Cerebral Cortex, June 2019;29: 2728–2736

doi: 10.1093/cercor/bhy142
Advance Access Publication Date: 25 May 2018
Original Article

ORIGINAL ARTICLE

Differential Contributions of Glutamatergic

Hippocampal→Retrosplenial Cortical Projections to the
Formation and Persistence of Context Memories
Naoki Yamawaki1, Kevin A. Corcoran2, Anita L. Guedea2,
Gordon M.G. Shepherd1 and Jelena Radulovic1,2
1
Department of Physiology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA and
2
Department of Psychiatry and Behavioral Sciences, Northwestern University, Feinberg School of Medicine,
Chicago, IL 60611, USA
Address correspondence to Jelena Radulovic, Northwestern University, Feinberg School of Medicine, 303 E Chicago Ave, Ward 13-130, Chicago, IL 60611,
USA. Email: j-radulovic@northwestern.edu

Abstract
Learning to associate stressful events with specific environmental contexts depends on excitatory transmission in the
hippocampus, but how this information is transmitted to the neocortex for lasting memory storage is unclear. We identified
dorsal hippocampal (DH) projections to the retrosplenial cortex (RSC), which arise mainly from the subiculum and contain
either the vesicular glutamate transporter 1 (vGlut1) or vGlut2. Both vGlut1+ and vGlut2+ axons strongly excite and
disynaptically inhibit RSC pyramidal neurons in superficial layers, but vGlut2+ axons trigger greater inhibition that spreads to
deep layers, indicating that these pathways engage RSC circuits via partially redundant, partially differentiated cellular
mechanisms. Using contextual fear conditioning in mice to model contextual associative memories, together with
chemogenetic axonal silencing, we found that vGlut1+ projections are principally involved in processing recent context
memories whereas vGlut2+ projections contribute to their long-lasting storage. Thus, within the DH→RSC pathway, engagement
of vGlut1+ and vGlut2+ circuits differentially contribute to the formation and persistence of fear-inducing context memories.

Key words: context fear conditioning, hippocampus, retrosplenial cortex, vGlut1, vGlut2

Introduction In animal models, learning to associate stressful events with

Episodic memories contain details of past events, including specific environmental contexts can be rapidly induced by contex-
where they occurred. The hippocampus, and in particular its tual fear conditioning (CFC). In CFC, exposure to a context is ter-
posterior (dorsal in rodents) subdivision, is critical for the forma- minated with brief footshock, resulting in a context/shock
tion of episodic memories, whereas its interactions with the cor- association and context-specific freezing upon re-exposure to the
tex are believed to provide a foundation for lasting memory context (Blanchard and Blanchard 1969; Fanselow 1990). To date,
storage through a process termed systems consolidation (Squire several neocortical areas have been implicated in the long-term
and Alvarez 1995). Understanding the mechanisms underlying processing of fear-inducing context memories including the ante-
these interactions will further our knowledge of basic principles rior cingulate (Frankland et al. 2004; Wiltgen and Tanaka 2013),
of memory but also provide novel targets for memory disorders, retrosplenial [RSC, (Corcoran et al. 2011)], and medial prefrontal
which range from impairments (dementia) to excessive persis- (Kitamura et al. 2017) cortices. Of these areas, dorsal hippocampal
tence (post-traumatic stress disorder) of episodic memories. (DH) projects directly only to RSC (Cenquizca and Swanson 2007).

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
 Hippocampal–Cortical Interactions Underlying Context Memory Yamawaki et al. | 2729

Moreover, glutamatergic neurotransmission in DH and RSC is breeding. Typically, we obtained 4–6 litters/breeding cycle with
required for CFC (Keene and Bucci 2008; Gao et al. 2010; Corcoran 5–8 mice/litter with similar distribution of males and females.
et al. 2011; Cowansage et al. 2014; Kwapis et al. 2014). All litters were used for behavioral experiments and randomly
Interestingly, RSC activity is required for processing of both recent selected mice were used for tracing and electrophysiological
and remote memories (Corcoran et al. 2011), suggesting that studies. Genotyping was confirmed by PCR with 3 primers. The
DH→RSC projections transmit essential contextual information common (5′-AAG AAG GTG CGC AAG ACG-3′) and endogenous
required for cortical memory processing from the early stages of (5′-CTG CCA CAG ATT GCA CTT GA-3′) primers generate a 299-
stimulus encoding (Van Groen and Wyss 2003; Aggleton 2010). bp PCR product from the endogenous locus, while the common
To provide experimental support for this assumption, we and mutant (5′-ACA CCG GCC TTA TTC CAA G-3′) primers gen-
sought to understand in more depth the cellular and circuit erate a 850-bp product from the targeted locus.
mechanisms underlying DH-RSC interactions, and to determine
their contributions to storage and retrieval of fear-inducing
Contextual Fear Conditioning
context memories. We show that vGlut1+ and vGlut2+ neuronal
populations in the DH, predominantly localized in the SUB, CFC was performed in an automated system (TSE Systems) as
project to the RSC and distinctively regulate RSC local cellular described previously (Corcoran et al. 2011). Briefly, mice were
networks. Using chemogenetic silencing of DH→RSC terminals, exposed for 3 min to a novel context, followed by a footshock
we found a major role of vGlut1+ projections in encoding and (2 s, 0.7 mA, constant current). Mice were tested for memory
retrieval of recent context memory, whereas vGlut2+ projec- retrieval 24 h later by returning them to the conditioning con-
tions, on the other hand contributed to its persistence. text for 3 min. Freezing was scored every 10 s during context
exposures and expressed as a percentage of the total number
of observations during which the mice were motionless. All
Materials and Methods behavioral experiments were performed between 10 a.m. and
2 p.m. Littermates were randomly assigned to the different
Animals treatment conditions. All behavioral tests and immunohisto-
We used male and female C57BL/6J mice, vGlut1-Cre, and chemical analyses were performed by experimenters who were
vGlut2-Cre male mice. Wild-type C57BL6/J mice were purchased blind to genotypes and drug treatments.
from Harlan, Indianapolis, IN. The vGlut1-Cre mouse line, also
known as Slc17a7-IRES2-Cre or Vglut1-IRES2-Cre-D, was created
Stereotaxic Surgeries and Infusions of Viral Vectors and
by the Hongkui Zeng lab, Allen Institute for Brain Science
(Harris et al. 2014), and obtained from the Jackson Laboratory
Drugs
(Bar Harbor, ME). These knockin mice express Cre recombinase Mice were anesthetized with 1.2% tribromoethanol (vol/vol,
in vGlut1+ cells, without disrupting endogenous vGlut1 expres- Avertin) for viral vector intracranial infusion and cannula implan-
sion. Cre recombinase expression (performed with in situ tation. The viral vector carrying a construct coding for the
hybridization using a Cre-specific probe) showed a pattern sim- Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-
ilar to that of the endogenous Slc17a7 gene (Allen Institute for mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD
Brain Science website Slc17a7-IRES2-Cre images), as also con- (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilater-
firmed with our immunohistochemical analyses. The vGlut2- ally infused into the dorsal hippocampus (1.8 mm posterior,
Cre knockin mice, also known as Slc17a6tm2(cre) and Lowl or ±1.0 mm lateral, 2.25 mm ventral to bregma). Infusions were per-
VGlut2-ires-Cre, express Cre recombinase in excitatory gluta- formed using an automatic microsyringe pump controller
matergic neuron cell bodies, without disrupting endogenous (Micro4-WPI) connected to a Hamilton microsyringe. The viral
vGlut2 expression, as described previously (Vong et al. 2011). vectors were infused in a volume of 0.5 μL per site over 2 min, and
All mice were 8 weeks of age at the beginning of the experi- syringes were left in place for 5 min prior to removal to allow for
ments. The mice were maintained under standard housing condi- virus diffusion. Bilateral 26 gauge guide cannulas (Plastics One)
tions (12/12 h light dark cycle with lights on at 7 a.m., temperature were placed in RSC (1.7 mm posterior, ±0.4 mm lateral, 0.75 mm
20–22 °C, humidity 30–60%) in our satellite behavioral facility. All ventral to bregma). Mice were allowed 6 weeks for virus expres-
animal procedures used in this study were approved by the sion prior to behavioral testing. Clozapine-N-oxide (Sigma; 0.3 μg/
Northwestern University IACUC and complied with federal regula- mL; 0.20 μL per side, at a rate of 0.5 μL/min) was infused through
tions set forth by the National Institutes of Health. the cannulas 30 min prior to either fear conditioning or memory
Heterozygous vGlut1-Cre mice [Jackson 023527, strain of ori- retrieval testing. Bicuculline (Sigma) was infused under the same
gin (129S6/SvEvTac x C57BL/6NCrl)F1, bred with C57BL/6 J wild- conditions at 3 doses (0.25 μg/μL, 0.5 μg/μL, and 1 μg/μL). After the
type mice for several generations in the Zeng laboratory and 3 completion of behavioral testing, all brains were collected and
generations at the Jackson Laboratory] were backcrossed with cannula placements and virus spread were confirmed by immu-
wild-type C57BL6/J for 6 generations in our facility to achieve nohistochemical analysis using anti-mCherry antibodies (1:1 000;
offspring with a genetic identity which is closer to the C57BL6/J Abcam, Ab167453). For retrograde tracing with hydroxystilbami-
strain. The colony was subsequently expanded by homozygous dine (Fluoro-Gold, Fluorohrome) we infused 0.2 μL of a 4%
breeding. Genotyping was confirmed by polymerase chain reac- Fluoro-Gold solution in artificial cerebrospinal fluid (aCSF) into
tion (PCR) with 3 primers. The common (5′-ATG AGC GAG GAG RSC and 5 days later collected and perfused the brains in 4%
AAG TGT GG-3′) and endogenous (5′-GTG GAA GTC CTG GAA paraformaldehyde.
ACT GC-3′) primers generate a 218-bp PCR product from the
endogenous locus, while the common and mutant (5′-CCC TAG
Immunohistochemistry and Immunofluorescence
GAA TGC TCG TCA AG-3′) primers generate a 344-bp product
from the targeted locus. Homozygous vGlut2-Cre (Jackson Mice were anesthetized with an i.p. injection of 240 mg/kg
016 963) mice were also backcrossed with wild-type C57BL6/J Avertin and transcardially perfused with ice-cold 4% parafor-
for 6 generations and the colony was expanded by homozygous maldehyde in phosphate buffer (pH 7.4, 150 mL per mouse).
 2730 | Cerebral Cortex, 2019, Vol. 29, No. 6

Brains were removed and postfixed for 48 h in the same fixative Responses to multiple trials were sampled with an interstimulus
and then immersed for 24 h each in 20% and 30% sucrose in interval of 20 s. Recordings with series resistance above 40 MΩ
phosphate buffer. Brains was frozen and 50 μm sections were were discarded. Data were acquired and hardware was con-
cut for use in free-floating immunohistochemistry (Jovasevic trolled using Ephus software (Suter et al. 2010). Signals were
et al. 2015) with primary antibodies against vGlut1 (1:4000; amplified using Axon Multiclamp 400B (Molecular Devices), fil-
Abcam AB104898), vGlut2 (1:2000; Millipore, Cat # MAB5504), tered at 4 kHz, and sampled at 10 kHz. Traces were analyzed
mCherry (1:1000; Abcam AB167453), and Fluoro-Gold using Matlab routines. Photo-evoked EPSCs or IPSCs from multi-
(Fluorochrome 1:40 000). Immunostaining with mCherry, vGlut1, ple trials in each neuron were averaged and the response was
vGlut2, and Fluoro-Gold antibodies was visualized with diami- computed as the mean current over a poststimulus interval of
nobenzidine (Sigma), fluorescein isothiocyanate (TSA systems, 50 ms. For comparison of DH input to superficial and deep layers
excitation 494 nm, emission 517 nm) or tetramethylrhodamine of RSC, responses recorded from each neuron in the same slice
(TSA systems, excitation 550 nm, emission 570 nm). Sections were normalized to mean responses of all recorded neurons.
were mounted using Vectashield (Vector) and observed with a Data were then pooled into superficial and deep layers based on
confocal laser-scanning microscope (Olympus Fluoview FV10i) position of the soma relative to the border of layers 3 and 5 A.
at 40×. Areas of mCherry immunostaining (red) were identified, Relative to EPSCs, the IPSCs were much larger in amplitude
marked, and superimposed on vGlut2 images to determine (reflecting the increased driving force for inhibitory conduc-
colocalization. tances), and slightly slower in onset (by 2.6 ms on average,
reflecting the disynaptic activation of the inhibitory responses),
as expected for this method of sampling EPSCs and IPSCs in the
Slice electrophysiology and Optogenetics
same neuron by voltage manipulations (e.g., Apicella et al. 2012;
Mice were anesthetized with isoflurane and head-fixed on stereo- Xue et al. 2014).
taxic frame. Craniotomy was performed above SUB (from For spike pattern analysis, whole-cell recordings were per-
bregma, in mm: 2.4 posterior, 1.5 lateral) and a beveled glass formed from vGlut1+ or vGlut2+ RSC-projecting neurons identi-
pipette loaded with Cre-dependent virus encoding eGFP (AAV1- fied by co-labeling of eGFP and red Retrobeads. After 3 min
CAG-Flex-eGFP-WPRE-bGH, Upenn, AV-1-ALL854) or hChR2 from break in, current steps (from −200 to 800 pA at 100 pA
(AAV5-EF1a-DIO-hChR2(E123T/T159C)-EYFP, Upenn, AV-5–35 509) increment) were injected into the soma. The voltage traces
was injected unilaterally into SUB (1.6-mm deep from surface) using obtained at the threshold response were analyzed offline to
a hydraulic displacement injector (Narishige MO-10). For experi- measure interspike intervals.
ments involving analysis of spiking properties, red RetroBeads To test the effectiveness of hM4D(Gi) in silencing synaptic
(Lumafluor) were additionally injected into RSC (in mm: 1.6 poste- transmission in slices, AAV8-DIO-hM4D(Gi) and AAV5-DIO-
rior, 0.2 lateral). Furthermore, to label RSC-projecting vGlut1+ neu- hChR2 were infused into DH of either vGlut1- or vGlut2-Cre
rons, Cre-dependent retrograde AAV encoding tdTomato was mice via cannula. The infusion of hM4D(Gi) preceded that of
injected into RSC of vGlut1-Cre mice (Fig. 2). After 3–5 weeks of viral hChR2 by 3 weeks, and 3 more weeks were then allowed for
expression, brain was removed and coronal slices (250 μm) contain- viral expression; thus, hM4D(Gi) was expressed for 6 weeks to
ing RSC were prepared using a vibratome (Leica VT1200S) in ice- be consistent with expression time used in behavioral experi-
cold choline-based cutting solution containing (in mM): 25 NaHCO3, ments. RSC slices were then prepared, and whole-cell record-
1.25 NaH2PO3, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 110 choline chloride, 11.6 ings were made from layer 3 pyramidal neurons. Photostimuli
sodium L-ascorbate, and 3.1 sodium pyruvate, aerated with 95% O2 were delivered every 30 s to depolarize vGlut1+ or vGlut2+ DH
and 5% CO2. Slices were subsequently stored in a holding chamber terminals, evoking excitatory synaptic transmission detected
filled with aCSF containing (in mM): 127 NaCl, 25 D-glucose, 2.5 KCl, as EPSCs in the recorded postsynaptic neuron. After 5 min of
1 MgCl2, 2 CaCl2, and 1.25 NaH2PO3, aired with 95% O2 and 5% CO2, stable baseline recording, clozapine-N-oxide (CNO) (0.03 μM or
at 34 °C for 30 min and then at room temperature (~21 °C) for at 0.1 μM) was bath-applied and recording was continued for at
least 1 h before recording. least 5 min. As a control, separate groups of mice from each
Electrophysiological recordings were performed using an Cre-line were injected with only ChR2 into DH using a glass
upright microscope (BX51WI, Olympus) equipped with gradient- pipette, and same slice experiment was repeated. A 1 mM stock
contrast and epifluorescence optics and a blue LED (M470L2, of CNO solution was made daily before its bath application
Thorlabs) for photostimulation. Whole-cell recordings were made from powder (Sigma-Aldech, C0832-5MG) using H2O as a sol-
using borosilicate glass pipettes (~4–6 MΩ). For circuit analysis, to vent. Data were normalized to the mean baseline to assess the
measure excitatory postsynaptic currents (EPSCs) and inhibitory time-dependent effect of CNO at each concentration. To com-
postsynaptic current (IPSCs) the pipette was filled with a cesium- pare pre- and post-CNO EPSCs, traces from 1 min immediately
based internal solution composed of (in mM): 128 cesium metha- before and the fifth minute after CNO (0.1 μM) application was
nesulfonate, 10 HEPES, 10 phosphocreatine, 4 MgCl2, 4 ATP, 0.4 averaged, and 50 ms mean current over a poststimulus interval
GTP, 3 ascorbate, 1 EGTA, 1 mM QX-314, pH 7.25, 290–295 mOsm. of 50 ms was calculated.
To examine spike patterns, cesium methanesulfonate was
replaced with potassium methanesulfonate. Photostimulation of
Quantification and Statistical Analyses
ChR2-expressing axons was performed using 4× objective lens
(UPlanSApo, N/A 0.16, Olympus) focused on the slice, by briefly Statistical analyses were performed using SPSS software and
(5 ms) gating an output of blue LED (1.00 mW/mm2 intensity in Matlab functions. For the behavioral studies, context freezing
the specimen plane). To measure photo-evoked synaptic input, data were analyzed for treatment (CNO or Veh) as a factor using
recordings were performed in voltage-clamp mode. The com- 2-tailed Student’s t tests. Significant F values were followed by
mand potential was set to −70 mV (the approximate reverse post hoc comparisons using Tukey’s test. Homogeneity of vari-
potential of GABAergic current) to record EPSCs, then to 0–10 mV ance was confirmed with Levene’s test for equality of var-
(the approximate reverse potential of glutamatergic current) to iances. Statistical differences were considered significant for all
measure IPSCs. In one neuron, only EPSCs were recorded. P values <0.05. Group sizes were determined using power
 Hippocampal–Cortical Interactions Underlying Context Memory Yamawaki et al. | 2731

analyses assuming a moderate effect size of 0.5. All key find- Results
ings were replicated at least twice.
Chemogenetic Silencing of DH→RSC Terminals Impairs
Details of statistical analyses are found in figure legends. All
source data for the preparation of graphs and statistical analy- Memory Encoding of CFC
sis are presented online. All other relevant data that support To determine the functional role of DH→RSC projections in CFC
the conclusions of the study are available from the authors we used a chemogenetic approach with the inhibitory Designer
upon request. Receptors Exclusively Activated by Designer Drugs (DREADD)

Figure 1. Chemogenetic silencing of DH→ RSC terminals impairs memory encoding of CFC. (a) Schematic of virus infusions and cannula implantations. The Cre-
independent viral vector AAV-hM4D(Gi)-mCherry was injected into the DH 6 weeks before behavioral experiments. During the same surgery, cannulae were
implanted into the RSC bilaterally. (b) Immunostaining for mCherry showing expression of hM4D(Gi) in DH and its projection throughout ventral RSC (RSCv) but not
in ventral hippocampus (VH). (c) Effect of pretraining infusion of CNO on activity (cm/s) during context and shock exposure at training (left) and freezing during con-
text test (right). Pretraining infusions of Veh and CNO did not affect locomotor activity (Veh: 14.7 ± 2.33; CNO: 16.6 ± 1.48) or activity burst (Veh: 71.5 ± 7.52; CNO: 70.1
± 5.69) to the footshock (activity before shock: t = 0.71, P = 0.49; activity during shock: t = 0.15, P = 0.89; Veh n = 6, CNO n = 7). However, CNO significantly impaired
freezing at test when compared to vehicle (Veh)-injected controls (t = 5.843, P 0.001; Veh: 62.3 ± 3.03; CNO: 25.3 ± 5.30).

Figure 2. 2 populations of vGlut1+ and vGlut2+ DH neurons project to RSC. (a) Schematic of Cre-dependent AAV-DIO-hM4D(Gi)-mCherry infusion in DH of vGlut1-Cre
and vGlut2-Cre mice (left). Labeling of vGlut1+ (middle) and vGlut2+ (right) DH neurons 6 weeks after virus expression. The mCherry labeling patterns were similar to
the one of endogenous transporters (Supplementary Fig. 2). (b) Immunofluorescent staining for mCherry RSC (top) in vGlut1-Cre mice and vGlut2-Cre mice revealing
terminal fields in RSC layers 1 and 3. Immunostaining in DH is shown below. (c) Retrograde labeling of DH neurons projecting to RSC using Fluoro-Gold (top and bot-
tom left) or CTB (top and bottom right). Fluoro-Gold injected into RSC (top left) was detected predominantly in the subiculum (SUB, bottom left). Sparse signals were
also found in dorsomedial CA1. Similarly, injection of CTB (blue) in RSC labels SUB neurons of vGlut1-Cre mice expressing Cre-dependent retrograde AAV-flex-
dtTomato (red) (bottom left).
 2732 | Cerebral Cortex, 2019, Vol. 29, No. 6

hM4D(Gi) (Zhu et al. 2014; Jovasevic et al. 2015). We expressed

the hM4D(Gi) construct in DH neurons, including those that are
the source of axonal projections to RSC, by bilaterally infusing
AAV-hSyn-HA-hM4D(Gi)-mCherry into DH (Fig. 1a). To suppress
activity specifically in the DH→RSC circuit, we focally delivered
CNO into RSC bilaterally via cannula (Fig. 1a). Thus applied, CNO
locally blocks presynaptic release from hM4D(Gi)-expressing
axon terminals, without affecting the spiking activity of the
labeled neurons (Stachniak et al. 2014). Immunohistochemical
amplification of the mCherry signal confirmed that prominent
hM4D(Gi) expression in RSC axons originate from DH and no
viral spread into ventral hippocampus (Fig. 1b). After virus
expression, we injected 0.2 μl/site of CNO solution (0.3 μg/μL) into
RSC 30 min before CFC to silence DH axonal outputs to RSC neu-
rons during memory encoding (Fig. 1c). The selection of virus
expression and drug infusion times and doses was based on
control experiments (Supplementary Fig. 1a–c). During training,
CNO did not adversely affect gross sensory-motor function or
activity during context exploration or in response to footshock
(Fig. 1c). At test however, CNO-injected mice froze less than
vehicle-injected controls, demonstrating an important contribu-
tion of DH→RSC inputs to the encoding of context memory.

Projections from DH to RSC are Molecularly Distinct

Figure 3. Firing pattern of vGlut1+ and vGlut2+ RSC-projecting neurons in the dor-
Our next goal was to identify the molecular identity of gluta-
sal SUB. (a) Schematic of injections. Retrograde tracer was injected into RSC and
matergic DH→RSC projections. Cortical and thalamic excitatory AAV-flex-eGFP was injected into SUB of either vGlut1- or vGlut2-cre mice.
inputs to RSC have been distinguished by the presence of the (b) Epifluorescence image showing a population of SUB neurons labeled with tracer
vesicular glutamate transporters vGlut1 and vGlut2, respec- (left) and eGFP (right). (c) An example bright-field (left) and epifluorescence (right)
tively (Ichinohe et al. 2008). Because hippocampal neurons are image of recorded SUB neuron labeled with tracer and eGFP in vGlut1-cre mouse.
Typical firing pattern of vGlut1-positive RSC-projecting neuron is also shown
mostly vGlut1+ (Fremeau et al. 2001; Herzog et al. 2006), we
(middle). Interspike interval (ISI) histogram of threshold response shown by
hypothesized that DH→RSC projections would mainly express
vGlut1-positive RSC-projecting neurons (right). (d) Same as (c), but in vGlut2-Cre
vGlut1. To test this, we used genetically modified mice expres- mouse. Kolmogorov–Smirnov (KS) test comparing ISI distributions showed a sig-
sing Cre recombinase driven by the vGlut1 (vGlut1-Cre mice) nificant difference (KS test, P < 0.001, vGlut1+ n = 6, and vGlut2+ n = 8 neurons).
(Harris et al. 2014) or vGlut2 (vGlut2-Cre mice) (Vong et al. 2011)
promoter (Fig. 2a), and infused a Cre-dependent AAV-hSyn-
DIO-hM4D(Gi)-mCherry bilaterally into DH to restrict hM4D(Gi)- and vGlut2+ neurons labeled by infusing virus with cannula
mCherry expression to vGlut1+ or vGlut2+ neurons. The pat- into DH (Supplementary Fig. 3) or with glass pipette into SUB
terns of mCherry expression in DH in both mouse lines were (Supplementary Fig. 4) showed similar patterns, except for indi-
generally similar to those seen with anti-vGlut1 and anti- cated thalamic nuclei. With respect to entorhinal cortex, both
vGlut2 antibodies in wild-type mice (Supplementary Fig. 2a). applications showed strong vGlut1+ but relatively weak vGlut2+
Surprisingly, not only vGlut1+, but also vGlut2+ neurons sent positive terminal fields.
dense projections to the ventral RSC (Fig. 2b) predominantly in
layer 3, but also in layer 1. To determine the origin of DH→RSC vGlut2+ RSC-Projecting Neurons in the Dorsal SUB
projections, we performed retrograde labeling using Fluoro-
Generate More Feedforward Inhibition than vGlut1+
Gold or Cholera Toxin B (CTB) and found, consistent with ear-
Neurons
lier work (Wyss and Van Groen 1992; Van Groen and Wyss
2003) that most signals were localized in the subiculum (SUB), We next tested the circuit mechanisms by which DH inputs
which contains both vGlut1+ and vGlut2+ neuronal populations influence RSC activity, focusing on direct excitation of pyramidal
(Supplementary Fig. 2–4). neurons and their feedforward inhibition through the recruit-
We next performed experiments in slices (Fig. 3a,b) to deter- ment of local inhibitory neurons. We used slice-based electro-
mine the firing patterns of subicular vGlut1+ and vGlut2+ neu- physiology combined with optogenetics. AAV-DIO-hChR2-EYFP
rons projecting to RSC. Both neurons exhibited burst firing in was injected into DH of vGlut1- or vGlut2-Cre mice, and slices
response to depolarizing current injection at rheobase, the containing RSC were prepared 3–5 weeks later (Fig. 4a, top). In
degree of which was significantly greater for vGlut2+ neurons slices from both mice, strong labeling of EYFP-expressing SUB
(Fig. 3c,d). To examine colocalization of vGlut1+ and vGlut2+ axons was observed in layer 3 of RSC (Fig. 4a, bottom), consis-
neurons, immunostaining for mCherry (indicating vGlut1) and tent with the projection pattern of vGlut1+ and vGlut2+ DH
vGlut2 was performed in DH slices from vGlut1-Cre mice axons labeled with hM4D(Gi)-mCherry (Fig. 2b). We next per-
injected with Cre-dependent hM4D(Gi)-mCherry. This did not formed whole-cell recordings of pyramidal neurons in different
reveal co-immunolabeling (Supplementary Fig. 2b), consistent layers within the same slices, and photostimulated ChR2-
with earlier evidence for nonoverlapping distribution of vGlut1 expressing vGlut1+ or vGlut2+ SUB axons. In each neuron, we
and vGlut2 (Heise et al. 2016), and demonstrating that these 2 sampled the photo-evoked EPSC and IPSC (Fig. 4b–e). In RSC
populations of excitatory subicular neurons project onto the slices from vGlut1-Cre mice, stimulation of vGlut1+ SUB axons
same superficial layers of RSC. Overall, projection from vGlut1+ generated significantly larger EPSC and IPSC in neurons in
 Hippocampal–Cortical Interactions Underlying Context Memory Yamawaki et al. | 2733

Figure 4. vGlut2+ RSC-projecting neurons in the dorsal SUB generate more feedforward inhibition than vGlut1+ neurons. (a) Schematic of injection performed. AAV-
DIO-hChR2 was injected into subiculum of either vGlut1- or vGlut2-cre mice (top). Example bright-field and epifluorescence images of RSC slice prepared from vGlut1-
cre mouse. Laminar borders are indicated with dotted line (bottom). Layers are further divided into superficial and deep layers. SUB, subiculum. (b) EPSC and IPSC
recorded from pyramidal neurons in superficial (green) and deep layers of RSC in slice from vGlut1-Cre mice. Traces from 2 neurons are shown (left and right).
(c) Cumulative distribution histogram of normalized EPSC (left) and IPSC (right) evoked by stimulating vGlut1-positive subicular axons. Each input was normalized to
average input recorded from all neurons in same slice. EPSC to superficial (2.1 ± 0.36) versus deep (0.47 ± 0.16): P < 0.01, Kolmogorov–Smirnov (KS), n = 7 and 15,
4 slices. IPSC to superficial (2.9 ± 0.37) versus deep (0.32 ± 0.15): P = 0.001, k–s, n = 7 and 15, 4 slices. (d) Same as (b), but from vGlut2-Cre mice. (e) Same as (c), but for
EPSC and IPSC evoked by stimulating vGlut2-positive subicular axons. EPSC to superficial (2.2 ± 0.65) versus deep (0.39 ± 0.21): P = 0.001, KS test, n = 12 superficial and
22 deep neurons, 6 slices. IPSC to superficial (0.9 ± 0.58) versus deep (1.05 ± 0.56): P = 0.46, KS test, n = 12 and 21 neurons, 6 slices.

superficial layers relative to neurons in deep layers (Fig. 4b,c). vGlut2+ DH→RSC axon terminals expressing hM4D(Gi) was con-
When this experiment was repeated with RSC slices from firmed in vitro, by co-expressing ChR2 in the same axons and
vGlut2-Cre mice, photostimulation of vGlut2+ SUB axons evoked bath-applying CNO while recording photostimulation-evoked
greater EPSC in pyramidal neurons in superficial layers relative responses from postsynaptic RSC neurons (Supplementary
to deep layers (Fig. 4d,e), similar to the excitation pattern gener- Fig. 5a–c). These control experiments showed potent CNO silenc-
ated by stimulation of vGlut1+ SUB axons (Fig. 4c). In contrast, ing of SUB→RSC synaptic transmission when either vGlut1 or
IPSC generated through vGlut2+ SUB axons were highly variable vGlut2 axons co-expressed ChR2 and hM4D(Gi) (Supplementary
between superficial and deep layers, with some neurons in both Fig. 5b–d), but no effect of CNO on evoked responses when axons
layers receiving IPSC and some receiving weak to no IPSC expressed only ChR2 and not hM4D(Gi) (Supplementary Fig. 5e–
(Fig. 4d,e). g). Inactivation of DH axon terminals in RSC by CNO infusion
These results show that in RSC, both vGlut1+ and vGlut2+ before conditioning significantly impaired freezing in vGlut1-Cre,
SUB axons strongly excite pyramidal neurons in superficial but not vGlut2-Cre mice tested 24 h later (Fig. 5a). We also exam-
layers as well as local interneurons that regulate their activity ined the effect of the same circuit manipulation on lasting mem-
through feedforward inhibition. vGlut2+ SUB axons additionally ory consolidation by measuring freezing behavior 24 h and 35
recruit local interneurons that provide feedforward inhibition days after CFC. Inactivation of the vGlut1+ pathway caused per-
onto excitatory neurons in deep layers. sistent freezing deficits during both recent and remote memory
tests (Fig. 5b), indicating silencing of vGlut1+ terminals impairs
Chemogenetic Silencing of vGlut1+ and vGlut2+ encoding of context memory.
Interestingly, inactivation of the vGlut2+ pathway, despite
DH→RSC Terminals Differentially Affects Encoding,
not affecting freezing during the recent memory test, resulted
Consolidation, and Retrieval of CFC
in significant freezing impairments during the remote memory
Given the different effects of vGlut1+ and vGlut2+ DH→RSC term- test (Fig. 5b). Thus, activity of vGlut2+ DH→RSC projections dur-
inals on RSC excitability, we next sought to determine whether ing encoding appears to be important for later context memory
these projections play similar or unique roles in the processing of consolidation. Possibly, this pathway provides a putative early
hippocampus-dependent associative memories. In the first series signal (“tagging”) to cortical neurons needed for systems con-
of experiments, we examined the roles of vGlut1+ and vGlut2+ solidation of memory (Lesburgueres et al. 2011; Kitamura et al.
DH→RSC projections in CFC. We used vGlut1- and vGlut2-Cre 2017). We next studied whether vGlut1+ and vGlut2+ DH inputs
mice and Cre-dependent virus to restrict hM4D(Gi) expression to to RSC contribute to memory retrieval. Local silencing of
vGlut1+ or vGlut2+ DH neurons. Effectiveness of CNO in reducing vGlut1+ DH terminals in RSC before the test for recent but not
photo-evoked synaptic transmission from both vGlut1+ and remote memory impaired freezing (Fig. 5c). In contrast, local
 2734 | Cerebral Cortex, 2019, Vol. 29, No. 6

Figure 5. Chemogenetic silencing of vGlut1+ DH→ RSC terminals impair encoding and retrieval of contextual fear conditioning. (a) Experimental design similar to
Figure 1 except for infusion of a Cre-dependent AAV8-DIO-hM4D(Gi)-mCherry. Freezing during the context test was significantly reduced in vGlut1-Cre mice injected
with CNO when compared to Veh (Veh: 61.5 ± 4.68; CNO: 37.13 ± 6.99; t = 2.87, P < 0.05 (n = 8/group)), but not in vGlut2-Cre mice (Veh: 49.48 ± 5.32; CNO: 45.66 ± 6.41; t
= 0.45, P = 0.65; Veh n = 12, CNO n = 14). (b) Within-subject design was used to determine the effect of pretraining CNO on recent and remote memory. Significant
treatment effects were found for each genotype (vGlut1-Cre: F1,16 = 43.78, P < 0.001; n = 9/group; vGlut2-Cre: F1,16 = 10.91, P < 0.01; Veh n = 12, CNO n = 14). However,
vGlut1-Cre mice receiving CNO before training showed reduced freezing both at recent (Veh: 54.3 ± 6.36; CNO: 7.56 ± 2.29; P < 0.05) and remote memory tests (Veh:
55.8 ± 6.09; CNO: 22.7 ± 5.79; P < 0.01), whereas similarly treated vGlut2-Cre mice showed freezing deficits only at the remote (Veh: 61.3 ± 6.35; CNO: 30.9 ± 5.96;
P < 0.01), but not recent test (Veh: 58.2 ± 3.86; CNO: 57.7 ± 5.06). (c) Infusion of CNO before the recent memory test impaired freezing to the conditioning context in
vGlut1-Cre mice (Veh: 64.6 ± 3.08; CNO: 37.2 ± 6.98; t = 3.14, P < 0.01; n = 9/group) without affecting freezing in vGlut2-Cre mice (Veh: 47.5 ± 7.73; CNO: 53.9 ± 8.46;
t = 0.56, P = 0.58; n = 7/group). Infusion of CNO before the remote memory test was ineffective in either mouse line (vGlut1-Cre, Veh: 36.2 ± 5.04; CNO: 38.2 ± 6.32;
t = 0.61, P = 0.54; n = 15/group; vGlut2-Cre: Veh: 49.9 ± 8.62; CNO: 54.8 ± 8.78; t13 = 0.39, P = 0.7; n = 7/group).

silencing of vGlut2+ DH terminals had no effect on either recent these synapses also exert different effects at the level of cellu-
or remote memory retrieval (Fig. 5c). lar circuits. Namely, vGlut1+ and vGlut2+pathways arose from
Together, these findings reveal a key role of vGlut1+ presynaptic neurons with distinct burst-firing properties, which
DH→RSC projections in the processing of recent context memo- furnished monosynaptic excitation to RSC excitatory neurons
ries as well as a contribution of vGlut2+ DH→RSC projections to in a similar way, preferentially innervating superficial- rather
systems consolidation of context memories. than deep-layer pyramidal neurons. However, feedforward (dis-
ynaptic) inhibition, while similarly restricted to pyramidal neu-
rons in superficial layers when triggered by vGlut1+ DH
Discussion terminals, was detected in both superficial- and deep-layer RSC
By combining optogenetic, chemogenetic, and behavioral analy- pyramidal neurons when triggered by vGlut2+ DH terminals.
ses, we delineate vGlut1+ and vGlut2+ DH→RSC circuits that play These similarities and differences are consistent with, and
distinct roles in the processing of contextual associative memo- likely contribute to the distinct roles of vGlut1+ and vGlut2+
ries. Encoding and retrieval of recent context memories required pathways in the formation and persistence of stress-related
vGlut1+ afferents, with vGlut2+ afferents contributing to the sys- context memories.
tems consolidation and persistence of these memories. Consistent with our findings with recent context memory,
Interestingly, although DH receives vGlut1+ and vGlut2+ pre- haploinsufficiency of vGlut1, but not vGlut2, impairs learning
synaptic terminals from different sources [vGlut1+ presynaptic and memory (Callaerts-Vegh et al. 2013). Although develop-
terminals from the trisynaptic hippocampal circuit and from mental effects of vGlut2 have been reported, dysfunction of
the cortex (Balschun et al. 2010; Zander et al. 2010) and vGlut2+ this transporter is accompanied by down-regulation of vGlut1
presynaptic terminals from the supramamillary nucleus or hil- as well (He et al. 2012), so the individual roles of vGlut1+ and
lar mossy cells (Halasy et al. 2004; Boulland et al. 2009)], DH vGlut2+ pathways could not be dissected with genetic tools.
neurons send both vGlut1+ and vGlut2+ projections to RSC. The Chemogenetic silencing, however, identified a discrete role of
importance of this overlap is revealed by the synergistic contri- vGlut2+ DH→RSC pathways in the persistence of fear-inducing
bution of these projections to the formation and persistence of context memories. Importantly, early silencing of vGlut2+ term-
fear-inducing context memories. inals during memory encoding was required for disruption of
The distinct contribution of vGlut1+ and vGlut2+ DH→RSC later consolidation, but the same manipulation was ineffective
projections to memory processing is not surprising given the during retrieval of remote memory. These findings are in line
known differences between vGlut1+ and vGlut2+ synapses in with the time-limited role of DH in context memory encoding
probability of transmitter release and synaptic plasticity (Kim and Fanselow 1992), and with previous observations
(Fremeau et al. 2004; Boulland et al. 2009). Here we show that showing that hippocampal input to the neocortex during early
 Hippocampal–Cortical Interactions Underlying Context Memory Yamawaki et al. | 2735

stages of memory encoding is needed for latter systems consol- threshold-spiking interneurons in mouse motor cortex.
idation of that memory (Lesburgueres et al. 2011; Kitamura J Neurosci. 32:7021–7033.
et al. 2017). The molecular basis of this phenomenon is not Balschun D, Moechars D, Callaerts-Vegh Z, Vermaercke B, Van
known beyond a hypothetical memory “tag”, however, our find- Acker N, Andries L, D’Hooge R. 2010. Vesicular glutamate
ings provide initial indication that such tag might be provided transporter VGLUT1 has a role in hippocampal long-term
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this view was recently challenged by findings demonstrating dif- and GABA transporters sort to distinct sets of vesicles in a
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projections in context memory encoding and retrieval, respec- Callaerts-Vegh Z, Moechars D, Van Acker N, Daneels G, Goris I,
tively (Roy et al. 2017). While we cannot rule out such possibility, Leo S, Naert A, Meert T, Balschun D, D’Hooge R. 2013.
given that our virus infusions targeted the entire DH, a similar Haploinsufficiency of VGluT1 but not VGluT2 impairs
scenario is not very likely because CA1 DH→RSC projections were extinction of spatial preference and response suppression.
very scarce. Behav Brain Res. 245:13–21.
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consolidation of memory downstream of the DH-entorhinal hippocampal field CA1 axonal projections to the rest of the
cortical circuit (Kitamura et al. 2017). It can be speculated, cerebral cortex. Brain Res Rev. 56:1–26.
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nisms of memory either upstream of the entorhinal cortex retrosplenial cortex are necessary for retrieval of recent and
(through a DH-RSC- entorhinal cortex circuit) or through a par- remote context fear memory. J Neurosci. 31:11655–11659.
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opportunity for different treatment approaches for memory- tical memory of context. Neuron. 84:432–441.
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Supplementary Material 2004. The involvement of the anterior cingulate cortex in
Supplementary material is available at Cerebral Cortex online. remote contextual fear memory. Science. 304:881–883.
Fremeau RT Jr., Kam K, Qureshi T, Johnson J, Copenhagen DR,
Storm-Mathisen J, Chaudhry FA, Nicoll RA, Edwards RH.
Authors’ Contributions 2004. Vesicular glutamate transporters 1 and 2 target to
J.R. designed the behavioral experiments. N.Y. and G.S. designed functionally distinct synaptic release sites. Science. 304:
and N.Y. conducted the in vitro electrophysiological experiments. 1815–1819.
K.C., J.K., and A.G. conducted the behavioral experiments and Fremeau RT Jr., Troyer MD, Pahner I, Nygaard GO, Tran CH,
the histology. N.Y. and A.G. conducted the tract-tracing studies; Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J,
N.Y., K.C., and J.R. analyzed the data. J.R. and G.S. secured the Edwards RH. 2001. The expression of vesicular glutamate
funding. N.Y., G.S., and J.R. wrote the manuscript. All authors transporters defines two classes of excitatory synapse.
discussed and commented on the manuscript. Neuron. 31:247–260.
Gao C, Gill MB, Tronson NC, Guedea AL, Guzman YF, Huh KH,
Corcoran KA, Swanson GT, Radulovic J. 2010. Hippocampal
Funding NMDA receptor subunits differentially regulate fear memory
This work was funded by NIMH grants to J.R. (MH108837 and formation and neuronal signal propagation. Hippocampus.
MH078064) and NINDS grant to G.S. (NS061963). 20:1072–1082.
Halasy K, Hajszan T, Kovacs EG, Lam TT, Leranth C. 2004.
Distribution and origin of vesicular glutamate transporter 2-
Notes immunoreactive fibers in the rat hippocampus. Hippocampus.
We thank Hongkui Zeng (Allen Brain Institute) for feedback on 14:908–918.
the breeding, genotyping, and overall characterization of Harris JA, Hirokawa KE, Sorensen SA, Gu H, Mills M, Ng LL,
vGlut1-Cre mice. Undergraduate students Jinhak Kim, Gabriel Bohn P, Mortrud M, Ouellette B, Kidney J, et al. 2014.
Hast, and Helen Chen for their help with the maintenance of Anatomical characterization of Cre driver mice for neural
mouse lines and animal surgery and care. Conflict of Interest: circuit mapping and manipulation. Front Neural Circuits. 8:
None declared. 76.
He H, Mahnke AH, Doyle S, Fan N, Wang CC, Hall BJ, Tang YP,
Inglis FM, Chen C, Erickson JD. 2012. Neurodevelopmental
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