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Abnormal wiring of CCK+ basket cells disrupts spatial information coding

Abstract

The function of cortical GABAergic interneurons is largely determined by their integration into specific neural circuits, but the mechanisms controlling the wiring of these cells remain largely unknown. This is particularly true for a major population of basket cells that express the neuropeptide cholecystokinin (CCK). Here we found that the tyrosine kinase receptor ErbB4 was required for the normal integration into cortical circuits of basket cells expressing CCK and vesicular glutamate transporter 3 (VGlut3). The number of inhibitory synapses made by CCK+VGlut3+ basket cells and the inhibitory drive they exerted on pyramidal cells were reduced in conditional mice lacking ErbB4. Developmental disruption of the connectivity of these cells diminished the power of theta oscillations during exploratory behavior, disrupted spatial coding by place cells, and caused selective alterations in spatial learning and memory in adult mice. These results suggest that normal integration of CCK+ basket cells in cortical networks is key to support spatial coding in the hippocampus.

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Figure 1: Subsets of CCK+ interneurons express ErbB4.
Figure 2: Deletion of Erbb4 from CCK+ interneurons decreases the number of synapses made on hippocampal pyramidal and parvalbumin cells at P30 in CA1 hippocampal region.
Figure 3: Functional impairment of inhibition in CA1 pyramidal cells of Cck-Cre;Erbb4F/F mice at P60–70.
Figure 4: Disturbed hippocampal oscillatory activity in Erbb4 conditional mutant mice in hippocampal CA1.
Figure 5: Deficits in recognition of spatial novelty in Erbb4 conditional mutant mice.
Figure 6: Delayed acquisition of spatial reference memory and deficits in hippocampal spatial organization in Erbb4 conditional mutant mice.
Figure 7: Impaired spatial representation by place cells in the hippocampus of Erbb4 conditional mutant mice.
Figure 8: Abnormal stability of place cells in Erbb4 conditional mutant mice.

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Acknowledgements

We are very thankful to C. Garcia-Frigola for scientific advice and support, M. Fernández, D. Baeza and V. Rodríguez-Millán for technical assistance, T. Gil and F. Navarrete for lab support, and J.Z. Huang (Cold Spring Harbor Laboratory) for mouse colonies (Cck-Cre and VIP-Cre). We thank C. Fernandes for guidance during an early phase of behavioral experiments at King's College London, S. Al Abed for technical advice and stimulating discussions on behavioral experiments performed at the Magendie Institute, and C. Leteneur for help with the behavioral experiments. We are grateful to L. Menéndez de la Prida and M. Maravall for critically reading early versions of this manuscript, and members of the Marín and Rico laboratories for stimulating discussions and ideas. Supported by grants from Fundación Alicia Koplowitz and the European Research Council (ERC-2012-StG 310021) to B.R., from the European Research Council (ERC-2011-AdG 293683) to O.M., from the Spanish Government (CONSOLIDER CSD2007-00023) and Lilly Research Awards Program to B.R. and O.M, and from the French government (ANR-10-EQX-008-1 to A.M. and LabEX BRAIN ANR-10-LABX-43 to A.F. and A.M.). O.M. and B.R. are Wellcome Trust Investigators.

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Authors and Affiliations

Authors

Contributions

I.d.P. performed cell, synaptic, biochemical and behavior experiments and analyzed data. J.R.B.-M. performed in vivo electrophysiology recordings and analyzed data. A.M.-S. carried out in vitro electrophysiology recordings and analyzed data. A.M. contributed to the behavior analysis. A.F. contribute with resources. I.d.P., O.M. and B.R. wrote the manuscript.

Corresponding authors

Correspondence to Oscar Marín or Beatriz Rico.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ErbB4 is expressed by CCK+ interneurons that express Cre recombinase in Cck-Cre mice. Parvalbumin-expressing interneurons receive innervation from subsets of CCK+ boutons (CB1+) that also express VGlut3.

(a) Double immunohistochemistry for ErbB4 and CCK (arrows) in a coronal section of the CA1 region of the hippocampus. (b) Percentage of ErbB4+ cells among CCK+ interneurons in the hippocampus, n = 160 neurons from 3 mice. (c) Triple immunohistochemistry for CCK (red) VGlut3 (green) and β-galactosidase (cyan) in the hippocampus of Cck-Cre;Rosa26 mice. Typical β-galactosidase dotty expression appeared inside the stratum pyramidale labeling putative mostly pyramidal cells and in some neurons sparsely located in the stratum radiatum (arrows) (d) Percentage of β-galactosidase+ cells among CCK+VGlut3+ neurons, n = 34 neurons from 2 Cck-Cre;Rosa26 mice. (e) Scheme of Erbb4 deletion from CCK+ interneurons and experimental design. The circle indicates the synapses analyzed in this experiment. (f) High magnification of the pyramidal cell layer showing a triple staining for PV (blue), CB1 (green; a marker of CCK+ boutons) and VGlut3 (red). or, stratum oriens; pc, stratum pyramidale; ra, stratum radiatum. Scale bar, 50 μm (a, c), 3 μm (f). Data are expressed as mean ± s.e.m. Scale bar.

Supplementary Figure 2 Deletion of Erbb4 from CCK+VGlut3+ interneurons

(bc) Double immunohistochemistry for ErbB4 (green, arrow) and CCK (red, arrow and arrowheads) in the CA1 region of the hippocampus in control (b) and mutant mice (c). (de) Double immunohistochemistry for ErbB4 (red, arrow and open arrowheads) and VGlut3 cells (green, open and full arrowheads) in the CA3 region of the hippocampus in control (d) and mutant mice (e). (f) Percentage of ErbB4+ cells among CCK+ or VGlut3+ interneurons in the hippocampus of control and mutant mice. t test, t (4) = 4.859, P < 0.01; t (6) = 13.368, P < 0.001, n = 218 and 110 CCK+ neurons from 3 controls and 3 mutants, respectively; n = 74 and 58 VGlut3+ neurons from 4 controls and 4 mutants, respectively; or, stratum oriens; pc, stratum pyramidale; ra, stratum radiatum. Scale bars, 50 μm (be) and 10 μm (high magnification). Data are expressed as mean ± s.e.m.

Supplementary Figure 3 Normal density and distribution of CCK+ and VGlut3+ interneurons in Erbb4 conditional mutant mice

(a) Quantification of the density of CCK and VGlut3 cells in the hippocampus of control and mutant mice. t test, (CCK+ control vs mutant t(8) = 0.443, p = 0367; Vglut3+ control vs mutant t(8) = 1.144, p = 0.286, n = 261 and 187 CCK+ neurons from 5 controls and 5 mutants, respectively. n = 196 and 154 VGlut3+ neurons from 5 mice controls and 5 mutants, respectively. (b,c) Coronal sections through the hippocampus of control (b) and Erbb4 conditional mutant mice (c) at P30 showing mRNA for CCK (left) and VGlut3 (right), with a short exposure to the colorimetric reaction to distinguish between pyramidal and interneurons. (d, g) Density of CCK+ and VGlut3+ cells in the CA1 (d) and CA3 (g) regions of the hippocampus. (e, h) Percentage of CCK+ cells in different hippocampal strata of the CA1 (e) and CA3 (h) regions. (f, i) Percentage of VGlut3+ cells in different hippocampal strata of the CA1 (f) and CA3 (i) regions. t test, CA1: n = 141 and 92 CCK+ neurons from 5 controls and 5 mutants, respectively; n = 91 and 76 VGlut3+ neurons from 5 controls and 5 mutants, respectively; CA3: n = 105 and 78 CCK+ neurons from 5 controls and 5 mutants, respectively; n = 85 and 65 VGlut3+ neurons from 5 controls and 5 mutants, respectively. or, stratum oriens; pc, stratum pyramidale; ra, stratum radiatum. Scale bar, 250 μm (b,c). ns, no significant difference, t test. Data are expressed as mean ± s.e.m.

Supplementary Figure 4 Excitatory inputs onto hippocampal CCK+ interneurons are reduced after Erbb4 deletion

(a) Scheme of Erbb4 deletion from CCK+ interneurons. The circle indicates the synapses analyzed in this experiment. (b) Density of VGlut1+ boutons apposed to PSP95+ clusters contacting the soma of CCK+ interneurons. t test, (t(6) = 2.638, p = 0.039, n = 87 and 66 neurons from 4 controls and 4 mutants, respectively. (c,d) Single confocal planes (top) and binary images (bottom) used for quantification, showing VGlut1 boutons (green) and PSD95 clusters (red) that are apposed (arrows) in the periphery of a CCK+ interneuron (blue) in control (c) and mutant mice (d). (e,f) Total density of VGlut1 boutons (e) and PSD95 clusters (f) per μm2 of field of view (1 field of view = 2792 μm2 in stratum radiatum) was not significantly different between control and Cck-Cre;Erbb4F/F mice. t test, t(6) = 1.139, p = 0.293, t (6) = 0.4169, p = 0.6913, (e) and (f) respective values, n = 59 and 74 fields of view from 4 controls and 4 mutants, respectively. Scale bar represents 5 μm (c,d). Data are expressed as mean ± s.e.m.

Supplementary Figure 5 Reduced GAD65 but not GAD67 protein levels in the hippocampus of Erbb4 conditional mutant mice

(a) Immunoblot and quantification of GAD65 in the hippocampus of control and mutant mice. t test, t(14)=2.581, p = 0.022, n = 8 control and 8 mutant mice. (b) Immunoblot and quantification of GAD67 in the hippocampus of control and mutant mice. ns, no significant difference, t test, t(14) = -0.01, p = 0.94, n = 8 control and 8 mutant. Data are expressed as mean ± s.e.m.

Supplementary Figure 6 Reduced connectivity of VGlut3+ cells onto hippocampal pyramidal and parvalbumin cells in hippocampal CA1 of Cck-Cre;Erbb4F/F mice at P60

(a) Schematic drawing of Erbb4 deletion from CCK+ interneurons. The circle indicates the synapses analyzed in this experiment. (b-c) Top panels, triple immunohistochemistry showing VGlut3 (red), CB1 (green) and NeuN staining (grey). Double positive VGlut3+/CB1+ boutons apposed to NeuN+ somas in the pyramidal cell layer are indicated by arrows. Bottom panels, binary images used for quantification; co-localization of VGlut3/CB1 in yellow. (d) Density of VGlut3+/CB1+ boutons contacting the soma of pyramidal cells in control and mutant mice. t test, t(7) = 3.264, p = 0.0138, n = 139 and 125 neurons in CA1 from 4 controls and 5 mutants respectively. (e) Scheme of Erbb4 deletion from CCK+ interneurons. The circle indicates the synapses analyzed in this experiment. (f,g) Representative single confocal images showing VGlut3+ boutons (green, arrows) contacting a PV+ interneuron (red) in control (f) and mutant mice (g). (h) Density of VGlut3+ boutons contacting the soma of PV+ interneurons. t test, t(7) = 3.098, p = 0.0174, n = 53 and 59 neurons from 4 controls and 5 mutants, respectively. (i) Confocal images showing neurobiotin (NB)-filled varicosities in red (white arrows) co-localizing with VGlut3+ boutons in green (red arrows). (j) Density of neurobiotin-positive varicosities per 100 μm of axon. t test, t(14) = 3.349, p = 0.0048, n = 6 and 10 neurons from 4 controls and 4 mutants, respectively. Scale bars, 3 μm (b,c,f,g), 2 μm (i). Data are expressed as mean ± s.e.m.

Supplementary Figure 7 Intrinsic properties of CCK+ interneurons and excitatory circuits in the hippocampus of Erbb4 conditional mutant mice at P60

(a) Schematic of experiment configuration and intrinsic properties. Membrane resistance (Rm), rheobase, delay to the first spike, action potential threshold (AP Thresh), action potential amplitude (AP Amp), action potential half-width (AP HW), fast afterhyperpolarization amplitude (fAhP Amp), fast afterhyperpolarization time (fAhP time), maximum firing frequency (Max FF), adaptation rate. ns, no significant, t test, n = 13 and 15 neurons from 6 controls and 7 mutants, respectively. (b) Schematic of Erbb4 deletion from CCK+ interneurons and recording site. (c) Sample traces and (d) mean sEPSC amplitude and frequency (cesium-based intracellular solution) in Control (grey) and Cck-Cre;Erbb4F/F (magenta) mice. t test, t (25) = 2.256, p = 0.033 (sEPSC amplitude); Mann-Whitney test, U = 72, p = 0.529 (sEPSC frequency); n = 26 and 22 neurons from 5 control and 5 mutant mice, respectively. Data are expressed as mean ± s.e.m.

Supplementary Figure 8 Schematic of stereotaxic injection, viral vector and hChR2-EYFP expression in the hippocampus

(a-d) Schematic of stereotaxic injection, viral vector and hChR2-EYFP expression in the hippocampus. (e) Current-voltage curve for ChR2 in CA1 pyramidal neurons (n = 21 cells). Inset shows example recording traces from one cell. (f) Peak photocurrent amplitude at a holding potential of +10 mV was negative for all cells recorded (-59.1 ± 10.2 pA). (g) At +10 mV, optogenetically-evoked positive GABAergic synaptic currents in ChR2+ pyramidal cells (green) showed opposite sign and were thus separable from negative direct optogenetic photocurrents (red), as revealed by pharmacological blockade of GABAA receptors with GABAzine. (h) Frequency distribution of the amplitude of IPSC events during minimal stimulation experiments suggests a shift towards lower amplitudes in conditional knockout mice. Inset: all-or-none IPSC amplitudes in an example cell during minimal stimulation experiments. (i) There were no statistically-significant differences in IPSC failure rate (Mann-Whitney test, U = 32, p >0.05) between Control (36.4 ± 2.9%) and Cck-Cre;Erbb4F/F mice (30.0 ± 10.3%). (j) There were no statistically-significant differences in photostimulation irradiance used for minimal stimulation experiments (Mann-Whitney test, U = 28, p >0.05) between Control (0.14 ± 0.04 mW/mm2) and Cck-Cre;Erbb4F/F mice (0.15 ± 0.06 mW/mm2).

Supplementary Figure 9 Absence of Cre recombination in subcortical theta-generating structures in Cck-Cre mice

(a) Schematic drawing of a sagittal section displaying the location of the medial septum (green). (b) Sagittal section through a similar level of the scheme showing the medial septal nuclei stained for ChAT (green) and tdTomato (red) in Cck-Cre;TdTomato mice. (c) High magnification showing the absence of co-localization between ChAT+ (green) neurons and tdTomato+ cells (red). (d) Schematic drawing of a sagittal section displaying the location of the pedunculopontine tegmental area (blue). (e) Sagittal section through a similar level of the scheme showing the pedunculopontine tegmental nuclei stained by ChAT (green) and tdTomato in Cck-Cre;TdTomato mice. (f) High magnification showing the absence of co-localization between ChAT+ neurons (green) and tdTomato+ cells. cc, corpus callosum; MS, medial septum; NCx, neocortex; ob, olfactory bulb; PPT, pedunculopontine tegmental area; Th, thalamus. Scale bars represent 1 mm (b, e) and 100 μm (c,f).

Supplementary Figure 10 Behavioral characterization of Erbb4 conditional mutant mice

ab) Locomotor activity in control and mutant mice. (a) Locomotor activity was tested in the open field arena in the first (D1) and fourth day of habituation (D4). The right panel represents the total distance travelled and the left panel shows the mean velocity during exploration; ns, no significant difference, Two-way ANOVA with Fisher’s LSD correction, t(56)= 1.785, p=0.0797, t test, P < 0.05, n = 16 controls and 14 mutant mice. (b) Number of entries in the Y-maze. t test, t(19)= 0.567, p = 0.577, n = 11 controls and 10 mutant mice. (cd) Anxiety behavior in control and mutant mice. (c) Permanence in the different zones on the elevated plus maze (expressed as percentage on the left panel) and ratio of the time spent in the closed versus the open arms (right panel); t test, (open: t (19) = -0.784, p = 0.44; close: t (19) = -0.007, p = 1; center: t (19) = 1.340, p = 0.19; ratio open vs close t(19) = 0.097, p = 0.92, n = 11 controls and 10 mutant)n = 11 controls and 10 mutant mice. (d) Ratio of the time spent in the dark versus the light chamber in the dark-light box test (left panel) and total number of transitions between the two chambers (right panel). t test, time ratio t(16) = 0.17, p = 0.87; transitions t(16) = -0.793, p = 0.44, n = 11 controls and 10 mutant mice. (ej) Motor behavior during LFP recordings was similar between control and CCK-ErbB4 mutants. (e) Theta power equally correlated with the speed of moment in control and mutant mice (t(11) = 1.811 p = 0.081) while the position of the animal presented a random relationship in both groups (t(11) = -1.104, p = 0.298) of the speed of movement and position (f). (g-j) Probability distribution of the speed of movement during spontaneous exploration for control and Cck-Cre;Erbb4F/F. (h) Cumulative immobility periods. (i) Speed of movement during the total spontaneous exploration and in the behavioral epochs used for analysis in the spontaneous exploration tests (periods above 5 cm/s, spontaneous speed t(11) = 0.539, p = 0.60; filtered epochs t(11) = 0.929, p = 0.372, t test). (j) Total time expended in the center vs the periphery of the open field (center t(11) = 0.2122 p = 0.83; periphery t(11) = -0.2122 p = 0.83). (e-j) No significant differences, P <0.05, t test were found for all these parameters or in the behavioral epochs used for analysis t test, n = 7 controls and 6 mutant mice (periods above 5 cm/s). ns, no significant difference, t test. Data are expressed as mean ± s.e.m.

Supplementary Figure 11 Behavioral assessment of attention and cognitive function

Behavioral assessment of attention and cognitive function. (a) The left panel shows baseline response to auditory-evoked startle stimulus (120 dB, t test, t (24) = 0.486, p = 0.631). The right panel shows percentage of prepulse inhibition (PPI) of the auditory startle reflex across different prepulse intensities. (80 dB t (24) = 2.085, p = 0.048; 85 dB t (24) = 2.882, p = 0.008, n=13 control and 13 mutant mice). (bc) Cognitive function in control and mutant mice. (b) Percentage of spontaneous alternation in the Y-maze; ns, no significant difference, t test, t (19) = -0.506, p = 0.5, n = 11 control and 10 mutant mice. (c) Mean percent of correct responses during spatial non-matching to place testing on the T-maze during four blocks of training with reward consisting of 20 trials per mouse. Two-way repeated measurements ANOVA, F(1) = 7.064, p = 0.01, n = 8 control and 10 mutant mice. Data are expressed as mean ± s.e.m.

Supplementary Figure 12 Spatial learning performance in Cck-Cre;Erbb4F/F mice assessed with the Morris water maze (MWM) and classical eight-arm radial maze

(af) MWM performance in a 150 cm tank along 6 days of training (2 trials/day). (ab) Erbb4F/F control mice decreased their path length (a) and latency to reach the escape platform (b) progressively from day 1 (H1) to day 5 (H5). Two-way ANOVA with Fisher’s LSD correction, path length main effects F(5, 115) = 11.63, p < 0.001; scape latency F (5, 115) = 16.66, p < 0.001, n = 8 control and 17 mutant mice. (c) Test for allocentric navigation. Latency to find the platform when both platform and distal visual cues were rotated one quadrant. t-test, t (23) = 0.9522, p = 0.3509, n = 8 control and 17 mutant mice. (de) Probe test in which the platform was removed from the tank and mice were allowed to explore the tank for 60 sec. Percent of distance in each quadrant (d) and tracking profile (e) during the probe test for control and Cre;Erbb4F/F mutant mice. One-way ANOVA with Fisher’s LSD correction, (control: NE vs NW t(21) = 2.104, p = 0.0476; NE vs SW t(21) = 2.172, p = 0.0476; NE vs SE t(21) = 1.679, p = 0.108. Mutant: NE vs NW t(48) = 1.998, p = 0.0514; NE vs SW t(48) = 3.847, p = 0.0004; NE vs SE t(48) = 3.136, p = 0.0029, n = 8 control and 17 mutant mice. (f) Average velocity for each group during the 6 days of training in the MWM. Two-way ANOVA with Fisher’s LSD correction, t(228) = 2.405, p = 0.0168, n = 8 control and 17 mutant mice. (gi) Spatial learning performance on the classical 8-arm radial maze. After two days of habituation to a fully open radial maze, 3 arms were baited and mice were allowed to explore until the 3 baits were collected (2 trials/day) for 7 consecutive days. Cre;Erbb4F/F mutant mice performed a higher number of reference (t(72) = 2.425, p = 0.0178 Fisher's LSD multiple comparisons) (g) and total errors (h), but not of working memory errors (i), during the second and third day of training before switching to a strategy of sequential entry (non-spatial) from day 4 onwards (data not shown). P < 0.05, two-way repeated measurements ANOVA with Fisher’s LSD correction, n = 13 control and 13 mutant mice. Data are expressed as mean ± s.e.m.

Supplementary Figure 13 Single unit isolation and quality sorting

LFP was bandpass and thresholded, for spike waveform extraction. (a, b) Principal components and other parameters were used to separate single unit activity. After automatic clustering, unit isolation was manually refined. Clusters in different colors correspond to different spike waveforms plotted in b where overlapping and mean waveforms are shown. To verify cluster quality isolation the overlapping probability of each pair of isolated clusters was calculated. (c) Comparison matrix for all units isolated in a. In red the principal component distribution of unit a, unit identity specified in the first row, and in blue, the distribution of unit b, unit identity specified in the top column. (d) Overlapping probability distribution between control and mutant groups was not significantly different, Mann-Whitney test, U = 99399.5, p = 0.73, n = 147 and 131 neurons from 7 controls and 6 mutant mice.

Supplementary Figure 14 Single unit characterization and criteria for cell inclusion

(a) Scatter plots relating spike widths and mean firing. Putative pyramidal units display a spike duration of around 500 μs and low firing rate, typically < 5 Hz. Putative interneurons tend to show shorter spike duration (e.g. 200 μs) and higher firing rates, typically > 7-8 Hz. Neurons displaying mixed characteristics were left out from the analysis to reduce ambiguity. (b) A putative pyramidal unit is shown in green, with a large spike duration (see waveform in E3), tendency to fire bursts (see auto-correlogram -50 to 50 ms) and lower firing frequency (see auto-correlogram -1000 to 1000 ms). (c) A putative interneuron is shown in blue, with short spike duration (see E2), no tendency to burst as demonstrated by the absence of counts in the short interval period of the auto-correlogram (see autocorrelogram -50 to 50 ms), high firing rate and a clear theta modulation (see recurrent peaks in the theta range of the -1000 to 1000 ms auto-correlogram). (d) Firing-rate maps were constructed by calculating the number of spikes fired in each pixel (red dots), divided by the dwelling time spent in that coordinate, tracking in black. The pixel map was converted to a 20 X 20 bin size, matrix 2.5 X 2.5 cm. Then firing maps were smoothed using a two-dimensional convolved matrix. Firing fields were contiguous areas of activity above the mean firing rate plus the SEM of the smoothed firing map. (e) Unit sorting criteria (red dashed line): the value expressed in % with respect to the maximal firing rate of the map is larger than the cumulative overlapping false positive for that cell. The histogram represents the probability of false positive Vs the sorting criteria. (f) Randomized firing maps, unsmoothed and smoothed. Original firing maps were use to generate a random distribution and the spatial coherence calculated (n=1000). (g) The mean value of the surrogate distribution for each cell was obtained and use to create a statistical framework to compare the original maps. Criteria for place cell inclusion (red dashed line): only units with a spatial coherence above r = 0.3, percentile > 99th of the random distribution were included.

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Supplementary Figures 1–14 (PDF 3006 kb)

Supplementary Methods Checklist (PDF 1126 kb)

Supplementary Software 1

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Supplementary Software 2

MaKPower3.m (TXT 3 kb)

Supplementary Software 3

PlotFiringFields2-2.m (TXT 19 kb)

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PowerVsSpeedVsPosition2.m (TXT 10 kb)

Supplementary Software 5

SpeedSelecModuleCoherence.m (TXT 11 kb)

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del Pino, I., Brotons-Mas, J., Marques-Smith, A. et al. Abnormal wiring of CCK+ basket cells disrupts spatial information coding. Nat Neurosci 20, 784–792 (2017). https://doi.org/10.1038/nn.4544

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