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Hadas E Sloin Hadas E Sloin
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2, * , Lior Bikovski Lior Bikovski
3 Myers Neuro-Behavioral Core Facility, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
4 School of Behavioral Sciences, Netanya Academic College, Netanya 4223587, Israel
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3, 4, * , Amir Levi Amir Levi
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2 , Ortal Amber-Vitos Ortal Amber-Vitos
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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2 , Tomer Katz Tomer Katz
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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2 , Lidor Spivak Lidor Spivak
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2 , Shirly Someck Shirly Someck
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2 , Roni Gattegno Roni Gattegno
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2 , Shir Sivroni Shir Sivroni
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
5 Department of Mathematics, Afeka Tel Aviv Academic College of Engineering, Tel Aviv 6910717, Israel
6 Department of Mathematics, The Open University, Raanana 4353701, Israel
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2, 5, 6 , Lucas Sjulson Lucas Sjulson
7 Department of Psychiatry and Behavioral Sciences, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461
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7 , Eran Stark Eran Stark
1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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1, 2, ✉
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1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
3 Myers Neuro-Behavioral Core Facility, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
4 School of Behavioral Sciences, Netanya Academic College, Netanya 4223587, Israel
5 Department of Mathematics, Afeka Tel Aviv Academic College of Engineering, Tel Aviv 6910717, Israel
6 Department of Mathematics, The Open University, Raanana 4353701, Israel
7 Department of Psychiatry and Behavioral Sciences, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461
The authors declare no competing financial interests.
Author contributions: L.B. and E.S. designed research; H.E.S., L.B., A.L., O.A.-V., T.K., L.Sp., S.So., R.G., S.Si., and E.S. performed research; L.B. and E.S. contributed unpublished reagents/analytic tools; H.E.S., L.B., A.L., and E.S. analyzed data; H.E.S., L.B., L.Sj., and E.S. wrote the paper.
This work was supported by the United States-Israel Binational Science Foundation (BSF) Grant 2015577; the European Research Council Grant 679253; the Israel Science Foundation Grant 638/16; the Israel Science Foundation FIRST Program Grant 1871/17; the Rosetrees Trust Grant A1576; the Canadian Institutes of Health Research (CIHR), the International Development Research Centre (IDRC), the Israel Science Foundation (ISF), and the Azrieli Foundation Grant 2558/18; and the Zimin Institute.
* H.E.S. and L.B. contributed equally to this work.
✉ Correspondence should be addressed to Eran Stark at eranstark@tauex.tau.ac.il .
✉ Corresponding author.
Received 2022 Jun 3; Revised 2022 Jun 28; Accepted 2022 Jul 11; Prepublished 2022 Jul 29; Collection date 2022 Jul-Aug.
Copyright © 2022 Sloin et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license , which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
PMC Copyright notice
PMCID: PMC9395247 PMID: 35906064
Abstract
C57BL/6 is the most commonly used mouse strain in neurobehavioral research, serving as a background for multiple transgenic lines. However, C57BL/6 exhibit behavioral and sensorimotor disadvantages that worsen with age.
Keywords: animal models, behavior, freely-moving, FVB/NJ, phenotyping, systems neuroscience
Significance Statement
Introduction
Many advances in behavioral and biomedical research rely on lab animals. In choosing an animal model, there are always two conflicting considerations. On the one hand, an organism as similar as possible to humans is desired. On the other hand, the least advanced organism for answering the research question is preferred because of ethical considerations. The lab mouse ( Mus musculus ) has emerged as a key model in balancing these demands, sharing multiple physiological systems and genes with humans while exhibiting simple mating. Over the years, numerous inbred mouse strains have been developed, allowing genetic modifications, labeling, and manipulation of specific proteins, cells, and organs ( Beck et al., 2000 ). However, there are phenotypic differences between inbred strains used as background for transgenic mice ( Upchurch and Wehner, 1988 ; Võikar et al., 2001 ; Wahlsten et al., 2003 ; Brown and Wong, 2007 ; O’Leary et al., 2011 , 2013 ; Kafkafi et al., 2017 ). Hence, mouse strain selection is essential when designing a scientific project.
The mouse strain used most often in neurobehavioral research is C57BL/6. The C57BL/6 Jackson Laboratory substrain, C57BL/6J, provided the first extensively sequenced mouse genome ( Mouse Genome Sequencing Consortium, 2002 ) and is among the most widely used inbred strains ( Altman and Katz, 1979 ; Mekada et al., 2009 ). An advantage of inbreeding is that differences between individuals of the same strain are minimal. The genetic similarity of inbred animals is especially useful for knock-out studies, which may require homozygous animals ( Silva et al., 1997 ). Indeed, a large variety of transgenic mice on the C57BL/6 background is available.
Here, we bred C57 males with FVB females to determine whether the first-generation hybrids (HYB) are a preferable model for neurobehavioral studies. We tested mice of the three strains (C57, FVB, and HYB) using a battery of standard phenotyping assays ( Crawley, 2008 ; Fuchs et al., 2011 ). Compared with either inbred strain, HYB exhibited similar sensorimotor performance, reduced anxiety-like behavior, faster learning, and improved memory, which were maintained or further improved on aging. HYB mice were physically larger, and during electrophysiological recordings exhibited enhanced running on a linear track compared with C57, while CA1 neurons had similar place coding properties. Furthermore, transgenic HYB supported optogenetic neuronal control in neocortex and CA1. Together, the results suggest that the hybrid mice constitute a preferable model for systems neuroscience studies combining behavioral tests, electrophysiology, and genetic targeting.
Materials and Methods
Experimental model and subject details
Table 1.
Mice used for phenotyping assays
C57, 3 M FVB, 3 M HYB, 3 M C57, 9 M FVB, 9 M HYB, 9 M Total
Battery A
12 12 11 19 14 19 87
Elevated plus-maze 8 8 7 19 14 19
Open field 7 8 7 19 14 15
Rotarod a
4 4 4 19 14 19
Treadmill 8 8 7 19 14 19
Optomotor drum 8 8 7 19 14 19
Forced swim test 6 8 7 19 14 19
Phenotyping cages b
- - - 8 8 6
Battery B
10 9 12 20 17 15 83
Weight c
- - - 20 17 15
Gait analysis 10 9 11 19 12 15
MWM 10 9 11 18 11 15
Phenotyping cages b
- - - 8 7 5
Total 22 21 23 39 31 34 170
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a Due to errors in the initial testing of three-month-old mice on the rotarod, an additional replacement cohort of 12 animals was used.
b Mice tested in the phenotyping cages were a subset of the animals used in test Batteries A and B.
c Mice were weighed at least once every week from the age of three months until subjected to test Battery B at nine months of age.
Elevated plus maze
The elevated plus maze is used to evaluate anxiety-like behavior based on the natural uneasiness of rodents toward open, elevated fields ( Rodgers and Dalvi, 1997 ; Crawley, 2000 ; Komada et al., 2008 ). The apparatus consists of a four-armed platform resembling a “+” shape, positioned 40 cm above the floor. Two arms are confined by walls (“closed”; L × W × H: 35 × 5 × 15 cm), whereas two other arms are not walled (“open”; 35 × 5 cm). Similar arms face one another. In the test, the mouse was initially placed at the center of the maze facing one of the closed arms and then allowed to move freely for 7 min.
Forced swim test
The forced swim test is used to evaluate depressive-like behavior based on induced “behavioral despair” ( Porsolt et al., 1977 ; Castagné et al., 2011 ; Can et al., 2012 ). The apparatus consists of a clear Plexiglas cylinder (24 cm in height, 19 cm in diameter) filled with 16 cm of water at 22°C. In the test, the mouse was placed in the cylinder for 7 min and then moved to a heated cage until the fur dried completely. Typically, the mouse gradually stopped swimming before being removed. Freeze (immobility) duration, defined as the time the mouse remained floating motionless in the water, was measured during the last 5 min as a manifestation of behavioral despair.
Gait analysis
The gait analysis apparatus (CatWalk XT, Noldus Information Technology) enables the assessment of voluntary gait and locomotion in mice ( Ängeby-Möller et al., 2000 ; Crowley et al., 2018 ). The apparatus consists of a hardware system with a glass walkway (L × W: 130 × 20 cm), a GigE video camera, and a software package for the quantitative assessment of animal footprints. The walkway is illuminated from above by red light and from the side by green light. The green light is internally reflected within the glass, except at touched points. The walkway is connected to a dark goal box and enclosed by an adjustable tunnel at one end. In the test, mice were placed at the walkway entrance and allowed to run freely.
Morris water maze
The MWM is used to evaluate learning and spatial long-term memory based on the natural tendency of rodents to attempt to escape a body of water ( Morris, 1981 , 1984 ; Vorhees and Williams, 2006 ; Bromley-Brits et al., 2011 ). The apparatus is a circular pool (150 cm diameter, 10 cm depth) filled with water and maintained at a temperature of 23°C. A transparent platform (5 × 5 cm) was fixed 1 cm below the water surface at a constant location. The pool was situated in a room containing distal visual cues, and proximal cues were placed on the inner walls of the pool. The mouse was introduced to the pool at different starting points and allowed 60 s to find the platform and additional 15 s to stay on the platform. If the mouse failed to find the platform within 60 s, the animal was guided to the platform and allowed to stay there for 15 s. The animal swam three times every day during four consecutive “learning days.”
On the fifth day of the assay (“probe day”), the platform was removed, and the mouse was allowed to swim for 60 s. The time spent in every quadrant was recorded. The fraction of time spent in the “target” quadrant (i.e., close to the location of the missing platform), the number of visits to the target quadrant, and the latency to platform were used to evaluate spatial long-term memory performance.
Open field
The open field test provides a way to systematically assess motivational behavior by quantifying exploration of a novel environment and general locomotor activity ( Christmas and Maxwell, 1970 ; Prut and Belzung, 2003 ). In the test, the mouse was initially placed in one of the corners of an open-top Plexiglas box (L × W: 50 × 50 cm, raised 40 cm above the floor), and behavior was recorded for 15 min. Activity, defined as the fraction of pixels that changed in the entire arena between consecutive frames (frame rate, 25 Hz), was measured throughout the run duration to evaluate exploratory behavior.
Optomotor visual test
The optomotor drum is based on an apparatus developed for immobile mice ( Mitchiner et al., 1976 ) and allows assessing visual behavior in freely-moving mice ( Abdeljalil et al., 2005 ). The apparatus consists of an elevated stationary platform (20 cm) surrounded by a drum (internal diameter, 39 cm) with vertically oriented black and white stripes on the inside, each spanning 10°. In the test, the mouse was initially habituated to the platform for 2 min, with the drum stationary. The drum then rotated at 2 rpm counterclockwise for 2 min, stopped for 30 s, and rotated clockwise for 2 min.
Phenotyping cages
The PhenoTyper (model 3000, Noldus Information Technology) is an instrumented home cage in which rodent behavior is automatically monitored through a video-based and event-based system ( De Visser et al., 2006 ; Maroteaux et al., 2012 ; Grieco et al., 2021 ). The cage (L × W × H: 30 × 30 × 35 cm) is made of transparent Plexiglas walls with an opaque Plexiglas floor. The cage is equipped with a watering station, a feeding station, a running wheel, and a shelter in one corner (H × D: 10 × 9 cm, transparent material). The feeding and watering stations are equipped with beam-breaking devices, allowing automatic recording of feeding behavior and water intake (number of feeds and licks). The lid of the cage lid is equipped with an infrared-sensitive video camera (768 × 576 pixels) and several infrared LEDs, allowing continuous recording of animal position. Bedding covered the floor, and food and water were provided ad libitum . The mouse was introduced to the cage at the beginning of the dark phase (8 A.M.). Testing lasted 3 d, during which no human interference took place.
Rotarod test
Mice were subjected to a five-lane accelerating rotarod (Ugo Basile) to evaluate motor learning and balance ( Jones and Roberts, 1968 ; Crawley, 2000 , 2003 ; Shiotsuki et al., 2010 ). The apparatus consists of a 3.2 cm (diameter) horizontal rod elevated 10 cm from the ground. The paradigm was comprised of five consecutive days. On every day, mice were subjected to five trials, of which the duration of the three longest trials were averaged. A trial began with the rod rotating at 4 rpm and gradually accelerating for 5 min, up to a maximum of 50 rpm.
Treadmill
The treadmill apparatus (Panlab, Harvard Apparatus) is used to assess maximal endurance in mice ( Marques-Aleixo et al., 2015 ). The apparatus consists of a five-lane motorized treadmill with an electric shock zone at one end of the track. Each lane is 38 × 7 × 7 cm (L × W × H). A current shock (0.2 mA) was used to encourage running; cumulative shock duration of 2 s defined the maximal ability of mice to run. The protocol included two training days and one testing day. On the first training day, treadmill speed was fixed at 5 cm/s. The second training day consisted of two parts: (1) walking on the treadmill for 300 s at a constant speed (5 cm/s); (2) subjecting the mice to 330 s of locomotion at a variable speed (increasing from 5 to 21 cm/s by 1 cm/s every 20 s). On the third (testing) day, speed was initially set to 5 cm/s and was gradually increased by 1 cm/s every 20 s. The trial ended when the cumulative shock duration was 2 s.
Indices for behavioral assays
When parameters are strictly positive, the difference divided by the sum, (a – b)/(a + b), provides a valid estimate. However, some parameters of interest can take negative values, for instance, the “contra-anxiety index” ranges from −1 to 1. Then, an increase from one negative value to another will yield a negative “aging index” (or “C57 index”). Thus, when the parameter of interest was itself an index, we replaced the ratio (a – b)/(a + b) with the a/b ratio.
Probes and surgery
Every animal used in electrophysiological experiments (Extended Data Fig. 5-1 ) was implanted with a multi-shank silicon probe attached to a movable microdrive, equipped with optical fibers following previously described procedures ( Stark et al., 2012 ; Noked et al., 2021 ). The probes used were Stark64 (Diagnostic Biochips), Buzaski32 (NeuroNexus), and Dual-sided64 (Diagnostic Biochips). The Stark64 probe consists of six shanks, spaced horizontally 200 μm apart, with each shank consisting of 10–11 recording sites spaced vertically 15 μm apart. The Buzaski32 probe consists of four shanks, spaced horizontally 200 μm apart, with each shank consisting of eight recording sites spaced vertically 20 μm apart. The Dual-sided64 probe consists of two dual-sided shanks, spaced horizontally 250 μm apart, with each shank consisting of 16 channels on each side (front and back), spaced vertically 20 μm apart.
Before probe implantation, the single-transgenic hybrid was injected with a DIO-hChR2 viral vector (rAAV5/EF1a-DIO-hChR2(H134R)-eYFP; 3.2 × 10 12 IU/ml; University of North Carolina viral core facility; courtesy of K. Deisseroth). The solution was injected stereotactically (Kopf) into the neocortex and hippocampus at 8 different depths (AP −1.6, ML 1.1, DV 0.4–1.8 at 0.2 mm increments; 25 nl/site; Nanoject III, Drummond).
Probes were implanted in the parietal neocortex above the right hippocampus (AP/ML, −1.6/1.1 mm; 45° angle to the midline) under isoflurane (1%) anesthesia. Following recovery from anesthesia, linear-track animals were placed on a water-restriction schedule that guaranteed at least 40 ml/kg of water (corresponding to 1 ml/25 g mouse) on every recording day. Recordings were conducted 5 d/week, and animals received free water on the sixth day. After every one to five recording sessions, the probe was translated vertically downwards by up to 70 μm.
Histology
After recordings have ended, the implanted mice were deeply anesthetized with pentobarbital (100 mg/kg) and perfused with 0.1 m phosphate buffered saline (PBS; pH 7.4) and 4% paraformaldehyde (PFA). The brains were removed and postfixed overnight in PFA. Coronal sections (70 μm) were cut on a vibratome (VT1000S, Leica) and collected in PBS. Sections were mounted in Fluoromount with DAPI (F6057-20ML, Sigma) and imaged with a wide-field fluorescence microscope (Axio Scope A1, Zeiss).
Linear track sessions
For the linear track analyses, neuronal activity was recorded in 4.5 [1.9 10.1]-h sessions (median [interquartile interval, IQR]). At the beginning of every session, neural activity was recorded while the animal was in the home cage. The animal was then placed on a 150 cm linear track that extended between two 10 × 10 cm square platforms. Each platform included a water delivery port. Mice were under water restriction and were trained to repeatedly traverse the track for a water reward of 3–10 μl. Over all sessions, mice ran 167 [132 200] one-direction trials over about 1 h (Extended Data Fig. 5-2 ). Trials with a mean running speed below 10 cm/s were excluded from analyses. Animals were equipped with a three-axis accelerometer (ADXL-335, Analog Devices) for monitoring head movements. Head position and orientation were tracked in real-time using two head-mounted LEDs, a machine vision camera (ace 1300-1200uc, Basler), and a dedicated system (“Spotter,” Gaspar et al., 2019 ).
Spike detection and sorting
Neural activity was filtered, amplified, multiplexed, and digitized on the headstage (0.1–7500 Hz, x192; 16 bits, 20 kHz; RHD2132 or RHD2164, Intan Technologies) and then recorded by an RHD2000 evaluation board (Intan Technologies). Offline, spikes were automatically detected and sorted into single units using KlustaKwik3 ( Kadir et al., 2014 ; Rossant et al., 2016 ) for shanks with up to 11 sites/shank or KiloSort2 ( Pachitariu et al., 2016 ) for 16 channel shanks. Automatic spike sorting was followed by manual adjustment of the clusters. Only well-isolated units were used for further analyses [amplitude >40 μV; L-ratio <0.05 ( Schmitzer-Torbert et al., 2005 ); ISI index <0.2 ( Fee et al., 1996 )].
Place field and phase precession analysis
Based on the linear track data, spatial information, place fields, and phase precession were determined for every PYR ( Sloin et al., 2022 ). Briefly, place fields were defined as regions spanning 15–100 cm in which the firing rate increased compared with the on-track spontaneous firing rate ( p < 0.05, Bonferroni-corrected Poisson test). Theta phase precession was quantified for each place field using a circular-linear analysis ( Schmidt et al., 2009 ; Kempter et al., 2012 ).
Optogenetic stimulation
To determine the effect of optogenetic stimulation on spiking, 50-ms blue-light pulses were administered. In every session, illumination was conducted for every shank separately. In CA1, pulses were given in a total of 131 shanks during 55 sessions, using light power of 2.43 [0.96 3.98] μW. In a given stimulation experiment, pulses were applied 137 [51 209] times. In the neocortex (Extended Data Fig. 6-3 ), pulses were given in a total of 63 shanks during 21 sessions, using light power of 11.07 [5.25 21.71] μW. In a given stimulation experiment, pulses were applied 150 [75 300] times.
Statistical analyses
Results
Hybrid mice are larger than C57 mice and exhibit similar sensorimotor performance
We decided to use offspring of C57 and FVB because the two inbred strains are of different genealogies ( Beck et al., 2000 ) and exhibit distinct phenotypic disadvantages ( Crawley, 2008 ). FVB dams produce large litters ( Silver, 1995 ) and were therefore selected as the maternal strain. To maximize the genetic similarity between individual mice, we focused on (FVB/NJ x C57BL/6J)F1, the first-generation offspring (HYB).
Figure 1.
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Hybrid mice weigh more than both progenitors. Mice of the three strains were weighted periodically at ages between 75 and 105 d (∼3-month-old; A ) or between 255 and 285 d (∼9-month-old; B ), and weights were averaged over measurements for every mouse. Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a plus indicates an outlier. P -value indicated by text is for a one-way Kruskal–Wallis test; *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons. See also Extended Data Figures 1-1 , 1-2 , and 1-3 .
Extended Data Figure 1-1 Hybrids are larger and exhibit distinct patterns of daily conduct in the home cage. A , HYB weigh more than both progenitors. Top, Mice of the three strains were weighed periodically over six months, from three to nine months old ( n = 20 C57, 17 FVB, and 15 HYB). Each line connects measurements of a single animal. P -values indicated by text are for a strain by day two-way Kruskal–Wallis test. Dashed lines, age ranges used for statistical analyses (center and bottom panels). Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a plus indicates an outlier. For every mouse, weights were averaged over measurements made in the age of 75–105 d (“3 months”; center) and 255–285 d (“9 months”; bottom); *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons. B , Hybrids are least active and spend more time in the shelter during the dark phase. Nine-month-old mice ( n = 16 C57, 15 FVB, and 11 HYB) were placed in individual phenotyping cages and the distance covered, time in the shelter, feeding, and licking were sampled every hour over 3 d. a , Top, During both dark (8 A.M. until 8 P.M.) and light phases, HYB cover less distance. Bands show SEM. Second row, During the dark phase, HYB spend more time in the shelter than C57 or FVB. Third row, During both dark and light phases, HYB feed less than either C57 or FVB. Bottom, The number of licks is not consistently different between the strains. b , Mean metric (distance covered, sheltering, feeding/licking rate) during the dark (top half) and light (bottom) phases. Error bars, SEM; ** p < 0.01, *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons. c , Diurnal indices, defined as the difference between the metric (e.g., distance covered) during the light phase (L) and the dark phase (D), divided by the sum; * p < 0.05, *** p < 0.001, bootstrap test. Mice of all strains exhibit diurnal changes in all metrics tested. d , Pie charts show the fraction of time spent by mice of every strain carrying three specific behaviors: sheltering (being in the shelter), feeding (breaking the photobeam of the feeding station), and wheeling (being on the wheel). “Other” corresponds to home cage roaming, including licking. The time mice dedicated to each behavior differs between strains (*** p < 0.001, strain, behavior, and interaction effects, two-way Kruskal–Wallis test). Compared to the dark phase, mice of all strains spend less time on the wheel and feeding during the light phase (*** p < 0.001, Kruskal–Wallis test). C , Gait patterns of the three strains. a , Gait regularity does not differ consistently between strains or between age groups. Here and in all other panels, p -values indicated in text in the top and center are for Kruskal–Wallis test (e.g., 0.93). Bottom, Aging indices; * p < 0.05, *** p < 0.001 bootstrap test. b , The front paws base of support (BOS) of HYB increases with aging. c , The hind paws BOS is distinct for the three strains; * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons. d–f , Track crossing duration, maximum variation, and cadence do not differ consistently between strains in either age group. Download Figure 1-1, EPS file (2.1MB, eps) .
Extended Data Figure 1-2 Results of phenotyping assays for every age group and strain. a Values indicate median [IQR] for every age group, strain, and metric. For learning on the MWM, values indicate mean ± SEM. Download Figure 1-2, DOCX file (31.1KB, docx) .
Extended Data Figure 1-3 Hybrid sensorimotor performance is maintained upon aging. A , Reaction to visual stimuli on the optomotor drum of HYB and C57 is similar and remains intact at an older age.
To assess the sensorimotor capabilities of HYB, we subjected three-month-old mice to three standard assays ( n = 12 C57, 12 FVB, and 11 HYB; Table 1 ; Extended Data Fig. 1-2 ). Visual behavior was quantified in an optomotor drum test ( Abdeljalil et al., 2005 ), in which mice follow vertical visual stimuli by making directional head movements. C57 and HYB made similar numbers of head turns (C57: 7.5 [3 11]; HYB: 7 [5 10], p = 0.98, Kruskal–Wallis test; Extended Data Fig. 1-3 A ).
Hybrid mice exhibit reduced anxiety and improved learning and memory
To characterize neuropsychiatric phenotypes and cognitive performance, we tested three-month-old mice using five assays ( Table 1 ). Anxiety-like behavior was studied using the elevated plus maze ( Rodgers and Dalvi, 1997 ) and quantified by a “contra-anxiety index” that ranges from −1 to 1, defined as time spent in (open – closed)/(open + closed) arms. Indices closer to one indicate that more time was spent in the two “open” arms, corresponding to reduced anxiety-like behavior. C57 spent consistently more time in the closed arms compared with the open arms (−0.59 [−0.66 −0.55]; p = 0.016, Wilcoxon’s signed-rank test), as did HYB (−0.34 [−0.55 −0.22], p = 0.016; Fig. 2 A ; Extended Data Fig. 2-1 A ).
Figure 2.
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Young hybrids exhibit reduced anxiety. A , At the age of three months, HYB exhibit reduced anxiety-like behavior compared with C57. Anxiety-like behavior was tested in an elevated plus maze and quantified using a “contra-anxiety index,” defined as the time spent in the open minus the time spent in the closed arms, divided by the sum. Lined * p < 0.05, Kruskal–Wallis test. Here, and in C , * p < 0.05, ** p < 0.01, Wilcoxon’s signed-rank test, comparing to chance level (horizontal dashed line). B , HYB exhibit improved learning in the MWM task compared with C57 and FVB, as measured by the latency to platform ( a ) and by the distance traveled ( b ) over days. Bands show mean and SEM; *** next to the top text indicate p < 0.001 for a two-way, strain by day, Kruskal–Wallis test; */**/*** next to strain names indicate p < 0.05/ p < 0.01/ p < 0.001 for a within-strain, across-day one-way Kruskal–Wallis test. C ,
Extended Data Figure 2-1 Hybrids exhibit reduced anxiety-like behavior, improved learning, and improved memory. A , HYB explore the open arms of an elevated plus maze more than C57 mice, particularly at an older age, indicating reduced anxiety-like behavior. Here and in subsequent panels, Top and center, Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a “+” sign indicates an outlier. Here and in Eb , Ec , P -values indicated by text are for a one-way Kruskal–Wallis test; lined * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, Wilcoxon’s signed-rank test, comparing indices to chance level. Bottom, Aging index. Bars and errors show mean and SEM, respectively; *** p < 0.001, bootstrap test, compared to a no-change null (index of zero). B , Depressive-like behavior is not consistently different among HYB and C57 mice, and is reduced among older FVB mice. C , Open field exploration increases at an older age for all strains. Top, In the three-month-old age group, HYB were more active in the open field than FVB; ** p < 0.01, Kruskal–Wallis test, corrected for multiple comparisons. Center, In the nine-month-old group, C57 were more active than FVB. Bottom, Mice of all three strains exhibited increased activity upon aging, evident as above-zero aging indices; ** p < 0.01, *** p < 0.001, bootstrap test. D , Learning on the MWM task is improved for HYB compared to C57, and is maintained at older age. a , Mean (and SEM) latency to finding the platform (limited to 60 s). Here and in c , **/*** next to the text indicate p < 0.01/ p < 0.001 in a two-way, strain by day, Kruskal–Wallis test; */**/*** at right indicate p < 0.05/ p < 0.01/ p < 0.001, one-way Kruskal–Wallis test, measuring the within-strain, across-day effect. In both age groups. only HYB showed consistent across-day learning. Bottom, Aging indices, averaged over all learning days. Learning performance of C57 deteriorates at an older age. b , Day-by-day “C57 indices” ( Fig. 1 D ), measuring the change in latency to the platform between FVB (blue) or HYB (green) and C57 mice. Bands show mean and SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, bootstrap test. Insets, Mean (and SEM) C57 indices, averaged over all learning days; p -values, geometric mean over all learning days. c , C57 and HYB travel progressively shorter distances over training days, indicating learning. All conventions are the same as in a . E , On the probe day of the MWM task, HYB spend the longest time in and the shortest latency to the target quadrant. a , Time spent in every quadrant; * p < 0.05, ** p < 0.01, *** p < 0.001, Wilcoxon’s signed-rank test, comparing time in each quadrant to chance level (0.25 of the total time). b , Time spent in the target quadrant, for three-month-old (top) and nine-month-old (center) mice. Here and in c , Horizontal dashed lines indicate chance level of 0.25, expected for random exploration; other conventions are the same as in A . Compared to C57 or FVB, nine-month-old HYB spend a larger fraction of time in the target quadrant. Bottom, Aging indices. Compared to younger HYB, older HYB spend more time in the target quadrant. c , In both age groups, the fraction of visits to the target quadrant is highest for HYB. d , Latency to the platform location, measured during the probe day, is the shortest for HYB. Download Figure 2-1, EPS file (1.4MB, eps) .
Depressive-like behavior was tested using a forced swim test, in which longer freeze duration indicates behavioral despair ( Porsolt et al., 1977 ). Mice of all three strains exhibited similar freeze durations (C57: 124 [94 135] s; FVB: 209 [130 261] s; HYB: 201 [85 290] s; p = 0.23, Kruskal–Wallis test; Extended Data Fig. 2-1 B ). Motivational behavior was quantified by activity levels in an open field ( Christmas and Maxwell, 1970 ). We did not observe consistent differences between C57 and HYB in overall activity in the field (C57: 0.23 [0.17 0.27] %; HYB: 0.28 [0.2 0.31] %; p = 0.36, Kruskal–Wallis test; Extended Data Fig. 2-1 C ). Thus, compared with C57, HYB exhibit similar depressive-like behavior and similar motivational behavior.
To assess learning and memory behavior, we trained three-month-old mice on the MWM task ( Morris, 1981 ; n = 10 C57, 9 FVB, and 11 HYB; Table 1 ). During four consecutive days, mice were placed in a pool with an invisible submerged platform. Over days, only HYB exhibited a decrease in the latency to platform, indicative of learning (C57: p = 0.67, FVB: p = 0.08, HYB: p = 0.03, intrastrain Kruskal–Wallis tests; Fig. 2 B ). Pooled over days, the mean ± SEM latency to the platform was shorter for HYB (19.3 ± 3.9 s), compared with C57 (21.7 ± 4.3 s) and to FVB (41.1 ± 3.8 s; p = 1.7 × 10 −13 , Kruskal–Wallis test; Fig. 2 Ba ). Similar results were obtained when examining the distance traveled ( Terry, 2009 ) instead of the latency to platform ( Fig. 2 Bb ).
To assess spatial long-term memory, the mice were tested in the MWM during a “probe” day, without a platform. Mice of the three strains spent similar fractions of time in the target quadrant ( p = 0.13, Kruskal–Wallis test; Fig. 2 Ca ). However, when spatial long-term memory was quantified by the fraction of visits to the target quadrant,
To quantify differences between HYB (or FVB) and the C57, we treated the C57 strain as a reference and computed “C57 indices” for the eight assays ( Fig. 2 D ). For a given metric, the C57 index is defined as the difference between the median value of HYB (or FVB) and the median value of C57, divided by the sum. Therefore, a C57 index above zero indicates higher values of the metric for the tested strain, compared with C57 performance. HYB performance was similar to C57 in assays testing visual behavior (mean ± SEM C57 index: −0.042 ± 0.19, p = 0.36, bootstrap test), motor endurance (−0.06 ± 0.2, p = 0.47), depressive-like behavior (0.21 ± 0.23, p = 0.19), learning (day-averaged latency to platform; 0.05 ± 0.2, p = 0.25), and spatial long-term memory (time in target quadrant; −0.011 ± 0.08, p = 0.47). In contrast, HYB exhibited reduced anxiety-like behavior (0.31 ± 0.1, p = 0.011) and increased exploratory behavior (0.089 ± 0.05, p = 0.031). Thus, at the age of three months, HYB motor endurance and visual behavior is similar to C57, whereas the HYB exhibit reduced anxiety-like behavior.
Older hybrids maintain sensorimotor performance and exhibit improved memory
To examine the suitability of the HYB for long-term studies that require stable behavioral performance, we applied all assays also to nine-month-old mice ( Table 1 ). Akin to the three-month-old HYB, nine-month-old HYB exhibited reduced anxiety-like behavior compared with C57 (contra-anxiety index: C57: −0.56 [−0.75 −0.46]; HYB: −0.15 [−0.27 0.15]; p = 4.1 × 10 −6 ; Kruskal–Wallis test; Fig. 3 A ; Extended Data Fig. 2-1 A ). The nine-month-old HYB exhibited improved learning compared with C57, as indicated by shorter latency to platform (C57: 33.3 ± 2.9 s; HYB: 17.8 ± 3 s; p = 1.7 × 10 −13 , Kruskal–Wallis test; Fig. 3 B ; Extended Data Fig. 2-1 D ). Furthermore, nine-month-old HYB displayed improved spatial long-term memory compared with C57, as indicated by a larger fraction of time spent in the target quadrant (C57: 0.3 [0.24 0.4]; HYB: 0.55 [0.36 0.62]; p = 0.002, Kruskal–Wallis test) and by a larger fraction of visits to the target quadrant (C57: 0.35 [0.27 0.4]; HYB: 0.45 [0.36 0.5]; p = 0.013; Kruskal–Wallis test; Fig. 3 C ; Extended Data Fig. 2-1 E ). Correspondingly, the C57 indices of HYB indicated reduced anxiety-like behavior (mean ± SEM C57 index: 0.49 ± 0.1, p = 0.00,001), improved learning (day-averaged latency to platform; 0.35 ± 0.14, p = 0.002), and improved spatial long-term memory (time in target quadrant; 0.3 ± 0.06, p = 0.0003; Fig. 3 D ). The performance of nine-month-old HYB was similar to C57 in assays testing visual behavior (−0.031 ± 0.1, p = 0.34, bootstrap test), motor endurance (0.14 ± 0.12, p = 0.09), balance (0.04 ± 0.05, p = 0.14), exploration (−0.01 ± 0.023, p = 0.35), and depressive-like behavior (0.04 ± 0.08, p = 0.32). Thus, nine-month-old HYB exhibit sensorimotor properties which are similar to nine-month-old C57, while exhibiting reduced anxiety-like behavior and improved learning and memory performance.
Figure 3.
Older hybrids exhibit improved learning and long-term memory.
To quantify performance differences between three- and nine-month-old mice of the same strain, we defined an “aging index” ( Fig. 4 ). For a given performance metric, the aging index is the difference between the median value obtained for nine- and three-month-old mice, divided by the sum. Thus, an aging index of zero indicates no age-related changes in performance, whereas positive indices indicate age-dependent increases. Visual behavior was maintained across age groups for all strains (mean ± SEM aging indices: C57, 0.03 ± 0.17, p = 0.41; FVB, not available; HYB, 0.04 ± 0.13, p = 0.28, bootstrap test; Fig. 4 Aa ). While C57 and FVB exhibited reduced motor endurance on aging (C57: −0.41 ± 0.1, p = 0.0001; FVB: −0.5 ± 0.13, p = 0.0014), HYB endurance was maintained (−0.23 ± 0.22, p = 0.11; Fig. 4 Ab ). Finally, while C57 and FVB balance performance declined between age groups (C57: −0.2 ± 0.06, p < 0.05; FVB: −0.31 ± 0.09, p < 0.05), HYB balance was maintained (0.1 ± 0.13, p = 0.75; Fig. 4 Ac ).
Figure 4.
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Hybrids maintain sensorimotor performance and exhibit improved long-term memory on aging. A , Sensorimotor behavior remains intact at an older age for HYB but not for C57 or FVB mice. Positive aging indices indicate improvement across three- and nine-month-old mice of the same strain. a , Visual behavior of HYB and C57 remains intact at an older age, whereas FVB do not react to rotating visual stimuli at any age. Here and in B , bars indicate mean; error bars, SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, bootstrap test compared with a no-change null. b , Motor endurance is reduced for older C57 and FVB and maintained for older HYB. c , Balance performance deteriorates for C57 and FVB on aging and is maintained for older HYB. B , Psychiatric-like and cognitive performance across age groups. All conventions are the same as in A . a , HYB, but not C57 or FVB, exhibit reduced anxiety-like behavior upon aging. b , Learning performance of C57, but not FVB or HYB, deteriorates upon aging. c , Spatial long-term memory performance of C57 and FVB is maintained upon aging, whereas HYB memory performance improves. C , Comparison of performance changes in eight behavioral assays using the “aging index.” All conventions are the same as in Figure 2 D .
Compared with C57, three-month-old HYB exhibited reduced anxiety-like behavior, improved learning, and improved long-term memory ( Fig. 2 ). We tested whether performance on the three assays was maintained across age groups in every strain. Compared with three-month-old mice of the same strain, anxiety-like behavior was reduced for nine-month-old HYB (mean ± SEM aging index: 0.26 ± 0.11, p = 0.0005, bootstrap test), but not for C57 (0.04 ± 0.12, p = 0.37) or FVB (0.22 ± 0.17, p = 0.07; Fig. 4 Ba ). Learning performance was reduced in older C57 mice (−0.28 ± 0.14, p = 0.03), but maintained for older HYB (0.04 ± 0.21, p = 0.43; Fig. 4 Bb ). Both C57 and FVB maintained spatial long-term memory performance between age groups (C57: 0.008 ± 0.13, p = 0.28; FVB: −0.07 ± 0.16, p = 0.16). In contrast, older HYB exhibited improved spatial long-term memory (0.31 ± 0.07; p = 0.0003; Fig. 4 Bc ).
Hybrid mice exhibit enhanced linear track running and typical CA1 place coding
The results reported in Figures 1 - 4 show that HYB exhibit certain advantages over C57. However, improved performance on behavioral assays spanning several minutes does not necessarily predict enhanced behavior over daily recording sessions that span hours and include the additional weight of an implanted apparatus. To assess the suitability of freely-moving HYB for electrophysiological experiments, we performed high-density recordings from two HYB and two C57 mice running back and forth on a 150-cm linear track (Extended Data Fig. 5-1 ). Behavior during the first 14 sessions on the track was assessed to quantify performance at similar experience levels.
Figure 5.
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Hybrid mice exhibit enhanced linear track running and typical CA1 place coding. A , Compared with C57 mice, HYB carry out more trials and run faster on a 150-cm-long linear track. Only the first 14 sessions from every mouse were employed (two C57 and two HYB mice); * p < 0.05, ** p < 0.01, Mann–Whitney test. Dashed vertical lines indicate group medians. a , The number of one-direction trials. b , Mean running speed over trials. B , Location of pyramidal cell (PYR) somata relative to the center of the CA1 pyramidal cell layer (dashed horizontal line) is not consistently different between strains. The estimated depths of PYR recorded by high-density silicon probes were partitioned into 5-μm bins. Positive numbers correspond to PYR closer to str. oriens. n.s.: p > 0.05, Mann–Whitney test. C , Units recorded from both strains exhibit increased firing rates within specific regions of the linear track. Each row represents a unit; firing rates on right (R) to left (L) runs are concatenated with L to R runs and scaled to the 0–1 (white-black) range for presentation purposes. Bar graphs (right) show peak on-track firing rates. D , A PYR recorded from a HYB exhibits a typical place field and theta phase precession. Top, Firing rate as a function of position (mean ± SEM over 82 right-to-left trials). Bottom, Theta phase and animal position at the time of every spike; phase of zero corresponds to theta peak. Running direction is presented from left to right, and vertical dashed lines indicate place field limits. a represents phase precession slope, and R represents the goodness of fit of spikes to the precession model; ** p < 0.01, permutation test. Left, Wide-band (0.1–7500 Hz) spike waveforms (mean ± SD) recorded on eight consecutive sites, vertically spaced by 20 μm. Bottom left, Autocorrelation histogram. E , HYB and C57 exhibit similar CA1 spatial rate coding. a , Spatial information rate. n.s.: p > 0.05, Mann–Whitney test. Here and in c , vertical dashed lines indicate group medians. b , Fraction of CA1 PYR with one or more place fields out of all units active and stable on the track; n.s.: p > 0.05, likelihood ratio test. Here and in Fa , *** p < 0.001, exact binomial test, compared with chance level (0.05; horizontal dashed line). Error bars, SEM. c , Place field size; n.s.: p > 0.05, Mann–Whitney test. F , HYB and C57 exhibit similar CA1 spatial phase coding. a , Fraction of fields exhibiting theta phase precession; n.s.: p > 0.05, likelihood ratio test. b , Precession slope size. c , Precession effect size; n.s.: p > 0.05, Mann–Whitney test. See also Extended Data Figures 5-1 and 5-2 .
Extended Data Figure 5-1 Mice used for electrophysiological recordings. a For C57 animals, the male parent was the reporter, and the female parent was the driver. b At time of implantation. Download Figure 5-1, DOCX file (31.1KB, docx) .
Extended Data Figure 5-2 Linear track behavior and place coding in individual mice. a Number of one direction trials, median [IQR] over sessions. b Well-isolated PYR, active and stable on the linear track. c SI, spatial information. Download Figure 5-2, DOCX file (31.1KB, docx) .
To examine whether CA1 pyramidal neurons (PYR) exhibit similar place coding in HYB and C57 mice tested under the same conditions, we analyzed the activity of CA1 pyramidal layer PYR active on the track (C57: n = 779, HYB: n = 960; Fig. 5 B ; Extended Data Fig. 5-2 ). In both strains, most active PYR exhibited increased firing rates in specific parts of the track ( Fig. 5 C ). An example PYR recorded in HYB exhibited typical spatial rate coding (place field; O’Keefe and Dostrovsky, 1971 ; Moser et al., 2008 ) and spatial phase coding (theta phase precession; O’Keefe and Recce, 1993 ; Fig. 5 D ). At the group level, there was no consistent difference in the spatial information carried by PYR, with 1.28 [0.47 3.18] bits/s for HYB PYR and 1.37 [0.63 2.87] bits/s for C57 PYR ( p = 0.56, Mann–Whitney test; Fig. 5 Ea ).
To determine whether HYB CA1 PYR exhibit typical phase coding of space, we examined theta phase precession within PYR place fields.
Transgenic hybrid mice enable optogenetic control of individual neurons
A major advantage of C57 mice for neurobehavioral research is the myriad transgenic strains available on the same background. To combine genetic control available for C57 with the enhanced behavior of the HYB, we first generated dual-transgenic mice on a C57 background by breeding CaMKII-Cre females with Ai32 males. We then crossed the male offspring with FVB females. Opsin expression in the resulting dual-transgenic and hybrid mice was verified using histology (Extended Data Fig. 6-1 A ). The dual-transgenic HYB are susceptible to blue-light optogenetic activation of projection neurons, including neocortical and CA1 PYR. Optogenetic PYR activation was conducted in transgenic HYB implanted with multi-site optoelectronic probes (Extended Data Fig. 5-1 ). A total of 5568 PYR and 1066 putative interneurons (INT) were recorded from the CA1 pyramidal layer during n = 55 sessions (Extended Data Fig. 6-2 ). In every recording session, we tested the response of the recorded units to 50-ms blue-light pulses. During illumination, PYR exhibited increased firing rates ( Fig. 6 A , purple). Of all recorded PYR, 1213 (22%) exhibited a consistent increase in firing rate during illumination ( p < 1.11 × 10 −16 , exact binomial test).
Figure 6.
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Transgenic hybrid mice enable optogenetic control of individual CA1 neurons. A , Directly activated PYR and an indirectly activated interneuron (INT) recorded in hippocampal region CA1 of a HYB dual-transgenic (CaMKII::ChR2) mouse. a , The units exhibit a cross-correlation histogram (center; no light condition) consistent with monosynaptic excitation ( p < 0.001, Poisson test). b , Local field and spiking responses to a single 50-ms blue-light pulse (0.67 μW). The PYR and the INT both spike during the light. c , Raster plots (top) and PSTHs (bottom) during blue light pulses for the PYR (left) and the INT (right). For visualization purposes, raster plots include 200 pulses. d , Top parts, Mean PSTHs of 22 PYR (purple) and four INT (green) recorded simultaneously on the illuminated shank (S1). Bottom parts, Greyscale, PSTHs of all 48 PYR and eight INT recorded simultaneously during the session. Each row shows the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Arrows indicate the example PYR and INT. B , Activation probability of CA1 units depends on the distance from the illuminated shank. Dataset includes a total of 5568 PYR and 1066 INT. a , Bars show the fraction of units with increased firing rate ( p < 0.05, Poisson test) during single-shank illumination. Error bars, SEM; *** p < 0.001, exact binomial test, comparing the fraction of light-activated units to chance level (horizontal dashed line). b , Median firing rate gain of activated PYR and INT. Error bars, IQR. Gain is shown only for distances with an above-chance number of optically activated units. P -values indicated in text are for a one-way Kruskal–Wallis test; *** p < 0.001, Kruskal–Wallis test. See also Extended Data Figures 6-1 , 6-2 , and 6-3 .
Extended Data Figure 6-1 Opsin expression in transgenic and injected hybrids. A , Opsin expression in neocortex and hippocampus of mA234, a double transgenic and hybrid mouse (FVB/NJ x CaMKII-Cre x Ai32) used in linear track ( Fig. 5 ) and optogenetic ( Fig. 6 ) experiments. EYFP fluorescence (green) is used as a reporter for ChR2 expression. Left, Low-magnification (2.5×) image of neocortex and hippocampus of a transgenic mouse. Right, High-magnification (20×) image, showing EYFP in neocortical somata. B , Opsin expression in neocortex and hippocampus of a HYB injected with a viral vector (rAVV5/CaMKII-hChR2(H134R)-mCherry) in the right hemisphere. mCherry fluorescence (red) is used as a reporter for ChR2 expression. All other conventions are the same as in A . Download Figure 6-1, EPS file (31.6MB, eps) .
Extended Data Figure 6-2 Optogenetic activation of individual neurons in individual mice. a Well-isolated PYR with increased firing rate during optogenetic stimulation ( p < 0.05, Poisson test), out of all PYR recorded in the same sessions. b Light-activated PYR recorded on the illuminated shank. Download Figure 6-2, DOCX file (31.1KB, docx) .
Extended Data Figure 6-3 Transgenic hybrids support robust optogenetic activation of individual neocortical neurons. A , B , Optogenetic activation in the neocortex of hybrid freely-moving dual-transgenic CaMKII::ChR2 mice. A , Example directly activated PYR and indirectly activated INT recorded in HYB neocortex. a , The units exhibit cross-correlation histogram (center; no light condition) consistent with monosynaptic excitation ( p < 0.001, Poisson test). Bottom, Auto-correlation histograms (ACHs). Wide-band (0.1–7500 Hz) spike waveforms (mean ± SD) recorded on the same shank; intersite vertical spacing is 20 μm. b , Local field and spiking responses of the units to a single 50-ms blue-light pulse (1.71 μW). Both units spike during the light. c , Raster plots (top) and PSTHs (bottom) during 230 blue light pulses for the PYR (left) and indirectly activated INT (right). The raster plot shows 200 representative repetitions. d , Top, Mean PSTHs of 26 PYR (purple) and seven INT (green) recorded simultaneously on the illuminated shank during light pulses. Bottom, Greyscale, PSTH of all unit recorded during the session (49 PYR and 21 INT). S1, S2, S3, and S4 denote different shanks. Each row corresponds to the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Arrows indicate the example units. B , Activation probability and firing rate gain of neocortical units depend on the distance from the illuminated shank. Dataset includes a total of 1546 PYR and 517 INT. a , Bars show the fraction of units with increased firing rate ( p < 0.05, Poisson test) during single-shank illumination as a function of the horizontal distance from the illuminated shank. Error bars, SEM; *** p < 0.001, exact binomial test, compared to chance level (0.05, horizontal dashed line). Neocortical PYR and INT recorded up to 600 μm away from the illuminated shank are activated. b , Median firing rate gain of activated PYR and INT as a function of distance from the illuminated shank. Error bars, IQR. Gain is considered only for distances with an above-chance number of activated units. P -values indicated in text are for a one-way Kruskal–Wallis test; *** p < 0.001, Kruskal–Wallis test, corrected for multiple comparisons. Gain differs between activated PYR recorded on the illuminated shank and activated PYR recorded 400 μm away. C , Parvalbumin-immunoreactive (PV) cell activation in a hybrid freely-moving single-transgenic PV-Cre mouse, injected with a Cre-dependent viral vector for ChR2. a , Subnetwork of a PYR (PYR1; purple) and a PV cell (green), recorded simultaneously using a high-density optoelectronic probe. ACHs and cross-correlations (no light condition), consistent with reciprocal monosynaptic excitation and inhibition ( p < 0.001, Poisson test). b , Wide-band (0.1–7500 Hz) traces and spikes of the PV cell and another unit, PYR2. Top three traces are neuronal recordings; fourth trace indicates instantaneous light power emitted by the fiber attached to the recording shank; and the bottom trace indicates head acceleration. During blue light pulses (62 μW), the PV cell spikes, whereas PYR2 spiking is suppressed. c , Raster plot (top) and PSTH (bottom) during blue light pulses for the light-activated PV cell (left) and the suppressed PYR (right). Arrows indicate the example units. d , PSTHs of six units recorded by the illuminated shank during blue light pulses. Bottom, Each row corresponds to the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Top, Mean PSTHs. Download Figure 6-3, EPS file (13MB, eps) .
Optogenetic stimulation led to the indirect activation of INT via monosynaptic inputs from excitatory neurons ( Fig. 6 A , green). A total of 241/1066 (23%) of CA1 INT exhibited light-induced activation ( p < 1.11 × 10 −16 , exact binomial test; Fig. 6 Ba , right). The fraction of light activated INT was 34% (92/273) on the illuminated shank and lower on shanks farther away (400 μm: 40/258, 16%, p = 1.3 × 10 −4 , Bonferroni-corrected likelihood ratio test). In contrast to PYR, the firing rate gain of light-activated INT did not differ consistently between shanks ( p = 0.38, Kruskal–Wallis test; Fig. 6 Bb , right).
To examine HYB optogenetic activation in a brain region with a distinct architecture, we performed optical stimulation in the neocortex of the same mice ( n = 21 sessions; Extended Data Fig. 6-3 ). Similar to CA1 units, neocortical PYR and INT exhibited induced spiking (Extended Data Fig. 6-3 A ), with activation probability that decreased with distance from the illuminated shank (Extended Data Fig. 6-3 Ba ). A total of 274/409 (67%) neocortical PYR recorded on the illuminated shank exhibited optical activation, compared with 722/1527 (47%) CA1 PYR ( p = 1.2 × 10 −4 , likelihood ratio test). Furthermore, 331/591 (56%) neocortical PYR exhibited optical activation on the adjacent shank (200 μm away), compared with 340/1984 (17%) CA1 PYR ( p < 3.5 × 10 −11 , likelihood ratio test). Compared with CA1, focal illumination in the neocortex activated PYR located farther away from the illuminated shank. Light-induced firing rate gain did not differ between the illuminated shank (7.6 [4.54 16.03]) and the adjacent shank (7.6 [4.08 13.94]; p = 0.79, Kruskal Wallis test; Extended Data Fig. 6-3 Bb , left).
An alternative approach for establishing optogenetic control in HYB is to cross FVB females with single-transgenic driver male mice and to inject the offspring with a viral vector that expresses a reporter gene. To demonstrate feasibility, we generated a PV-Cre x FVB mouse and injected the animal with a viral vector that allows Cre-dependent expression of ChR2. Focal illumination in the transgenic HYB induced direct PV activation and indirect PYR silencing (Extended Data Fig. 6-3 C ).
Discussion
Mice have emerged as a dominant model for biomedical research, mainly because of their small size, high reproductive rate, and genetic similarities to humans. The same properties facilitated the engineering of multiple transgenic lines. The maintenance of transgenic lines, particularly the generation of double-transgenic or multitransgenic mice, is greatly facilitated by inbred mice ( Silva et al., 1997 ). However, inbred mice are suboptimal for neurobehavioral studies because of idiosyncratic recessive traits. Furthermore, C57 exhibit age-dependent deterioration, limiting the duration of prolonged experiments.
To provide a quantitative description of the HYB, we supplemented the array of standard assays with gait assessment and home cage behavior. The gait of all strains was inconsistent with abnormalities seen in motor disorders ( Preisig et al., 2016 ; Rahn et al., 2021 ), yet every strain exhibited distinct gait patterns that were largely preserved upon aging. In addition, we observed apparent interstrain differences of daily conduct in the home cage, including distinct time division between sheltering, wheel running, and feeding.
For studies that do not involve genetic manipulations, HYB can be used as-is, providing improved performance while maintaining minimal intersubject differences. Specifically, HYB can be implanted with high-density electrode arrays for neocortical and hippocampal recordings over multiple months. To combine transgenic control with enhanced behavior, we used two distinct strategies. One simple strategy involves using a homozygous parent of a C57-background, e.g., a driver line, yielding heterozygous HYB offspring that present one transgenic allele. For conditional expression of a reporter gene, HYB can be injected with a viral vector (as in Extended Data Figs. 6-1 B , 6-3 C ). A second possibility is for one of the parents to be multi-transgenic, e.g., derived by crossing C57-based driver and reporter lines (as in Fig. 6 ; Extended Data Figs. 6-1 A , 6-3 A , B ). Yet a third strategy is to use a C57-based driver line and backcross the reporter onto an FVB/NJ background. Because of the large body of transgenic mice developed on the C57 background, the number of possible combinations is very large, allowing to harness the behavioral potential of the hybrids for a wide range of studies.
To conclude, we showed that a single breeding step produces a hybrid mouse strain that exhibits improved behavioral performance while maintaining genetic control.
Acknowledgments
Acknowledgments: We thank Ronni Cohen for constructive comments.
Synthesis
Reviewing Editor: Mark Laubach, American University
Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: Sebastien Royer. Note: If this manuscript was transferred from JNeurosci and a decision was made to accept the manuscript without peer review, a brief statement to this effect will instead be what is listed below.
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Also, please move the Methods section to where it normally is in J Neurosci/eNeuro style articles.
Reviewer 1
The manuscript compare motor and cognitive performances of two age groups (3 and 9 months) of C57, FVB and C57/FVB hybrid mouse strains. The hybrids tend to outperform both C57 and FVB strains for several parameters in 9-month old mice and are therefore well indicated experimental models for neurobehavioral research with genetic tools. In addition, the authors demonstrate that optogenetic photo-stimulation experiments using optoelectronic probes can successfully be carried in C57/FVB hybrid strains expressing ChR2.
Overall the data are clearly presented and the manuscript well written. My main critic is that the manuscript is somewhat misleading regarding the nature of the differences between species. The major claim is that hybrids outperform both progenitor strains in several learning and motor tasks, and, in addition, show less aging-related deficits. The overall impression from title, abstract and main text is that hybrids outperform progenitor strains regardless of age. However, according to the data (supplementary figures 2 and 3), there is not much difference between hybrid and C57 strains for the 3-month old mice. The differences are mostly significant for 9-month old mice. Hence, hybrid and C57 mice start at the same level during early adulthood (3-months) and then C57 mice show reduced properties as they degrade faster with age.
EDITOR COMMENT: Please revise the manuscript in response to these comments. Addressing these points is key for your resubmission.
The data of the 9-month old mice is presented before the data of the 3-month old mice (which is introduced as to characterize the effect of aging), which is a bit confusing. I suggest presenting the 3-month old mice data first (or altogether with the 9-month old mice data, like in the supplementary figures).
EDITOR COMMENT: Please reorganize the presentation of your data following the advice above
minor comments:
Introduction . “On the one hand, an organism as similar as possible to the object of interest is desired.”
maybe “an organism as similar as possible to human is most often desired"
Introduction . “On the other hand, the least advanced organism for answering the research question is preferred. “
maybe adding an explanation such as “for ethical considerations” at the end
FigS4: the “b” for Ab is missing
EDITOR COMMENT: Please revise to address the above three points.
Reviewer 2
The manuscript by Sloin et al., entitled “Hybrid offspring of C57BL/6J mice exhibit improved properties for neurobehavioral research” produced and extensively tested the first-generation hybrid offspring of FVB/NJ x C57BL/6J mouse strains.
This study is well designed, nicely conducted and excellently written. I believe that this manuscript will be worth publishing in eNeuro following minor revision as suggested below.
1. Authors need to discuss that aging index for HYB mice is significant thus what age of mouse suitable for anxiety studies. In addition, it would be interesting to know that how much anxiety resistance could persists in stressful situations in 9-month-old mice.
EDITOR COMMENT: Please add comments to address these points in the revised Discussion section.
2. Authors used Kruskal Wallis test for several group's analysis. They need to explicitly mention in methods that why they used Non-parametric test.
EDITOR COMMENT: Please clarify why non-parametric tests were used in the Methods section. Report results from exploratory data analysis to justify use of non-parametrics or state another reason.
3. The LED power used in optogenetic study was 11.07 μW, which seems very low. Is the unit used herein is correct?
EDITOR COMMENT: Please confirm or revise the level of light used in the opto part of the study.
4. Since differential expression of viral vectors expressions observed in different neurons, authors need to show the pictures of their viral vector expression, for opto study. Also, briefly discuss the possibility of affected viral expressions, if any, in the HYB mouse brains.
EDITOR COMMENT: Please add an image of a representative tissue as requested and add text to address these issues in the Methods or Discussion.
5. Give the information on statistical analysis software used in the study.
EDITOR COMMENT: Please review terms used for statistical tests throughout the paper. It might not be clear to many readers what you mean by, e.g., U and G tests.