Molecular Psychiatry (2021) 26:5097–5111

https://doi.org/10.1038/s41380-020-0796-3

ARTICLE

Cell-type specific modulation of NMDA receptors triggers

antidepressant actions
Santosh Pothula 1 Taro Kato1 Rong-Jian Liu1 Min Wu1 Danielle Gerhard1,2 Ryota Shinohara 1
● ● ● ● ● ●

Alexa-Nicole Sliby1 Golam M. I. Chowdhury1 Kevin L. Behar1 Gerard Sanacora1 Pradeep Banerjee3
● ● ● ● ●

Ronald S. Duman 1

Received: 30 April 2020 / Revised: 13 May 2020 / Accepted: 18 May 2020 / Published online: 2 June 2020
© The Author(s), under exclusive licence to Springer Nature Limited 2020

Abstract
Both the NMDA receptor (NMDAR) positive allosteric modulator (PAM), and antagonist, can exert rapid antidepressant
effects as shown in several animal and human studies. However, how this bidirectional modulation of NMDARs causes
similar antidepressant effects remains unknown. Notably, the initial cellular trigger, specific cell-type(s), and subunit(s) of
NMDARs mediating the antidepressant-like effects of a PAM or an antagonist have not been identified. Here, we used
1234567890();,:
1234567890();,:

electrophysiology, microdialysis, and NMR spectroscopy to evaluate the effect of a NMDAR PAM (rapastinel) or NMDAR
antagonist, ketamine on NMDAR function and disinhibition-mediated glutamate release. Further, we used cell-type specific
knockdown (KD), pharmacological, and behavioral approaches to dissect the cell-type specific role of GluN2B, GluN2A,
and dopamine receptor subunits in the actions of NMDAR PAM vs. antagonists. We demonstrate that rapastinel directly
enhances NMDAR activity on principal glutamatergic neurons in medial prefrontal cortex (mPFC) without any effect on
glutamate efflux, while ketamine blocks NMDAR on GABA interneurons to cause glutamate efflux and indirect activation
of excitatory synapses. Behavioral studies using cell-type-specific KD in mPFC demonstrate that NMDAR-GluN2B KD on
Camk2a- but not Gad1-expressing neurons blocks the antidepressant effects of rapastinel. In contrast, GluN2B KD on Gad1-
but not Camk2a-expressing neurons blocks the actions of ketamine. The results also demonstrate that Drd1-expressing
pyramidal neurons in mPFC mediate the rapid antidepressant actions of ketamine and rapastinel. Together, these results
demonstrate unique initial cellular triggers as well as converging effects on Drd1-pyramidal cell signaling that underlie the
antidepressant actions of NMDAR-positive modulation vs. NMDAR blockade.

Introduction neurobiology of depression is unknown and currently

Depression is a widespread, debilitating illness with
devastating personal and economic consequences, and is a
leading cause of disability world-wide [1]. In addition, the
Supplementary information The online version of this article (https://
doi.org/10.1038/s41380-020-0796-3) contains supplementary
material, which is available to authorized users.
* Santosh Pothula
santosh.pothula@yale.edu
1
Department of Psychiatry, Yale School of Medicine, New Haven,
CT 06511, USA
2
Department of Psychiatry, Weill Cornell Medicine, New York,
NY 10065, USA
3
Allergan Inc., Madison, NJ, USA
available medications have significant limitations, including
a time-lag of weeks to months for a therapeutic response,
low rates of response and remission, and a significant
subpopulation of depressed patients considered treatment
resistant [2, 3]. The discovery that a single sub-anesthetic
dose of ketamine, an NMDA receptor (NMDAR) antago-
nist, exerts rapid and sustained antidepressant effects in
treatment-resistant patients represents a fundamental
breakthrough and major advance for the treatment of
depression, including difficult to treat and suicidal patients
[4, 5].
However, ketamine produces dissociative and psycho-
tomimetic effects and is a drug of abuse [6, 7]. Recent
studies have shown that NMDAR positive allosteric mod-
ulators (PAMs) also exerts rapid and sustained anti-
depressant effects without the dissociative and
psychotomimetic-like side effects of ketamine [8–10].
5098
Due to these attractive properties, there is immense interest
in developing NMDAR PAMs as rapid acting anti-
depressants. Preclinical studies demonstrate that while
ketamine and rapastinel have opposing effects on
NMDARs, they both require activation of glutamate AMPA
receptors, and have convergent downstream signaling cas-
cades, including mTORC1, BDNF release, and synaptic
plasticity in medial prefrontal cortex (mPFC) [11–13]. The
actions of ketamine are mediated by blockade of NMDAR
on GABA interneurons, resulting in disinhibition of gluta-
mate transmission [14–17], but the cellular targets of
NMDAR PAMs have not been determined. Further, it is
unclear how NMDAR antagonists and PAMs exert similar
antidepressant effects, and the cellular trigger and neuronal
cell-types involved in the actions of NMDAR PAMs remain
elusive. Here, we used ketamine and rapastinel as tool
compounds of NMDAR antagonists and PAMs, respec-
tively, to address these questions.
In the current study, we investigate the role of NMDAR
subunits on Camk2a-excitatory neurons vs. Gad1-inhibitory
interneurons in the mPFC. We target GluN2B, one of the
major NMDAR subunits expressed on both inhibitory and
excitatory neurons in mPFC, as preclinical and clinical stu-
dies have reported that selective GluN2B antagonists pro-
duce rapid antidepressant effects [11, 18]. In addition, we
further investigate a subtype of excitatory neurons, char-
acterized by expression of dopamine receptor Drd1, as well
as the role of Drd1 and Drd2 dopamine receptors (Drd1R
and Drd2R) in the actions of rapastinel [19–23].
The results demonstrate that GluN2B knockdown on
glutamatergic, but not GABAergic neurons in mPFC blocks
the antidepressant effects of rapastinel. Conversely, the
actions of ketamine are blocked by GluN2B KD on
GABAergic but not glutamatergic neurons, in agreement
with a recent study [17], indicating unique initial cellular
triggers for the actions of NMDAR PAM vs. NMDAR
blockade. The results also demonstrate a role for Drd1-
expressing excitatory neurons in the actions of rapastinel, as
well as ketamine, indicating a convergent mechanism for
these two types of rapid antidepressants.
Materials and methods
Animals
Rodents used in the current study included adult
(8–12 week old) male mice. Wild-type C57BL/6 male mice
were used (Jackson Laboratories) for electrophysiology,
and Drd1R and Drd2R antagonist studies. For viral-
mediated knockdown and behavioral experiments,
Camk2a-cre (male; obtained from Dr. Günter Schütz,
German Cancer Research Center, Heidelberg, Germany),
S. Pothula et al.
Gad1-cre (male; obtained from Dr. Marina Picciotto, Yale
University) mice, and WT littermates bred on a C57BL/6J
background (Jackson Laboratories, Bar Harbor ME). Sst-
tdTomato male mice were used for NMDA- or AMPA-
inward currents after rapastinel treatment. Sst-Cre mice
were bred with Ai9(RCL-tdT) mice (catalog 007909;
Jackson Laboratories) to obtain Sst-tdTomato mice. Adult
male Sprague Dawley (SD) rats (Charles River, Raleigh,
NC, USA) of 180–220 g weight range were used for
microdialysis and NMR spectroscopy analysis of 13C
incorporation. Animals were housed under 12 h light–dark
cycle with ad libitum access to water and rodent chow.
Animals were group housed until surgery. All experiments
were performed during the 12-h light cycle. Animal use and
procedures were in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Ani-
mals and approved by the Yale University Animal Care and
Use Committees. Sample sizes for this study were deter-
mined based on our previous experience [17] with the types
of viral infusion studies conducted in this project.
Drugs
Rapastinel was procured from Sai Life Sciences (India).
(S)-ketamine and ketamine were purchased from Sigma-
Aldrich (St. Louis, MO) and Tocris Biosciences (USA).
SCH39166 and L741,626 were purchased from Tocris
Biosciences (USA). Rapastinel and (S)-ketamine or keta-
mine were dissolved in sterile saline (0.9% NaCl). For
microdialysis and NMR spectroscopy studies in rats, drugs
were administered via s.c. (rapastinel) or i.p. ((S)-ketamine
or ketamine) routes, respectively. For behavioral studies in
mice, rapastinel and ketamine were administered via i.v.
and i.p. routes.
Electrophysiology
The mPFC brain slices were prepared from male mice
(8–12 weeks old). Coronal slices of 300-μm thickness
containing the mPFC were prepared and placed in artificial
cerebrospinal fluid (ACSF) (pH 7.35–7.38) equilibrated
with 95% O2/5% CO2 before transferring them to recording
chamber fixed with an Olympus BX50WI scope for elec-
trophysiology recordings. The chamber was continuously
perfused with normal ACSF at a rate of 2–3 ml/min and
its temperature maintained at 33 ± 0.5 °C. Patch pipettes
(3–5 MΩ) were pulled from glass tubing with a Flaming-
Brown Horizontal Puller. The pipette solution contained the
following: 115 mM K gluconate, 5 mM KCl, 2 mM MgCl2,
2 mM Mg-ATP, 2 mM Na2ATP, 10 mM Na2-phospho-
creatine, 0.4 mM Na2GTP, and 10 mM Hepes, pH 7.33.
Pyramidal neurons and interneurons in mPFC were visua-
lized by video microscopy using microscope (40× IR lens)
Cell-type specific modulation of NMDA receptors triggers antidepressant actions
with infrared differential interference contrast (IR/DIC).
Whole-cell recordings were made with an Axoclamp-2B
amplifier. NMDA- and AMPA-induced inward currents
were measured in magnesium free ACSF upon bath appli-
cation of 5 or 10 µM NMDA, or 5 µM AMPA alone, before
and after application of rapastinel or ketamine.
Immunohistology
Brains were collected from all virus infused mice after
transcardiac perfusion with sterile PBS and 4% paraf-
ormaldehyde (PFA). They were postfixed in 4% PFA for
24 h and incubated in 30% sucrose for an additional 24 h.
30-µm thick sections were made from postfixed frozen
brains using a Microm HM550 cryostat and mounted on
slides for further immunofluorescence visualization. Images
were acquired using confocal laser scanning microscope
(Olympus FV1000).
Microdialysis
Microdialysis studies were performed as previously
described [24]. Using coordinates: +3.2 mm anterior-
posterior (AP); +0.5 mm medial–lateral (ML); −1.5 mm
dorsal–ventral (DV) from bregma, guide cannulae were
implanted to target mPFC. A microdialysis probe (3 mm
membrane length; 50 kDa cutoff) was inserted via guide
cannula to dialyze mPFC region (V, −4.5 mm) with ACSF
(148 NaCl, 4 KCl, 0.8 MgCl2, 1.4 CaCl2, 1.2 Na2HPO4,
and 0.3 NaH2PO4; in mM; pH 7.2) at a flow rate of 1 μl/
min. After a 3-h stabilization period, samples were collected
at every 20 min intervals for 1 h for baseline and up to 4 h
after drug administration. Glutamate concentrations were
measured by ultra high-performance liquid chromatography
tandem mass spectrometry as previously described [25].
[1,6-13C2]glucose infusions and NMR spectroscopy
analysis
[1,6-13C2]glucose infusions and NMR spectroscopy ana-
lysis were performed as previously described [26]. Rats
were briefly anethesized with isoflurane and catheters were
placed into tail vein. Drugs/vehicle were injected following
recovery for at least 30 min after anesthesia. Ten minute
after administration of ketamine or rapastinel or vehicle, a
solution of [1,6-13C2]glucose (99 atom%; Cambridge Iso-
topes, Andover, MA, USA) dissolved in water (0.75 mol
l−1 per 200 g body weight) was infused for 8 min. Imme-
diately following the 8 min infusion of [1,6-13C2]glucose,
the rats were quickly sacrificed and brains were flash fro-
zen. Metabolites in the mPFC were extracted from frozen
tissue (65–85 mg) and brain samples were loaded into
5 mm tubes for NMR analysis. 1H-[13C]-NMR spectra were
5099
acquired at 11.7 T (1 H frequency of 500.13 MHz; Bruker
AVANCE; Bruker Instruments, Billerica, MA, USA) to
determine total concentrations, and 13C enrichments of
amino acids and metabolites in mPFC as previously
described [27].
Surgical procedures for viral/drug infusions
Mice were anesthetized with a cocktail of ketamine/xyla-
zine (100/10 mg/kg, i.p.), head fixed in a stereotaxic appa-
ratus (David Kopf, Tujunga, CA) and the incision site was
sterilized. Ophthalmic ointment was applied on eyes. A pair
of craniotomies were made at following coordinates from
bregma: +1.9 mm AP, ±0.4 mm ML, and –2.8 mm DV,
and bilateral viral infusions into the mPFC (0.5 μl per side;
0.1 μl/min) were performed using Hamilton syringe (Reno,
NV) fitted with a 30 gauge needle. Incision sites were
closed with sutures, an antibiotic was applied, and carprofen
(5 mg/kg, i.p.) was injected immediately after surgery and
daily for the next 2 days. We followed the same procedure
for drug infusions, where bilateral stainless steel guide
cannulae (0.8 mm center-to-center, 26 gauge, Plastics One,
Roanoke, VA) were placed in the mPFC (coordinates from
bregma: +1.9 AP, ±0.4 ML, −2.5 DV), fixed to the skull
using dental cement and skull screws. Mice were indivi-
dually housed to prevent dislodging of cannula, resulting in
a social isolation period. Infusion needles (2.8 mm long)
were inserted via guide cannulae to infuse drugs into
the mPFC.
Viral constructs
The following shRNA sequences were designed to
target GluN2B or GluN2A subunits. 5′-Tgtaccaacagg
tctcaccttaaacTTCAAGAGAgtttaaggtgagacctgttggtacTTTT
TTC-3′ (GluN2B) or 5′-TtgacagaacgcgaacttcgaaatcTTCA
AGAGAgatttcgaagttcgcgttctgtcaTTTTTTC-3′ (GluN2A;
Integrated DNA Technologies). They were ligated into a
TATAloxP-flanked CMV-EGFP cassette containing pSico
plasmid (from Tyler Jacks, Massachusetts Institute of
Technology, Boston, Massachusetts, USA; Addgene
plasmid catalog 11578), designed to restrict shRNA
expression to cells that express Cre-recombinase. The
resulting pGluN2BshRNA or pGluN2AshRNA constructs
were subcloned into pAAV-mCherry (Virovek) to gen-
erate the plasmid, which allows ubiquitous mCherry
expression and conditional EGFP expression driven by
floxed CMV cassette. These constructs were packaged into
adeno-associated virus 2 (AAV2). AAV-DIO-EmGFP-
D1miR was used for cell-type-specific knockdown of
Drd1Rs as previously described [28]. AAV-Camk2a-
EGFP was used a control for Drd1R knockdown
experiment.
5100
Behavioral tests
All tests were conducted at least three weeks after virus
infusion or one week after cannulation surgeries. During
this time mice were single housed, resulting in social iso-
lation. Time lines of experimental approaches were detailed
in figures. Animals were habituated at least 30 min to
experimental conditions prior to behavioral tests. Animals
were randomly assigned to the treatment groups and
observers were blinded to treatments and genotypes during
the experiments and behavioral analysis.
Forced swim test (FST)
Mice were placed in a clear cylinder filled with water (24 ±
1 °C, 18 cm depth) for 6 min. Tests were video recorded and
scored for immobility time, defined as a lack of activity
except the minimal amount of effort required to keep head
above water, by an experimenter blinded to treatment groups.
Immobility time during 2–6 min window was scored. Pre-
swim is the FST conducted prior to drug administrations.
Female urine sniffing test (FUST)
FUST, a validated test of reward-seeking behavior in
rodents, was performed as previously described [29]. Each
mouse was placed in individual novel cage for acclimati-
zation (30 min) followed by habituation to a cotton-tipped
applicator for 1 h. Subsequently, a fresh water-soaked cot-
ton-tipped applicator was placed on the inner wall of the
cage, and video recorded for 5 min. The cotton-tipped
applicator was removed. After 45 min, a cotton-tipped
applicator with fresh female urine was placed in the cage,
and video recorded for 5 min. Only the time spent sniffing
water- or female urine-soaked cotton-tipped applicator were
scored.
Novelty-suppressed feeding test (NSFT)
Mice were food deprived for 18 h prior to exposure to a
novel open arena (40 × 40 × 25 cm) with chow pellet in the
center. The latency to feed was assessed. Later, home cage
feeding was assessed as a feeding control, where mice were
allowed ad libitum access to a preweighed chow pellet in
their home cages.
Open field test (OFT)
Each mouse was placed in the center of a plexiglass test
apparatus (40 × 40 × 25 cm) and video recorded for 10 min.
The time spent in the center zone and total distance traveled
in the open field were analyzed using an automated analysis
software (ANY-maze, Stoelting).
S. Pothula et al.
Locomotor activity (LMA)
Mice were placed in clean home cages (25 × 15 × 12 cm) for
30 min. LMA was tracked using an automated infrared
beam break system and monitored with Med-PC software
(Med Associates). The number of beam breaks were
recorded as a measure of locomotion.
Statistics
Data are expressed as the mean ± standard error of mean
and analyzed using Graphpad prism v8. For multi-group
comparisons and to determine treatment or genotype effects
or interaction, one-way ANOVA or two-way ANOVA post
hoc Tukey’s multiple comparison tests were used to deter-
mine statistical significance. For comparisons between two
groups, either unpaired or paired Student’s t test (two-tailed)
was performed to determine statistical significance. p < 0.05
was considered as statistically significant. The details of
statistical tests and their outcomes were presented in the
figure legends. The sampling distribution of the data was
assumed to be normal and the variances were assumed to be
equal, but this was not formally tested for every analysis.
Any value >2 standard deviation from the group mean was
considered as an outlier (a criteria decided on prior to
initiating the experiments) and excluded from the analysis.
Results
Rapastinel, but not ketamine, directly enhances
NMDA-inward current, but has no effect on
glutamate release
We first determined the effects of rapastinel and ketamine on
NMDAR activity by patch clamp recordings of layer V
pyramidal neurons in slices of mPFC. Incubation with
rapastinel enhanced NMDA-induced inward currents and
displayed an inverted-U-dose response relationship: 0.1 µM
rapastinel significantly enhanced the NMDA-inward current
in layer V neurons and 1 µM produced a relatively smaller,
nonsignificant enhancement, while a higher concentration
(10 µM) had no effect compared with vehicle (Fig. 1a, b).
Analysis of GABA interneurons, tested in Sst-reporter mice
revealed that rapastinel (0.1 µM) produced a nonsignificant
trend for increase in NMDA-induced inward currents (Sup-
plementary Fig. 1a, b), with no effect on AMPA-induced
inward currents. In line with its NMDAR antagonistic
activity, ketamine produced a near complete blockade of the
NMDA-induced inward current (Fig. 1c, d).
Ketamine has been shown to increase glutamate release
via disinhibition, which is required for its downstream
signaling and antidepressant behavioral actions [15, 17, 30].
Cell-type specific modulation of NMDA receptors triggers antidepressant actions
Fig. 1 Rapastinel directly enhances NMDAR activity without any
changes in glutamate release. NMDA-inward currents were recorded
from layer V pyramidal neurons of mPFC before and after bath
application of NMDA alone or in combination with rapastinel or
ketamine. a, c Representative traces showing inward currents in
response to NMDA (5 µM) before and after bath application of
0.1–10 µM rapastinel or 10 µM ketamine. b Rapastinel increased the
NMDA-inward current at 0.1 and 1 µM, respectively, while there was
no effect observed at 10 µM compared with baseline; results are the
mean ± SEM, n = 14–23 neurons (One-way ANOVA and post hoc
Tukey’s test, F3,74 = 6.09, p < 0.01; **p < 0.01). d Ketamine sig-
nificantly reduced the inward currents in response to NMDA (10 µM)
compared with baseline (n = 6 neurons, paired t-test, **p < 0.01).
e Microdialysis was performed in rats and dialysate samples were
collected from mPFC at 20 min intervals starting 40 min before
To determine if rapastinel enhances glutamate release, we
used in vivo microdialysis and 1H-[13C]-nuclear magnetic
resonance (NMR) spectroscopy. For comparison in micro-
dialysis studies, we used (S)-ketamine, which has higher
affinity for the NMDAR than (R)-ketamine [31]. The results
show that (S)-ketamine (10 mg/kg) significantly increased
glutamate efflux as measured by percent change of basal
levels of glutamate in rat mPFC (Fig. 1e), consistent with
previous reports [14, 15]. In contrast, rapastinel at 3, 10, and
30 mg/kg, doses previously shown to exert antidepressant-
like effects in rodents [13, 32, 33], had no significant effects
on extracellular glutamate (Fig. 1e). In line with the
microdialysis results, ketamine but not rapastinel sig-
nificantly increased the percent change in glutamate-C4,
GABA-C2 and glutamine-C4 fractional enrichments of 13C
as measured by 1H-[13C]-NMR spectroscopy in rat PFC
(Fig. 1f). These findings highlight the differential effects of
rapastinel and ketamine on glutamate release.
5101
rapastinel or (S)-ketamine administration up to 240 min post-treatment.
(S)-ketamine (30 mg/kg) significantly increased the % change in basal
levels of glutamate in mPFC at 60 min and remained elevated up to
180 min. No significant differences in glutamate levels were observed
with 3, 10, and 30 mg/kg doses of rapastinel (n = 8–9 SD rats per
group, one-way ANOVA and post hoc Dunnett’s multiple comparison
tests, *p < 0.05). f NMR spectroscopy was performed to analyze 13C
fraction enrichment of glutamate-C4, GABA-C2, and glutamine-C4 in
mPFC of rats 30 min after ketamine or rapastinel administration. 13C
fraction enrichment of glutamate-C4, GABA-C2, and glutamine-C4
significantly increased in mPFC after ketamine (10 mg/kg) adminis-
tration, but no changes were observed with 3, 10, and 30 mg/kg doses
of rapastinel (n = 7–13 SD rats per group, one-way ANOVA and post
hoc Dunnett’s test, *p < 0.05; **p < 0.01).
GluN2B knockdown on glutamatergic, but not
GABAergic, neurons blocks the antidepressant-like
effects of rapastinel
To identify the initial cellular trigger and specific NMDAR
subunit which mediates the antidepressant-like behavioral
actions of rapastinel, we used an AAV driven shRNA-
targeting NMDAR subunits and cell-specific Cre-
recombinase (Cre+) transgenic mouse lines to achieve
cell-type-specific knockdown. Based on reports that selec-
tive GluN2B modulators produce rapid antidepressant
actions in rodent models and in clinical trials [30], we first
focused on knockdown of this subunit in Camk2a-expres-
sing pyramidal neurons. The GluN2B shRNA construct was
packaged into AAV-2 (referred to as AAV2GluN2BshRNA); the
viral construct included dsRed and EGFP and upon Cre-
mediated recombination, EGFP is excised, resulting in the
expression of only dsRed as a marker of recombination
5102 S. Pothula et al.

a GluN2B knockdown in Camk2a-neurons: b

TATA-lox TATA-lox

CMV dsRed U6 CMV EGFP Sh-GluN2B hGH polyA

+ Camk2a-Cre recombinase
CMV dsRed U6 Sh-GluN2B hGH polyA
TATA-lox GluN2B
c WT d NMDA +Rps 0.1 μM e ACSF Rps 100nM Glutamatergic
neuron
2500 **

NMDA- inward current

WT 2000

dsRED/EGFP 1500
#

(pA)
Camk2a cre+
GluN2B KD

1000

500 pA
500
1 min
0
dsRED/EGFP WT GluN2B KD
f
WTAAV2B-Sal
WTAAV2B-Rps or Ket
Camk2aCre+/AAV2B-Sal
Camk2aCre+/AAV2B-Rps or Ket

g FST-Rps h FUST-Rps i NSFT-Rps j FST-Ket

** ** * ** 250 *
80
*

Immobility time (sec)

200 1000
Latency to feed (sec)
Immobility time (sec)

** *
Sniffing time (sec)

60 800 200
150
600 150
40
100
400 100
20
50 200 50
0
0 0 0
Water Urine

Fig. 2 Knockdown of GluN2B in the mPFC of Camk2a-Cre mice
blocks the antidepressant-like actions of rapastinel. a Schematic
showing pGluN2BshRNA construct before and after Cre-recombinase
to generate the active shRNA construct and b removal of NMDARs
(GluN2B) on glutamatergic neuron. c Representative images of
AAV2GluN2BshRNA virus expression showing fluorescent labeling of
WT neurons (yellow: colocalization of EGFP and dsRed fluorescence)
and Cre-recombination dependent pyramidal neurons (subset of
only dsRed-labeled neurons) in the mPFC of WTCre−/AAV2B and
Camk2aCre+/AAV2B mice, respectively. d Representative traces of
NMDA (10 µM) induced inward currents in WT and GluN2B KD
glutamatergic neurons of WTCre−/AAV2B and Camk2aCre+/AAV2B mice
before and after rapastinel (100 nM) treatment. e NMDA-inward cur-
rent was significantly reduced in neurons of Camk2aCre+/AAV2B
compared with WTAAV2B mice (n = 8–11 neurons, unpaired t-test,
#p < 0.05); rapastinel significantly enhanced NMDA-inward current in
WTCre−/AAV2B but not in Camk2aCre+/AAV2B mice (n = 8–11 neurons,
paired t-test, *p < 0.05). f Timeline for experimental approach.
Rapastinel significantly reduced immobility time in the FST
(g), increased female urine sniffing time in the FUST (h), and reduced
latency to feed in the NSFT (i) in WTCre−/AAV2B but these effects were
blocked in Camk2aCre+/AAV2B mice. Data expressed as the Mean ±
SEM and analyzed by two-way ANOVA with Tukey’s multiple
comparisons (n = 8–10 per group; FST: treatment: F1,34 = 7.18, p =
0.01, genotype: F1,34 = 6.02, p = 0.02, interaction: F1,34 = 5.83,
p = 0.02; NSFT: treatment: F1,34 = 9.01, p < 0.01, genotype:
F1,34 = 2.23, p = 0.14, interaction: F1,34 = 5.9, p = 0.02; *p < 0.05;
**p < 0.01). j Ketamine significantly reduced immobility time in FST
in both WTCre−/AAV2B and Camk2aCre+/AAV2B mice (n = 9–10; FST:
treatment: F1,34 = 15.6, p < 0.001).

(Fig. 2a). We have recently validated this viral construct by antidepressant effects similar to systemic administration
demonstrating a reduction of GluN2B immunoreactivity and [32, 34]. AAV2GluN2BshRNA was bilaterally infused into the
reduction of NMDA-induced inward current in recombined mPFC of adult, littermate wild-type controls (WTCre−/AAV2B)
layer V pyramidal neurons of mPFC [17]. The mPFC was and Camk2aCre+ mice (Camk2aCre+/AAV2B) and three-weeks
chosen as a target region as prior studies have reported that post-surgery, to allow for viral expression and recombina-
infusion of rapastinel or ketamine into the mPFC exerts tion, behavioral testing was conducted (Fig. 2f).
Cell-type specific modulation of NMDA receptors triggers antidepressant actions
Histological and electrophysiological approaches con-
firmed AAV2GluN2BshRNA expression, recombination, and
cell-type-specific GluN2B KD. NMDA-induced inward
currents were measured in virus transduced cells of
WTCre−/AAV2B (EGFP + dsRed-expressing cells) and
recombined cells of Camk2aCre+/AAV2B mice (dsRed only
expressing cells) (Fig. 2c–e). WTCre−/AAV2B mice displayed
cells expressing both EGFP and dsRed (labeled in yellow),
while slices from Camk2aCre+/AAV2B mice displayed a
subset of cells expressing only dsRed (Fig. 2c), confirming
cell-type-specific recombination. Consistent with our recent
report validating NMDAR KD [17], NMDA-induced
inward current was significantly decreased in GluN2B KD
cells in Camk2aCre+/AAV2B mice compared with non-
recombined cells, and rapastinel enhancement of NMDA-
induced inward currents was partially blocked (Fig. 2d, e).
WTCre−/AAV2B and Camk2aCre+/AAV2B mice were admi-
nistered with saline or rapastinel (day 0), and evaluated for
rapid and sustained antidepressant-like behavioral actions.
This included the FST (a measure of behavioral despair) on
day 1, LMA on day 2, the FUST (a measure of reward
behavior) on day 3, and the NSFT (a measure of anxiety-
like behavior) on day 4. A 1 mg/kg (i.v.) dose of rapastinel
was chosen based on our previous work demonstrating
rapid and sustained antidepressant-like effects in mice [13].
Rapastinel significantly decreased immobility time in the
FST in WTCre−/AAV2B mice, but the effect was completely
blocked in Camk2aCre+/AAV2B mice (Fig. 2g). In FUST,
rapastinel significantly increased female urine sniffing
time in WTCre−/AAV2B mice without altering water
sniffing time, and the effect was completely blocked in
Camk2aCre+/AAV2AB mice (Fig. 2h). Rapastinel significantly
decreased latency to feed in the NSFT in WTCre−/AAV2B
mice, while the effect was blocked in Camk2aCre+/AAV2B
mice (Fig. 2i). There were no significant differences
observed in home cage feeding or LMA (Supplementary Fig.
2a, b). For comparison, we tested the effects of ketamine (10
mg/kg, i.p.) in these same mice after a 9-day washout period
(Fig. 2f). In contrast to blockade of the rapastinel response,
ketamine significantly decreased the immobility time in
both WTCre−/AAV2B and Camk2aCre+/AAV2B mice in the FST
(Fig. 2j).
To examine the role of GluN2B subunits on GABAergic
interneurons, we used the same design as above, infusing
AAV2GluN2BshRNA into WTCre−/AAV2B littermate controls or
Gad1-Cre mice (Gad1Cre+/AAV2B) (Fig. 3a–c). Sections of
mPFC from WTCre−/AAV2B mice displayed cells expressing
both EGFP and dsRed (labeled in yellow), while slices from
Gad1Cre+/AAV2B mice displayed a subset of cells expressing
only dsRed (Fig. 3b), confirming cell-type-specific recom-
bination. We have also validated this construct on func-
tional knockdown of NMDA-induced inward current in
recombined GABA interneurons of mPFC slices [17]. In
5103
both WTCre−/AAV2B and Gad1Cre+/AAV2B mice, rapastinel
significantly decreased immobility time and increased
female urine sniffing time in the FST and FUST, respec-
tively (Fig. 3d, e). Rapastinel also significantly decreased
the latency to feed in the NSFT in both WTCre−/AAV2B and
Gad1Cre+/AAV2B mice (Fig. 3f). There were no significant
effects on water sniffing time (Fig. 3e), or home cage
feeding and LMA (Supplementary Fig. 2c, d). As described
above for the Camk2aCre+/AAV2B mice, after a washout
period we also evaluated the effect of ketamine in the same
mice. Ketamine significantly decreased the immobility time
in WTCre−/AAV2B mice, but this effect was blocked in
Gad1Cre+/AAV2B mice (Fig. 3g). Together, these results
demonstrate that GluN2B containing NMDARs on gluta-
matergic neurons act as the initial cellular trigger for
rapastinel, while blockade of NMDAR-GluN2B on
GABAergic neurons triggers ketamine’s antidepressant-like
effects.
To demonstrate the specificity of the GluN2B shRNA
knockdown, we also tested an AAV-scrambled shRNA
construct in WT littermate and Camk2a-Cre mice
(WTCre−/AAV-Scr and Camk2aCre+/AAV-Scr mice) (Supple-
mentary Fig. 3a, b). As expected, yellow-labeled (EGFP +
dsRed) cells were observed in mPFC slices from
WTCre−/AAV-Scr mice and a subset of dsRed-expressing cells
were found only in Camk2aCre+/AAV-Scr mice, indicating
Cre-dependent recombination, EGFP excision and expres-
sion of scrambled shRNA only in Camk2aCre+/AAV-Scr mice
(Supplementary Fig. 3c). In both WTCre−/AAV-Scr and
Camk2aCre+/AAV-Scr mice, rapastinel significantly decreased
immobility time, increased female urine sniffing time and
decreased latency to feed in the FST, FUST and NSFT,
respectively (Supplementary Fig. 3d–f). There were no
significant differences observed in home cage feeding or
LMA (Supplementary Fig 3g, h).
GluN2A knockdown on glutamatergic neurons does
not block the antidepressant-like effects of
rapastinel
To determine if there is specificity for GluN2B vs. another
major subunit, GluN2A on Camk2a neurons in the
antidepressant-like actions of rapastinel, we used the
same approach but infused AAV2GluN2AshRNA into WT
littermates and Camk2a-cre mice (WTCre−/AAV2A and
Camk2aCre+/AAV2A mice) (Fig. 4a–f). For this construct,
mCherry was substituted for dsRed to enhance the visuali-
zation of recombined cells. Immunohistochemistry revealed
yellow (EGFP + mCherry) labeled cells in mPFC slices
from WTCre−/AAV2A mice, while a subset of mCherry only
expressing cells were found in Camk2aCre+/AAV2A mice,
confirming recombination (Fig. 4c). Further, there was a
significant reduction in NMDA-inward current in
5104 S. Pothula et al.

a GluN2B knockdown in Gad1-neurons: b WT

TATA-lox TATA-lox

CMV dsRed U6 CMV EGFP Sh-GluN2B hGH polyA

+ Gad1-Cre recombinase
CMV dsRed U6 Sh-GluN2B hGH polyA

TATA-lox

c Gad1 cre+

dsRED/EGFP
WTAAV2B-Sal Gad1Cre+/AAV2B-Sal
WTAAV2B-Rps or Ket Gad1Cre+/AAV2B-Rps or Ket

d FST-Rps e FUST-Rps
f NSFT-Rps
g FST-Ket
100 800 250

Latency to feed (sec)

250
**

Immobility time (sec)

** **
Immobility time (sec)

**
Sniffing time (sec)

200 * 80 ** ** 200
600
150 60 150
400
100 40 100
200
50 20 50

0 0 0 0
Water Urine

Fig. 3 Knockdown of GluN2B in the mPFC of Gad1-cre mice does
not block the antidepressant-like actions of rapastinel. a Schematic
showing pGluN2BshRNA construct before and after Gad1-Cre− to
generate the active construct in GABA neurons. b Representative
images of AAV2GluN2BshRNA virus expression, demonstrating fluor-
escent labeling of WT neurons (yellow: colocalization of EGFP and
dsRed) and Cre-recombined GABA neurons (subset of only dsRed-
expressing cells) in the mPFC of WTCre−/AAV2B and Gad1Cre+/AAV2B
mice, respectively. c Timeline for experimental approach. Rapastinel
significantly reduced immobility time (d), increased female urine
sniffing time (e), and reduced latency to feed (f) in the FST, FUST, and
NSFT, respectively, both in WTCre−/AAV2B and Gad1Cre+/AAV2B mice
(n = 8–10; FST: treatment: F1,30 = 15.1, p < 0.001, genotype:
F1,30 = 0.5, p = 0.49; NSFT: treatment: F1,30 = 24.4, p < 0.001,
genotype: F1,30 = 0.38, p = 0.54; *p < 0.05; **p < 0.01). g Ketamine
significantly reduced immobility time in FST in WTCre−/AAV2B but this
effect was blocked in Gad1Cre+/AAV2B mice (n = 8–9; FST: treatment:
F1,29 = 8.04, p < 0.01; interaction: F1,29 = 4.48, p = 0.04).

recombined, mCherry only expressing pyramidal neurons in Fig. 4e). These results demonstrate that GluN2A subunits
Camk2aCre+/AAV2A mice compared with yellow-labeled on glutamatergic neurons are not involved in the rapid and
cells in WTCre−/AAV2A mice, demonstrating functional sustained antidepressant-like effects of rapastinel.
knockdown of GluN2A. In contrast to the results from
GluN2B KD, rapastinel significantly increased the Drd1-expressing pyramidal neurons are required for
NMDA-inward current in virus infected glutamatergic the antidepressant-like effects of rapastinel and
neurons of Camk2aCre+/AAV2A as well as WTCre−/AAV2A ketamine
mice (Fig. 4d, e).
Baseline behavioral tests conducted before rapastinel Pyramidal neurons in the mPFC display distinct molecular,
administration revealed that there were no significant dif- cellular, and morphological characteristics, with two well-
ferences between WTCre−/AAV2A and Camk2aCre+/AAV2A characterized subtypes, referred to as types 1 and 2, which
mice in preswim immobility time or center or total distance express Drd2 and Drd1, respectively [19–23]. Chronic
traveled in an OFT (Supplementary Fig. 4a–c). Rapastinel unpredictable stress or social defeat stress alters the activity
administration resulted in significant antidepressant-like and morphology of Drd1-expressing neurons, indicating a
actions in both WTCre−/AAV2A and Camk2aCre+/AAV2A role for this pyramidal cell subtype in stress responses
mice, including decreased immobility time in the FST [28, 35, 36]. Here we examined the role of Drd1-expressing
(Fig. 4g) and latency to feed in the NSFT (Fig. 4h) without pyramidal neurons in the actions of rapastinel. In pre-
any significant change in home cage feeding (Supplemen- liminary studies we found that deletion of GluN2B on
tary Fig. 4d). As expected, we also found that GluN2A KD Drd1-expressing type 2 neurons altered baseline behaviors
in Camk2aCre+/AAV2A mice had no effect on the response to (not shown), so as an alternative approach we examined the
ketamine in the FST or NSFT (Fig. 4i, j); there was no role of Drd1-expressing neurons by knockdown of Drd1 in
effect on home cage feeding in either group (Supplementary Camk2a-expressing pyramidal neurons. We used a
Cell-type specific modulation of NMDA receptors triggers antidepressant actions 5105

GluN2A knockdown in Camk2a-neurons:

a
TATA-lox TATA-lox
b
CMV mCherry U6 CMV EGFP sh-GluN2A hGH polyA

+Camk2a-Cre recombinase
CMV mCherry U6 sh-GluN2A hGH polyA
TATA-lox
GluN2A
c WT
d NMDA +Rps 0.1 μM
e Glutamatergic
neuron

2500
*

NMDA- inward current

ACSF

WT
2000 * Rps 100nM
1500

(pA)
Camk2a cre+ #
1000

500
GluN2A-KD

500 pA
0
WTAAV2A Camk2aCre+/AAV2A
mCherry/EGFP 1 min

AAV2-
f GluN2A- WTAAV2A-Sal
shRNA WTAAV2A-Rps or Ket
infusion Camk2aCre+/AAV2A-Sal
Camk2aCre+/AAV2A-Rps or Ket

g FST-Rps h NSFT-Rps i FST-Ket NSFT-Ket

j ** *
250 ** ** * * * * 600
*
Immobility time (Sec)

Latency to feed (Sec)

600 250
Immobility time (Sec)
Latency to feed (Sec)

200
** **
200
400 400
150 150
100 100
200 200
50 50
0 0 0 0
WT Camk2a-Cre WT Camk2a-Cre WT Camk2a-Cre WT Camk2a-Cre

Fig. 4 Knockdown of GluN2A in the mPFC of Camk2a-Cre paired t-test, *p < 0.05). f Schematic showing time line for experi-
mice does not block the antidepressant-like actions of rapastinel. mental approach. Rapastinel significantly reduced immobility time
a, b Schematics showing pGluN2AshRNA construct and removal of (g) and latency to feed (h) in the FST and NSFT, respectively in both
NMDARs (GluN2A) on glutamatergic neuron. c Representative ima- WTCre−/AAV2A and Camk2aCre+/AAV2A mice compared with saline
ges of AAV2GluN2AshRNA virus expression showing fluorescent labeling controls. Data expressed as Mean ± SEM (n = 8–10 per group, two-
of WT neurons (yellow: colocalization of EGFP and mCherry fluor- way ANOVA with Tukey’s multiple comparisons; FST: treatment:
escence) and Cre-recombined pyramidal neurons (subset of only F1,32 = 20.6, p < 0.001, genotype: F1,32 = 0.185, p = 0.67, interac-
mCherry-labeled neurons) in the mPFC of WTCre−/AAV2A and tion: F1,32 = 0.183, p = 0.67; NSFT: treatment: F1,32 = 17.5, p <
Camk2aCre+/AAV2A mice, respectively. d Representative traces of 0.001, genotype: F1,32 = 0.21, p = 0.65, interaction: F1,32 = 0.025,
NMDA-inward currents in WT and GluN2A KD glutamatergic neu- p = 0.88; *p < 0.05; **p < 0.01). Ketamine significantly reduced
rons of WTCre−/AAV2A and Camk2aCre+/AAV2A mice before and after immobility time (i) and latency to feed (j) in the FST and NSFT,
rapastinel (100 nM) treatment. e NMDA-inward current was sig- respectively, in both WTCre−/AAV2A and Camk2aCre+/AAV2A mice (n =
nificantly reduced in neurons of Camk2aCre+/AAV2A compared with 8–9 per group, two-way ANOVA with Tukey’s multiple comparisons;
WTCre−/AAV2A mice (n = 10–11 neurons, unpaired t-test, #p < 0.05), FST: treatment: F1,31 = 26.9, p < 0.001; NSFT: treatment: F1,31 =
and rapastinel significantly enhanced NMDA-inward current in both 25, p < 0.001).
WTCre−/AAV2A and Camk2aCre+/AAV2A mice (n = 10–11 neurons,

previously described Cre-dependent Drd1 miRNA virus, of pyramidal neurons in Camk2aCre+/AAV-Drd1 mice (Fig. 5b,
AAV-DIO-EmGFP-Drd1amiR, infused into Camk2aCre+ c). Analysis of baseline behavior showed no significant
mice (referred to as Camk2aCre+/AAV-Drd1 mice) (Fig. 5a); differences in Camk2aCre+/AAV-Drd1 mice in preswim
functional knockdown of Drd1 using this virus was vali- immobility time or total distance traveled in the OFT,
dated in an earlier study [28]. Control, WT mice were although there was a significant reduction of time spent in
infused with AAV-Camk2a-EGFP (referred as WTCre−/AAV). center zone (Supplementary Fig. 5a–c). Rapastinel sig-
Immunohistochemistry shows Cre-independent EGFP nificantly reduced the immobility time and latency to feed
labeling in WTCre−/AAV and Cre-dependent EmGFP labeling in WTCre−/AAV mice, but these effects were completely
5106
a Drd1 knockdown in Camk2a-neurons:
Double floxed inverted ORF (DIO)
miRNA
EmGFP
EF1a Drd1a-
lox2272 + Cre recombinase
loxP
Drd1a-
EF1a EmGFP
miRNA
c
WTAAVDrd1-Sal Camk2aCre+/AAVDrd1-Sal
WTAAVDrd1-Rps or Ket Camk2aCre+/AAVDrd1-Rps or Ket
d FST-Rps e NSFT-Rps
200 600
150
400
100
200
50
0 0
Fig. 5 Knockdown of Drd1 in the mPFC of Camk2a-Cre mice
blocks antidepressant-like actions of rapastinel. a Schematic show-
ing the viral construct of AAV-DIO-EmGFP-Drd1amiR.
b Representative images of AAV-Camk2a-EGFP and AAV-DIO-
EmGFP-Drd1amiR virus expression, showing EGFP and
EmGFP fluorescent labeling of neurons in the mPFC of WTcre−/AAV
and Camk2aCre+/AAV-Drd1 mice, respectively. c Schematic showing the
timeline for experimental approach. Rapastinel significantly reduced
immobility time (d) and latency to feed (e) in the FST and NSFT,
respectively, in WTcre−/AAV but these effects were blocked in Cam-
k2aCre+/AAV-Drd1 mice. Data expressed as Mean ± SEM, two-way
blocked in Camk2aCre+/AAV-Drd1 mice (Fig. 5d, e); there
were no significant differences in home cage feeding
(Supplementary Fig. 5d). After a two-week washout, we
found that the antidepressant actions of ketamine on
immobility time in the FST and latency to feed in the NSFT
were also completely blocked in the Camk2aCre+/AAV-Drd1
mice (Fig. 5f, g); there were no significant differences in
home cage feeding (Supplementary Fig. 5e). These findings
are consistent with recent optogenetic and chemogenetic
studies demonstrating that Drd1-expressing neurons are
necessary and sufficient for the rapid antidepressant actions
of ketamine [37].
Previous studies report that rapastinel [38] as well as
ketamine increase dopamine efflux in the mPFC [14, 15],
and we have reported that the actions of ketamine are
blocked by infusion of a Drd1 antagonist into the PFC [37].
Based on these findings, we further tested the role of Drd1
S. Pothula et al.
b WT Camk2a cre+
EmGFP
f FST-Ket g NSFT-Ket
250 600
200
400
150
100
200
50
0 0
ANOVA with Tukey’s multiple comparisons (n = 7–12 per group;
FST: treatment: F1,32 = 6.03, p < 0.05, genotype: F1,32 = 5.06,
p = 0.03, interaction: F1,32 = 4.44, p = 0.04; NSFT: treatment:
F1,32 = 15.3, p < 0.001, genotype: F1,32 = 3.4, p = 0.07, interaction:
F1,32 = 2.12, p = 0.16; *p < 0.05; **p < 0.01; ***p < 0.001). Keta-
mine significantly reduced immobility time (f) and latency to feed (g) in
the FST and NSFT, respectively, in WTcre−/AAV but these effects were
blocked in Camk2aCre+/AAV-Drd1 mice (FST: treatment: F1,32 = 18.3,
p < 0.001, genotype: F1,32 = 12.9, p = 0.001, interaction: F1,32 =
8.38, p = 0.007; NSFT: treatment: F1,32 = 11.4, p = 0.002, genotype:
F1,32 = 3.87, p = 0.06, interaction: F1,32 = 0.92, p = 0.34).
in the actions of rapastinel using a pharmacological
approach. Pre-infusion of a selective Drd1 (SCH39166,
500 ng per side), but not Drd2 (L741,626, 700 ng per side)
antagonist into mPFC blocked the antidepressant-like
behavioral actions of rapastinel in the FST and NSFT
(Fig. 6a–e). There were no significant differences in home
cage feeding or LMA in response to rapastinel with or
without SCH39166 (Supplementary Fig. 6a, b) or L741,626
(Supplementary Fig. 6c, d).
Discussion
Research in the past decade has generated tremendous
interest in the utility of NMDAR modulators in depression.
Both positive and negative modulation of NMDARs by
rapastinel and ketamine, respectively, exert rapid and
Cell-type specific modulation of NMDA receptors triggers antidepressant actions 5107

a Cannulaon
Drug treatment FST LMA NSFT
surgery
//
-15/-16 Day 1 2 Food 3
days SCH39166/L-741626 10-15 min Rapasnel (i.v)
deprivaon
(mPFC)

Sal SCH39166 / Sal L741,626 / Sal

Rps SCH39166 / Rps L741,626 / Rps
FST NSFT FST NSFT
b c d e * * *
0.1
200 600 ** ** 200 * ** 600

Latency to feed (Sec)

Latency to feed (Sec)

Immobility time (Sec)

Immobility time (Sec)

** *
150 150
400 400
100 100
200 200
50 50

0 0 0 0
Saline SCH39166 Saline SCH39166 Saline L741626 Saline L741626

Fig. 6 Infusion of dopamine Drd1R, but not Drd2R, antagonists
prevent the antidepressant-like actions of rapastinel. a Schematic
for the timeline for experimental approach. Prior infusion of Drd1R
antagonist SCH39166 (500 ng per side) into mPFC blocked the
antidepressant-like effects of rapastinel on immobility time (b) and
latency to feed (c) in the FST and NSFT, respectively. Data expressed
as Mean ± SEM; two-way ANOVA with Tukey’s multiple compar-
isons (n = 6–8 mice per group; FST: treatment: F1,27 = 11.9, p <
0.01, infusion: F1,27 = 0.805, p = 0.38, interaction: F1,27 = 6.74,
p = 0.02; NSFT: treatment: F1,23 = 13.4, p = 0.001, infusion:
F1,23 = 4.75, p = 0.04, interaction: F1,23 = 5.81, p = 0.02; *p < 0.05;
**p < 0.01). d In the FST, rapastinel significantly reduced immobility
time in mice pre-infused with vehicle and showed a strong reduction
in mice pre-infused with Drd2R antagonist 741,626 (700 ng per side)
(n = 6 mice per group; treatment: F1,20 = 16.2, p < 0.001, infusion:
F1,20 = 2.58, p = 0.12, interaction: F1,20 = 0.29, p = 0.6). e In the
NSFT, rapastinel significantly reduced latency to feed in mice pre-
infused with either vehicle or L471,626 (treatment: F1,20 = 19.4, p <
0.001, infusion: F1,20 = 0.04, p = 0.84, interaction: F1,20 = 0.035,
p = 0.85). g Proposed model of direct vs. indirect actions of rapastinel
and ketamine. The NMDAR antagonist ketamine blocks NMDARs on
GABAergic interneurons to induce a disinhibition-mediated glutamate
burst and indirect activation of glutamatergic neurons in mPFC. In
contrast, the NMDAR positive modulator rapastinel directly activates
the NMDARs on glutamatergic neurons without causing a glutamate
burst, demonstrating distinct initial cellular triggers for these rapid
acting antidepressants. Indirect and direct activation of excitatory
mPFC-VTA projection neurons by NMDAR antagonists and PAMs,
respectively, could likely induce dopamine efflux in mPFC, which
could activate the Drd1-expressing pyramidal neurons (in mPFC), a
point of convergence for rapastinel and ketamine, resulting in rapid
and sustained antidepressant-like actions of these agents.
5108
sustained antidepressant effects by activating convergent
downstream signaling cascades [11–13], but NMDAR
antagonists and NMDAR PAMs with opposing actions on
NMDARs exert similar antidepressant effects and the initial
cellular target for NMDAR PAMs have not been deter-
mined. To address these questions, here we used ketamine
and rapastinel as tool compounds for these two types of
compounds. The results of the current study demonstrate
that NMDAR PAM, rapastinel enhances NMDAR inward
currents without effecting glutamate release in the mPFC
and that GluN2B subunits on glutamatergic, but not
GABAergic neurons in mPFC are required for the
antidepressant-like effects of rapastinel (Fig. 6g). In con-
trast, ketamine inhibits NMDAR activity and enhances
glutamate release, and selective knockdown of GluN2B
subunits on GABAergic but not glutamatergic neurons in
mPFC blocks the antidepressant-like effects of ketamine, in
agreement with a recent report [17]. The results also show
that Drd1-expressing subtype pyramidal neurons, and Drd1
receptors are required for the actions of rapastinel, as well as
ketamine, indicating that Drd1 is a point of convergence for
these two agents.
Here we show that rapastinel exhibits an inverted-U dose
response on NMDA-induced inward current in mPFC pyr-
amidal neurons, with low concentrations (100 nM) enhan-
cing NMDA-inward current, indicating a direct activation of
excitatory neurons. A recent study reported that rapastinel
(1 µM), as well as ketamine disinhibits pyramidal neurons
by acting on GABAergic neurons in hippocampus [39]. The
possible explanation for this discrepancy is the higher
concentration of rapastinel used in their study because
30–100 nM of rapastinel was previously shown to be
detected in mPFC after systemic injection and is sufficient
to induce antidepressant effects [32]. We have reported a
similar disinhibition by ketamine [34], but not rapastinel
(current study). In contrast, a trend for increase in NMDA
current observed in Sst interneurons even at 10 μM (data not
shown). Further, rapastinel concentrations ≥1 μM have been
shown to partially inhibit NMDAR activity [32], although
in the current study there was no effect observed on
NMDAR activity of pyramidal neurons with 10 μM
rapastinel.
Here we confirm that ketamine produces a rapid, but
transient increase in extracellular glutamate release in
mPFC [14] and also provide evidence that rapastinel, at
doses which produce an antidepressant-like response in
rodents, has no effect on extracellular glutamate. Consistent
with microdialysis results, we also found that ketamine, but
not rapastinel increased glutamate signaling measured by
NMR spectroscopy in mPFC. It has been suggested that
disinhibition-mediated glutamate efflux underlies the psy-
chotomimetic and dissociative effects of ketamine [14], and
absence of these side effects for rapastinel may be due to its
S. Pothula et al.
lack of effect on glutamate release. Together, these results
are consistent with an indirect and direct hypothesis for the
actions for ketamine and rapastinel [30], respectively
(Fig. 6g).
Previous studies have reported that rapastinel non-
selectively enhances the activity of NMDAR GluN2
subunits (A–D) and blockade of NMDARs prevents the
antidepressant activity of rapastinel [8, 32]. However, the
specific GluN2 subunits and cell type (excitatory or inhi-
bitory neurons) mediating the antidepressant actions of
rapastinel are unknown. Using a brain region- and cell-type-
specific approach we show that knockdown of GluN2B
subunits on glutamatergic but not GABAergic interneurons
in the mPFC blocks the antidepressant-like behavioral
actions of rapastinel in the FST, FUST, and NSFT. In
contrast, the results show that after a washout period in the
same mice the antidepressant actions of ketamine are
blocked by GluN2B KD in GABAergic, but not glutama-
tergic neurons consistent with our recent study [17]. The
lack of baseline reduction in immobility time in FST after
GluN2B knockdown in our study differs from earlier stu-
dies [40, 41] reporting a reduction in baseline immobility
time in FST after constitutive deletion of GluN2B in the
forebrain [40]. This could be due to the robust increase in
locomotion reported in mice with constitutive forebrain
deletion of GluN2B [40], which would produce an
antidepressant-like decrease in immobility time in the FST
and TST. In contrast, in the current study there were no
effects of GluN2B KD on LMA in Camk2a-Cre mice.
Another study reported that viral-mediated deletion of
GluN2B in thalamocortical prelimbic mPFC circuits, which
differs from the region and circuits targeted in the current
study, also results in a baseline antidepressant phenotype
[41].
The results also demonstrate a lack of off-target effects of
AAV viral-GluN2B shRNA as infusion of an AAV-
scrambled virus did not block the antidepressant-like
effects of rapastinel. In addition, our study demonstrates
that knockdown of GluN2A subunits in glutamatergic
neurons does not block the antidepressant-like behavioral
actions of rapastinel. We have also reported that the actions
of ketamine on GABAergic neurons are mediated by
GluN2B but not GluN2A [17]. In support of these beha-
vioral results, electrophysiology studies demonstrate that
knockdown of GluN2B, but not GluN2A partially blocks
rapastinel enhancement of NMDA-induced inward currents
in layer V pyramidal neurons of mPFC. The reason for this
in vivo selectivity, in spite of nonselective GluN2 subunit
effects in vitro [32] is unknown but could be due to higher
expression levels of GluN2B and/or different subunit
combinations or higher calcium influx mediated by GluN2B
or activity patterns of principal neurons in vivo. Previous
studies have reported reductions of GluN2B levels and
Cell-type specific modulation of NMDA receptors triggers antidepressant actions
NMDAR binding in PFC of depressed individuals [42, 43].
In addition, previous studies report that rapastinel increases
GluN2B levels and GluN2B-mediated metaplasticity in
mPFC [8], providing further evidence for a specific role of
GluN2B in the sustained antidepressant-like effects of
rapastinel. It will be interesting in future studies to deter-
mine how NMDAR PAMs specifically increases GluN2B
levels and whether this increase occurs in a cell-type-
specific manner.
Optogenetic activation of principal neurons in mPFC
induces rapid and sustained antidepressant effects [34].
More recent optogenetic and chemogenetic studies
demonstrate that activity of Drd1-, but not Drd2-expressing
neurons in the mPFC is necessary and sufficient for the
antidepressant actions of ketamine, presumably via disin-
hibition and subsequent glutamate and dopamine burst [37]
which activate these neurons to induce synaptic plasticity.
In support of this possibility, we have recently reported that
chemogenetic inhibition of Drd1-expressing neurons blocks
the effects of ketamine [37]. To further characterize the
mechanisms underlying the actions of NMDAR antagonists
and PAM, we also examined the role of the Drd1-expres-
sing, type 2 subtype pyramidal neurons in the mPFC. Here
we show that knockdown of Drd1 on Camk2a-neurons in
the mPFC blocks the antidepressant-like behavioral actions
of rapastinel, as well as ketamine. Projection targets of these
mPFC Drd1-expressing neurons, including the basolateral
amygdala (BLA) and bed nucleus of stria terminalis, have
been implicated in regulation of anxiety and depression-like
behaviors [37, 44]. Moreover, optogenetic activation of
mPFC Drd1-neurons projecting to BLA induces anti-
depressant effects [37], consistent with the possibility that
BLA projecting Drd1-pyramidal neurons could mediate the
actions of rapastinel, as well as ketamine.
Both ketamine and rapastinel increase dopamine efflux in
mPFC [14, 38] and identification of a role for Drd1-
expressing neurons in the current as well as earlier studies
[37] suggests that dopamine signaling is involved in the
actions of ketamine and rapastinel. In support of this pos-
sibility, we have recently reported that pharmacological
blockade of Drd1 receptors blocks the antidepressant
actions of ketamine [37]. Here we show that pharmacolo-
gical blockade of Drd1, but not Drd2, in the mPFC blocks
the antidepressant-like actions of rapastinel. The mechan-
isms underlying the increase in dopamine efflux is unclear
but it could likely be possible that ketamine indirectly
activate excitatory mPFC-VTA neuronal projections,
implicated in depression [45], via disinhibition in mPFC
and rapastinel could directly activate these projections
(Fig. 6g). Also, mechanisms underlying these Drd1R-
mediated actions are unclear, but previous studies demon-
strate that Drd1-signaling increases GluN2B and AMPAR
expression [46, 47] and increases mPFC pyramidal neurons
5109
excitability [48] and synaptic plasticity [49, 50]. These
findings suggest that Drd1-signaling could contribute to
upregulation of GluN2B expression, activity, and plasticity
observed after rapastinel, as well as ketamine administration
[8]. Together the results indicate that Drd1-signaling is a
point of convergence for the molecular and behavioral
actions of both NMDAR positive modulation (rapastinel)
and inhibition (ketamine) (Fig. 6g). It will be interesting in
future studies to identify the role of the mesolimbic dopa-
mine system and downstream signaling pathways in the
synaptic and antidepressant-like behavioral actions of these
rapid acting agents.
Previous studies report that NMDAR PAM, rapastinel
exerts rapid and sustained antidepressant effects in both
stress models and naïve, non-stressed animals
[8, 12, 13, 33]. Although the current study does not employ
stress models per se, the mice were single housed/socially
isolated (at least 2–3 weeks post-surgery), and social iso-
lation is reported to cause depressive-like molecular and
behavioral alterations [51–54]. Future studies are needed to
determine if GluN2B and/or Drd1 on glutamatergic neurons
mediate the actions of rapastinel and ketamine in patholo-
gical stress models, such as social defeat and chronic
unpredictable stress. Nevertheless, the current findings
provide crucial mechanistic insights into the role of differ-
ent neuronal cell-types and receptor subtypes in initiating
and mediating the antidepressant-like behavioral actions of
NMDAR enhancing vs. blocking agents.
In conclusion, the findings of the current study provide
direct evidence for previously unknown mechanisms
underlying the initial cellular trigger, neuronal cell-types
and specific NMDA and dopamine receptor subtypes
involved in the antidepressant-like actions of NMDAR
PAMs. Given the significant unmet need for safe and effi-
cacious rapid acting antidepressants, ongoing clinical trials
of NMDAR PAMs like AGN-241751 which are more
potent and longer acting than rapastinel, and the need for
additional rapid acting antidepressants without the side
effect profile of ketamine, it is important to understand the
cellular trigger and mechanisms underlying the actions of
NMDAR PAMs. The results reveal both unique as well as
convergent mechanisms underlying the antidepressant-like
actions of NMDAR positive modulation vs. antagonism.
Previous reports of altered mPFC dopamine and
Drd1 synaptic transmission in chronic stress models com-
bined with the current findings indicate that Drd1-expres-
sing neurons could play a crucial role in both the
pathophysiology of depression, as well as the response to
rapid acting antidepressants. Further characterization of the
mPFC neuronal cell-types and circuits using cellular and
molecular approaches will provide novel and promising
molecular targets for the development of safer and effica-
cious rapid antidepressants.
5110
Acknowledgements This research was supported by National Institute
of Mental Health grants MH093897 and MH105910 (RSD) and a
research grant from Allergan Inc. New Jersey. We thank Xiao Yuan Li
for her help with genotyping of mouse lines. We thank Jan Kehr
(Pronexus Analytical AB, Stockholm, Sweden) for performing the
microdialysis experiments. Dr. Duman passed away on February 1st
2020. This article is dedicated to Dr. Duman in memory of his men-
torship and scientific leadership.
Author contributions SP, TK, PB, and RSD designed the study. SP
wrote the manuscript and RSD contributed to writing the manuscript.
SP, TK, RJL, and MW conducted experiments, analyzed data, and
interpreted the results. GS, GMIC, and KLB designed glutamate
cycling experiment using MRS spectroscopy. GMIC and KLB con-
ducted glutamate cycling experiment. DG and RS are involved in
design and preparation of pGluN2BshRNA (dsRed version) and AAV-
DIO-EmGFP-D1miR viruses. A-NS helped with experiments. All
authors reviewed and approved the final manuscript.
Compliance with ethical standards
Conflict of interest RSD has received consulting fees from Taisho,
Johnson & Johnson, and Naurex, and grant support from Taisho,
Johnson & Johnson, Naurex, Allergan, Navitor, Lundbeck, Relmada,
and Lilly. PB is an employee of Allergan Inc, New Jersey. All other
authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
1. Hasin DS, Sarvet AL, Meyers JL, Saha TD, Ruan WJ, Stohl M, et al.
Epidemiology of adult DSM-5 major depressive disorder and Its
specifiers in the United States. JAMA Psychiatry. 2018;75:336–46.
2. Fava M. Diagnosis and definition of treatment-resistant depres-
sion. Biol Psychiatry. 2003;53:649–59.
3. Trivedi M, Rush AJ, Wisniewski SR, Nierenberg AA, Warden D,
Ritz L, STAR*D Study Team, et al. Evaluation of outcomes with
citalopram for depression using measurement-based care in
STAR*D: implications for clinical practice. Am J Psychiatry.
2006;163:28–40.
4. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR,
Charney DS, et al. Antidepressant effects of ketamine in depressed
patients. Biol Psychiatry. 2000;47:351–4.
5. Abdallah CG, Sanacora G, Duman RS, Krystal JH. Ketamine and
rapid-acting antidepressants: a window into a new neurobiology
for mood disorder therapeutics. Annu Rev Med. 2015;66:509–23.
6. Sleigh J, Harvey M, Voss L, Denny B. Ketamine—more
mechanisms of action than just NMDA blockade. Trends Anaesth
Crit Care. 2014;4:76–81.
7. Morgan CJA, Curran HV. Ketamine use: a review. Addiction.
2012;107:27–38.
8. Moskal JR, Burch R, Burgdorf JS, Kroes RA, Stanton PK, Dis-
terhoft JF, et al. GLYX-13, an NMDA receptor glycine site
functional partial agonist enhances cognition and produces anti-
depressant effects without the psychotomimetic side effects of
NMDA receptor antagonists. Expert Opin Investig Drugs. 2014;
23:243–54.
9. Burch R, Preskorn S, Bastin L, Yu W, Burgdorf J, Moskal J.
Adjunctive GLYX-13 induces prolonged efficacy in subjects with
major depressive disorder (MDD). Neuropsychopharmacology.
2014;39:S335.
S. Pothula et al.
10. Preskorn S, Macaluso M, Mehra DO, Zammit G, Moskal JR,
Burch RM, et al. Randomized proof of concept trial of GLYX-13,
an N-methyl-D-aspartate receptor glycine site partial agonist, in
major depressive disorder nonresponsive to a previous anti-
depressant agent. J Psychiatr Pract. 2015;21:140–9.
11. Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, et al. mTOR-
dependent synapse formation underlies the rapid antidepressant
effects of NMDA antagonists. Science. 2010;329:959–64.
12. Liu RJ, Duman C, Kato T, Hare B, Lopresto D, Bang E, et al.
GLYX-13 produces rapid antidepressant responses with key
synaptic and behavioral effects distinct from ketamine. Neu-
ropsychopharmacology. 2017;42:1231–42.
13. Kato T, Fogaca M, Deyama S, Li X, Fukumoto K, Duman R.
BDNF release and signaling are required for the antidepressant
actions of GLYX-13. Mol Psych. 2017;23:2007–17.
14. Moghaddam B, Adams B, Verma A, Daly D. Activation of glu-
tamatergic neurotransmission by ketamine: a novel step in the
pathway from NMDA receptor blockade to dopaminergic and
cognitive disruptions associated with the prefrontal cortex. J
Neurosci. 1997;17:2921–7.
15. Lorrain DS, Baccei CS, Bristow LJ, Anderson JJ, Varney MA.
Effects of ketamine and N-methyl-D-aspartate on glutamate and
dopamine release in the rat prefrontal cortex: modulation by a
group II selective metabotropic glutamate receptor agonist
LY379268. Neuroscience. 2003;117:697–706.
16. Homayoun H, Moghaddam B. NMDA receptor hypofunction
produces opposite effects on prefrontal cortex interneurons and
pyramidal neurons. J Neurosci. 2007;27:11496–500.
17. Gerhard D, Pothula S, Liu RJ, Wu M, Li XY, Girgenti MJ, et al.
GABA interneurons are the initial trigger for the rapid anti-
depressant actions of ketamine. J Clin Invest. 2020;130:1336–49.
18. Preskorn S, Baker B, Kolluri S, Menniti FS, Krams M, Landen
JW. An innovative design to establish proof of concept of the
antidepressant effects of the NR2B subunit selective N-Methyl-D-
Aspartate antagonist, CP-101,606, in patients with treatment-
refractory major depressive disorder. J Clin Psychopharmacol.
2008;28:631–7.
19. Yang CR, Seamans JK, Gorelova N. Electrophysiological and
morphological properties of layers V-VI principal pyramidal cells
in rat prefrontal cortex in vitro. J Neurosci. 1996;16:1904–21.
20. Santana N, Mengod G, Artigas F. Quantitative analysis of the
expression of dopamine D1 and D2 receptors in pyramidal and
GABAergic neurons of the rat prefrontal cortex. Cereb Cortex.
2009;19:849–60.
21. Wei X, Ma T, Cheng Y, Huang CCY, Wang X, Lu J, et al.
Dopamine D1 or D2 receptor-expressing neurons in the central
nervous system. Addict Biol. 2018;23:569–84.
22. Anastasiades PG, Boada C, Carter AG. Cell-Type-Specific D1
dopamine receptor modulation of projection neurons and inter-
neurons in the prefrontal cortex. Cereb Cortex 2018;29:3224–42.
23. Gee S, Ellwood I, Patel T, Luongo F, Deisseroth K, Sohal VS.
Synaptic activity unmasks dopamine D2 receptor modulation of a
specific class of layer V pyramidal neurons in prefrontal cortex. J
Neurosci. 2012;32:4959–71.
24. Kehr J, Yoshitake T. Monitoring brain chemical signals by
microdialysis. In: Grimes CA, Dickey EC, Pishko MV, editors.
Encyclopedia of sensors, Vol. 6. USA: American Scientific Pub-
lishers; 2006. p. 287–312.
25. Kehr J, Yoshitake T. Derivatization chemistries for improved
detection of monoamine neurotransmitters and their metabolites in
microdialysis samples by liquid chromatography with fluores-
cence detection and mass spectrometry. In: Wilson G, Michael A,
editors. Compendium of in-vivo monitoring in real-time molecular
neuroscience, Vol. 2, microdialysis and sensing of neural tissues.
Singapore: World Scientific Publishing; 2017. p. 193–216.
Cell-type specific modulation of NMDA receptors triggers antidepressant actions
26. Chowdhury GM, Behar KL, Cho W, Thomas MA, Rothman DL,
Sanacora G. (1)H-[(1)(3)C]-nuclear magnetic resonance spectro-
scopy measures of ketamine’s effect on amino acid neuro-
transmitter metabolism. Biolo Psychiatry. 2012;71:1022–25.
27. Chowdhury GM, Zhang J, Thomas M, et al. Transiently increased
glutamate cycling in rat PFC is associated with rapid onset of
antidepressant-like effects. Mol Psychiatry. 2017;22:120–6.
28. Shinohara R, Taniguchi M, Ehrlich AT, Yokogawa K, Deguchi Y,
Cherasse Y, et al. Dopamine D1 receptor subtype mediates acute
stress-induced dendritic growth in excitatory neurons of the
medial prefrontal cortex and contributes to suppression of stress
susceptibility in mice. Mol Psychiatry. 2018;23:1717–30.
29. Wohleb ES, Wu M, Gerhard DM, Taylor SR, Picciotto MR,
Alreja M, et al. GABA interneurons mediate the rapid
antidepressant-like effects of scopolamine. J Clin Invest.
2016;126:2482–94.
30. Duman RS, Shinohara R, Fogaça MV, Hare B. Neurobiology of
rapid-acting antidepressants: convergent effects on GluA1-
synaptic function. Mol Psychiatry. 2019;24:1816–32.
31. White PF, Schüttler J, Shafer A, Stanski DR, Horai Y, Trevor AJ.
Comparative pharmacology of the ketamine isomers: studies in
volunteers. Br J Anaesth. 1985;57:197–203.
32. Donello JE, Banerjee P, Li XY, Guo YX, Yoshitake T, Zhang XL,
et al. Positive N-Methyl-D-Aspartate receptor modulation by
rapastinel promotes rapid and sustained antidepressant-like
effects. Int J Neuropsychopharmacol. 2019;22:247–59.
33. Burgdorf J, Zhang XL, Nicholson KL, Balster RL, Leander JD,
Stanton PK, et al. GLYX-13, an NMDA receptor glycine-site
functional partial agonist, induces antidepressant-like effects
without ketamine-like side effects. Neuropsychopharmacology.
2013;38:729–42.
34. Fuchikami M, Thomas A, Liu R, Wohleb ES, Land BB, DiLeone
RJ, et al. Optogenetic stimulation of infralimbic PFC reproduces
ketamine’s rapid and sustained antidepressant actions. Proc Natl
Acad Sci USA. 2015;112:8106–11.
35. Tanaka K, Furuyashiki T, Kitaoka S, Senzai Y, Imoto Y, Segi-
Nishida E, et al. Prostaglandin E2-mediated attenuation of
mesocortical dopaminergic pathway is critical for susceptibility to
repeated social defeat stress in mice. J Neurosci.
2012;32:4319–29.
36. Anderson EM, Gomez D, Caccamise A, McPhail D, Hearing M.
Chronic unpredictable stress promotes cell-specific plasticity in
prefrontal cortex D1 and D2 pyramidal neurons. Neurobiol Stress.
2019;10:100152.
37. Hare BD, Shinohara R, Liu RJ, Pothula S, DiLeone RJ, Duman
RS. Optogenetic stimulation of medial prefrontal cortex Drd1
neurons produces rapid and long-lasting antidepressant effects.
Nat Commun. 2019;10:223.
38. Rajagopal L, Huang M, Li J, He W, Soni D, Banerjee P, et al.
Rapastinel, a novel NMDA receptor modulator, produces pro-
longed rescue of subchronic phencyclidine-induced deficits in
episodic memory as well as other beneficial effects on cognitive
function in a rapamycin sensitive manner. 341.12 / VV3. 2017
Neuroscience Meeting Planner. Washington, DC: Society for
Neuroscience; 2017.
5111
39. Widman AJ, McMahon LL. Disinhibition of CA1 pyramidal cells
by low-dose ketamine and other antagonists with rapid anti-
depressant efficacy. Proc Natl Acad Sci USA. 2018;115:E3007–16.
40. Miller OH, Yang L, Wang CC, Hargroder EA, Zhang Y,
Delpire E. et al. GluN2B-containing NMDA receptors regulate
depression-like behavior and are critical for the rapid anti-
depressant actions of ketamine. Elife. 2014;3:e03581. https://doi.
org/10.7554/eLife.03581.
41. Miller OH, Bruns A, Ben Ammar I, Mueggler T, Hall BJ.
Synaptic regulation of a thalamocortical circuit controls
depression-related behavior. Cell Rep. 2017;20:1867–80.
42. Feyissa AM, Chandran A, Stockmeier CA, Karolewicz B.
Reduced levels of NR2A and NR2B subunits of NMDA receptor
and PSD-95 in the prefrontal cortex in major depression. Prog
Neuro-Psychopharmacol Biol Psychiatry. 2009;33:70–5.
43. Nowak G, Ordway GA, Paul IA. Alterations in the N-methyl-d-
asparatate (NMDA) receptor complex in the frontal cortex of
suicide victims. Brain Res. 1995;675:157–64.
44. Land BB, Narayanan NS, Liu RJ, Gianessi CA, Brayton CE,
Grimaldi D, et al. Medial prefrontal D1 dopamine neurons control
food intake. Nat Neurosci. 2014;17:248–53.
45. Chaudhury D, Walsh JJ, Friedman AK, Juarez B, Ku SM, Koo
JW, et al. Rapid regulation of depression-related behaviors by
control of midbrain dopamine neurons. Nature. 2013;493:532–6.
46. Gao C, Wolf ME. Dopamine receptors regulate NMDA receptor
surface expression in prefrontal cortex neurons. J Neurochem.
2008;106:2489–501.
47. Sun X, Zhao Y, Wolf ME. Dopamine receptor stimulation mod-
ulates AMPA receptor synaptic insertion in prefrontal cortex
neurons. J Neurosci. 2005;25:7342–51.
48. Lavin A, Grace AA. Stimulation of D1-type dopamine receptors
enhances excitability in prefrontal cortical pyramidal neurons in a
state-dependent manner. Neuroscience. 2001;104:335–46.
49. Gurden H, Takita M, Jay TM. Essential role of D1 but not D2
receptors in the NMDA receptor‐dependent long‐term potentiation
at hippocampal‐prefrontal cortex synapses in vivo. J Neurosci.
2000;20:RC106.
50. Jay TM. Dopamine: a potential substrate for synaptic plasticity
and memory mechanisms. Prog Neurobiol. 2003;69:375–90.
51. Berry A, Bellisario V, Capoccia S, Tirassa P, Calza A, Alleva E,
et al. Social deprivation stress is a triggering factor for the
emergence of anxiety- and depression-like behaviours and leads to
reduced brain BDNF levels in C57BL/6J mice. Psychoneur-
oendocrinology. 2012;37:762–72.
52. Ieraci A, Mallei A, Popoli M. Social isolation stress induces
anxious-depressive-like behavior and alterations of
neuroplasticity-related genes in adult male mice. Neural Plast.
2016;2016:6212983.
53. Mumtaz F, Khan MI, Zubair M, Dehpour AR. Neurobiology and
consequences of social isolation stress in animal model-A com-
prehensive review. Biomed Pharmacother. 2018;105:1205–22.
54. Haj-Mirzaian A, Amini-Khoei H, Haj-Mirzaian A, Amiri S,
Ghesmati M, Zahir M, et al. Activation of cannabinoid receptors
elicits antidepressant-like effects in a mouse model of social iso-
lation stress. Brain Res Bull. 2017;130:200–10.