CCK+ interneurons contribute to thalamus-evoked feed-forward inhibition in prelimbic prefrontal cortex

Abstract

Interneurons in the medial prefrontal cortex (PFC) regulate local neural activity to influence cognitive, motivated, and emotional behaviors. Parvalbumin-expressing (PV+) interneurons are the primary mediators of thalamus-evoked feed-forward inhibition across the mouse cortex, including the anterior cingulate cortex, where they are engaged by inputs from the mediodorsal (MD) thalamus. In contrast, in the adjacent prelimbic (PL) cortex, we find that PV+ interneurons are scarce in the principal thalamorecipient layer 3 (L3), suggesting distinct mechanisms of inhibition. To identify the interneurons that mediate MD-evoked inhibition in PL, we combine slice physiology, optogenetics, and intersectional genetic tools in mice of both sexes. We find interneurons expressing cholecystokinin (CCK+) are abundant in L3 of PL, with cells exhibiting fast-spiking (fs) or non-fast-spiking (nfs) properties. MD inputs make stronger connections onto fs-CCK+ interneurons, driving them to fire more readily than nearby L3 pyramidal cells and other interneurons. CCK+ interneurons in turn make inhibitory, perisomatic connections onto L3 pyramidal cells, where they exhibit cannabinoid 1 receptor (CB1R) mediated modulation. Moreover, MD-evoked feed-forward inhibition, but not direct excitation, is also sensitive to CB1R modulation. Our findings indicate that CCK+ interneurons contribute to MD-evoked inhibition in PL, revealing a mechanism by which cannabinoids can modulate MD-PFC communication.

Full text

Cellular/Molecular
CCK+ Interneurons Contribute to Thalamus-Evoked
Feed-Forward Inhibition in the Prelimbic Prefrontal
Cortex
Aichurok Kamalova,*Kasra Manoocheri,*Xingchen Liu,*Sanne M. Casello, Matthew Huang, Corey Baimel,
Emily V. Jang, Paul G. Anastasiades, David P. Collins, and Adam G. Carter
Center for Neural Science, New York University, New York, New York 10003
Interneurons in the medial prefrontal cortex (PFC) regulate local neural activity to influence cognitive, motivated, and emotional
behaviors. Parvalbumin-expressing (PV+) interneurons are the primary mediators of thalamus-evoked feed-forward inhibition
across the mouse cortex, including the anterior cingulate cortex, where they are engaged by inputs from the mediodorsal (MD)
thalamus. In contrast, in the adjacent prelimbic (PL) cortex, we find that PV+ interneurons are scarce in the principal thalamore-
cipient layer 3 (L3), suggesting distinct mechanisms of inhibition. To identify the interneurons that mediate MD-evoked inhibition in
PL, we combine slice physiology, optogenetics, and intersectional genetic tools in mice of both sexes. We find interneurons express-
ing cholecystokinin (CCK+) are abundant in L3 of PL, with cells exhibiting fast-spiking (fs) or non–fast-spiking (nfs) properties. MD
inputs make stronger connections onto fs-CCK+ interneurons, driving them to fire more readily than nearby L3 pyramidal cells and
other interneurons. CCK+ interneurons in turn make inhibitory, perisomatic connections onto L3 pyramidal cells, where they
exhibit cannabinoid 1 receptor (CB1R) mediated modulation. Moreover, MD-evoked feed-forward inhibition, but not direct excita-
tion, is also sensitive to CB1R modulation. Our findings indicate that CCK+ interneurons contribute to MD-evoked inhibition in PL,
revealing a mechanism by which cannabinoids can modulate MD-PFC communication.
Key words: cannabinoids; CCK+ interneurons; inhibition; prefrontal cortex; thalamus
Significance Statement
Here, we use anatomy, slice physiology, and optogenetics to examine how mediodorsal (MD) thalamus evokes feed-forward
inhibition in Layer 3 of the prelimbic subregion of the mouse prefrontal cortex (PFC). We first show that PV+ interneurons
are relatively sparse in Layer 3 of prelimbic PFC, whereas CCK+ interneurons are prevalent, and we describe the properties of
these cells. We then show how MD thalamus preferentially drives a subpopulation of fast-spiking CCK+ interneurons to fire
more readily than nearby pyramidal cells and other interneurons. Lastly, we show that CCK+ interneurons make inhibitory
connections onto pyramidal cells and both CCK+ and thalamus-evoked inhibition are modulated by CB1 receptors. Our find-
ings identify a novel and unexpected role for CCK+ interneurons in thalamus-evoked inhibition in the PFC.
Introduction
The medial prefrontal cortex (PFC) and mediodorsal (MD) thal-
amus are reciprocally connected brain regions that control cog-
nition, motivation, and emotion (Euston et al., 2012;
Parnaudeau et al., 2018;Halassa and Sherman, 2019).
GABAergic interneurons in the mouse PFC are activated by
excitatory inputs from the thalamus and mediate feed-forward
inhibition (FFI) onto pyramidal cells (Delevich et al., 2015;
Collins et al., 2018;Anastasiades et al., 2021). These interneurons
thus play a critical role in controlling activity within corticotha-
lamic loops and contribute to associated behaviors such as work-
ing memory (Bolkan et al., 2017;Schmitt et al., 2017;Mukherjee
et al., 2021). Dysfunction of PFC interneurons is also linked to
Received May 23, 2023; revised April 12, 2024; accepted April 18, 2024.
Author contributions: A.K., K.M., X.L., P.G.A., D.P.C., and A.G.C. designed research; A.K., K.M., X.L., S.M.C., M.H.,
C.B., E.V.J., P.G.A., and D.P.C. performed research; A.K., K.M., X.L., S.M.C., P.G.A., and D.P.C. analyzed data; A.K.,
K.M., X.L., P.G.A., and A.G.C. wrote the paper.
We thank the members of the Carter Lab for their helpful discussions and comments on the manuscript. We
thank Sumaita Mahmood and Jane Choe for their technical help with histology and viral injections. This work was
supported by NIH T32 MH019524 (K.M.), NIH R01 MH085974 (A.G.C.), and NIH U19 NS123714 (A.G.C.).
*A.K., K.M., and X.L. contributed equally to this work.
The authors declare no competing financial interests.
P.G.A.’s present address: Department of Translational Health Sciences, University of Bristol, Whitson Street,
Bristol BS1 3NY.
Correspondence should be addressed to Adam G. Carter at adam.carter@nyu.edu.
https://doi.org/10.1523/JNEUROSCI.0957-23.2024
Copyright © 2024 the authors
1–14 •The Journal of Neuroscience, June 5, 2024 •44(23):e0957232024

neuropsychiatric disorders, including schizophrenia, stress dis-
orders, and depression (Lewis et al., 2012;Dienel and Lewis,
2019). However, the composition of interneurons in the PFC
may differ from other cortical areas (Whissell et al., 2015;Kim
et al., 2017), particularly in the prelimbic (PL) and infralimbic
(IL) cortices, and the cell types responsible for MD-evoked FFI
in these regions remain unclear.
The PFC and other cortices contain a variety of GABAergic
interneurons, which occupy specific layers, express distinct
markers, and display different morphology, physiology, and local
connectivity (McGarry and Carter, 2016;Tremblay et al., 2016;
Anastasiades et al., 2018;Cummings and Clem, 2020;Liu et al.,
2020;Anastasiades and Carter, 2021). Three major populations
of interneurons include cells expressing parvalbumin (PV+),
somatostatin (SOM+), and 5HT3a receptors, with the latter
including interneurons that express vasoactive intestinal peptide
(VIP+; Rudy et al., 2011). In most cases, PV+ cells mediate feed-
forward inhibition, SOM+ cells mediate feedback inhibition, and
VIP+ cells mediate disinhibition (Gabernet et al., 2005;
Cruikshank et al., 2007;Silberberg and Markram, 2007;Atallah
et al., 2012;Gentet et al., 2012). However, the density of these
cells is distinct in the PFC, with fewer PV+ interneurons and
more SOM+ interneurons (Whissell et al., 2015;Kim et al.,
2017), hinting at distinct connectivity and functional roles.
The mediodorsal (MD) thalamus sends robust glutamatergic
projections to several subregions of the PFC, including the PL
and anterior cingulate cortex (ACC; Krettek and Price, 1977;
Kuramoto et al., 2017). Within both PL and ACC, MD axons
are primarily concentrated in Layer 1b (L1b) and Layer 3 (L3;
Delevich et al., 2015;Collins et al., 2018). MD inputs make
strong, excitatory synapses onto L3 pyramidal cells, which they
can drive to fire, and also engage inhibitory networks. In PL,
MD inputs to L1b preferentially engage VIP+ interneurons
that mediate disinhibition (Anastasiades et al., 2021). In ACC,
MD inputs to L3 activate PV+ interneurons to mediate FFI
(Delevich et al., 2015), as found in sensory systems. However,
the number of PV+ interneurons in PL appears to be much
less than in ACC (Whissell et al., 2015;Kim et al., 2017), suggest-
ing another class of interneuron may mediate MD-evoked FFI
onto pyramidal cells in PL.
Cholecystokinin-expressing (CCK+) interneurons are a sub-
population of inhibitory cells that also express 5-HT3a receptors
(Rudy et al., 2011). CCK+ interneurons are unique among corti-
cal interneurons in expressing cannabinoid type 1 receptors
(CB1R) on their axon terminals, with inhibitory output strongly
modulated by cannabinoids (Katona et al., 1999;Wilson et al.,
2001;Wilson and Nicoll, 2001). However, because pyramidal
cells can also express low levels of CCK, CCK+ interneurons
have been difficult to study (Taniguchi et al., 2011). This techni-
cal challenge is overcome using intersectional labeling methods
to target interneurons over pyramidal cells (Dimidschstein et
al., 2016). In IL, ventral hippocampal inputs to L5 robustly
engage CCK+ interneurons to mediate cannabinoid-sensitive
FFI (Liu et al., 2020). In PL, recent work indicates that CCK+
interneurons are present in L3 and play an important role in
working memory (Nguyen et al., 2020). One possibility is that
these cells may compensate for the lack of PV+ interneurons
and may confer unique properties, such as cannabinoid sensitiv-
ity, to MD-evoked FFI.
Here, we use slice electrophysiology and optogenetics to
examine the contributions of PV+, SOM+, VIP+, and CCK+
interneurons in MD-evoked FFI at L3 pyramidal cells in PL.
We first determine that PV+ interneurons are sparse in L3 of
PL, whereas SOM+, VIP+, and CCK+ interneurons are abun-
dant. We characterize two classes of CCK+ interneurons, which
display either fast-spiking (fs) or non–fast-spiking (nfs) proper-
ties. We show that MD inputs drive robust action potential firing
in fs-CCK+ interneurons more readily than pyramidal cells and
other interneurons. We also establish that CCK+ interneurons
robustly inhibit the soma of L3 pyramidal cells and that these
connections are potently suppressed by CB1R modulation.
Lastly, we show that MD-evoked FFI is also sensitive to CB1R
modulation, indicating that CCK+ interneurons are likely major
sources of FFI. These results highlight the physiology, connectiv-
ity, and functional properties of CCK+ interneurons in superfi-
cial layers of PL, which both contribute to MD-evoked FFI at
L3 pyramidal cells and exhibit prominent sensitivity to cannabi-
noid signaling.
Materials and Methods
Animals
Experiments used wild-type and transgenic mice of either sex in a
C57BL/6J background (all breeders from The Jackson Laboratory).
Homozygote male breeders: PV-Cre = JAX 008069 (Hippenmeyer et al.,
2005); SOM-Cre= JAX 013044 (Taniguchi et al., 2011); VIP-Cre = JAX
010908 (Taniguchi et al., 2011); CCK-Cre = JAX 012706 (Taniguchi et
al., 2011); and PV-2A-Cre = JAX 012358 (Madisen et al., 2010) were
paired with female wild-type or Ai14 (Cre-dependent tdTomato) breeders,
JAX 007914 (Madisen et al., 2010), to yield heterozygote offspring used for
experiments. All experimental procedures were approved by the University
Animal Welfare Committee of New York University.
Viruses
Adeno-associated viruses (AAVs) used in this study were as follows:
AAV1-hSyn-hChR2(H134R)-eYFP-WPRE-hGH (UPenn AV-26973P),
AAV1-EF1a-DIO-eYFP-WPRE-hGH (UPenn AV-1-27056), AAV1-
Dlx-Flex-EGFP (Addgene #83895; a gift from Jordane Dimidschstein
and Gord Fishell; Dimidschstein et al., 2016), and AAV1-Dlx-
Flex-ChR2-mCherry (a gift from Jordane Dimidschstein and Gord
Fishell; Liu et al., 2020).
Stereotaxic injections
Mice aged 4–6 weeks were deeply anesthetized with isoflurane and then
head-fixed in a stereotaxic frame (David Kopf Instruments). A small cra-
niotomy was made over the injection site, using these coordinates relative
to the bregma: PL PFC = ±0.4, −2.3, +2.1 mm; MD = −0.4, −3.5,
−0.7 mm (mediolateral, dorsoventral, and rostrocaudal axes).
Borosilicate pipettes with 5–10-µm-diameter tips were backfilled with
virus, and a volume of 130–550 nl was pressure-injected using a
Nanoject III (Drummond) every 30 s. The pipette was left in place for
an additional 5 min, allowing time to diffuse away from the pipette tip,
before being slowly retracted from the brain. For both retrograde and
viral labeling, animals were housed for 2–3 weeks before slicing. We ver-
ified viral expression in the PFC and MD before use in slice electrophys-
iology recordings. Care was taken to avoid leakage into neighboring
structures, but we cannot rule out some minor spread into PVT.
Histology and fluorescence microscopy
Mice were anesthetized with a lethal dose of ketamine and xylazine and
then perfused intracardially with 0.01 M phosphate-buffered saline
(PBS) followed by 4% paraformaldehyde (PFA) in 0.01 M PBS. Brains
were fixed in 4% PFA in 0.01 M PBS overnight at 4°C. Slices were pre-
pared at a thickness of 70 µm for directly imaging fluorescent proteins
or tracers and 40 µm for antibody staining (Leica Vibratome Vt1000s).
For immunohistochemistry, slices were incubated with blocking solution
(1% bovine serum albumin and 0.2% Triton X-100 in 0.01 M PBS) for
1 h at room temperature before primary antibodies were applied in
blocking solution [mouse anti-parvalbumin antibody (Millipore,
MAB1572)] at 1:2,000 overnight at 4°C. Slices were then incubated
with secondary antibodies in blocking solution [goat anti-mouse 647 at
2•J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

1:200 (Invitrogen)] for 1.5 h at room temperature before mounting
under glass coverslips on gelatin-coated slides using ProLong Gold anti-
fade reagent with DAPI (Invitrogen). Images were acquired using a con-
focal microscope (Leica SP8). Image processing involved adjusting
brightness, contrast, and manual cell counting using ImageJ (NIH).
Fluorescence in situ hybridization (RNAscope)
Mice were anesthetized with a lethal dose of ketamine and xylazine and
then perfused intracardially with chilled 0.01 M PBS. Brains were imme-
diately submerged in cold isopentane on dry ice after dissection. Solid
brains were then embedded in OCT media (Tissue-Tek), frozen in iso-
pentane, and stored in an airtight container at −80°C until sectioning.
Sectioning was performed on a cryostat at −13°C, and 16 µm slices
were mounted on Superfrost Plus microscope slides (Fisher Scientific)
and stored at −80°C until staining. We followed a standardized
RNAscope protocol for flash-frozen tissue from ACDBio, using
Mm-Cck-C2, EGFP-C2, Mm-Pvalb-C3, Mm-VIP-C3, Mm-Cnr1-C3,
and Mm-SST-C3 probes. Slides were mounted using ProLong Gold anti-
fade reagent with DAPI (Invitrogen) before being covered under
coverslips.
Slice preparation
Mice aged 6–8 weeks were anesthetized with an intraperitoneal injection
of a lethal dose of ketamine and xylazine and perfused intracardially with
an ice-cold external solution containing the following (in mM):
65 sucrose, 76 NaCl, 25 NaHCO
3
, 1.4 NaH
2
PO
4
, 25 glucose, 2.5 KCl,
7 MgCl
2
, 0.4 Na-ascorbate, and 2 Na-pyruvate (295–305 mOsm) and
bubbled with 95% O
2
/5% CO
2
. Coronal slices (300 µm thick) were cut
on a VS1200 vibratome (Leica) in ice-cold external solution and trans-
ferred to ACSF containing the following (in mM): 120 NaCl,
25 NaHCO
3
, 1.4 NaH
2
PO
4
, 21 glucose, 2.5 KCl, 2 CaCl
2
, 1 MgCl
2
,
0.4 Na-ascorbate, and 2 Na-pyruvate (295–305 mOsm), bubbled with
95% O
2
/5%CO
2
. Slices were kept for 30 min at 35°C and recovered
for 30 min at room temperature before starting recordings. All record-
ings were conducted at 30–32°C.
Electrophysiology
Whole-cell recordings were obtained from pyramidal cells or interneu-
rons located in L3 of PL, defined as 225–300 µm from the pial surface
(Collins et al., 2018). Cells were identified by infrared-differential inter-
ference contrast or fluorescence, as previously described (Chalifoux and
Carter, 2010). Pairs of adjacent cells were chosen for sequential record-
ing, ensuring they received similar inputs (typically <50 µm between
cells). Borosilicate pipettes (3–5MΩ) were filled with one of three inter-
nal solutions. For current-clamp recordings (in mM): 135 K-gluconate,
7 KCl, 10 HEPES, 10 Na-phosphocreatine, 4 Mg
2
-ATP, 0.5 EGTA, and
0.4 Na-GTP, 290–295 mOsm, pH 7.3, with KOH. For voltage-clamp
recordings (in mM): 135 Cs-gluconate, 10 HEPES, 0.5 EGTA, 10
Na-phosphocreatine, 4 Mg
2
-ATP, and 0.4 Na-GTP, 10 TEA-chloride,
and 2 QX-314, 290–295 mOsm, pH 7.3, with CsOH. For DSI and
CB1R modulation experiments (in mM): 130 K-gluconate, 10 HEPES,
1.1 EGTA, 10 Na-phosphocreatine, 1.5 MgCl
2
,2Mg
2
-ATP, 0.4
Na-GTP. In some experiments studying cellular morphology, 5% biocy-
tin was also included in the recording internal solution. After allowing
biocytin to diffuse through the recorded cell for at least 30 min, slices
were fixed with 4% PFA before staining with streptavidin conjugated
to Alexa Fluor 647 (Invitrogen).
Electrophysiology recordings were made with a MultiClamp 700B
amplifier (Axon Instruments), filtered at 4 kHz for current-clamp, and
2 kHz for voltage-clamp, and sampled at 10 kHz. The initial series resis-
tance was <20 MΩ, and recordings were ended if the series resistance
rose above 25 MΩ. In many experiments, 10 µM CPP was used to block
NMDA receptors. In current-clamp experiments characterizing intrinsic
properties, 10 µM NBQX, 10 µM CPP, and 10 µM gabazine were used to
block excitation and inhibition. In some experiments, 10 µm AM-251
was used to block CB1 receptors, or 1 µM WIN 55,212–2 (WIN) was
used to activate CB1 receptors. All chemicals were purchased from either
Sigma or Tocris Bioscience.
Optogenetics
Channelrhodopsin-2 (ChR2) was expressed in presynaptic cells and acti-
vated with a 2 ms light pulse from a blue LED (473 nm; Thorlabs). For
wide-field illumination, the light was delivered via a 10 × 0.3 NA objec-
tive (Olympus) centered on the recorded cell, as previously described
(Collins et al., 2018;Liu et al., 2020;Anastasiades et al., 2021;Baimel
et al., 2022;Manoocheri and Carter, 2022). LED power was routinely cal-
ibrated at the back aperture of the objective. LED power was adjusted to
obtain reliable responses, with typical values of 0.4–10 mW. Subcellular
targeting recordings utilized a digital mirror device (Mightex Polygon
400 G) to stimulate a 10 × 10 grid of 75 µm squares at a power range
of 0.05–0.2 mW per square at 1 Hz, with the first row aligned to the
pia, as previously described (Manoocheri and Carter, 2022).
Data analysis
Electrophysiology and imaging data were acquired using National
Instruments boards and custom software written in MATLAB
(MathWorks). Offline analysis was performed using custom software
written in Igor Pro (WaveMetrics). Intrinsic properties were determined
as follows. Input resistance was calculated from the steady-state voltage
during a −50 pA, 500 ms current step. The maximum firing rate was
determined by increasing the current injection in a stepwise fashion until
the firing rate plateaued. Rheobase was determined by the average min-
imum current injection that elicited firing. For experiments with a single
optogenetic stimulation, the PSC amplitude was measured as the average
value across 1 ms around the peak subtracted by the average 100 ms
baseline value prior to the stimulation. For experiments with a train of
optogenetic stimulation, each PSC amplitude was measured as the aver-
age value in a 1 ms window around the peak, minus the average 2 ms
baseline value before each stimulation. Most summary data are reported
in the text and figures as arithmetic mean ± SEM. Ratios of responses at
pairs of cells are reported as geometric mean in the text and with ± 95%
confidence interval (CI) in the figures, unless otherwise noted.
Comparisons between unpaired data were performed using the nonpara-
metric Mann–Whitney Utest. Comparisons between data recorded in
pairs were performed using the nonparametric Wilcoxon test. For
unpaired comparisons of more than two groups, Kruskal–Wallis tests
with Dunn’s multiple-comparisons tests were performed. Two-tailed
pvalues < 0.05 were considered significant.
Results
Parvalbumin-expressing interneurons are sparse in L3 of the
prelimbic cortex
MD thalamus generates direct excitation and feed-forward inhi-
bition (FFI) in both the PL and ACC subregions of the medial
PFC (Delevich et al., 2015;Collins et al., 2018). To examine
MD-evoked synaptic responses in PL, we injected
AAV-ChR2-eYFP into MD, waited for expression and transport
(Fig. 1A, Extended Data Fig. 1-1A,B). We then recorded light-
evoked EPSCs at −60 mV, the reversal potential for inhibition,
and IPSCs at +10 mV, the reversal potential for excitation,
from L3 pyramidal cells (Fig. 1B). Consistent with our previous
work (Collins et al., 2018), we found that MD inputs evoke prom-
inent excitation and FFI (EPSC = 335 ± 22 pA, IPSC = 391 ±
101 pA; n= 8 cells, three animals; Fig. 1B,C). MD-evoked FFI
exhibited a delay (EPSC to IPSC = 2.8 ± 0.8 ms, n= 8 cells, three
animals) and displayed a pronounced reduction of amplitude
[paired-pulse ratio (PPR) = IPSC
5
/IPSC
1
= 0.04 ± 0.01, n= 8 cells,
three animals; Fig. 1B,C], as observed for FFI mediated by PV+
interneurons in other cortical regions (Gabernet et al., 2005;
Cruikshank et al., 2010).
PV+ interneurons have been shown to mediate MD-evoked
FFI in ACC (Delevich et al., 2015), but whether this occurs in
PL is unknown. While PV+ interneurons are the most abundant
GABAergic cell type in most of the cortex (Rudy et al., 2011),
recent work suggests a relative paucity of PV+ interneurons in
Kamalova et al. •Thalamus-Evoked Inhibition in the PFC J. Neurosci., June 5, 2024 •44(23):e0957232024 •3

PL compared with other cortical areas (Kim et al., 2017).
Whether this relative lack of PV+ interneurons in PL occurs
within the main thalamorecipient L3 was unclear. Therefore,
we next used three separate methods to visualize PV+ interneu-
rons and compared their distributions across cortical layers in
both PL and ACC. First, we labeled PV+ interneurons using
PV-Cre mice crossed with Ai14 reporter mice to express
Cre-dependent tdTomato in PV+ cells (Madisen et al., 2010).
We found significantly fewer PV+ interneurons labeled in PL
L3 relative to ACC L3 (PL L3 PV x Ai14 = 1.6 ± 0.5 cells per slice,
ACC L3 PV x Ai14 = 19 ± 1.4, p= 0.001; n= 11 slices, three ani-
mals; Fig. 1D, Extended Data Fig. 1-1C,D). To confirm the lack
of PV+ interneurons is not due to reduced expression of Cre in
these mice, we also compared PV labeling with immunohisto-
chemistry and fluorescence in situ hybridization (RNAscope;
Wang et al., 2012; Fig. 1E,F). We found that both immunolabeled
(PL L3 PV = 4.1 ± 0.6 cells per slice, ACC L3 PV = 15.4 ± 1.3,
p= 0.0005; n= 12 slices, three animals) and mRNA labeled
(PL L3 PV = 1.6 ± 0.7 cells per slice, ACC L3 PV = 4.6 ± 1.1,
p= 0.035; n= 11 slices, three animals) PV+ interneurons were
significantly less abundant in PL L3 compared with ACC L3.
However, there was significantly higher PV labeling in PL L5
compared with L3 (Extended Data Fig. 1-1E–G). These results
show a relative lack of PV+ interneurons in PL L3, suggesting
another interneuron is responsible for MD-evoked FFI.
CCK-expressing interneurons are prominent in L3 of the
prelimbic cortex
While PV+ interneurons are common across the cortex, several
other cell types are also abundant and could contribute to
MD-evoked FFI. We next used transgenic mice and viruses to
establish the distributions of SOM+, VIP+, and CCK+ interneu-
rons in L3 of PL (Fig. 2A). We labeled SOM+ and VIP+ interneu-
rons by crossing Cre lines with a Cre-dependent tdTomato
reporter line (Ai14; Madisen et al., 2010). We labeled CCK+
interneurons by injecting a Cre-dependent, interneuron-specific
virus expressing EGFP (AAV-Dlx-Flex-EGFP) into the PFC of
CCK-Cre mice, which minimizes expression in pyramidal cells
that can express low levels of CCK (Dimidschstein et al., 2016).
We observed higher numbers of SOM+, VIP+, and CCK+ inter-
neurons compared with PV+ interneurons in L3 of PL (PV+ =
2.3 ± 0.5 cells per slice, SOM+ = 13.5 ± 0.9, VIP+ = 14.3 ± 0.7,
CCK+ = 9.5 ± 1; PV vs SOM, p< 0.0001, PV vs VIP, p< 0.0001;
PV vs CCK, p= 0.032; n= 12 slices, three animals each; Fig. 2B,C).
Figure 1. PV+ interneurons are sparse in L3 of PL. A, Left, Schematic of AAV-ChR2-eYFP injection into MD and recording from the ipsilateral PFC. Right, Labeling of MD axons in L1b and L3 of
the PFC, including the prelimbic (PL) and anterior cingulate cortex (ACC). Dashed lines = subregion borders. Scale bar = 200 µm. B, Average MD-evoked EPSCs (at E
GABA
=−60 mV) and IPSCs (at
E
AMPA
= +10 mV) at L3 pyramidal cells (PYR; n= 8 cells, three animals). Blue arrow = 2 ms light pulse. C, Left, Summary of MD-evoked EPSC and IPSC amplitudes, where gray dots are individual
cells. Right, Summary of paired-pulse ratio (PPR = PSC
n
/PSC
1
) for MD-evoked responses as a function of pulse number (n). D, Left, Confocal image of labeled cells (blue) in PV-Cre x Ai14 mice,
across the dorsoventral axis of the PFC. Dashed lines = subregion borders. Scale bar = 200 µm. Right top, Expansion of ACC (left) and PL (right), showing differential labeling. Scale
bar = 100 µm. Right bottom, Summary of labeling in L3 of ACC and PL, with minimal labeling in the latter. Gray lines = individual slices (n= 11 slices, three animals). E, Similar to Dfor
anti-PV antibody labeling (n= 12 slices, three animals). F, Similar to Efor PV RNA labeling (n= 11 slices, three animals).
4•J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

These results suggest all three classes of GABAergic interneurons
are well positioned to mediate MD-evoked FFI in L3 of PL.
We were particularly interested in the CCK+ interneurons,
which we recently showed contribute to hippocampal-evoked
FFI in Layer 5 of the IL PFC (Liu et al., 2020). To validate our
ability to specifically label CCK+ interneurons, we examined
mRNA expression of known interneuron markers using fluores-
cence in situ hybridization (RNAscope; Fig. 2D,E). In CCK-Cre
mice injected with AAV-Dlx-Flex-EGFP, we found that
EGFP+ cells exhibited significant overlap with CCK+ mRNA
(EGFP+/CCK+ = 0.90 ± 0.03, n= 9 slices, three animals) but min-
imal overlap with either PV+ mRNA (EGFP+/PV+ = 0.08 ± 0.03,
n= 9 slices, three animals) or SOM+ mRNA (EGFP+/SOM+ =
0.03 ± 0.03, n= 8 slices, three animals). We also found a small
population of EGFP+ cells that overlapped with VIP+ mRNA
(EGFP+/VIP+ = 0.29 ± 0.05, n= 11 slices, five animals), consis-
tent with previous reports (Bodor et al., 2005;Tasic et al., 2018;
Gouwens et al., 2020;(BICCN), 2021;Scala et al., 2021), and sug-
gesting heterogeneity in the CCK+ population. These results
indicate our approach reliably labels CCK+ interneurons, with
minimal labeling of other cell types, enabling us to assess how
they are engaged by MD inputs and contribute to FFI in PL.
PL L3 interneurons have characteristic intrinsic properties
Having identified several interneurons as possible candidates for
mediating inhibition in PL L3, we next sought to characterize
their intrinsic properties. While we previously assessed the
intrinsic properties of CCK+ interneurons in deep layers of the
neighboring IL PFC (Liu et al., 2020), the intrinsic properties
of PL L3 CCK+ interneurons have remained unknown. We
used whole-cell current-clamp recordings in coronal slices to
characterize the morphology and physiology of PV+, SOM+,
VIP+, and CCK+ interneurons in PL L3 (Fig. 3A,B). We found
that PV+, SOM+, and VIP+ interneurons had similar intrinsic
properties to those previously reported in the PFC and other cor-
tices (McGarry et al., 2010;Lee et al., 2013;Karnani et al., 2016),
consistent with studies of the diversity of inhibitory cell types
throughout the cortex (Tasic et al., 2018;Gouwens et al., 2020;
(BICCN), 2021;Scala et al., 2021). Interestingly, CCK+ interneu-
rons in L3 of PL showed two strikingly divergent subtypes
(Fig. 3B,C): (1) fast-spiking CCK+ (fs-CCK+) interneurons
with a high max firing rate, high rheobase, and low input resis-
tance (max firing rate = 248 ± 18 Hz; rheobase = 351 ± 67 pA;
input resistance = 103 ± 13 MΩ,n= 7 cells, four animals) and
(2) non–fast-spiking CCK+ (nfs-CCK+) interneurons with low
max firing rate, low rheobase, and high input resistance (max
firing rate = 64 ± 11 Hz; rheobase = 98 ± 29 pA; input resistance =
377 ± 84 MΩ,n= 7 cells, four animals). We distinguished
fs-CCK+ from nfs-CCK+ interneurons by determining their
maximal firing rate and applying a cutoffof 100 Hz. These results
are consistent with previous studies of CCK+ interneurons in
other cortical areas, which identified both fast-spiking basket
Figure 2. CCK+ interneuronsare prominent in L3 of PL. A, Confocal images of GABAergic interneurons across layers of PL. From left toright, PV+ interneurons labeled in PV-Crex Ai14 mice; SOM+
interneurons labeled in SOM-Cre x Ai14 mice; VIP+ interneurons labeled in VIP-Cre x Ai14 mice, and CCK+ interneurons labeled by injecting AAV-Dlx-Flex-EGFP virus into CCK-Cre mice. Dashed
lines = layer borders. Scale bar = 100 µm. B, Average number of each interneuron across cortical depth (n= 12 slices, three animals each). C, Summary of the average number of labeled cells in each
PL L3 slice. Open circles =individual slices. D, Confocal images of RNA staining in L3 PL of CCK-Cre mice injected with AAV-Dlx-Flex-EGFP virus. Each slice was stained for EGFP, PV, SOM, VIP, and/or
CCK mRNA. Scale bar = 100 µm. E, Summary of overlap between EGFP+ (white) and PV+ (blue), SOM+ (green), VIP+ (red), or CCK+ (magenta) mRNA (EGFP+/CCK+: n= 9 slices, three animals;
EGFP+/PV+: n=9 slices, three animals; EGFP+/SOM+: n= 8 slices, three animals; EGFP+/VIP+: n=11slices,five animals). Open circles = individual slices. Values are mean ± SEM. *p<0.05.
Kamalova et al. •Thalamus-Evoked Inhibition in the PFC J. Neurosci., June 5, 2024 •44(23):e0957232024 •5

cells and a population of highly excitable bipolar cells
(Kawaguchi and Kubota, 1998;He et al., 2016). Notably, the
intrinsic properties of fs-CCK+ interneurons are broadly similar
to PV+ interneurons that mediate thalamus-evoked inhibition in
other parts of the cortex (Porter et al., 2001).
Thalamic inputs preferentially drive fs-CCK+ interneurons in
PL L3
While PV+ interneurons in other cortical areas receive strong
thalamic input and often act as the primary mediator of FFI
(Cruikshank et al., 2007), their low numbers in PL L3 suggest
other interneuron cell types could be fulfilling that role in this cir-
cuit. We next examined which population of interneurons are
preferentially activated by MD inputs, assessing which cell types
are activated more readily than neighboring L3 pyramidal cells,
which is the hallmark of FFI. To study MD-evoked activity, we
injected AAV-ChR2-eYFP into MD while also labeling PV+,
SOM+, VIP+, or CCK+ interneurons. After waiting for expres-
sion and transport, we prepared coronal PFC slices and made
sequential recordings from neighboring pairs of L3 pyramidal
cells and either PV+, SOM+, VIP+, or CCK+ interneurons at
their resting membrane potential (Fig. 4A). We activated MD
inputs with a stimulus train (five pulses at 10 Hz, 2 ms pulse
duration) at several light intensities (1–8mW; Fig. 4B,
Extended Data Fig. 4-1). To compensate for variation in viral
labeling, we compared firing probability at the lowest intensity
Figure 3. Distinct intrinsic properties of L3 interneurons. A, Representative biocytin fills showing the morphology of recorded PV+, SOM+, VIP+, fast-spiking CCK+ (fs-CCK+), and non–
fast-spiking CCK+ (nfs-CCK+) interneurons. Scale bar = 50 µm. B,APfiring in response to current injection at rheobase (gray traces) and max firing rate (colored traces), in addition to a
hyperpolarizing current step for each cell type shown in A(note current steps not drawn to scale). C, Summary of key intrinsic properties, including resting membrane potential (Vm),
max firing rate, rheobase current, and input resistance (Rin; PV+: n= 8 cells, four animals; SOM+: n= 13 cells, three animals; VIP+: n= 10 cells, four animals; fs-CCK+: n= 7 cells, four
animals; nfs-CCK+: n= 7 cells, four animals). Values are mean ± SEM. *p< 0.05.
6•J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

that reliably drove firing in at least one cell type in the pair
(Fig. 4C). Because each pair used a different light intensity, the
firing probability was variable across recordings, leading to
different firing of pyramidal cells. However, we found that only
fs-CCK+ interneurons were activated at lower intensities relative
to L3 pyramidal cells, whereas PV+ interneurons were activated
at similar intensities to L3 pyramidal cells, and SOM+, VIP+, and
nfs-CCK+ interneurons were only engaged when L3 pyramidal
cells were already active (Fig. 4C;firing probabilities: L3 PYR
vs L3 PV+ = 0.59 ± 0.14 vs 0.46 ± 0.13, p= 0.43, n= 11 pairs, five
animals; L3 PYR vs L3 SOM+ = 0.83 ± 0.08 vs 0.43 ± 0.13, p=
0.02, n= 12 pairs, four animals; L3 PYR vs L3 VIP+ = 0.94 ±
0.06 vs 0.12 ± 0.10, p= 0.008, n= 8 pairs, four animals; L3 PYR
vs L3 fs-CCK+ = 0.27 ± 0.12 vs 0.91 ± 0.07, p= 0.03, n= 7 pairs,
five animals; L3 PYR vs L3 nfs-CCK+ = 0.72 ± 0.14 vs 0.14 ±
0.08, p= 0.008, n= 7 pairs, four animals). Examining responses
over stimulus trains, we observed that MD-evoked firing was
also highly adapting at fs-CCK+ interneurons (Extended Data
Fig. 4-1), resembling the MD-evoked FFI observed at L3 pyrami-
dal cells (Fig. 1B,C). Together, these results indicate MD input
influences multiple interneuron populations in L3 of the PL
PFC but preferentially drives fs-CCK+ interneurons to fire before
L3 pyramidal cells, which could be replacing PV+ interneurons
as the primary contributor to thalamus-evoked FFI.
We previously found that poor PV+ interneuron labeling in
other brain regions can be overcome using the PV-2A-Cre trans-
genic line (Madisen et al., 2010;Scudder et al., 2018;Baimel et al.,
2022). To test if this approach might also allow us to target a pre-
viously undetectable population of fast-spiking cells in the PFC,
we injected AAV-Dlx-Flex-EGFP into the PL of PV-2A-Cre mice
(Fig. 5). With this strategy, we observed a substantially greater
number of labeled cells across layers of PL (Fig. 5A). However,
in situ hybridization showed low colabelling of EGFP+ cells
with PV+ mRNA and substantial labeling with CCK+ mRNA
(Fig. 5B). Interestingly, current-clamp recordings showed these
cells displayed a fast-spiking phenotype (max firing rate = 212 ±
13.6 Hz; rheobase = 300 ± 28.2 pA; input resistance = 105.7 ±
7.5 MΩ,n= 10 cells, four animals; Fig. 5C,D). Moreover, optoge-
netic experiments showed these cells could be driven to fire, similar
to our PV-Cre recordings (Fig. 5E). However, unlike fs-CCK+
interneurons, there were no significant differences in firing prob-
ability compared with nearby L3 pyramidal neurons in response
to MD inputs (Fig. 5F;firing probability: L3 PYR vs L3 PV-2A+ =
0.57 ± 0.14 vs 0.51 ± 0.16, p= 0.81, n= 7 pairs, three animals).
Figure 4. MD preferentially activates fast-spiking CCK ± interneurons. A, MD-evoked EPSPs and APs at pairs of interneurons (color, top) and corresponding PYR (black, bottom), showing PV+,
SOM+, VIP+, fs-CCK+, and nfs-CCK+ interneurons, with five example traces offset for each cell. Replicates are denoted by different colors. Blue arrow = 2 ms light pulse. B, Summary of AP
probability at any stimulus pulse during trains across five different light intensities, color-coded as in A(PV+: n= 11 pairs, five animals; SOM+: n= 12 pairs, four animals; VIP+: n= 8 pairs, four
animals; fs-CCK+: n= 7 pairs, five animals; nfs-CCK+: n= 7 pairs, four animals). C, Summary of AP probability during trains at the lowest light intensity that drove firing at either the interneuron
or PYR in a pair, color-coded as in (A). Values are mean ± SEM. *p< 0.05 (Extended Data Fig. 4-1).
Kamalova et al. •Thalamus-Evoked Inhibition in the PFC J. Neurosci., June 5, 2024 •44(23):e0957232024 •7

Together, these results suggest PV-2A-Cre labels a complex
selection of interneurons, which are unlikely to be the main
mediator of MD-evoked FFI in PL.
CCK+ interneurons robustly inhibit L3 pyramidal cells in PL
Having identified CCK+ interneurons as a key target of MD,
we next characterized their output onto L3 pyramidal cells in
PL. Previous studies in the hippocampus and cortex indicate
that CCK+ basket cells, analogous to fs-CCK+ interneurons,
share some properties with PV+ basket cells, and thus could
be performing a similar role in the PFC (Kawaguchi and
Kubota, 1998;Armstrong and Soltesz, 2012). To test this
idea, we first examined whether CCK+ output to PL L3 is
GABAergic, using a Cre-dependent, interneuron-specificvirus
expressing ChR2 (AAV-Dlx-Flex-ChR2) injected into the PFC
of CCK-Cre mice (Fig. 6A). In slice recordings, we stimulated
CCK+ interneurons with brief light pulses (2 ms) and recorded
L3 pyramidal cells held at −50 mV with a low chloride [Cl−]
internal solution, which increases the driving force for
GABA
A
receptor currents and simultaneously captures both
EPSCsandIPSCs(Fig. 6B;Glickfeld and Scanziani, 2006).
We found robust CCK-evoked IPSCs but not EPSCs, which
were blocked by wash-in of the GABA
A
receptor antagonist,
gabazine (10 µM; baseline IPSC = 218 ± 60 pA, gabazine
IPSC=1.5±0.3pA, p= 0.0078, n= 8 pairs, three animals; Fig. 6B).
These results confirm that CCK+ interneuron output is
Figure 5. PV-2A-Cre labels both PV ± and CCK+ interneurons. A, Left, Confocal images of labeled cells in PV-2A-Cre animal injected withAAV-Dlx-Flex-EGFP across PL layers. Dashed lines =layer
borders. Scale bar = 100 µm. Right, Average number of PV-2A+ (blue) interneurons across cortical depth relative to PV+ interneuron distribution from PV x Ai14 (gray, same dataset as Fig. 2A–C).
B, Left, Confocal images of EGFP+, PV+, and CCK+ mRNA labelingin L3 of PL. Scale bar = 100 µm. Right, Summary of the overlap between EGFP+ and eitherCCK+ or PV+ mRNA (n= 9 slices, three
animals). C,Left, Representative biocytin fill showing morphology of L3 PV-2A+ interneuron. Scalebar = 100 µm. Right, Example firing pattern in response to current injection at rheobase (gray trace)
and max firing rate (blue trace) in addition to a hyperpolarizing current step for PV-2A+ interneurons (note current steps are not drawn to scale). D, Summary of key intrinsic properties, including
resting membrane potential (Vm), max firing rate, rheobase current, and input resistance (Rin; n= 10 cells, four animals). E, Examples of MD-evoked EPSPs and AP firing at pairs of PV-2A+
interneurons (blue) and PYR (gray) in L3, with five example traces shown for each cell. Blue arrow = 2 ms light pulse. F, Left, Summary of AP probability at any stimulus pulse during trains across
five different light intensities. Right, Summary of AP probability per pair of PV-2A+ interneuron and PYR in L3 during the stimulus train (n= 7 pairs, three animals).
8•J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

exclusively inhibitory in PL L3 and that our approach targets
inhibitory interneurons.
Another important property that affects CCK output to PL L3
is short-term synaptic dynamics, including whether they facilitate
or depress (Zucker and Regehr, 2002). To examine the dynamics
of CCK+ output to L3 pyramidal cells in PL, we next recorded
pairs of CCK+ interneurons expressing ChR2 and a nearby L3
pyramidal cells, calibrating our stimulus intensity so that the
CCK+ interneuron fired a single action potential per pulse
(Fig. 6C). We found CCK+ synapses on PL L3 pyramidal cells
exhibited marked depression (PPR = EPSC
5
/EPSC
1
= 0.62 ± 0.05,
n = 7 cells, four animals; Fig. 6D), similar to PV+ interneuron out-
put in many other circuits (Reyes et al., 1998;Gabernet et al., 2005)
and mirroring the depressing FFI we observed in the MD to PFC
circuit. Lastly, the subcellular targeting of CCK+ synapses on pyra-
midal cells could affect their influence on activity in PL L3 (Marlin
and Carter, 2014). Using subcellular channelrhodopsin-assisted
circuit mapping (sCRACM; Petreanu et al., 2009), we recorded
L3 pyramidal cells while stimulating across the layers of PL using
a pseudorandom grid, under conditions that restrict synaptic
transmission to direct terminal release (Fig. 6E). We found that
CCK-evoked IPSCs were primarily targeted to the peri-somatic
region (n= 8 cells, three animals; Fig. 6E,F), consistent with a sub-
stantial CCK+ basket cell population in PL L3, which may act anal-
ogously to L3 PV+ interneurons in ACC and other areas of the
cortex. Together, these results show that CCK+ interneurons
Figure 6. Properties of CCK ± synapses onto L3 pyramidal cells. A, Left, Schematic of injection of AAV-Dlx-Flex-ChR2 into PL of CCK-Cre mice to express opsin in CCK+ interneurons. Right,
Confocal image of CCK+ interneurons across layers. Dashed lines = layer borders. Scale bar = 100 µm. B, Left, Average CCK-evoked IPSCs in PL L3 PYR at baseline (black) and after gabazine (red),
recorded with low [Cl−] internal at −50 mV. Blue arrow = 2 ms light pulse. Right, Summary of IPSC amplitude before and after gabazine wash-in. The gray lines are individual cells (n= 8 cells,
three animals). C, Left, Example traces of CCK+ interneuron firing in response to 10 Hz light pulses. Blue arrow = 2 ms light pulse. Right, Summary of the number of APs per pulse of stimulation
(n= 7 cells, four animals). D, Left, Average CCK+-evoked IPSCs recorded at L3 PYRs at −50 mV with 10 Hz stimulation. Blue arrow = 2 ms light pulse. Right, Summary of paired-pulse ratio
(PPR = IPSC
n
/IPSC
1
) across the stimulus train (n= 7 cells, four animals). E, Left, Schematic for sCRACM experiment, with PL L3 PYR recorded at −50 mV in the presence of TTX and 4-AP and CCK
+ interneuron terminals stimulated in a 10 × 10 grid of 75 µm squares via patterned light. Right, Example CCK+-evoked IPSCs along the somatodendritic axis. Blue arrow = 2 ms light pulse.
F, Left, Summary of average IPSC amplitudes evoked across the 10 ×10 grid (n= 8 cells, three animals). Right, Summary of IPSC amplitudes as a function of distance to the pial surface (n=8
cells, three animals). The dotted lines indicate L3. Values are mean ± SEM. *=p< 0.05.
Kamalova et al. •Thalamus-Evoked Inhibition in the PFC J. Neurosci., June 5, 2024 •44(23):e0957232024 •9

target L3 pyramidal cells with robust, depressing, perisomatic
inhibition.
CCK+ output and MD-evoked FFI are strongly modulated by
cannabinoids
Akeydifference between CCK+ interneurons and PV+ interneu-
rons is that the former often express CB1R on their axon termi-
nals (Katona et al., 1999;Marsicano and Lutz, 1999;Tsou et al.,
1999), enabling cannabinoid modulation of CCK+-evoked inhi-
bition. However, CB1R expression levels vary with brain region,
the subtype of CCK+ interneuron, and the specific postsynaptic
target (Lee et al., 2010;Varga et al., 2010;Vogel et al., 2016). To
determine if CCK+ interneurons in PL express CB1R, we exam-
ined mRNA expression using fluorescence in situ hybridization
(RNAscope) and found that a substantial proportion of
EGFP+ cells exhibited strong overlap with CB1R+ mRNA
(EGFP+/CB1R+ = 0.62 ± 0.05, n= 14 slices, five animals; Fig. 7A).
We previously found that cannabinoids selectively modulated
CCK+ outputs onto a subset of intratelencephalic (IT) pyramidal
cells in L5 of IL (Liu et al., 2020). One possibility is that CCK+
connections onto L3 pyramidal cells in PL, which represent
another population of IT cells, could also be cannabinoid sensi-
tive. We evoked CCK+-mediated IPSCs at L3 pyramidal cells
every 15 s, held at −50 mV with a low chloride [Cl-] internal
solution, which increases the driving force for GABA
A
receptor
currents and simultaneously captures both EPSCs and IPSCs.
We bath applied the CB1R agonist WIN 55,212-1 (WIN,
1 µM) by itself, or in the presence of the CB1R inverse agonist
AM-251 (10 µM). We found that WIN significantly reduced
CCK+-evoked IPSCs at L3 pyramidal cells compared with base-
line after 10 min [Fig. 7B–D; baseline ratio WIN = 0.45 (0.28–
0.72); WIN vs baseline, p= 0.0078; n= 8 cells, four animals].
However, in the presence of AM-251, WIN had no effect on
CCK+-evoked IPSCs at L3 pyramidal cells [Fig. 7B–D; baseline
ratio WIN + AM-251 = 1.00 (0.85–1.18); WIN + AM-251 vs
baseline AM-251 IPSC, p= 0.81; n= 7 cells, four animals]. In
separate recordings, we also sequentially applied WIN followed
by AM-251 and again found that WIN significantly reduced
CCK+-evoked IPSCs at L3 pyramidal cells, whereas the applica-
tion of AM-251 effectively reversed these changes (Extended
Data Fig. 7-1A,B). These results demonstrate that CCK+ output
onto L3 pyramidal cells in PL undergoes robust CB1R-dependent
suppression of synaptic transmission that leads to reduced
inhibition.
Lastly, having established that CCK+-evoked inhibition is
sensitive to CB1R modulation, we examined if this is also the
case for MD-evoked FFI. As described above, we expressed
ChR2 in MD terminals and recorded L3 pyramidal cells at
−50 mV to record compound EPSCs and IPSCs. We found
WIN application significantly reduced MD-evoked IPSCs, but
not MD-evoked EPSCs [Fig. 7E,F: baseline ratio WIN IPSC =
0.48 (0.24–0.98); WIN vs baseline IPSC, p= 0.031; WIN
EPSC = 0.92 (0.76–1.11); WIN vs baseline EPSC, p= 0.58; n=7
cells, four animals]. However, in separate recordings in the pres-
ence of AM-251, we found WIN had no effect on either IPSCs or
EPSCs [Fig. 7G,H; baseline ratio WIN + AM-251 IPSC = 0.93
(0.67–1.29); WIN + AM-251 vs baseline AM-251 IPSC, p=
0.16; WIN + AM-251 EPSC = 0.91 (0.78–1.06); WIN + AM-251
vs AM-251 EPSC, p= 0.43; n= 9 cells, four animals). In another
set of recordings, we also performed sequential application of
WIN followed by AM-251 and again observed that WIN signifi-
cantly reduced MD-evoked IPSCs at L3 pyramidal cells, whereas
application of AM-251 reversed these changes (Extended Data
Fig. 7-1C,D). Given the comparable magnitude of IPSC suppres-
sion by WIN in both CCK+-evoked IPSCs and MD-evoked FFI,
these results suggest that CCK+ interneurons are a major con-
tributor to MD-evoked FFI at pyramidal cells in PL L3.
Discussion
We determined that CCK+ interneurons are major contributors
to MD-evoked feed-forward inhibition in PL L3. While PV+
interneurons are relatively sparse in L3 of PL, SOM+, VIP+,
and CCK+ interneurons are abundant. There are two electro-
physiologically distinct CCK+ populations, with the fast-spiking,
basket-like population more strongly activated by MD inputs
before other interneuron types. CCK+ interneurons inhibit L3
pyramidal cells perisomatically, exhibit depressing short-term
dynamics and are cannabinoid sensitive. Similarly, MD-evoked
feed-forward inhibition, but not direct excitation, is also sensitive
to CB1R modulation. Our findings shed light on the inhibitory
network in PL L3 and highlight how a distinct subset of CCK+
interneurons contribute to MD-evoked FFI. They also suggest
how PL is unique compared with other thalamocortical circuits
in both sensory systems and the more dorsal, neighboring
ACC, with feed-forward inhibition exhibiting prominent sensi-
tivity to cannabinoid signaling.
Throughout the cortex, PV+ interneurons are a major inter-
neuron population and the primary mediator of thalamus-
evoked inhibition. While this circuit is present in ACC
(Delevich et al., 2015), we found significantly fewer PV+ inter-
neurons in PL L3. Our anatomy is consistent with and extends
upon recent analyses of PV+ interneurons across the cortex
(Whissell et al., 2015;Kim et al., 2017). Within the PFC, we
found, using a variety of approaches, there was a sharp drop-off
in L3 PV+ interneurons from ACC to the PL, highlighting distinct
cellular makeup. We observed a similar abundance of VIP+ and
SOM+ interneurons in L3, unlike previous studies that found
fewer VIP+ interneurons than SOM+ interneurons when pooling
across layers (Kim et al., 2017), which is likely due to the fact that
VIP+ interneurons appear to be enriched in superficial L1-L3
across the cortex (Tremblay et al., 2016). We also found that
CCK+ interneurons are enriched in L3 relative to PV+ interneu-
rons, consistent with other studies using different approaches
(Kawaguchi and Kubota, 1998;Nguyen et al., 2020). Our results
on the distributions of interneuron populations in PL build on
previous broad-scale studies on PFC interneurons to highlight
laminar specificity.
The two subpopulations of CCK+ interneurons that we found
in PL L3 are consistent with CCK+ interneuron diversity in other
brain regions. Recent studies have highlighted multiple tran-
scriptionally defined subgroups of CCK+ interneurons in other
parts of the cortex (Tasic et al., 2018;Gouwens et al., 2020;
(BICCN), 2021;Scala et al., 2021). Similarly, in the hippocampus,
CCK+ interneurons encompass multiple subtypes with distinct
electrophysiology, morphology, and synaptic connectivity
(Daw et al., 2009;Lee et al., 2010). Two of these CCK+ popula-
tions, which coexpress either VIP or VGLUT3, are also found
in the cortex and the amygdala (Somogyi et al., 2004;Bodor et
al., 2005;Omiya et al., 2015;Pelkey et al., 2020). The nfs-CCK
+ population that we recorded, which displays high input resis-
tance and low maximum firing rate, may be the CCK+/VIP+ sub-
type that we observed with fluorescence in situ hybridization.In
contrast, the fs-CCK+ population that we recorded may be the
fast-spiking, basket-like CCK+/VGLUT3+ subtype, correspond-
ing to CCK+/CB1R+ fluorescent population. Notably, we find
10 •J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

that PL L3 CCK+ interneuron output is fully GABAergic and
lacks a glutamatergic component, indicating our approach exclu-
sively targets inhibitory interneurons. While PV+ and CCK+
basket cells are classically considered separate cell types
(Freund and Katona, 2007;Tremblay et al., 2016), recent studies
have shown an overlap between these two populations (Nguyen
Figure 7. Cannabinoid modulation of CCK ± and MD-evoked FFI. A, Left, Confocal images of EGFP+, CB1R+, and CCK+ mRNA staining in L3 PL of CCK-Cre mice injected with
AAV-Dlx-Flex-EGFP. Scale bar = 100 µm. Right, Summary of overlap between EGFP+ (gray) and CCK+ (magenta) or CB1R+ (orange) mRNA (n= 14 slices, five animals). B, Left, Average
CCK+-evoked IPSCs in L3 PYRs measured at −50 mV at baseline (black) and after wash-in of WIN (red). Blue arrow = 2 ms light pulse. Right, Average CCK+-evoked IPSCs measured at
−50 mV, at baseline with AM-251 (black) and after wash-in of WIN (red) in the presence of AM-251. Blue arrow = 2 ms light pulse. C, Time course of CCK+-evoked IPSCs during drug wash-ins,
as indicated by colored bars, where IPSCs are normalized to baseline (n= 8 cells, four animals). D, Summary of IPSC amplitude ratios during wash-in of WIN (left) or WIN + AM-251 (right). E,
Left, Average MD-evoked EPSCs and IPSCs at baseline (black) and after wash-in of WIN (red). Right, Time course of MD-evoked EPSCs (black) and IPSCs (gray) during WIN wash-in, as indicated by
the colored bar, where PSCs are normalized to baseline (n= 7 cells, four animals). Blue arrow = 2 ms light pulse. F, Summary of EPSC and IPSC amplitude ratio after wash-in of WIN. G,H, Similar
to Eand Fin the presence of AM-251 during both baseline and WIN (n= 9 cells, four animals). Values are geometric mean and 95% CI. *=p< 0.05 (Extended Data Fig. 7-1).
Kamalova et al. •Thalamus-Evoked Inhibition in the PFC J. Neurosci., June 5, 2024 •44(23):e0957232024 •11

et al., 2020). The intersectional viral strategy we used to label
CCK+ cells does not label PV+/CCK+ coexpressing interneurons
in CCK-Cre mice. Interestingly, the same strategy may label
some of these interneurons in PV-2A-Cre mice, which could
be exploited to study this population further. Further exploration
of CCK+ subtypes will be essential in disentangling their roles in
feed-forward circuits in other regions of the PFC and other cor-
tical areas and determining whether the role of CCK+ interneu-
rons in the PFC is unique or generalizes across other higher
cortical regions.
Several of our experiments indicate that CCK+ interneurons
make a major contribution to MD-evoked FFI in L3 of PL. MD
inputs preferentially activate fs-CCK+ cells over neighboring
pyramidal cells but are unable to activate nfs-CCK+ cells. The
engagement of fs-CCK+ cells resembles the activation of PV+
basket cells in L3 of ACC and L4 of sensory cortices
(Cruikshank et al., 2007;Delevich et al., 2015). It is also similar
to hippocampal activation of CCK+ interneurons onto IT cells
in L5 of IL (Liu et al., 2020). In contrast, the lack of activation
of nsf-CCK+ cells suggests that they are unlikely to contribute
to MD-evoked FFI. We recently found MD inputs also engage
VIP+ interneurons in L1b of PL, triggering a disinhibitory circuit
(Anastasiades et al., 2021). In contrast, MD inputs only weakly
targeted VIP+ interneurons in L3, suggesting MD projections
to L1b and L3 exhibit markedly different connectivity and func-
tion. Previous work from our lab and others also indicates that a
variety of long-range inputs can also engage SOM+ interneurons
via facilitating synapses (Beierlein et al., 2003;Tan et al., 2008;
McGarry and Carter, 2016). While MD inputs also contact
SOM+ interneurons in L3, this occurred in tandem with activa-
tion of pyramidal cells, making it hard to distinguish from feed-
back inhibition via activation of the local circuit (Reyes et al.,
1998). Taken together, our results suggest that MD inputs
strongly engage CCK+ interneurons in L3, highlighting a key
role for these cells in prefrontal inhibition.
The study of CCK+ interneurons in the cortex has been ham-
pered by CCK expression in pyramidal cells, which we overcame
with intersectional viral tools (Dimidschstein et al., 2016;Nguyen
et al., 2020). Our results indicate that CCK+ interneurons syn-
apse strongly near the soma region of L3 pyramidal cells and pro-
vide depressing inhibition. These results are again consistent
with fs-CCK+ interneurons fulfilling a similar niche to PV+
interneurons in other cortical areas (Karube et al., 2004;
Cruikshank et al., 2007). We also determined that CCK+-evoked
inhibition is cannabinoid sensitive, which we have previously
examined for similar connections in IL L5 (Liu et al., 2020).
However, in contrast to CCK+-evoked and hippocampal-evoked
inhibition in IL L5, we did not observe robust DSI (Extended
Data Fig. 7-1E,F), consistent with the lack of DSI in other L3
pyramidal cells in the absence of additional pharmacological
modulation (Yoshino et al., 2011). One possibility is that L3 pyra-
midal cells may not produce endocannabinoids as readily as L5
IT cells (Best and Regehr, 2010;Yoshino et al., 2011). In the
future, it will be interesting to identify the source of endocanna-
binoids, including identifying the locations of their synthesis
enzymes (Best and Regehr, 2010;Tanimura et al., 2010) and
how their expression can be influenced by behavioral experience
(Vogel et al., 2016).
The finding that MD-evoked FFI is also suppressed by CB1Rs
suggests that CCK+ interneurons contribute to FFI in PL. Other
studies have used cell-type specific optogenetic or pharmacoge-
netic inhibition to link the suppression of a specific interneuron
population with the reduction in inhibition (Xu et al., 2013;
Delevich et al., 2015;Anastasiades et al., 2021). We used CB1R
modulation of CCK+ interneuron output to perform an equiva-
lent loss-of-function experiment (Liu et al., 2020), finding a
similar magnitude of reduction for both MD-evoked and
CCK+-evoked inhibition at L3 pyramidal cells. This experiment
was possible because CB1R is predominantly expressed in CCK+
interneurons in the cortex, and CB1Rs are not found on MD ter-
minals (Herkenham et al., 1990;Matsuda et al., 1993;Marsicano
and Lutz, 1999). Consequently, we did not observe
CB1R-mediated modulation of MD-evoked excitation at L3
pyramidal cells. While our results cannot rule out the presence
of a separate population of CB1R-expressing interneurons,
both transcriptomic and immunohistochemical studies of the
PFC suggest that there is unlikely to be another major population
of CB1R-expressing interneurons (Marsicano and Lutz, 1999;
Paul et al., 2017). Together, the activation of CCK+ interneurons,
their inhibition of pyramidal cells, and their modulation by
CB1Rs support the idea that these cells contribute to
MD-evoked FFI in L3 of PL.
Our results suggest CCK+ interneurons in L3 of PL assume
some of the functional roles of PV+ interneurons in L4 of the sen-
sory cortex (Gabernet et al., 2005;Cruikshank et al., 2007;
Delevich et al., 2015). However, the potent CB1R modulation
of both CCK+ and MD-evoked inhibition suggests this circuit
may be uniquely modulated. Moreover, the sparsity of PV+ inter-
neurons in L3 of PL suggests they may play a different functional
role than is often assumed. Our findings appear to contrast with
several in vivo studies showing a prominent role for PV+ inter-
neurons in the PFC (Courtin et al., 2014;Cummings and
Clem, 2020;Mukherjee et al., 2021;Cho et al., 2023). It may be
that in vivo recordings were made from the small subset of
PV+ cells in L3 of PL, from the larger pool of PV+ cells in L5
of PL, or from PV+ cells located in either L3 or L5 of more dorsal
areas like ACC (Delevich et al., 2015). Given that fs-CCK+ cells
have indistinguishable physiological properties from PV+ cells, it
may be challenging to identify them based on firing alone. While
our data suggests there are at least two populations of CCK+
interneurons with distinct firing and response properties, in
the future, it will be interesting to parse additional subpopula-
tions (Tasic et al., 2018;Gouwens et al., 2020;(BICCN), 2021;
Scala et al., 2021).
Lastly, our results suggest that CB1R modulation may play
an important role in shaping PFC-dependent behaviors via
CCK+ interneurons. Reciprocal loops between the MD and
PFC mediate working memory (Bolkan et al., 2017;Schmitt
et al., 2017;Mukherjee et al., 2021), and we predict that changes
in cannabinoid signaling, mediated in part by CCK+ interneu-
rons, could contribute to disruptions in a variety of cognitive
tasks mediated by the PFC. In support of this idea,
Δ9-tetrahydrocannabinol (THC), a CB1R agonist, can cause
impairments in working memory (Adam et al., 2020).
Conversely, reduced endocannabinoid activity could dampen
activity in PFC thalamocortical loops via increased output
from CCK+ interneurons. These shifts in CCK+ inhibition
could be clinically relevant, given that chronic pain leads to
excessive synaptic inhibition in the PFC (Woodhams et al.,
2017) and reduced endocannabinoid signaling (Mecca et al.,
2021), whereas acute stress elevates cannabinoid signaling in
the PFC (Hill et al., 2011). In future studies, it will be important
to assess the contributions of CCK+ interneurons, their engage-
ment by MD, and their modulation by cannabinoids, in both
behaviors and neuropsychiatric disorders associated with the
PFC.
12 •J. Neurosci., June 5, 2024 •44(23):e0957232024 Kamalova et al. •Thalamus-Evoked Inhibition in the PFC

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