appear to involve PFCterm dysfunction, it is unclear whether specific circuit elements within the PFCterm contribute differentially to symptom complexity.
Post-mortem studies of schizophreniaterm patients have suggested that PFCterm dysfunction involves alterations of the inhibitory circuitry of the PFCterm 6 , 7 , 8 . In particular reduced mRNA levels of the 67 kD isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD67term) have been consistently reported 9 , 10 , 11 , 12 , 13 . These changes seem to dominantly affect a subclass of GABAergic interneurons, that expresses the calcium-binding protein PV, which itself shows reduced expression in schizophreniaterm 11 , 13 , 14 , 15 , 16 . In addition, genes associated with increased susceptibility to schizophreniaterm like DISC1, NRG1/ERBB4 or dysbindin, play crucial roles for circuit integration and function particularly of PVIsterm in rodents 17 , 18 , 19 , 20 , 21 . PVIsterm are also preferentially damaged by drugs of abuse such as phencyclidine and ketamine, which produce schizophrenia-like symptoms 22 , 23 . Accordingly impaired signaling of PVIsterm may form a common endpoint for different genetic and environmental factors leading to PFCterm malfunction and schizophrenia-associated deficits 19 , 24 .
In the isocortex PVIsterm mainly comprise basketterm and chandelier cellsterm, which control network activity by targeting the soma and proximal dendrites or the axon initial segment of principal cells, respectively 25 . They are therefore well placed to exert rigorous control over PFCterm activity. However, little is known about the behavioral functions of these interneurons within the PFCterm circuitry.
To directly test how chronic impairment of PVIterm signaling affects PFC-dependent behavior, we selectively blocked output from PVIsterm in the mouse PFCterm by cell-type- and region-selective expression of tetanus toxin light chain (TeLC)term·def. To distinguish PFC-dependent behaviors which require intact PVIterm signaling from those that do not, we compared PVI-selective interference with non-cell-type-selective perturbations of the same circuitry in behavioral assays that were chosen to detect alterations that map onto symptom categories (positiveterm, negativeterm, cognitiveterm) in schizophreniaterm.
Results PV-cell-specific disruption of synaptic transmission in the PFCterm We made stereotaxic injections of adeno-associated viruses carrying a GFP-tagged TeLC (or GFP alone as control, Fig. S1 ) reading frame inverted (3’ to 5’) in a flip-excision cassetteterm (AAV-FLEX-TeLC and AAV-FLEX-GFP) 26 into the PFCterm of PV-Cre knock-in mice 27 . As Cre-recombinase is required to flip the reading frame into the correct orientation, transcription of TeLC can only occur in PVIsterm 26 . Once expressed, TeLC efficiently stops transmitter release by cleaving VAMP2term, a protein required for synaptic vesicle docking 26 ( Fig. 1A ). Bilateral infusions of AAV-FLEX-TeLC resulted in expression of TeLC in over 75% of PVIsterm in prelimbic and infralimbic regions of the PFCterm ( Fig. 1C,D,F ). Additionally, TeLC expression was found in the cingulate and medial orbital cortices, demonstrating functional removal of PVIsterm across an area of mouse PFCterm thought to integrate functions of the dorsolateral, medial and orbital PFCterm in humans 28 , 29 , 30 . Transgene-expression in PV-negative cells was scarce (4.3%) and likely includes PVIsterm containing PV levels below detection threshold. Similar results were obtained for control AAV-FLEX-GFP injections (PFC-PV-GFP mice, data not shown). As expected, immunoreactivity for VAMP2term was strongly reduced in TeLC-positive terminals of PFC-PV-TeLC mice when compared with GFP-positive terminals of PFC-PV-GFP mice ( p < 0.0001; t = 22.2; n = 4 animals per group; Fig. 1G ). The residual VAMP2term immunoreactivity is likely due to low affinity recognition of VAMP2term cleavage products by the antibody 31 . However, using the same AAV-FLEX-TeLC we have previously shown that transmission of hippocampal PVIsterm was fully abolished 10 days after virus injection despite residual VAMP2term immunoreactivity 26 . In principle, loss of PVI-mediated inhibition could induce hyper-activity in pyramidal cells and induce ictal discharges in the PFCterm, which in turn could complicate the interpretation of behavioral data. However, local field potentialterm (LFP) recordings from PFCterm showed no indication of enhanced synchronous activity in PFC-PV-TeLC mice. PFC-PV-TeLC and PFC-PV-GFP mice displayed similar absolute power at different spectral frequency bands ( Fig. S2 ). In a second set of experiments we also made non-cell-type-specific PFC lesionsterm·var, which have previously been used to investigate the behavioral role of the PFCterm, by injection of the fiber-sparing neurotoxin ibotenic acidterm 32 , 33 , 34 , 35 , 36 , 37 (or saline as control; PFC-Lesion and PFC-Saline mice; Fig. 1B,E,H ). We reasoned that this approach would confirm the PFC-dependence of our behavioral tests and that qualitative comparison of the behavioral deficits between PV-cell-selective and non-cell-type-selective circuit lesions would allow us to dissociate general PFCterm functions from those that specifically involve PVIsterm. To produce perturbations of comparable location and extent we titrated ibotenic acidterm injections according to viral spread.
Figure 1. Cell-type-selective and general inactivation in the prefrontal cortex.
( A ) Injection of AAV-FLEX-TeLC into the PFCterm selectively blocks synaptic transmission in PVIsterm (grey shade in circuit cartoon) by cleaving VAMP2term. Transmission in all other neurons remains intact. ( B ) Injection of ibotenic acidterm into the PFCterm causes general disruption of neuronal circuitry (grey shade). ( C ) Schematic of AAV spread in the mPFC of AAV-FLEX-TeLC-injected PV-Cre animals showing minimum (all animals >50% infected PVIsterm) and maximum (>50% infected PVIsterm only in most affected animals) infection area. ( D ) Immunohistochemistry illustrating co-localization of TeLC-GFP and PV in the mPFC ( E ), Schematic showing minimum (common to all animals) and maximum (in most affected animals) extent of ibotenic acidterm lesion in the mPFC. ( F ) Percentage of TeLC-GFP-expressing PVIsterm in subregions of the PFCterm. ( G ) Quantification of VAMP2term immunofluorescence intensity shows a substantial reduction in GFP-TeLC + /vGAT + puncta in PFC-PV-TeLC animals compared with GFP + /vGAT + puncta in PFC-PV-GFP animals. ( H ) Percentage of lesioned area in subfields of the PFCterm after ibotenic acidterm injection. PrL Prelimbic cortex; IL Infralimbic cortex; M2 Secondary motor cortex; CgL Cingulate; MO Medial orbital cortex. Scale bar in D = 20 µm. Data are mean ± s.e.m. ***p < 0.001.
PVIsterm in the PFCterm are required for spatial working memoryterm Neither PFC-PV-TeLC nor PFC-Lesion mice showed obvious neurological deficits or alterations in locomotion and anxiety-related behavior ( Fig. 2A-D ). As deficits in working memoryterm are a dominant cognitiveterm symptom in both schizophreniaterm and PFCterm lesions 4 , 35 , 38 , 39 we investigated the involvement of PVIsterm in the production of working memoryterm. We implemented a hole-board-based working memoryterm test by analyzing repeat investigationsterm·var of previously explored holes. Although all groups of mice showed similar overall levels of nose pokes ( Fig. 2E,F ), indicating similar exploratory activity, both PFC-PV-TeLC and PFC-Lesion animals showed increased levels of repeat investigationsterm·var (PFC-PV-TeLC vs. PFC-PV-GFP, p = 0.04; t = 1.97; PFC-Lesion vs. PFC-Saline, p = 0.04; t = 1.98) ( Fig. 3A,B ). This deficit was confirmed in a spontaneous alternationterm task in the Y-maze, where alternation levels were high in PFC-PV-GFP and PFC-Saline animals (70.0 ± 3.0% and 74.3 ± 4.1% respectively), but significantly lower in PFC-PV-TeLC and PFC-Lesion animals (56.9 ± 2.7% and 60.0 ± 2.3% respectively) ( Fig. 3C,D ). Both reinvestigation and spontaneous alternationterm probe a spontaneous on-line processing of information. To assess whether PVIsterm also contribute to temporally expanded forms of working memoryterm, which are known to be impaired after PFCterm lesions 40 , 41 , we used a delayed (60s) match-to-placeterm test in a Y-maze configuration in the water maze, where the percentage of correct responses in match trials represents trial unique short-term memory 26 . Whereas PFC-PV-GFP animals showed significant improvement in match trials, PFC-PV-TeLC animals did not ( Fig. 3E ).
Figure 2. Neither PFC-PV-TeLC nor PFC-Lesion animals showed alterations during baseline behavioral assessment.
Analysis of path lengthterm·var in the open field for ( A ), PFC-PV-GFP (n = 8) and PFC-PV-TeLC (n = 10) and ( B ), PFC-Saline (n = 9) and PFC-Lesion (n = 13) animals suggests normal baseline motor activity. ( C,D ) Time spent in the centre of an open field arena indicates no alterations in anxiety-related thigmotaxis. ( E,F ) The number of nose pokes during exploration of a hole-boardterm arena is similar in all groups of mice indicating normal exploratory activity. Data are mean ± s.e.m.
Figure 3. Working memoryterm is impaired in PFC-Lesion and PFC-PV-TeLC animals.
( A ) PFC-PV-TeLC (n = 10) animals make significantly more repeat investigationsterm·var in the hole-boardterm test than PFC-PV-GFP (n = 8) and ( B ), PFC-Lesion (n = 13) mice make significantly more repeat investigationsterm·var than PFC-Saline (n = 9) animals. ( C ) In the same cohorts of mice spontaneous alternationterm in a Y-maze is reduced in PFC-PV-TeLC animals and in ( D ), PFC-Lesion animals compared to respective controls. ( E ) The same PFC-PV-TeLC and PFC-PV-GFP mice were subjected to a match to placeterm protocol in the water Y-maze. PFC-PV-GFP animals are able to retain the platform position during the 1 minute delay period, whereas PFC-PV-TeLC animals are not. Data are mean ± s.e.m. * P < 0.05.
Thus various modalities of PFC-dependent working memoryterm critically depend on PVIterm integrity.
Cognitiveterm flexibility depends on prefrontal PVIsterm Cognitiveterm flexibility is impaired in schizophreniaterm and after PFCterm lesion 41 , 42 , 43 , 44 . We investigated the relevance of prefrontal PVIsterm for cognitiveterm flexibility in a reversal learningterm task in the water maze 36 , 37 . After initial spatial training (5 days, 4 trials/day) the platform was re-located to the opposite quadrant and mice underwent another 4 days (4 trials/day) of reversal training. During initial training PFC-PV-TeLC and PFC-Lesion mice learned the platform location as well as their respective controls as indicated by the reduction in path lengthterm·var (PFC-PV-TeLC/PFC-PV-GFP, day effectterm·stat: F(4,120) = 45; p < 0.0001, no effect with group as factor: F’s < 1; PFC-Lesion/PFC-Saline, day effectterm·stat: F(4,80) = 28; p < 0.0001; no effect with group as factor: F’s < 1) ( Fig. 4A,B ; see also Fig. 5 ) and the amount of time spent in the target quadrant during probe trials ( t’s < 1.4) ( Fig. 4 insets in A,B), indicating that spatial reference memoryterm per se does not depend on the PFCterm 35 , 36 . In contrast, during reversal, PFC-PV-TeLC and PFC-Lesion mice needed more time to learn the new platform location although this was only significant for PFC-PV-TeLC mice (PFC-PV-TeLC vs. PFC-PV-GFP, day effectterm·stat F(3,84) = 24; p < 0.001; group effect F(4,84) = 8.8; p = 0.006; and PFC-Lesion vs. PFC-Saline, F(3,60) = 2.1; p = 0.16) ( Fig. 4C–F ). The deficit was transient as PFC-PV-TeLC mice performed as well as controls in the probe trial after over-training (t’s < 1.1) ( Fig. 4 insets in C,D). We confirmed these deficits by analyzing two additional parameters. 1) The number of trials to attain floor level (4m path lengthterm·var, derived from Fig. 4 ) in two consecutive trials was not different between PFC-PV-TeLC or PFC-Lesion groups and their controls ( Fig. 5A,B ; t’s < 1.4) during initial training, but was significantly higher in reversal trials ( Fig. 5A,B , t’s > 1.7; p ’s ≤ 0.05). 2) We established the percentage of subjects meeting criterion (4m path lengthterm·var) for each trial during both training phases ( Fig. 5C–F ). During initial training we detected small differences between PFC-PV-TeLC or PFC-Lesion animals and their respective controls (a mild delay or acceleration in learning, respectively; Fig. 5C,D ). However, all subjects met criterion at completion of acquisition. During reversal both PFC-PV-TeLC and PFC-Lesion mice showed a deficit in learning the new platform location ( Fig. 5E,F ). However, whereas PFC-PV-TeLC mice met reversal criterion upon overtraining, more than 30% of PFC-Lesion mice failed to do so. These results suggest that PVIsterm contribute to prefrontal operations involved in the inhibition of previously learned responses.
Figure 4. PFC-PV-TeLC and PFC-Lesion animals show impaired reversal learningterm.
Both PFC-PV-GFP (n = 15) and PFC-PV-TeLC (n = 17) ( A ) and PFC-Saline (n = 9) and PFC-Lesion (n = 13) animals ( B ) learn the location of the hidden platform in an open field water maze as indicated by 1) the reduction in path lengthterm·var to platform over the 5 day training period and 2) the amount of time spent in the target quadrant in a probe trial after training (insets). ( C ) PFC-PV-TeLC and ( D ), PFC-Lesion animals show a deficit in reversal learningterm as indicated by a delay in path lengthterm·var reduction during 4 days of reversal training (R1-4). All groups spent significantly more time in the new target quadrant in a probe trial after reversal learningterm (insets in C and D ). ( E,F ) Example traces of path taken by PFC-PV-GFP ( E , green), PFC-PV-TeLC (E, blue), PFC-Saline ( F , grey) and PFC-Lesion ( F , red) animals on the third day of reversal training. The original platform location is indicated as a hollow circle, the new platform location as filled circle. Note the increased length around the original platform location of PFC-PV-TeLC and PFC-Lesion animals compared to respective controls. Data are mean ± s.e.m.
Figure 5. Criterion analysis confirms deficits in PFC-PV-TeLC and PFC-Lesion animals during reversal learningterm.
The average number of trials to criterion for PFC-PV-TeLC (blue) ( A ) and for PFC-Lesion (red) animals ( B ) was similar to respective controls (green and grey) during acquisition (left) but significantly different during reversal (right) learning. ( C,D ) During initial water maze training PFC-PV-TeLC ( C ; n = 17) and PFC-Lesion ( D ); n = 13) animals show small differences when compared to their respective controls (n = 15 and 9), but all animals acquire the task to the same standard. ( E,F ) After re-location of the platform both PFC-PV-TeLC ( E ) and PFC-Lesion ( F ) animals are significantly impaired at learning the new platform location. Data are mean ± s.e.m. *P < 0.05; ***p < 0.001.
PVIsterm are dispensable for PFC-dependent behavior in a social preferenceterm test Impairments in social function are common after prefrontal lesions and a component of the negativeterm symptomatology in schizophreniaterm 2 , 45 , 46 . Similarly, interference with PFCterm function in rodents has been shown to reduce social interaction 47 , 48 . We tested PFC-PV-TeLC, PFC-Lesion and control animals for sociability and social recognition in a 3-chamber social preferenceterm test 49 . During sociability testing PFC-Lesion animals showed strongly reduced interaction with the conspecific animal ( p = 0.003; t = 3.57) ( Fig. 6B ). However, PFC-PV-TeLC mice displayed similar levels of social interaction as PFC-PV-GFP control animals ( p = 0.15; t = 1.59) ( Fig. 6A ). A similar pattern evolved in the second phase, where both control groups and PFC-PV-TeLC mice spent significantly more time with the unfamiliar than with the familiar animal ( p = 0.003; t = 3.57 for PFC-PV-TeLC; p = 0.04; t = 1.97 for PFC-PV-GFP; p = 0.04; t = 2.00 for PFC-Saline), indicating intact social memory and a preference for social novelty ( Fig. 6C,D ). In contrast PFC-Lesion animals did not discriminate between familiar and unfamiliar mice ( p = 0.3; t < 1) ( Fig. 6D ).
Figure 6. PFC-Lesion but not PFC-PV-TeLC mice show impaired social behavior and amphetamineterm hypersensitivity.
( A,B ) Quantification of time spent interacting with a conspecific stranger (S1) and an empty chamber ( E ) in a 3-chamber social preferenceterm test. ( A ) PFC-PV-GFP (n = 8) and PFC-PV-TeLC (n = 10) animals spent significantly more time interacting with a conspecific mouse than with an empty chamber. ( B ) PFC-Lesion animals (n = 13) do not show a significant preference for social interaction. Example traces in ( A , B ) illustrate the travelled path. Increased density in the stranger compartment of PFC-PV-GFP, PFC-PV-TeLC and PFC-Saline (n = 9) but not Lesion animals indicates preference for social interaction. ( C,D ) Discrimination between a novel (S2) and a familiar (S1) conspecific. PFC-PV-GFP, PFC-PV-TeLC and PFC-Saline animals spent significantly more time with the novel mouse indicating social recognition. PFC-Lesion animals did not distinguish between novel and familiar animals. Dashed line indicates level where equal time is spent with familiar and stranger animals. ( E,F ) Response to amphetamineterm. ( E ) Path lengthterm·var in an open field of PFC-PV-GFP (n = 8) and PFC-PV-TeLC (n = 10) animals. Dashed line indicates amphetamineterm administration. Both groups show enhanced locomotor activity after amphetamineterm administration. Insets show cumulated path lengthterm·var during the amphetamineterm activity period. ( F ) PFC-Saline (n = 9) and PFC-Lesion (n = 13) animals before and after amphetamineterm administration. Both groups show enhanced locomotor activity after receiving amphetamineterm but PFC-Lesion animals show a hypersensitivity as indicated by increased peak locomotor activity (inset) and slower decay kinetics. Data are mean ± s.e.m. * P < 0.05; ** p < 0.01; ***p < 0.001.
Accordingly social approach/avoidance behavior as well as social recognition memory/novelty preference depends on the PFCterm circuitry. PV-positive interneurons, however, do not seem to be a crucial component of this circuit.
Impaired signaling of PV-positive PFCterm interneurons does not alter behavioral sensitivity to psychostimulants Increased sensitivity to psychostimulants, such as amphetamineterm, is common in schizophreniaterm patients and considered a rodent correlate of the positiveterm symptoms 45 , 50 , 51 . It is thought that both positiveterm symptoms and increased amphetamineterm sensitivity are due to enhanced subcortical dopaminergic tone, which in turn correlates with and might be secondary to PFCterm dysfunction in schizophreniaterm 3 , 50 , 52 . Consistent with this idea, lesions of the PFCterm in rodents also increase amphetamineterm sensitivity and subcortical dopamine presumably through dysregulation of dopaminergic neurons in the ventral brainstem after loss of glutamatergic PFCterm afferent control 33 , 53 . Intraperitoneal injection of amphetamineterm caused a substantial increase in locomotor activity in all groups of mice with peak activityterm·var 10 to 15 minutes after injection ( Fig. 6E,F ). Compared with controls, PFC-Lesion animals showed significantly higher levels of locomotor activity ( Fig. 6F ), with increased peak activityterm·var ( p = 0.04; t = 1.97) and slower decay kinetics ( p = 0.005; t = 3.25), confirming the importance of the PFCterm for amphetamineterm sensitivity. In contrast, PFC-PV-TeLC animals did not differ from controls in amphetamineterm response (peak activityterm·var, t < 1; activity decay, t < 1) ( Fig. 6E ).
These results support the idea that the PFCterm controls subcortical dopamine release and may thus be causally involved in the generation of positiveterm symptoms 53 . However, PVIsterm do not seem to be required for this regulatory function of the PFCterm.
Discussion Schizophreniaterm presents with a complex blend of positiveterm, negativeterm and cognitiveterm symptoms and PFCterm dysfunction may be causally involved in the origin of all three symptom categories. Within the PFCterm, alterations of the inhibitory circuitry, which appear to preferentially involve PVIsterm, are among the most robust histological findings. To detect PFCterm functions that depend on intact signaling of PVIsterm we contrasted PV-cell-specific with general perturbations of the PFCterm circuitry. We found that PFCterm lesions produced behavioral alterations in all three symptom categories, whereas selective inactivation of PVIsterm caused deficits only in the cognitiveterm domain.
Working memoryterm represents a short-lasting on-line memory buffer that holds information relevant to ongoing tasks 54 . Although it is generally accepted that short-term memory relies on a distributed network of brain regions connected to and orchestrated by the PFCterm 35 , 55 , the physiological correlates of working memoryterm are not clear. At the systems level synchronous network activity within the PFCterm and across functionally coupled brain structures may aid information processing during working memoryterm 56 , 57 . Generation of network oscillations in turn is thought to depend on the activity of fast-spiking putative PVIsterm 58 , 59 , 60 , 61 . Here we show that PFC-dependent working memoryterm indeed fully depends on intact transmission from PVIsterm.
Behavioral flexibilityterm, which permits the individual to appropriate behavioral strategies to a changing environment, is another core function of the PFCterm that is impaired in schizophreniaterm 43 , 44 , 62 . The process may be mediated by PFCterm driven desynchronisation of network activity and thus functional uncoupling across task-relevant brain structures 63 . We have assessed behavioral flexibilityterm in a water maze reversal task. Whereas both PFC-Lesion and PFC-PV-TeLC animals showed little difference in acquisition learning of a reference memory task, they needed more time to re-learn the platform position after its relocation. Analysis of reversal performance by subject revealed that this deficit was transient in PFC-PV-TeLC animals, where all mice eventually acquired the new location. By contrast, more than 30% of PFC-Lesion mice did not reach floor level after platform relocation suggesting that complete inactivation of the PFCterm circuitry induced more persistent impairments in reversal learningterm. Overall, our results indicate that impaired signaling of PFCterm PVIsterm leads to a transient failure to adjust behavioral responses to altered cue-reward associations.
Among the negativeterm symptoms in schizophreniaterm social withdrawal and impairments in social cognition represent core elements and are amenable to modeling in rodents 2 , 46 , 64 . We found that general PFCterm dysfunction impairs both social-approach avoidance behavior and social recognition memory. Similar results were obtained after PFCterm perturbations in rats and in DISC1 and neuregulin-1 mouse models of schizophreniaterm (for review see 47 , 64 ). Accordingly, optogenetic stimulation of the PFCterm has been shown to enhance social interaction in stressed mice 65 , presumably through prefrontal regulation of dopaminergic projections between ventral tegmental area (VTA) and nucleus accumbens and/or serotonergic raphe neurons 53 , 66 , 67 . Similarly, positiveterm symptoms and enhanced amphetamineterm sensitivity in schizophreniaterm may result from deranged prefrontal control of brain stem dopamine neurons 3 , 33 , 50 , 52 , 53 . In line with this theory we found an enhanced peak and decelerated decay of the locomotor response to amphetamineterm in PFC-Lesion animals.
The results we obtained after non-cell-type-selective inactivation in mice thus confirm that PFCterm dysfunction may be causally involved not only in cognitiveterm symptoms but also in the emergence of positiveterm and negativeterm symptoms, presumably by affecting the complex mono- and polysynaptic prefrontal regulation of subcortical monaminergic pathways 53 , 66 . Interestingly, complete disruption of GABA A -receptor-mediated inhibition in the PFCterm by bicuculline infusion has been shown to cause similar behavioral alterations as those we report here for PFC-lesion animals 68 , 69 , 70 . In contrast, selective inactivation of one sub-population of GABAergic interneurons – PVIsterm – caused deficits only in the cognitiveterm domain, suggesting that these interneurons may subserve specific circuit functions in the PFCterm. Such a “division of labor” scenario between different populations of interneurons is further supported by our finding that analogous tetanus toxin-based targeting of a different, somatostatinterm (Sst)-expressing, population of interneurons in the PFCterm of Sst-Cre mice 71 caused no change in working memoryterm performance in the same task that was clearly impaired after inactivation of PVIsterm ( Fig. S3 ).
In summary, impaired signaling of PVIsterm in the PFCterm, which is thought to form a core pathology in schizophreniaterm, may be causally involved in the development of cognitiveterm symptoms. However, amphetamine-hypersensitivity and deficits in social-approach avoidance behavior as indicators of positiveterm and negativeterm symptoms, respectively, are not precipitated by PVIterm dysfunction. Based on these data we suggest that different aspects of the composite symptomatology of PFCterm dysfunction in schizophreniaterm can be allocated to specific neuronal elements.
Materials and Methods All procedures involving experimental mice were in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Aberdeen.
Production of recombinant adeno-associated viral vectors AAV vectors were produced as described previously 26 , 72 . Briefly, AAV virions were produced that contained a 1:1 ratio type 1 and type 2 capsid proteins by transfecting human embryonic kidney (HEK) 293 cells with the AAV backbone plasmids pAM-FLEX-TeLC or pAM-FLEX-GFP 26 and AAV1 (pH21), AAV2 (pRV1) helper plasmids along with the adenovirus helper plasmid pFdelta6 using the calcium phosphate method 72 . 48 hours post transfection the cells were harvested and AAVs purified using 1 ml HiTrap heparin columns (Sigma) and concentrated using Amicon Ultra centrifugal filter devices (Millipore). Infectious AAV particles (viral titre) were calculated by serially infecting HEK293 cells stably expressing Cre-recombinase 26 and counting GFP-positive cells.
Animals, surgery and recording of local field potentialsterm Homozygous PV-Cre 27 and Sst-Cre 71 mice were purchased from Jackson laboratories (Repository number: PV 008069; SST 013044) and maintained as homozygous colonies. All stereotaxic injections and behavioral testing were carried out on male and female homozygous PV-Cre or SST-Cre littermates starting between P42 and P56. Animals were housed in a temperature-controlled environment with a 12 hour light/dark cycle and free access to food and water. Stereotaxic surgery was carried out as in 26 . Briefly, anesthesia was induced with 3% isoflurane in O 2 by inhalation and maintained on 1.5–2% isoflurane throughout surgery. Heads of mice were fixed in a stereotaxic frame (Stoelting, USA), an incision was made on the skin above the skull and the skull exposed. Small holes were drilled relative to Bregma. The coordinates used were AP +2.0 mm, L ±1.0 mm, angle 17°, depth −2.6 mm. Lesions were made by injecting 1 μl of 1 mg/ml ibotenic acidterm, control animals received an injection of 1 μl vehicle (PBS). PFC-PV-GFP and PFC-PV-TeLC animals were injected with AAV-FLEX-GFP and AAV-FLEX-TeLC respectively. Both AAVs had a titer of about 6 × 10 6 infectious particles per μl. 2 μl of rAAV along with 1 μl of 20% mannitol solution (total 3 μl volume) were injected over a 20 minute period. Stereotaxic injections were made using a pulled glass pipette (VWR) with an 8 μm bore width. After removal of the glass pipette the burr hole was filled with bone wax, the skin replaced and fixed using Vetbond tissue adhesive (3M).
For LFP recordings double twisted insulated gold wire depth electrodes (ADVENT Research Materials, Oxford, UK) were lowered bilaterally into prefrontal cortex (and an additional set into hippocampal CA1) at the same coordinates as viral deposition. Wires were externalized and a head-stage was formed of gold contact pins. Two gold plated watchmaker screws (TSE Systems GmbH, Germany) also connected to pins served as reference and ground respectively and were located above the posterior parietal cortex on either side of Lambda. Electrodes were anchored to the scull using dental cement and pins were covered with a low weight dummy connector.
Post-operative analgesia was given as needed. Behavioural analysis began 10 days after stereotaxic injections. All animals were housed individually after surgery and during behavioural testing periods.
Local field potentialsterm (LFPs) from PFCterm were recorded while animals were freely moving in their holding cage. Low weight dummies were removed and replaced by a wireless data logger for continuous recording of 4 channels and on board memory (NeuroLogger, New Behavior, Zurich, Switzerland), set at a sampling rate of 200 Hz. A total of 10 minutes were recorded, files were extracted from the Neurologgers using the customised CommSW application and further converted into .txt format using an in-house MatLAB script (MatLAB 8.3, The MathWorks Inc., Natick, USA). Artefact free continuous recordings (120s) of LFPs were chosen and spectral power analysis conducted using Fast Fourier Transformterm·stat in BrainVision Analyzer 2.0 software (BrainProducts GmbH, Germany) using ASCII (American standard code for information interchange) data format with multiplexed orientation, time domain, and sampling interval of 5000 μs (200 Hz).
Histology Mice were deeply anaesthetised by intraperitoneal injection of medetomidine/ketamine (1 and 6 mg per kg, respectively) and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS (PFA). After removal brains were post-fixed in PFA overnight at 4 °C. 50 μm coronal sections were taken on a VT1200S vibratome (Leica). Sections were permeabilised in 0.4% Triton X-100 for 30 minutes at room temperature and incubated with primary antibody overnight at 4°C in PBS containing 2% normal goat serum and 0.1% Triton X-100. Sections were washed three times in PBS containing 1% NGS for 10 minutes at room temperature and incubated with secondary antibody for 2.5 hours at room temperature. Sections were washed twice in PBS containing 1% NGS and once in PBS only for 10 minutes at room temperature, mounted on Superfrost glass slides (VWR International) and cover-slipped in Mowiol. Primary antibodies used were rabbit polyclonal GFP (1:1000) (Invitrogen), rabbit polyclonal PV (1:1000) (Swant), rabbit polyclonal VAMP2term (1:500) (Synaptic Systems), guinea pig polyclonal vesicular GABA transporter (vGAT; 1:500) (Synaptic Systems), mouse monoclonal GFP (1:1000) (Invitrogen). Secondary antibodies used were Alexa Fluor goat anti mouse 488, Alexa Fluor goat anti rabbit 488, Alexa Fluor goat anti mouse 647 (all Invitrogen; 1:1000) and goat anti rabbit Cy3 and goat anti guinea pig Cy5 (Jackson Immunoresearch; 1:500).
For analyses of GFP-TeLC-expessing PV-positive neurons (n = 9344 cells) sections were taken from at least 3 points along the rostro-caudal extent of the PFCterm and immunostained for GFP and PV. XY scans of the entire coronal section were taken on an epifluorescent Zeiss Axio Imager M1 microscope. Images of GFP and PV immunostaining were acquired separately and merged digitally in Adobe Photoshop (version CS3). The appropriate image of the mouse brain atlas 73 was digitally overlayed to delineate the brain section into regions. The total numbers of PV-positive and GFP-TeLC-positive neurons were counted in the following regions: prelimbic cortex, infralimbic cortex, cingulate cortex area 1, secondary motor cortex, ventral orbital cortex, medial orbital cortex. Water maze experiments were replicates of two cohorts. Only cohort 1 was quantitatively assessed (n = 10/17). For cohort 2 and all PFC-PV-GFP animals correct viral expression was confirmed visually.
For analysis of ibotenic acidterm lesions, brains were flash frozen in liquid nitrogen and 40 μm sections were cut on a cryostat (Leica) and mounted on superfrost plus slides. Sections were stained with cresyl violet and images captured with a Zeiss Axioimager M1 microscope. The mouse brain atlas was digitally overlayed and the lesion in each brain area calculated as a percentage of the total brain area in ImageJ.
For analysis of VAMP2term expression three sections from four animals from both the PV-PFC-GFP and PV-PFC-TeLC groups were randomly selected and immunostained for VAMP2term, vGAT and GFP. Images were taken using a Zeiss LSM 510 confocal microscope at equal exposure times for each image. Analysis was carried out blind to the experimental group. GFP or GFP-TeLC and vGAT-positive bouton-like structures were randomly chosen and traced using Zeiss Zen 2009 image software. Fluorescence intensity for VAMP2term within the traced area was measured as a mean grey scale value in ImageJ. In total 87 GFP-positive and 65 TeLC-positive puncta were analysed.
Behavioural analysis Behavioral testing of Experiment 1 was performed in PFC-PV-TeLC mice (n = 10) first to detail the phenotypes relative to PFC-PV-GFP (n = 8) mice. In a second follow-on experiment PFC-Lesion mice (n = 13) were tested against PFC-Saline mice (n = 9). This experimental design precludes a direct 2 × 2 statistical comparison. The general sequence of testing was open field, hole boardterm, Y-maze alternation, social interaction, water maze and amphetamineterm challenge. Water maze experiments of PFC-PV-TeLC and PFC-PV-GFP mice were replicates of two cohorts with n = 7 for each group in the second cohort.
A separate cohort of PFC-Sst-GFP (n = 8) and PFC-Sst-TeLC (n = 11) was tested in a spontaneous alternationterm paradigm.
Open field Motor activity was analysed in a square white Perspex arena (60 × 60 cm). Animals were placed in the arena and tracked for 10 minutes with an overhead CCTV camera, data was recorded onto a PC and analysed using Ethovision XT software (Noldus). The arena was cleaned thoroughly between animals to eliminate odour cues. We analysed path lengthterm·var travelled during the trial, time spent in periphery or centre (equi-area) and habituation, as measured by change in path lengthterm·var over time.
Hole-boardterm and reinvestigation The 40 cm by 40 cm hole board-arena was devoid of side walls and elevated 10 cm above the bench. It had 16 equi-distant holes containing infra-red sensors to record “nose-pokes” into the hole. The number of nose-pokes over a 5 minute period was recorded for each animal. Each trial was simultaneously recorded using an overhead CCTV camera for post-hoc analysis of reinvestigation. Reinvestigation was analyzed manually by an experimenter blind to the experimental group. The position of each nose-poke was noted, and the number of repeat pokes into previously explored holes was divided by the number of initial pokes. An animal that did not reinvestigate any holes would receive a score of 0.
Match to placeterm Y-maze The match to placeterm Y-maze was carried out as detailed in 26 . Briefly, trials were carried out in a circular white Perspex pool 150 cm in diameter and 50 cm deep filled with water to a depth of 35 cm with water at 21 ± 1 °C. The arena was equipped with 8 radiating arms (arm length 55 cm, arm width 15 cm, central area 40 cm diameter). The protocol was a delayed matching to place test with 3 trials per day (max trial time 60s) and inter-trial intervals of 60 s. The mice were released into the start arm facing the wall. The first trial was a free swim/extinction trial with both the start arm and one possible goal arm open but without an accessible platform. In the second trial, a second goal arm containing the submerged platform was open (sample trial) and the animal released again from the start arm and allowed to find the hidden platform. If it did not find the platform within 60s it was guided to it by the experimenter. Trial 3 was a match trial, where the animal was again released from the start arm and was monitored for performance with the platform remaining in the same position. A perfect trialterm·def was defined as the animal leaving the start arm and immediately entering the goal arm. Entering the wrong arm or re-entering the start arm was counted as errors. Training was continued on consecutive days until control animals reached a criterion of >70% correct match trials. Locations of arms and platform were varied pseudo-randomly each day to prevent spatial reference memoryterm bias.
Y-maze spontaneous alternationterm Spontaneous alternationterm was analysed in a white Perspex arena consisting of three equally sized arms in a “Y” configuration. The animal was placed at the end of one of the arms facing the wall and allowed to freely explore the arena for 10 minutes. Each trial was recorded using an overhead CCTV camera for post-hoc analysis. Trials were analysed manually by an experimenter blind to the experimental group and the sequence of arm entries noted. A complete alternationterm·def was defined as an animal entering each of the three arms in sequence without re-entering a previously explored arm. Percentage alternationterm·var was calculated as the number of total complete alternationsterm·def, divided by the total number of arm entries minus two and expressed as a percentage.
Water maze and reversal learningterm Open field water maze and reversal learningterm was carried out in a white Perspex pool 150 cm in diameter and 50 cm deep filled with water to a depth of 35 cm with water at 21 ± 1 °C. The extra-maze environment contained numerous spatial cues. On days one to five animals were trained to locate a hidden platform with four trials per day and an inter-trial interval of 30 minutes. Animals were released from a position at one of the four cardinal compass points in a pseudorandom fashion with one release from each point per day. Maximum trial length was 90 seconds, if an animal failed to locate the platform within the trial period it was guided to it by the experimenter. Following training on day 5 the animals received a probe trial, where they were released opposite the “platform” location and allowed to swim for 60 seconds. On day 8 the animals received a “refresher” day where they were again trained to the original platform location. On day 9 the platform location was switched to the quadrant opposite to the original location and training continued for three further days. Following training on day 12 the animals again underwent a probe trial. Throughout all trials animals were tracked via an overhead CCTV camera connected to the PC running Any-Maze (Ugo Basile) software. Primary parameters analysed were path lengthterm·var, time in target quadrant, swim speed. As a secondary parameter we calculated the number of trials required to attain floor level performance (2 consecutive trials with ≤4 m path lengthterm·var in each trial). It provides a best performance scenario and contingency plots reveal the percentage of mice that met floor level for each trial.
Social interaction Social interaction was examined essentially as described in 49 . The testing apparatus consisted of a three-compartment white Perspex box. Each compartment was 20 cm long, 42 cm wide and 22 cm high. Dividing walls were made from clear Perspex with small circular openings, 8 cm in diameter allowing free access to each chamber. The two outer compartments contained metal wire cages where conspecific animals were held, trials were recorded via an overhead CCTV camera connected to a PC and analyzed using Ethovision XT (Noldus). Test animals were first allowed a habituation period of 10 minutes where they could freely explore the apparatus without any stranger animals present. The test animal was removed from the apparatus for five minutes. Next the animals were tested for sociability, when a stranger junior conspecific (S1) was introduced to one of the side compartment cages (randomly selected and counterbalanced). The test animal was introduced to the central compartment and allowed to freely explore for 10 minutes. Time spent with the nose point of the test mouse in the immediate vicinity of the cage containing the stranger animal was taken as social interaction. The test animal was then removed from the apparatus for 5 minutes and a second stranger (S2) was introduced to the opposing cage, with the design counterbalanced so that stranger 1 could remain in the same position or move to the opposite location. The test animal was again introduced to the central compartment and allowed to freely explore for 10 minutes. Time spent with stranger 2 was divided by time spent with stranger 1 to give a discrimination indexterm·def, where 1 (dashed line in Fig. 6C,D ) indicates equal time spent with each stranger. The apparatus was cleaned thoroughly between animals to eliminate odor cues.
Amphetamineterm challenge Amphetamineterm challenge took place in the open field arena. Animals were placed in the centre of the arena and allowed to freely explore for 10 minutes. They were then given an intraperitoneal injection of 5 mg/kg D-amphetamine sulphate salt (Sigma-Aldrich) dissolved in saline. Animals were returned to the arena and monitored for a further 40 minutes, the arena was cleaned thoroughly between trials to minimise odor cues. Movements were recorded by an overhead CCTV camera and movements tracked using Ethovision XT software (Noldus).
Statistical analysis Statistical analyses were performed using Minitab version 15 (Minitab, Inc.) or GraphPad Prism 5.0. Unless otherwise stated data were analysed with two-tailed paired or unpaired Student’s t-testterm·stat as appropriate. We also used two-way ANOVA with treatment as between-subject and trial/day as within-subject factorsterm·stat. Contingency analysis applied Fisher’s exact or Chi-Square teststerm·stat. The null hypothesis was rejected for alpha greater than 5%.
Additional Information How to cite this article : MurraypersName, A. J. et al . Parvalbumin-positive interneurons of the prefrontal cortex support working memoryterm and cognitiveterm flexibility. Sci. Rep . 5 , 16778; doi: 10.1038/srep16778 (2015).
Supplementary Material Supplementary Information srep16778-s1.docx (190.8KB, docx) Acknowledgments We were supported by the Biotechnology and Biological Sciences Research Council grant BB/H001123/1 (P.W.), the Medical Research Council grants G0601498 and G1100546/2 (P.W.), Tenovus Scotland Grant G09/17 (A.J.M.) and the University of Aberdeen (P.W.). We thank O. Tüscher for discussion, P. Teismann and the microscopy core facility at the University of Aberdeen for the use of microscopy equipment, L. Strachan, A. Plano, S. Deiana for help with behavioral testing.
Footnotes Author Contributions A.J.M., G.R. and P.W. designed experiments. A.J.M., L.A.-B., M.W.-F., K.L.H.C., A.F. and B.C. performed experiments. Data were analyzed by A.J.M., M.W.-F., B.C., G.R. and P.W.; A.J.M., G.R. and P.W. wrote the manuscript.