Article

Cortical regulation of neurogenesis and cell

proliferation in the ventral subventricular zone
Graphical abstract Authors
Moawiah M. Naffaa, Rehan R. Khan,
Chay T. Kuo, Henry H. Yin

Correspondence
moawiah.naffaa@duke.edu (M.M.N.),
hy43@duke.edu (H.H.Y.)

In brief
Naffaa et al. identify a frontal cortical
circuit regulating postnatal SVZ
neurogenesis. The circuit includes
calretinin+ GABAergic interneurons and
anterior cingulate glutamatergic neurons,
which provide bidirectional control of the
activity of subep-ChAT+ neurons and
modulate NSC proliferation and
neurogenesis in the ventral SVZ.

Highlights
d VGlut1+ anterior cingulate cortical neurons excite subep-
ChAT+ neurons

d Local calretinin+ interneurons inhibit subep-ChAT+ neurons

d The ACC circuit regulates neurogenesis and NSC

proliferation in the ventral SVZ

d Subep-ChAT+ and inhibitory CR+ neurons control the circuit’s

impact on SVZ neurogenesis

Naffaa et al., 2023, Cell Reports 42, 112783

July 25, 2023 ª 2023 The Author(s).
https://doi.org/10.1016/j.celrep.2023.112783 ll
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Article
Cortical regulation of neurogenesis and cell proliferation
in the ventral subventricular zone
Moawiah M. Naffaa,1,2,* Rehan R. Khan,2 Chay T. Kuo,2 and Henry H. Yin1,3,4,*
1Department of Psychology and Neuroscience, Duke University, Durham, NC, USA
2Department of Cell Biology, Duke University School of Medicine, Durham, NC, USA
3Department of Neurobiology, Duke University School of Medicine, Durham, NC, USA
4Lead contact

*Correspondence: moawiah.naffaa@duke.edu (M.M.N.), hy43@duke.edu (H.H.Y.)

https://doi.org/10.1016/j.celrep.2023.112783

SUMMARY

Neurogenesis and differentiation of neural stem cells (NSCs) are controlled by cell-intrinsic molecular path-
ways that interact with extrinsic signaling cues. In this study, we identify a circuit that regulates neurogenesis
and cell proliferation in the lateral ventricle-subventricular zone (LV-SVZ). Our results demonstrate that direct
glutamatergic projections from the anterior cingulate cortex (ACC), as well as inhibitory projections from
calretinin+ local interneurons, modulate the activity of cholinergic neurons in the subependymal zone
(subep-ChAT+). Furthermore, in vivo optogenetic stimulation and inhibition of the ACC-subep-ChAT+ circuit
are sufficient to control neurogenesis in the ventral SVZ. Both subep-ChAT+ and local calretinin+ neurons play
critical roles in regulating ventral SVZ neurogenesis and LV-SVZ cell proliferation.

INTRODUCTION Subep-ChAT+ neurons release acetylcholine (ACh) into the SVZ

The rodent subventricular zone (SVZ) of the lateral ventricles
(LVs) is a major site of postnatal neurogenesis.1 Neurogenesis
in SVZ provides a useful model system for studying neuronal
regeneration and tissue remodeling in the adult brain.2,3 LV neu-
ral stem cells (NSCs) divide asymmetrically for self-renewal or
differentiate to become transient amplifying intermediate pro-
genitors.4–6 These intermediate progenitors divide and differen-
tiate into doublecortin-positive neuroblasts,7,8 which migrate to
the olfactory bulb,7,9,10 where they become mature interneurons
that are incorporated into the local circuitry.11,12 The generation
of adult-born neurons from the SVZ contributes to experience-
dependent plasticity in the postnatal brain13,14 and has been
implicated in social behavior.12,15,16
The LV NSCs receive inputs from a variety of sources, in-
cluding neighboring NSCs, TAPs (transit-amplifying progeni-
tors), and neuroblasts.17,18 Different neurotransmitters, including
gamma-aminobutyric acid (GABA), dopamine, and serotonin,
released by neurons in different brain regions, are also known
to regulate postnatal and adult SVZ neurogenesis.19–24 However,
little is known about the local and distal circuitry that directly or
indirectly influences the SVZ neurogenesis.
Recent work has identified a small population of cholinergic
neurons in the subependymal space to the LV. These neurons,
which express choline acetyltransferase (ChAT+), can modulate
the proliferation of LV NSCs and neuroblasts in an activity-
dependent manner.25 Optogenetic stimulation of subep-ChAT+
neurons with concurrent electrophysiological recording from
local NSCs revealed that LV NSCs respond to the activity of local
ChAT+ neurons.
niche to promote the proliferation of LV NSCs.25 We hypothesize
that subependymal ChAT+ (subep-ChAT+) neurons in the ventral
SVZ provide a key node in the neural circuit regulation of SVZ neu-
rogenesis and LV NSCs proliferation. Using viral tracing, electro-
physiology, and in vivo optogenetics and chemogenetics, we
have identified a neural circuit involving anterior cingulate glutama-
tergic projections and local calretinin-positive (CR+) GABAergic in-
terneurons. That circuit can regulate the activity of subep-ChAT+
neurons. We showed that this circuit directly modulates SVZ neuro-
genesis, LV NSC proliferation, and cell division in the ventral SVZ.
These findings reveal an unprecedented neural circuit through
which frontal cortical inputs can influence cholinergic signaling in
the ventral SVZ to modulate neurogenesis and cell proliferation.
RESULTS
Excitatory and inhibitory presynaptic inputs to the
subep-ChAT+ neurons
Unlike striatal cholinergic neurons, subep-ChAT+ neurons do not
show tonic activity in the absence of synaptic inputs, suggesting
that excitatory inputs are needed to activate these neurons.26
However, it is currently unknown where the excitatory inputs
come from and how they influence the activity of subep-ChAT+
neurons. We hypothesized that glutamatergic inputs from
cortical projection neurons may excite the subep-ChAT+ neu-
rons. The cortical neurons mainly express vesicular glutamate
transporter 1 protein (VGlut1).27 To selectively stimulate these
glutamatergic inputs, we crossed VGlut1-Cre mice with ChAT-
eGFP and ChR2-tdTomato transgenic mouse line (Ai27) that ex-
presses channelrhodopsin in a Cre-dependent manner.28 The

Cell Reports 42, 112783, July 25, 2023 ª 2023 The Author(s). 1
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resulting VGlut1-Cre; ChAT-eGFP; Ai27 mice express channelr-
hodopsin in VGlut1+ neurons (Figure 1A). Whole-cell recordings
from these mice showed robust firing in response to 473-nm light
activation of VGlut1+ axon terminals (firing frequency = 5.38 ±
0.32 Hz) (Figure 1B). In voltage-clamp mode (holding at

60 mV), photo-stimulation evoked excitatory postsynaptic cur-
rents (EPSCs) in the subep-ChAT+ neurons (current amplitude =
98.2 ± 10.7 pA and current latency = 8.6 ± 0.62 ms). The re-
corded currents can be blocked by glutamate receptors
blockers AP5 (NMDA receptor antagonist) and CNQX (AMPA re-
ceptor antagonist) (Figure 1C). These findings demonstrate the
existence of cortical glutamatergic inputs that drive and regulate
the activity of subep-ChAT+ neurons.
To examine the inhibitory inputs (i.e., VGAT+; the vesicular
GABA transporter) to subep-ChAT+ neurons, we crossed
VGat-ChR2-eYFP (i.e., channelrhodopsin expressed in
GABAergic neurons only) with ChAT-Cre and Ai9. VGat-ChR2-
eYFP; ChAT-Cre; Ai9 mice allow us to record from subep-
ChAT+ neurons in animals with channelrhodopsin expressed
in GABAergic neurons (Figure 1D). Whole-cell recordings
from subep-ChAT+ neurons in voltage-clamp mode (holding at

60 mV) and activating VGAT+ axon terminals consistently eli-
cited inhibitory postsynaptic currents (IPSCs) in the subep-
ChAT+ neurons. These IPSCs were blocked by picrotoxin
(GABAA receptors antagonist) (current amplitude = 196.1 ±
24.13 pA and current latency = 4.5 ± 0.88 ms) (Figure 1E).
Developing rabies virus strategy for tracing a neural
circuit
Our initial electrophysiological recording results established the
presence of excitatory and inhibitory synaptic inputs that can
regulate the activity of subep-ChAT+ neurons (Figure 1F). How-
ever, it remains unclear exactly where these inputs originated.
To answer this question, we generated Cre-dependent R26R-
FLEX-TVA-2A-RabiesG-2A-tdTomato-FLEX (R26F-RTT) mice
to trace the connectivity of subep-ChAT+ neurons using a single
rabies viral injection (Figures 1G and 1H). This method allows for
the expression of TVA, RabiesG, and tdTomato in all Cre-ex-
pressing neurons. The R26F-RTT mice were first tested by in-
jecting EnvA G-deleted rabies-eGFP virus into the striatum. We
did not detect non-specific rabies infections at the injection
site (Figures S1A and S1B).
To determine the expression of TVA, RabiesG, and tdTomato
in Cre-dependent mice, R26F-RTT mice were crossed with
ChAT-Cre, VGat-Cre, and D2-Cre. In R26F-RTT; ChAT-Cre
mice, ChAT+ neurons were found to express tdTomato (Fig-
ure S1C), which indicates that R26F-RTT mice are selectively ex-
pressed in Cre-positive neurons. To assess the retrograde trans-
synaptic spread of rabies with R26F-RTT, EnvA G-deleted
rabies-eGFP virus was injected into the striatum of R26F-RTT;
VGat-Cre and R26F-RTT; D2-Cre mice (Figures S2A and S2C).
After 7 days, the cortical excitatory neurons in cingulate and sec-
ondary motor cortices projecting to the striatal neurons were
found to be labeled with GFP (Figures S2B and S2D). Together,
these results demonstrated that R26F-RTT mice can express
TVA, RabiesG, and tdTomato selectively in Cre-expressing neu-
rons and allows for retrograde tracing.
To discover the inputs to the subep-ChAT+ neurons, we gener-
ated ChAT-Cre; R26F-RTT mice to express the TVA receptor,
RabiesG protein, and tdTomato in all cholinergic neurons.
We first injected EnvA G-deleted rabies-eGFP virus into the
striatum of P30 ChAT-Cre; R26F-RTT mice (Figure S3A). As

Figure 1. Glutamatergic and GABAergic inputs to subep-ChAT+ neurons and rabies virus infection of these cholinergic neurons in SVZ
(A) Illustration of electrophysiological recording of excitatory inputs (VGlut1+) to the subep-ChAT+ neuron from the wholemount of P30-50 VGlut1-Cre; ChAT-
eGFP; Ai27 mice. Scale bar, 10 mm.
(B) Representative trace from whole-cell current clamp recording of evoked action potential (APs) from subep-ChAT+ neurons upon blue (473-nm) light stim-
ulation for 5 s (left). Mean frequency (right). n = 4 (biological replicates), one-sample t test. Each dot represents data from one subep-ChAT+ neuron.
(C) Representative trace of EPSCs that were obtained in whole-cell voltage-clamp recordings from subep-ChAT+ neurons after photo-stimulation for 500 ms
(black) and the application of AMPA and NMDA receptors antagonists; CNQX and AP-5, respectively (red) (left). Mean evoked EPSC amplitude and latency (right).
n = 4 (biological replicates), one-sample t test. Each dot represents the amplitude (upper) and latency (lower) of a single EPSC recorded from subep-ChAT+
neuron.
(D) Illustration of electrophysiological recording of inhibitory inputs (VGat+) to the subep-ChAT+ neuron from P30-50 VGat-ChR2-eYFP; ChAT-Cre; Ai9 mice.
Scale bar, 10 mm.
(E) Representative trace of electrophysiological recordings of IPSCs that were obtained in whole-cell recordings from subep-ChAT+ neurons upon blue (473 nm)
light stimulation for 500 ms (black), and the application of GABAAR antagonist; picrotoxin (blue) (left). Mean evoked IPSC amplitude and latency (right). n = 4
(biological replicates), one-sample t test.
(F) Schematic representation of presynaptic (excitatory and inhibitory) inputs to the subep-ChAT+ neuron.
(G) Schematic representation of R26R-FLEX-TVA-2A-RabiesG-2A-tdTomato-FLEX (R26F-RTT) mice before and after Cre recombinase.
(H) Schematic of EnvA G-deleted rabies-eGFP virus injection into the brain of -Cre; R26F-RTT mice. It represents monosynaptic retrograde tracing using R26F-
RTT mouse tool.
(I) Experimental representation of EnvA G-deleted rabies-eGFP virus injection into lateral ventricle (LV) of P30 Chat-Cre; R26F-RTT mice. Mice were sacrificed
7 days after injection.
(J and K) Immunofluorescence staining for GFP (green) and ChAT (purple) in SVZ and striatum of ipsilateral (injected) side of Chat-Cre; R26F-RTT mice (7 days
post injection). Scale bar, 30 mm.
(L) Experimental representation of EnvA G-deleted rabies-eGFP virus injection into LV of P30 Chat-Cre; R26F-RTT mice. Mice were sacrificed 14 days after
injection.
(M and N) Immunofluorescence staining for GFP (green) and ChAT (purple) in SVZ and striatum of the ipsilateral (injected) side of Chat-Cre; R26F-RTT mice
(14 days post injection). Scale bar, 30 mm.
(O) Schematic representation of the connectivity of striatal cholinergic and subep-ChAT+ neurons. All blue bars represent the duration of light stimulation. All data
presented as mean ± SEM.
See also Figures S1, S2, and S3.

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the striatal cholinergic neurons receive synaptic inputs from
each other,29 an infection of a striatal cholinergic neuron with
EnvA G-deleted rabies-eGFP virus results in labeling of neigh-
boring striatal cholinergic neurons, which subsequently pass
rabies to other connected neurons (Figures S3B and S3C).
Then, EnvA G-deleted rabies-eGFP virus was injected into the
LV of ChAT-Cre; R26F-RTT mice to target subep-ChAT+ neu-
rons, immediately following mannitol injection to disrupt the
ependymal layer locally (Figures 1I and 1L). Using this strategy,
we were able to selectively infect subep-ChAT+ neurons and
trace their presynaptic inputs (Figures 1J and 1M). The local con-
nectivity of subep-ChAT+ neurons and their projections were
checked at 7 and 14 days after rabies injection (Figures 1J, 1K,
1M, and 1N). Unlike striatal cholinergic neurons, subep-ChAT+
neurons in the SVZ niche are not connected with neighboring
striatal cholinergic neurons (Figure 1O).
Local inhibitory input to subep-ChAT+ neurons
Injection of EnvA G-deleted rabies-eGFP virus into the LV of
ChAT-Cre; R26F-RTT mice revealed local interneurons (GFP+)
adjacent to the infected subep-ChAT+ neurons (Figures 2A and
2B). There was colocalization of GFP+ interneurons and CR, sug-
gesting that CR+ interneurons provide local inhibitory inputs to
subep-ChAT+ neurons (Figure 2C). To test this possibility, we per-
formed whole-cell patch-clamp recording from subep-ChAT+
neurons of Cr-Cre; ChAT-eGFP; Ai27 mice (Figure 2D). We stimu-
lated local CR+ interneurons with blue light and evoked IPSCs,
which were completely blocked by picrotoxin (current amplitude =
122.3 ± 9.0 pA and current latency = 4.9 ± 0.33 ms) (Figure 2E). Op-
togenetic stimulation of other common inhibitory interneurons in
the cortex (calbindin+, somatostatin+, or parvalbumin+) did not
elicit IPSCs in subep-ChAT+ neurons (Figures S4A and S4B).
To further confirm local CR+ interneuron inputs to the subep-
ChAT+ neurons, we used a Patterned Light Stimulator LED
controller (Mightex) to target CR+ labeled neurons in SVZ whole-
mount of Cr-Cre; ChAT-eGFP; Ai27 mice. Focal optogenetic
activation of a single CR+ interneuron generated IPSCs in the
subep-ChAT+ neuron (current amplitude = 40.14 ± 3.78 pA and
current latency = 4.7 ± 0.4 ms) (Figures 2F and 2G). We were
able to identify a local inhibitory circuit with CR+ interneurons
that inhibits subep-ChAT+ neurons. Interestingly, our staining re-
sults reveal two to four CR+ interneurons surrounding each
subep-ChAT+ neuron in the LV-SVZ (Figures 2H and 2I).
Excitatory inputs from anterior cingulate cortex Cg1
area to subep-ChAT+ neurons
To determine connections for subep-ChAT+ neurons in the brain,
EnvA rVSV-eGFP (EnvA/RABVG-eGFP) virus30 was injected into
the LV of ChAT-Cre; R26F-RTT mice (Figure 3A). Distal GFP+
projection neurons from the anterior cingulate cortex (ACC)
were observed in the ipsilateral hemisphere only, showing ipsi-
lateral projections from the cortex to subep-ChAT+ neurons (Fig-
ure 3B). To confirm the cortical input to subep-ChAT+ neurons,
pAAV-CaMKIIa-hChR2(E123A)-mCherry virus was first injected
into the ACC region to express mCherry in the GFP+ neurons
from Figure 3B (Figure 3C). By tracing their projections in the
SVZ region, we observed them adjacent to the subep-ChAT+
neurons (Figure 3D).
To examine the direct projection from the ACC to the subep-
ChAT+ neurons, a general ChR2 viral vector (pAAV-CaMKIIa-
hChR2(E123T/T159C) -EYFP) was injected into the ACC region
of Chat-Cre; Ai9 mice to express ChR2 in the projections of
ACC neurons (Figure 3E). Optogenetic stimulation of the ACC
projections reliably evoked action potentials (APs) in subep-
ChAT+ neurons (firing frequency = 2.5 ± 0.327 Hz) (Figure 3F).
Stimulation induced reliable EPSCs in subep-ChAT+ neurons,
which were blocked by the glutamate antagonists (AP5 and
CNQX) (current amplitude = 64.86 ± 2.424 pA and current la-
tency = 9.1 ± 0.7 ms) (Figure 3G). These results confirm the pres-
ence of glutamatergic neurons in the ipsilateral ACC region that
directly excite subep-ChAT+ neurons.
To further confirm the ACC-subep-ChAT+ circuit, pAAV-Ef1a-
DIO hChR2(E123T/T159C)-mCherry virus was injected into the
ACC region of VGlut1-Cre; ChAT-eGFP mice to express ChR2
in ACC VGlut1+ neurons (Figure 3H). EPSCs were recorded in
subep-ChAT+ neurons upon optogenetic stimulation of ACC
VGlut1+ neurons (Figure 3I, left). The currents were completely
blocked by AP5 and CNQX (current amplitude = 53.29 ± 2.96 pA
and current latency = 8.7 ± 0.44 ms) (Figure 3I, right). These find-
ings show that a specific population of cortical neurons in the ACC
region provides excitatory drive to subep-ChAT+ neurons.
Cr-Cre mice label the ACC excitatory input to the subep-
ChAT+ neurons
The ACC-subep-ChAT+ circuit was then further tested by inject-
ing AAVrg viruses (i.e., retrograde tracing using adeno-associ-
ated viruses [AAVs]) into LVs of C57BL/6J, VGlut1-Cre and

Figure 2. Local CR+ GABAergic interneurons provide inhibitory inputs to subep-ChAT+ neurons
(A) Experimental design of EnvA G-deleted rabies-eGFP virus injection into LV of P30 Chat-Cre; R26F-RTT mice.
(B and C) Immunofluorescence staining for GFP (green), CR (purple), and DAPI (blue) in ipsilateral SVZ wholemount (injected) of P37 Chat-Cre; R26F-RTT mice.
Scale bar, 30 mm (B) and 5 mm (C).
(D) Illustration of electrophysiological recording of CR+ inhibitory inputs to the subep-ChAT+ neurons from SVZ wholemount of P30-50 Cr-Cre; ChAT-eGFP; Ai27
mice. Scale bar, 10 mm.
(E) Representative trace of evoked IPSCs from subep-ChAT+ neurons upon photo-stimulation for 500 ms (black) and after application of GABAAR antagonist
picrotoxin (green) (left). Mean current amplitude and latency of the IPSCs upon stimulation (right). n = 5 (biological replicates), one-sample t test.
(F) Representation of subep-ChAT+ neuron recording from SVZ wholemount of P30-50 Cr-Cre; ChAT-eGFP; Ai27 mice. (Left) subep-ChAT+ neuron (473-nm light).
(Middle) subep-CR+ neurons (590-nm light). Right: Bright-field image showing both subep-ChAT+ and -CR+ neurons.
(G) Representative trace of evoked IPSCs in subep-ChAT+ neurons following photo-stimulation for 100 ms (left). Mean current amplitude and latency of the IPSCs
upon stimulation (right). n = 3 (biological replicates), one-sample t test.
(H and I) Immunofluorescence staining for CR (purple) and ChAT (green) in the SVZ wholemount (H) and coronal section (I) of P28 C57BL/6J mice. Scale bar,
10 mm (H) and 20 mm (I). All blue bars represent the duration of light stimulation. All data presented as mean ± SEM.
See also Figure S4.

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Figure 3. Anterior cingulate neurons (VGlut1+ and CR+) project directly to subep-ChAT+ neurons
(A) Schematic: EnvA rVSV-eGFP virus injection into LV of P30 Chat-Cre; R26F-RTT mice.
(B) Immunofluorescence: GFP (green) in anterior cingulate cortex of injected side of P33 Chat-Cre; R26F-RTT mice. Scale bar, 100 mm (left) and 30 mm (right).
(C) Schematic: AAV-CaMKII-hChR2(E123A)-mCherry virus injection into ACC region of P30 C57BL/6J mice.
(D) Immunofluorescence: ChAT (green) and infected projections (red) of the ipsilateral ACC neurons in striatum and SVZ (left), and SVZ (right) of mice in (C). Scale
bar, 100 mm (left) and 30 mm (right).

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CR-Cre mice. Upon injection of pAAVrg-hSyn-mCherry and
pAAVrg-hSyn-DIO-mCherry viruses into LVs of C57BL/6J and
VGlut1-Cre mice, respectively (Figures S5A and S5C), we
observed mCherry+ neurons in the ipsilateral ACC region
(Figures S5B and S5D). Surprisingly, injecting pAAVrg-hSyn-
DIO-mCherry virus into the LV of Cr-Cre mice labels neurons in
the ipsilateral ACC region (Figures 3J and 3K). The detected
mCherry+ neurons in the ACC of C57BL/6J, VGlut1-Cre, and
CR-Cre mice post pAAVrg virus injections and the GFP+ ACC
neurons in Figure 3B are located along the same rostro-caudal
axis of the ACC region. These findings strongly suggest that
the ACC glutamatergic drivers of subep-ChAT+ neurons are
CR+ during the postnatal period. To test this possibility, we per-
formed whole-cell recordings from subep-ChAT+ neurons in Cr-
Cre; ChAT-eGFP; Ai27 mice (Figure 3L). In voltage mode at

60 mV, optogenetic stimulation of CR+ terminals reliably
evoked EPSCs (current amplitude = 106 ± 6.68 pA and current
latency = 5.13 ± 0.44 ms), which were completely blocked by
AP5 and CNQX (Figure 3M). This observation supports that
cortical glutamatergic neurons that excite subep-ChAT+ neurons
are CR+ during the postnatal period. In addition, we injected
AAV-EF1a-DIO-hChR2(E123T/T159C)-mCherry virus into the
ACC region of Cr-Cre; ChAT-eGFP mice (Figure 3N). Whole-
cell recording from subep-ChAT+ neurons in voltage-clamp
mode induced EPSCs upon optogenetic stimulation (current
amplitude = 33 ± 2.46 pA, and latency = 5.13 ± 0.44 ms)
(Figure 3O).
The total number of labeled ACC cortical neurons with Cr-Cre
mice during postnatal period must be lower than the number of
labeled neurons with VGlut1-Cre mice. Thereby, the Cr-Cre
mouse is a reliable tool to study the population of glutamatergic
neurons from the ACC region that control the activity of subep-
ChAT+ neurons (Figure 3P).
In vivo ACC-subep-ChAT+ circuit stimulation regulates
neurogenesis in the ventral domain of SVZ
After determining that the ACC area contains presynaptic excit-
atory drivers of subep-ChAT+ neurons, we studied the in vivo
functional role of these projections on SVZ neurogenesis and
LV cell proliferation. We hypothesized that this circuit regulates
SVZ neurogenesis surrounding subep-ChAT+ neurons by modu-
lating the cell proliferation in the ventral SVZ. AAV-EF1a-DIO-
hChR2 (E123T/T159C)-mCherry virus was injected into ipsilat-
eral ACC neurons to express ChR2 in the projections of
Cre-driver line, and optical fibers were implanted into the ACC
regions of Cr-Cre mice (Figure 4A). The optogenetic stimulation
was performed for 2 or 3 days and delivered by TTL (tran-
sistor-transistor logic) control of 473-nm laser, 10-ms pulses at
10 Hz, lasting 10 s, given once every 1 min (Figures 4B and S6A).
In the 2 days of circuit stimulation experiments, the mice were
perfused immediately after stimulation cessation, and brain sli-
ces of the stimulated ACC sections were stained against
ChAT, phospho-S6 ribosomal (P-S6), and doublecortin (DCX)
antibodies. To confirm circuit activation in the studied coronal
sections, activation of subep-ChAT+ neurons was determined
by quantifying the intensity of P-S6 protein (Figure S6B, B0 and
B00 , upper). The subep-ChAT+ neurons on the ipsilateral side
showed significantly higher P-S6 intensity than those on the
contralateral side (Figure S6C). In these sections, the doublecor-
tin (DCX+) neuroblasts surrounding subep-ChAT+ neurons on the
activated side were not clustered and were mainly disordered,
unlike their counterpart DCX+ neuroblasts on the contralateral
side (Figure S6B, B0 and B00 ; lower). This observed disorganized
pattern of the clustered local DCX+ cells adjacent to the acti-
vated subep-ChAT+ neurons may be due to the continuous stim-
ulation of the circuit, which resulted in tonic release of ACh.
Although the circuit was stimulated for 2 days, the total number
of DCX+ neuroblasts did not differ between the ipsilateral (acti-
vated) and contralateral (control) sides of these coronal sections
(Figure S6D). We also quantified the intensity of DCX staining on
the ipsilateral SVZ (stimulated) vs. contralateral SVZ (control)
(Figure S6E). The intensity of DCX was significantly higher in
the activated SVZ compared to the control SVZ (Figure S6F).
Optogenetic stimulation for 3 days (Figures 4A and 4B) shows
that there does not appear to be a significant difference in the in-
tensity of DCX staining between the stimulated and control SVZ
wholemounts. (Figures 4C and 4D). Interestingly, DCX clusters
were observed to accumulate in a less organized manner in
the area surrounding subep-ChAT+ neurons of the stimulated
ventral SVZ compared to the control ventral SVZ (Figure 4C,

(E) Experimental design: pAAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP virus injection into ACC region of P30 Chat-Cre; Ai9 mice.
(F) Whole-cell recording: evoked APs from subep-ChAT+ neurons upon photo-stimulation for 5 s (left). n = 5 (biological replicates), one-sample t test (right).
(G) Whole-cell recordings: evoked EPSCs in response to light stimulation in subep-ChAT+ neurons (black) and after blocking with CNQX and AP-5 (red) (left).
Mean current amplitude and latency (right). n = 5 (biological replicates), one-sample t test.
(H) Experimental design: pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry virus injection into ACC region of P30 VGlut1-Cre; ChAT-eGFP mice.
(I) Whole-cell recordings: evoked EPSCs in response to light stimulation in subep-ChAT+ neurons (black) and after blocking with CNQX and AP-5 (red) (left). Mean
current amplitude and latency (right). n = 4 (biological replicates), one-sample t test.
(J) Schematic: pAAVrg-hSyn-DIO-mCherry virus injection into LV of P28 Cr-Cre mice.
(K) Immunofluorescence: RFP (red) in ACC region of injected side in P50 Cr-Cre mice in (J). Scale bar, 100 mm (left) and 30 mm (right).
(L) Electrophysiological recording: CR+ excitatory inputs to subep-ChAT+ neurons from SVZ wholemount of P30-50 Cr-Cre; ChAT-eGFP; Ai27 mice. Scale
bar, 10 mm.
(M) Whole-cell recordings: evoked EPSCs in response to light stimulation in subep-ChAT+ neurons (black) and after blocking (red) (left). Mean EPSC amplitude
and latency (right). n = 5 (biological replicates), one-sample t test.
(N) Experimental design: AAV-EF1a-DIO-hChR2(E123T/T159C)-mCherry virus injection into ACC of P28 Cr-Cre; ChAT-eGFP mice.
(O) Whole-cell recordings: EPSCs specifically evoked by ACC CR+ input were recorded from subep-ChAT+ neurons upon light stimulation (black) and after
blocking (red) (left). Mean current amplitude and latency (right). n = 4 (biological replicates), one-sample t test.
(P) Schematic summary: ACC-subep-ChAT+ circuit regulation of LV NSCs. All blue bars represent the duration of light stimulation. All data presented as
mean ± SEM.
See also Figure S5.

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Figure 4. In vivo optogenetic excitation of the ACC-subep-ChAT+ circuit increases neurogenesis and cell proliferation in the SVZ niche
(A) Schematic representation of in vivo optogenetic stimulation experiment in (B) and (J). Immunofluorescence staining for RFP (red) in the ipsilateral (injected)
ACC of Cr-Cre mice. Scale bar, 100 mm (right).
(B) Experimental design of in vivo optogenetic stimulation for 3 days post AAV-EF1a-DIO-hChR2(E123T/T159C)-mCherry virus injection into ipsilateral ACC and
optical fibers implantation into ACC regions of P28 Cr-Cre mice.

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upper image, yellow dotted circle). For further functional studies,
the neuronal activity of subep-ChAT+ neurons and number of
DCX+ neuroblasts were then examined in the coronal sections
following stimulation (Figure 4E, E0 and E00 , upper). The stimu-
lated sections showed significantly higher P-S6 intensity in the
ipsilateral (activated) subep-ChAT+ neurons compared to the
subep-ChAT+ neurons in the contralateral (control) side of
the same brain (Figure 4F). The DCX+ neuroblasts surrounding
the subep-ChAT+ neurons on the activation side were highly ran-
domized and not clustered like DCX+ cells on the contralateral
side (Figure 4E, E0 and E00 , lower, red arrows). The continuous
stimulation of subep-ChAT+ neurons resulted in the observed
continuous generation of DCX+ neuroblasts in the surrounding
area of these neurons (Figure 4E, red arrows). Notably, the higher
rate of newly produced DCX+ neuroblasts in the activated side
compared to the control side led to migration of more neuro-
blasts to the dorsal SVZ domain (Figure 4G and 4I, blue arrows).
Generally, all generated neuroblasts in SVZ migrate to the rostral
migratory stream (RMS), passing across the dorsal SVZ.31 The
total number of DCX+ cells in the stimulated coronal section of
the ipsilateral side was higher than on the contralateral side
(Figure 4H).
In vivo ACC-subep-ChAT+ circuit stimulation regulates
cell proliferation in the ventral domain of SVZ
In the previous section, we demonstrated the effect of selective
circuit stimulation on the activity of ventral SVZ neurogenesis.
Subsequently, we studied the impact of this circuit on the
regional LV cell proliferation around subep-ChAT+ neurons in
the ventral SVZ. To achieve this goal, AAV-EF1a-DIO-hChR2
(E123T/T159C)-mCherry virus was injected into ipsilateral
ACC, and optical fibers were implanted into the ACC regions
of Cr-Cre mice. To test the stimulation effect of cortical inputs
on LV-SVZ cell proliferation, we stimulated the circuit for 1 day
and 5-ethynyl-20 -deoxyuridine (EdU) was intraperitoneally (i.p.)
injected 2–3 h before mice were sacrificed (Figure 4J). Due to
the spatial arrangements of proliferative cells and subep-
ChAT+ neurons in the LV-SVZ neurogenic niche, we used SVZ
wholemount to observe the cell proliferation in the ventricle-
SVZ (V-SVZ) and SVZ layers. V-SVZ is a 3- to 5-mm-thick layer
underneath the ependymal layer where LV glial fibrillary acidic
protein (GFAP+) NSCs reside, whereas the SVZ is a 4- to
6-mm-thick layer located above the striatum where subep-
ChAT+ neurons reside (Figure 4K).
We first examined the activity of subep-ChAT+ neurons in the
stimulated SVZ wholemount compared to the control SVZ
wholemount. In the stimulated sections, the activity of subep-
ChAT+ neurons were higher than in the control sections
(Figures S7A and S7B). A combination of EdU with either
GFAP or Ki67 (nuclear protein) markers were used to assess
cell proliferation adjacent to subep-ChAT+ neurons in the ventral
SVZ (Figures 4L and 4N). While GFAP+ cells, a marker of quies-
cent NSCs and early activated NSCs,6 were observed mainly in
the V-SVZ (Figure 4L), the number of spatially localized EdU+/
GFAP+ cells surrounding the subep-ChAT+ neurons in the ipsilat-
eral V-SVZ (activated) were higher than in the contralateral
V-SVZ (control) (Figure 4M). This shows that the ACC-subep-
ChAT+ circuit regulates the proliferation of LV NSCs in the
ventral SVZ.
Ki67 is a known marker for labeling both the early-activated
NSCs and transit amplifying cells (TACs)6 and was utilized here
to study the cell proliferation in the V-SVZ and SVZ after circuit
stimulation (Figure 4N). In the ipsilateral (activation) side, the
number of EdU+/Ki67+ cells around the subep-ChAT+ neurons

(C) DCX (green) immunofluorescence staining of ipsilateral (activation) vs. contralateral (control) of SVZ wholemounts from stimulated mice in (B). The yellow
dotted circle represents an area where subep-ChAT+ neurons are found in the ventral SVZ. Scale bar, 200 mm.
(D) Analysis of DCX intensity in ipsilateral (activation) vs. contralateral (control) SVZ wholemounts. p = 0.76 (non-significant), t3 = 0.32, n = 4 (biological replicates),
paired t test. Each dot represents a normalized total DCX protein per SVZ wholemount.
(E) DCX (gray) and ChAT (green) immunofluorescence staining of (E0 ) contralateral (control) vs. (E0 0 ) ipsilateral (activation) sides of SVZ from the coronal sections
from mice in (B). Red arrows represent DCX+ neuroblasts around subep-ChAT+ neurons. Scale bar, 10 mm (left and right) and 100 mm (middle).
(F) P-S6 intensity analysis of subep-ChAT+ neurons in ipsilateral side (activation) vs. contralateral side (control). p = 0.0097, t4 = 4.65, n = 5 (biological replicates),
paired t test. Each dot represents subep-ChAT+ neurons per mouse.
(G) DCX (gray) immunofluorescence staining of contralateral SVZ (control) vs. ipsilateral SVZ (activation) from coronal section of mice in (E). Blue arrows show
DCX+ neuroblasts in dorsal domains. Red arrow shows DCX+ neuroblasts adjacent to subep-ChAT+ neurons in the ventral SVZ.
(H) Analysis of DCX+ neuroblasts where subep-ChAT+ neurons are found on the ipsilateral side (activation) vs. contralateral side (control). ****p < 0.0001, t4 = 16.3,
n = 5 (biological replicates), paired t test. Each dot represents a total of DCX+ cells in stimulated coronal sections per mouse.
(I) DCX (gray) immunofluorescence staining of contralateral SVZ (control) vs. ipsilateral SVZ (activation) from coronal section of mice in (E) and (G). Blue arrows
show DCX+ neuroblasts in dorsal domains of SVZ. Scale bar, 20 mm.
(J) Experimental design of in vivo optogenetic stimulation for 1 day post AAV-EF1a-DIO-hChR2(E123T/T159C)-mCherry virus injection into ipsilateral ACC and
optical fibers implantation into ACC regions of P28 Cr-Cre mice.
(K) Schematic representation of the organization of the LV-SVZ.
(L) EdU (purple), ChAT (green), and GFAP (gray) immunofluorescence staining of ipsilateral (activation) vs. contralateral (control) SVZ wholemounts from stim-
ulated mice in (J). Yellow arrows show EdU+/GFAP+ cells surrounding subep-ChAT+ neurons. Scale bar, 10 mm.
(M) Analysis of EdU+/GFAP+ cells surrounding subep-ChAT+ neurons in ipsilateral (activation) V-SVZ vs. contralateral (control) of SVZ wholemounts. p = 0.0015,
t19 = 3.7, n = 20 (biological replicates), paired t test. Data collected from five stimulated Cr-Cre mice. Each dot represents total EdU+/GFAP+ cells surrounding a
subep-ChAT+ neuron.
(N) EdU (purple), ChAT (green), and Ki67 (gray) immunofluorescence staining of ipsilateral (activation) vs. contralateral (control) SVZ wholemounts from stimulated
mice in (A). Yellow arrows show EdU+/Ki67+ cells surrounding subep-ChAT+ neurons. Scale bar, 10 mm.
(O) Analysis of EdU+/Ki67+ cells surrounding subep-ChAT+ neurons in ipsilateral (activation) V-SVZ vs. contralateral (control) of SVZ wholemounts. p = 0.0002,
t19 = 4.5, n = 20 (biological replicates), paired t test. Data collected from five stimulated Cr-Cre mice. Each dot represents total EdU+/Ki67+ cells surrounding a
subep-ChAT+ neuron. All data presented as mean ± SEM.
See also Figures S6 and S7.

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Figure 5. In vivo optogenetic inhibition of the ACC-subep-ChAT+ circuit reduces SVZ neurogenesis and cell proliferation
(A) Schematic: in vivo optogenetic inhibition experiment in Cr-Cre mice.
(B) Experimental design: in vivo optogenetic inhibition for 2 days in Cr-Cre mice.
(C) immunofluorescence: DCX (gray) and ChAT (green) staining in control (C0 ) vs. ipsilateral (C0 0 ) sides of SVZ. Red arrows represent DCX+ neuroblasts around
subep-ChAT+ neurons. Scale bar, 10 mm (left and right) and 100 mm (middle).
(D) P-S6 intensity analysis of ipsilateral vs. control subep-ChAT+ neurons. p = 0.0013, t4 = 8.1, n = 5 (biological replicates), paired t test.
(E) DCX (gray) immunofluorescence: ipsilateral vs. contralateral sides of SVZ. The red arrow shows DCX+ cells adjacent to subep-ChAT+ neurons of the stimulated
and control sides.
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Article
in V-SVZ and SVZ were notably higher than their counterparts in
contralateral (control) side (Figures 4N and 4O). This strongly
suggests that the ACC-subep-ChAT+ circuit has a role in the
regulation of LV Ki67+ cells. Overall, the presence of more prolif-
erative cells in both the V-SVZ and SVZ after stimulation sug-
gests that this circuit is involved in modulating the activity of
NSCs and cell division in the LV-SVZ.
In vivo ACC-subep-ChAT+ circuit inhibition reduces SVZ
neurogenesis and LV cell proliferation in the ventral SVZ
Our previous results predicted that the ACC-subep-ChAT+ cir-
cuit controls neurogenesis and cell proliferation surrounding
subep-ChAT+ neurons in the ventral SVZ. We also tested the ef-
fect of in vivo optogenetic inhibition of ACC-subep-ChAT+ circuit
on SVZ neurogenesis around subep-ChAT+ neurons. pAAV_
hSyn1-SIO-stGtACR2-FusionRed virus was injected into ipsilat-
eral ACC, and optical fibers were implanted into the ACC regions
of Cr-Cre mice (Figure 5A). The inhibition of ACC (CR+) neurons
was continuously conducted for 2 days and were delivered by
TTL control of a 473-nm laser (Figure 5B). The mice were
perfused directly after the circuit inhibition was terminated and
brain slices of the targeted sections were stained against DCX,
ChAT, and P-S6 antibodies (Figure 5C, C0 and C00 ). The activity
of subep-ChAT+ neurons on the ipsilateral (inhibited) side was
reduced compared to the activity of subep-ChAT+ neurons on
the control side within the same brains (Figure 5C, C0 and C00 , up-
per, and 5D). Adjacent to the subep-ChAT+ neurons on the ipsi-
lateral (inhibited) side, the number of DCX+ neuroblasts was also
significantly lower compared with the control side (Figure 5C, C0
and C00 , lower). Furthermore, the total number of DCX+ neuro-
blasts on the ipsilateral side was lower than their comparative
neuroblasts on the contralateral ones (Figures 5E and 5F). All
previous findings proposed a crucial role for ACC-subep-
ChAT+ circuit in managing neurogenesis of areas surrounding
subep-ChAT+ neurons in the ventral SVZ.
We also tested the effect of circuit inhibition on the LV-SVZ
cell proliferation around subep-ChAT+ neurons. We injected
pAAV_hSyn1-SIO-stGtACR2-FusionRed virus into ipsilateral
ACC and implanted optic fibers in the ACC regions of Cr-Cre
mice (Figure 5A). The ACC-subep-ChAT+ circuit was continu-
ously inhibited for 1 day and EdU injected 2–3 h before the
mice were sacrificed (Figure 5G). The subep-ChAT+ neurons in
the ipsilateral SVZ wholemounts show significantly lower activity
than their contralateral controls (Figure S8B). Using GFAP and
EdU markers, we showed that the number of spatially localized
EdU+/GFAP+ cells around subep-ChAT+ neurons in the ipsilat-
eral SVZ is lower than in the contralateral (control) SVZ
(F) Analysis of DCX+ cells where subep-ChAT+ neurons are found on the ipsilateral vs. control. p = 0.0010, t4 = 8.5, n = 5 (biological replicates), paired t test.
(G) Experimental design: in vivo optogenetic inhibition for 1 day in Cr-Cre mice.
(H) immunofluorescence: EdU (purple), ChAT (green), and GFAP (red/gray) staining in ipsilateral vs. control SVZ wholemounts. Yellow arrows show EdU+/GFAP+
cells surrounding subep-ChAT+ neurons in SVZ wholemount. Scale bar, 10 mm.
(I) Analysis of EdU+/GFAP+ cells in ipsilateral vs. control SVZ wholemounts. p = 0.0035, t19 = 3.3, n = 20 (biological replicates), paired t test. Data collected from
five stimulated Cr-Cre mice.
(J) Immunofluorescence: EdU (purple), ChAT (green), and Ki67 (red/gray) staining in ipsilateral vs. control SVZ wholemounts. Yellow arrows show EdU+/Ki67+
cells surrounding subep-ChAT+ neurons in SVZ wholemount. Scale bar, 10 mm.
(K) Analysis of EdU+/Ki67+ cells in ipsilateral vs. control SVZ wholemounts. p = 0.025, t15 = 2.5, n = 16 (biological replicates), paired t test. Data collected from four
stimulated Cr-Cre mice. All data presented as mean ± SEM.
See also Figure S8.
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(Figures 5H and 5I). This suggests that ACC-subep-ChAT+ circuit
modulates the proliferation of LV NSCs. Furthermore, both Ki67
and EdU markers were used together to study the effect of circuit
inhibition on the proliferation of LV NSCs and TAC in the SVZ.
The number of EdU+/Ki67+ cells surrounding the subep-ChAT+
neurons in the ipsilateral SVZ was also lower than in the contra-
lateral (control) SVZ (Figures 5J and 5K). Together these findings
suggest that the ACC-subep-ChAT+ circuit regulates SVZ neuro-
genesis and proliferation of LV NSCs in the ventral SVZ.
Subep-ChAT+ neurons play a key role in the ACC-subep-
ChAT+ circuit modulation of SVZ neurogenesis and
proliferation of LV NSCs in ventral SVZ
Our results in the previous section showed that activation of the
ACC-subep-ChAT+ circuit is sufficient to regulate SVZ neuro-
genesis by modulating the proliferation of LV NSCs in the ventral
SVZ. Next, we tested whether subep-ChAT+ neurons are neces-
sary for such regulation. We combined in vivo chemogenetics
and optogenetics to simultaneously stimulate ACC-subep-
ChAT+ circuit and inhibit subep-ChAT+ neurons.
To confirm the validity of the chemogenetic approach, we first
injected pAAV-hSyn-hM3D(Gq)-mCherry virus into ipsilateral
ACC regions of P30 C57BL/6J mice (Figures S9A and S9B).
The ACC neurons around the injection site (including those inner-
vating subep-ChAT+ neurons) expressed Gq-coupled hM3D
DREADD fused with mCherry protein, which are activated by
compound 21 (Cpd21; a potent hM3D(Gq) agonist, i.p. injection).
To activate the ACC neuronal input to subep-ChAT+ neurons,
Cpd21 was injected for 1–2 days (Figure S9B). Two days after
chemogenetic stimulation, the activity of subep-ChAT+ neurons
(indicated by intensity of P-S6 protein) was higher in ipsilateral
(activation) side compared to their counterpart cholinergic neu-
rons in contralateral (control) side (Figures S9C and S9D).
SVZ neurogenesis was then studied by quantifying DCX+ neu-
roblast in the coronal sections where subep-ChAT+ neurons are
located within the SVZ. Consistent with our optogenetic stimula-
tion results, chemogenetic circuit stimulation increased ipsilat-
eral SVZ neurogenesis in comparison to the contralateral SVZ
(control) (Figures S9C and S9E). Furthermore, the number of
active proliferative LV NSCs (indicated by EdU+/GFAP+ and
EdU+/Ki67+ cells) adjacent to subep-ChAT+ neurons was higher
in ipsilateral compared to contralateral sides after chemogenetic
stimulation (Figure S9G–S9I). These results are consistent
with our previous in vivo optogenetic circuit stimulation and
confirmed that hM3D(Gq) can be used to study ACC-subep-
ChAT+ circuit regulation of LV NSCs proliferation and SVZ
neurogenesis.
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We injected pAAV-hSyn-hM3D(Gq)-mCherry virus into both
ACC regions and implanted optic fibers in P30 Ai40D; ChAT-
Cre mice (Figures 6A and 6B). In these mice, all ChAT+ neurons
expressed an improved Archaerhodopsin-3/EGFP fusion protein
(ArchT-EGFP). Upon photo-stimulation (550 nm), these neu-
rons are inhibited. We first used chemogenetics to activate the
ACC-subep-ChAT+ circuit in both hemispheres (Cpd21 injec-
tions for 2 days). Concurrently, subep-ChAT+ neurons on the
ipsilateral side were optogenetically inhibited continuously (Fig-
ure 6B). The chemogenetic ACC-subep-ChAT+ circuit stimula-
tion was validated as previously reported in Figure S9, and the
activity of subep-ChAT+ neurons was confirmed using P-S6 pro-
tein intensity. On the ipsilateral side (i.e., ACC-subep-ChAT+ cir-
cuit is activated and subep-ChAT+ neurons are inhibited), the ac-
tivity of subep-ChAT+ neurons was significantly lower than their
counterpart on the contralateral side where ACC-subep-ChAT+
circuit is activated only (Figures 6C and 6D). On the coronal sec-
tions where subep-ChAT+ neurons reside, SVZ neurogenesis
was significantly reduced, as indicated by the number of DCX+
cells on the ipsilateral side compared to the contralateral side
(Figures 6C and 6E).
To assess cell proliferation of LV NSCs, the previous study was
conducted for 1 day only and EdU was injected 2 h prior to the end
of the experiment (Figures 6A and 6F). We also quantified the
active proliferative LV NSCs that are adjacent to subep-ChAT+
neurons in the ventral SVZ. There were very few, if any, EdU+/
GFAP+ cells on the ipsilateral side (ACC-subep-ChAT+ excitation
and subep-ChAT+ neurons inhibition) compared to EdU+/GFAP+
cells on the contralateral side (ACC stimulation only) (Figures 6G
and 6H). The number of EdU+/Ki67+ cells surrounding subep-
ChAT+ neurons in ipsilateral side was lower compared to the
contralateral side (Figures 6I and 6J). Overall, these results
demonstrated that subep-ChAT+ neurons are essential for the
regulatory function of ACC neurons on the activity of LV NSCs
and SVZ neurogenesis in the ventral domain of SVZ.
Previous studies suggested that glutamate receptors can pro-
mote proliferation and differentiation of LV NSCs.32–34 In the cur-
rent study, we confirmed that subep-ChAT+ neurons are neces-
sary intermediates for the ACC input that regulates the
proliferation activity of LV GFAP+ NSCs in ventral SVZ. It is still
unknown whether the ACC region directly innervates ventral LV
GFAP+ NSCs and modulates their activity. To test this possibility,
we generated GFAP-Cre; R26F-RTT mice to allow for retrograde
tracing from LV GFAP+ NSCs in the ventral domain. EnvA
G-deleted rabies-eGFP virus was injected into the ventral LV of
P30 GFAP-Cre; R26F-RTT mice (Figure S11A). After virus incu-
bation for 7 days, no GFP+ neurons were detected in the ACC re-
gion. Thus, the glutamatergic input from ACC does not directly
project to LV GFAP+ NSCs (Figure S11B).
Local CR+ neurons regulate the effect of ACC-subep-
ChAT+ circuit on the SVZ neurogenesis and proliferation
of LV NSCs in ventral SVZ
We found that local CR+ neurons in SVZ can inhibit the activity of
subep-ChAT+ neurons (Figure 2). To test whether these CR+ neu-
rons can regulate the activity of LV NSCs, we used optogenetics
to activate the ACC-subep-ChAT+ circuit in both hemispheres
and local CR+ neurons only in one hemisphere (Figures 7A and
7B). Briefly, we injected AAV-EF1a-DIO-hChR2(E123T/T159C)-
mCherry virus into both ACC regions (Figure 7A, left) and ipsilat-
eral LV only (Figure 7A, right), and implanted optic fibers to LV
regions of P28 Cr-Cre mice (Figure 7A, middle). Light stimulation
was used to activate ACC-subep-ChAT+ circuit in both hemi-
spheres and local CR+ neurons on the ipsilateral side to
study neurogenesis and cell proliferation in SVZ, respectively
(Figures 7B and 7F, respectively). Activity of subep-ChAT+ neu-
rons 1–2 days post stimulation was significantly decreased on
the ipsilateral side in comparison to the contralateral side
(Figures 7C, 7D, S12A, and S12B). This observation indicates
that the optogenetic stimulation of local CR+ neurons inhibits
subep-ChAT+ neurons on the ipsilateral side (Figure 7A, right).
As a result of subep-ChAT+ neuronal inhibition in ipsilateral
sides, neurogenesis was also reduced in the coronal sections
where subep-ChAT+ neurons are located compared to neuro-
genesis in contralateral control sections (Figures 7C and 7E).
This observation suggests that local CR+ neurons surrounding
subep-ChAT+ neurons can modulate the effects of ACC-
subep-ChAT+ projections on SVZ neurogenesis.

Figure 6. In vivo chemogenetic stimulation of ACC-subep-ChAT+ circuit and optogenetic inhibition of subep-ChAT+ neurons modulate SVZ
neurogenesis and LV NSCs proliferation
(A) Schematic: in vivo chemogenetic ACC-subep-ChAT+ circuit stimulation and optogenetic subep-ChAT+ neurons inhibition experiment in (B) and (F).
(B) Experimental design: in vivo chemogenetic ACC-subep-ChAT+ circuit stimulation and optogenetic subep-ChAT+ neurons inhibition for 2 days in Ai40D; ChAT-
Cre mice.
(C) Immunofluorescence: DCX (gray) and ChAT (green) staining in control (C0 ) vs. Ipsilateral (C0 0 ) sides of SVZ. Red arrows represent DCX+ neuroblasts sur-
rounding subep-ChAT+ neurons. Scale bar, 10 mm (left and right) and 100 mm (middle).
(D) P-S6 intensity analysis of ipsilateral vs. control subep-ChAT+ neurons. p = 0.0017, t3 = 10.8, n = 4 (biological replicates), paired t test.
(E) Analysis of DCX+ neuroblasts on ipsilateral vs. control sides. p = 0.0008, t4 = 9.2, n = 5 (biological replicates), paired t test.
(F) Experimental design: in vivo chemogenetic ACC-subep-ChAT+ circuit stimulation and optogenetic subep-ChAT+ neurons inhibition for 1 day post in Ai40D;
ChAT-Cre mice.
(G) Immunofluorescence: ChAT (green), EdU (purple), and GFAP (red/gray) staining in ipsilateral vs. control SVZ wholemounts. Yellow arrows show EdU+/GFAP+
cells surrounding subep-ChAT+ neurons. Scale bar, 10 mm.
(H) Analysis of EdU+/GFAP+ cells per subep-ChAT+ neuron in SVZ wholemounts of ipsilateral vs. control. ****p < 0.0001, t14 = 7.6, n = 15 (biological replicates),
paired t test. Data collected from four stimulated Ai40D; ChAT-Cre mice.
(I) Immunofluorescence: ChAT (green), EdU (purple), and Ki67 (gray) staining in ipsilateral and control SVZ wholemounts. Yellow arrows show EdU+/Ki67+ cells
surrounding subep-ChAT+ neurons. Scale bar, 10 mm.
(J) Analysis of EdU+/Ki67+ cells per subep-ChAT+ neuron in SVZ wholemounts of ipsilateral vs. control. ****p < 0.0001, t14 = 7.2, n = 15 (biological replicates),
paired t test; collected from four stimulated Ai40D; ChAT-Cre mice. All data presented as mean ± SEM.
See also Figures S9–S11.

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Figure 7. In vivo optogenetic ACC-subep-ChAT+ circuit and local CR+ neuron stimulation modulate SVZ neurogenesis and LV NSCs pro-
liferation
(A) Schematic representation: in vivo optogenetic ACC-subep-ChAT+ circuit and CR+ neurons stimulations in ipsilateral side experiment in (B) and (F).
(B) Experimental design: in vivo optogenetic ACC-subep-ChAT+ circuit and CR+ neurons stimulation for 2 days in CR-Cre mice.

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To study the influence of local CR+ neurons on the proliferation
of adjacent LV NSCs to subep-ChAT+ neurons post 1 day of op-
tic stimulation, EdU was i.p. injected 2 h prior the end of the
experiment (Figure 7F). The EdU+/GFAP+ and EdU+/Ki67+ cells
were rarely found in the area surrounding subep-ChAT+ neurons
in ipsilateral sides compared to their counterparts in contralat-
eral sides (Figures 7G–7J). These findings demonstrate that local
CR+ neurons in SVZ can regulate the impact of excitatory cortical
inputs to subep-ChAT+ activity and thereby the activity of LV
NSCs in the ventral SVZ.
DISCUSSION
Postnatal and adult LV NSCs proliferation and SVZ neurogenesis
can be modulated by neural activity.18 LV NSCs express recep-
tors for many neurotransmitters that are known to play a role in
regulating neurogenesis.32 A population of cholinergic (subep-
ChAT+) neurons was found to release ACh into the subependy-
mal space to modulate the proliferation of LV NSCs and neuro-
blasts in an activity-dependent manner.25 Unlike striatal ChAT+
neurons, subep-ChAT+ neurons do not show spontaneous firing
activity. They also have a different pattern of neural connectivity
compared to the striatal cholinergic neurons, which are often
connected with their adjacent cholinergic neurons.33 However,
it is unclear how subep-ChAT+ neurons are regulated by inputs
from other brain regions.
Our results reveal the crucial roles of subep-ChAT+ and local
CR+ inhibitory interneurons in regulating LV NSC proliferation
and SVZ neurogenesis within the area surrounding subep-
ChAT+ neurons in the ventral SVZ. Using rabies transneuronal
tracing and in vitro electrophysiological recording, we uncov-
ered the distal and local components of the ACC-subep-
ChAT+ circuit (Figures 1, 2, and 3). By modulating CR+ ACC
neurons in vivo, we regulated the proliferation and neurogene-
sis in the area surrounding their postsynaptic inputs in the
ventral SVZ (Figures 4 and 5). Moreover, the simultaneous
in vivo manipulation of the ACC-subep-ChAT+ circuit, involving
either subep-ChAT+ or CR+ neurons, further corroborated their
important roles in NSC proliferation and SVZ neurogenesis in
the LV (Figures 6 and 7).
Several studies suggested that glutamate receptors are ex-
pressed on the LV NSCs and play a role in NSC proliferation,34–36
but the source of glutamatergic projections to the LV NSCs was
not known. We found that glutamatergic (VGlut1+) neurons in the
ACC project directly to subep-ChAT+ neurons and regulate their
activity in the SVZ niche.
The subep-ChAT+ neurons are spatially localized in the ventral
domain of the SVZ niche. In vivo optogenetic manipulation of the
ACC-subep-ChAT+ circuit is sufficient to regulate the activity of
LV NSCs proliferation and SVZ neurogenesis surrounding the
subep-ChAT+ neurons in the ventral SVZ. While this strongly
suggests that subep-ChAT+ neurons release ACh locally in the
ventral SVZ, further research will be needed to understand the
role of ACh signaling, including the postsynaptic receptors
involved, in the SVZ niche.
In previous studies, GABAergic signaling has been implicated
in various functions within the SVZ, such as enhancing neuro-
blast maturation37 and preserving postnatal/adult LV NSCs by
inhibiting their proliferation and differentiation.21,38 However,
the specific source of GABA has remained unclear. In this study,
we show that a subset of CR+ interneurons, positioned in close
proximity to the subep-ChAT+ neurons, release GABA and
directly exert inhibitory control over subep-ChAT+ neurons.
In humans, there is evidence of active neurogenesis in the wall
of the LVs that generates migratory neuroblasts for up to 2 years
after birth.39 However, the mechanism underlying circuit modu-
lation of neurogenesis in the human SVZ remains unknown.
The analogous process in rodents, as revealed in our study,
may shed light on how neurogenesis during postnatal brain
development is influenced by frontal cortical projections and
cortical regulation of cholinergic signaling. Our results thus
promise to open an avenue of research into cortical-activity-
dependent SVZ neurogenesis in postnatal and adult animals.
Limitations of the study
In our study, we attempted to perform in vivo functional analysis
of a frontal cortical circuit, focusing on its impact on the anterior
ventral domain of the SVZ, where subep-ChAT+ neurons are
located. However, it is important to acknowledge that the pre-
sent study did not explore whether the ACC-subep-ChAT+ cir-
cuit also influences neurogenesis in the posterior ventral and
dorsal SVZ. To determine the regulatory impact of the ACC-
subep-ChAT+ circuit on neurogenesis in these regions, further
investigation is required, specifically examining the ACh-
releasing terminals.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE

(C) Immunofluorescence: DCX (gray) and ChAT (green) staining in control (C0 ) vs. ipsilateral (C0 0 ) sections of SVZ. Red arrows represent DCX+ neuroblasts
surrounding subep-ChAT+ neurons. Scale bar, 100 mm (left) and 10 mm (right).
(D) P-S6 intensity analysis of ipsilateral vs. control subep-ChAT+ neurons. p = 0.0058, t3 = 7.1, n = 4 (biological replicates), paired t test.
(E) Analysis of DCX+ neuroblasts on the ipsilateral vs. control sides. p = 0.0002, t4 = 13.3, n = 5 (biological replicates), paired t test.
(F) Experimental design: in vivo optogenetic ACC-subep-ChAT+ circuit and CR+ neurons stimulations for 1 day in CR-Cre mice.
(G) Immunofluorescence: ChAT (green), EdU (purple), and GFAP (red/gray) staining in ipsilateral vs. control SVZ wholemounts. Scale bar, 10 mm.
(H) Analysis of EdU+/GFAP+ cells per subep-ChAT+ neuron in SVZ wholemounts. ****p < 0.0001, t15 = 8.1, n = 16 (biological replicates), paired t test. Data collected
from four stimulated Cr-Cre mice.
(I) Immunofluorescence: ChAT (green), EdU (purple), and Ki67 (gray) staining in ipsilateral vs. control SVZ wholemounts. Scale bar, 10 mm.
(J) Analysis of EdU+/Ki67+ cells per subep-ChAT+ neuron in SVZ wholemounts. ****p < 0.0001, t15 = 5.95, n = 16 (biological replicates), paired t test; collected from
four stimulated Cr-Cre mice. All data presented as mean ± SEM.
See also Figure S12.

Cell Reports 42, 112783, July 25, 2023 15

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OPEN ACCESS Article
d RESOURCE AVAILABILITY mammalian brain. Cell 97, 703–716. https://doi.org/10.1016/s0092-
B Lead contact 8674(00)80783-7.
B Materials availability 5. Obernier, K., Cebrian-Silla, A., Thomson, M., Parraguez, J.I., Anderson, R.,
B Data and code availability Guinto, C., Rodas Rodriguez, J., Garcia-Verdugo, J.M., and Alvarez-
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE- Buylla, A. (2018). Adult Neurogenesis Is Sustained by Symmetric Self-
Renewal and Differentiation. Cell Stem Cell 22, 221–234.e8. https://doi.
TAILS
org/10.1016/j.stem.2018.01.003.
B Animals
6. Codega, P., Silva-Vargas, V., Paul, A., Maldonado-Soto, A.R., Deleo,
d METHOD DETAILS
A.M., Pastrana, E., and Doetsch, F. (2014). Prospective identification
B Rabies virus retrograde tracing and purification of quiescent adult neural stem cells from their in vivo
B Stereotaxic injections niche. Neuron 82, 545–559. https://doi.org/10.1016/j.neuron.2014.
B SVZ wholemount preparation and whole-cell patch- 02.039.
clamp recording 7. Lois, C., Garcı́a-Verdugo, J.M., and Alvarez-Buylla, A. (1996). Chain
B Immunofluorescence staining and imaging migration of neuronal precursors. Science 271, 978–981. https://doi.org/
B In vivo optogenetic stimulation and inhibition 10.1126/science.271.5251.978.
B In vivo chemogenetic 8. Ponti, G., Obernier, K., and Alvarez-Buylla, A. (2013). Lineage progression
B In vivo EdU labeling from stem cells to new neurons in the adult brain ventricular-subventricular
d QUANTIFICATION AND STATISTICAL ANALYSIS zone. Cell Cycle 12, 1649–1650. https://doi.org/10.4161/cc.24984.
9. Lois, C., and Alvarez-Buylla, A. (1994). Long-distance neuronal migration
in the adult mammalian brain. Science 264, 1145–1148. https://doi.org/
SUPPLEMENTAL INFORMATION
10.1126/science.8178174.

Supplemental information can be found online at https://doi.org/10.1016/j. 10. Luskin, M.B. (1993). Restricted proliferation and migration of postnatally
celrep.2023.112783. generated neurons derived from the forebrain subventricular zone. Neuron
11, 173–189. https://doi.org/10.1016/0896-6273(93)90281-u.

ACKNOWLEDGMENTS
We thank Scott Soderling and Shawn Je for helpful comments on the manu-
script; the Transgenic and Knockout Mouse Facility at Duke University for
assistance with generation of R26R-FLEX-TVA-2A-RabiesG-2A-tdTomato-
FLEX mice; and E. Adlaf, J. Erb, B. Asrican, D. Fromme, P. Paez-Gonzalez,
K. Abdi, G. Neves, S. Ramamoorthy, J. David, and K. Woldemichael for project
assistance. This work was supported by NIH grant R01MH105416.
AUTHOR CONTRIBUTIONS
M.M.N., C.T.K., and H.H.Y. conceived the project and participated in research
design. M.M.N. performed all experiments and analyzed data. R.R.K. helped
with preparation of SVZ wholemounts for electrophysiology experiments.
M.M.N. and H.H.Y wrote the paper.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: October 26, 2022
Revised: April 13, 2023
Accepted: June 25, 2023
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Green Fluorescent Protein (GFP) chicken polyclonal AVES GFP-1020; PRID: AB_10000240
antibody
Choline Acetyltransferase Antibody Millipore sigma AB-144P
tdTomato [16D7] Kerafast EST-203
RFP rabbit Rockland 600-401-379
Calretinin Abcam ab-702
Calretinin mouse Swant 6B3
Calretinin mouse monoclonal antibody EnCor Biotechnology MCA-3G9
RFP mouse Abcam ab-62341
Doublecortin Millipore AB-2253
Doublecortin rabbit Cell Signaling 4604S
Technology
Ki-67 Rat Invitrogen 14-5698-95 (SolA15)
Ki-67 Rabbit Invitrogen MA5-14520
GFAP Chicken Aves Labs GFAP
Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 Invitrogen A-21206
Donkey anti-Rabbit IgG (H + L) Alexa Fluor 594 Invitrogen A-21207
Donkey anti-Rabbit IgG (H + L) Alexa Fluor 647 Invitrogen A-31573
Donkey anti-Mouse IgG (H + L) Alexa Fluor Plus 488 Invitrogen A-32766
Donkey anti-Mouse IgG (H + L) Alexa Fluor Plus 594 Invitrogen A-32744
Donkey anti-Mouse IgG (H + L) Alexa Fluor Plus 647 Invitrogen A-32787
Donkey anti-Rat IgG (H + L) Alexa Fluor 488 Invitrogen A-21208
Donkey anti-Rat IgG (H + L) Alexa Fluor 594 Invitrogen A-21209
Donkey anti-Rat IgG (H + L) Alexa Fluor Plus 647 Invitrogen A-48272
Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H + L) Jackson ImmunoResearch Inc 703-545-155
Alexa Fluor 594 Donkey Anti-Chicken IgY (IgG) (H + L) Jackson ImmunoResearch Inc 703-585-155
Alexa Fluor 647 Donkey Anti-Chicken IgY (IgG) (H + L) Jackson ImmunoResearch Inc 703-605-155
Bacterial and virus strains
EnvA G-deleted Rabies-eGFP Salk, USA N\A
EnvA rVSV-eGFP (EnvA/RABVG-eGFP) Salk, USA N\A
AAV-CaMKII-hChR2(E123A)-mCherry Karl Deisseroth addgene #35506
pAAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP Karl Deisseroth addgene #35509
pAAV-Ef1a-DIO hChR2(E123T/T159C)- mCherry Karl Deisseroth addgene #35510
pAAVrg-hSyn-DIO-mCherry Bryan Roth addgene #50459-AAVrg
pAAVrg-hSyn-mCherry Karl Deisseroth addgene # 114472-AAVrg
pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry Karl Deisseroth addgene #35510
pAAV_hSyn1-SIO-stGtACR2-FusionRed Ofer Yizhar addgene #105677
pAAV-hSyn-hM3D(Gq)-mCherry Bryan Roth addgene #50474
Normal Donkey Serum Jackson ImmunoResearch Inc 017-000-121
Chemicals, peptides, and recombinant proteins
DAPI Sigma-Aldrich D9542
DL-AP5 R&D Systems 3693/50
CNQX Tocris 0190
Picrotoxin Millipore Sigma 124-87-8
(Continued on next page)

18 Cell Reports 42, 112783, July 25, 2023

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Article OPEN ACCESS

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Aqua-Poly/Mount Polysciences 18606–20
Apex Safe DNA Gel Stain Genesee Scientific 20–278
EdU in vivo Kits baseclick GmbH, Germany BCK647-IV-IM
Bovine Serum Albumin Sigma-Aldrich 9048-46-8
Paraformaldehyde Sigma-Aldrich P6148-1KG
Software and algorithms
FIJI NIH, USA https://imagej.net/Fiji RRID:SCR_002285
Prism 9 GraphPad https://www.graphpad.com/scientific-
software/prism/RRID:SCR_002798
pClamp 10 Molecular Devices RRID:SCR_011323
AxoGraph AxoGraph https://axograph.com/
Zen Zeiss https://www.zeiss.com/microscopy/us/
products/microscope-software/zen.html

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Henry
Yin (hy43@duke.edu).

Materials availability
All unique reagents in this paper will be shared by the lead contact upon request.

Data and code availability

d All data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon
request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals
All experiments in this study were conducted in mice and were approved by the Institutional Animal Care and Use Committee at Duke
University. Mice were group housed on a standard 12 h light/dark cycle (lights on at 7 a.m.) with a controlled average ambient tem-
perature of 21 C and 45% humidity. The following mouse lines were purchased from JAX: C57BL/6J (000664); VGlut1-Cre (023527);
ChAT-eGFP (007902); ChR2(RCL-hChR2(H134R)/tdT)-D (Ai27) (012567); ChAT- Cre (006410); Cr-Cre (010774); VGat-Cre (016962);
Cb-Cre (028532); SST-Cre (013044); PV-Cre (017320); Ai9(RCL-tdT) (007905); Ai40D (021188). We generated the R26R-FLEX-TVA-
2A-RabiesG-2A-tdTomato-FLEX (R26F-RTT) mouse line for rabies monosynaptic circuit tracing (Figure 1G).

METHOD DETAILS

Rabies virus retrograde tracing

An intraventricular approach via a Cre-dependent viral strategy was employed to avoid labeling striatal ChAT+ neurons. In the begin-
ning we used a monosynaptic circuit tracing with Rabies strategy40 by injecting the first virus into LV of ChAT-Cre mice to express
TVA, Rabies-G protein, and tdTomato in subep-ChAT+ neurons. The infected subep-ChAT+ neurons were later targeted with the sec-
ond EnvA G-deleted rabies virus to enable efficient mono-synaptic tracing. It was extremely difficult to infect the same subep-ChAT+
neurons via two separate viral injections. To overcome this obstacle, R26R-FLEX-TVA-2A-RabiesG-2A-tdTomato-FLEX (R26F-RTT)
mice were successfully generated and validated (Figures 1G, S1, S2, and S3). These mice allow for monosynaptic tracing of Cre-tar-
geted neurons via a single EnvA G-deleted Rabies-eGFP (Salk, USA) or EnvA rVSV-eGFP (EnvA/RABVG-eGFP) (Salk, USA) viral
injections.

Cell Reports 42, 112783, July 25, 2023 19

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OPEN ACCESS Article

Stereotaxic injections
Stereotaxic injections were performed as mice were kept deeply anesthetized in a stereotaxic frame (David Kopf Instruments) with
isoflurane. To perform circuit tracing using rabies virus, 300 nL of (EnvA G-deleted Rabies-eGFP or EnvA rVSV-eGFP (Salk, USA))
virus were injected into striatum or lateral ventricle (LV) of P30 ChAT-Cre; R26F-RTT mice. Viruses were infused slowly over
10 min into striatum as the following coordinates relative to bregma (AP: +1, ML ± 2.0, DV: 2.2 from brain surface) or LV
(AP: +0.8, ML ± 0.65, DV: 2.1 from brain surface) using a microdriver with a 10 mL Hamilton syringe. For electrophysiological testing
of ACC inputs to the subep-ChAT+ neurons (optogenetic-light stimulation), adeno-associated viral viruses were used for Cre-depen-
dent expression of the excitatory channelrhodopsin. pAAV-CaMKIIa-hChR2(E123A)-mCherry (addgene #35506) (300nL) virus or
pAAV-CaMKIIa-hChR2(E123T/T159C)-EYFP (addgene #35509) (300nL) virus were injected into P30 C57BL/6J or P30 ChAT-Cre;
R26F-RTT mice, respectively. Also, pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry (addgene #35510) (300nL) virus was injected
into P30 VGlut1-Cre; ChAT-eGFP and P28 Cr-Cre; ChAT-eGFP mice. Viruses were infused slowly over 10 min into the ACC using
the following coordinates relative to bregma (AP: +0.8, ML ± 0.3, DV: 0.5 from brain surface). For pAAV retrograde tracing,
pAAVrg-hSyn-DIO-mCherry (addgene #50459-AAVrg) and pAAVrg-hSyn-mCherry (addgene # 114472-AAVrg) viruses (300 nL)
were injected into the LV of P28 Cr-Cre, P30 C57BL/6J (P30) and P30 VGlut1-Cre. Viruses were infused slowly over 10 min into
the ACC as the following coordinates relative to bregma (AP: +0.8, ML ± 0.65, DV: 2.1 from brain surface). For in vivo optogenetic
testing of ACC inputs to the subep-ChAT+ neurons, pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry (addgene #35510) (300nL)
and pAAV_hSyn1-SIO-stGtACR2-FusionRed (addgene #105677) (300nL) viruses were injected into P28 Cr-Cre mice. Viruses
were infused slowly over 10 min into the ACC (AP: +0.8, ML ± 0.25, DV: 0.5 from brain surface).
All viruses were infused slowly for over 10 min using a Nanoject (Drummond Scientific) connected to a glass pipette. The injection
pipette was left in place for 10 min post-injection before it was retracted.

SVZ wholemount preparation and whole-cell patch-clamp recording

For electrophysiology experiments both male and female mice (4- to 10-week-old) were used. They were anesthetized with isofluor-
ane and transcardially perfused; the ventricular wall was then dissected as SVZ wholemounts in ice-cold NMDG artificial cerebro-
spinal fluid (ACSF; containing 92 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 2 mM glucose,
5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4, 0.5 mM CaCl2), and bubbled with 5% CO2/
95% O2. Tissues were then bubbled in same solution at 37 C for 15 min, transferred to bubbled, modified-HEPES ACSF at 23–
25 C (92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 2 mM glucose, 5 mM sodium ascorbate,
2 mM thiourea, 3 mM sodium pyruvate, 2 mM MgSO4, 2 mM CaCl2) for at least 45 min before start experimentation. For NMDG arti-
ficial cerebrospinal fluid and modified-HEPES ACSF solutions, pH was adjusted to 7.5 and osmolarity to 290 mOsm. Recordings
were performed in submerged chamber, superfused with continuously bubbled ACSF (125 mM NaCl, 2.5 mM KCl, 1.25 mM
NaH2PO4, 26 mM NaHCO3, 20 mM glucose, 2 mM CaCl2, 1.3 mM MgCl2) at 2.5–5 mL/min at 23–25 C.
Patch electrodes with a resistance of 3–6 MU were pulled from borosilicate glass capillaries using a horizontal puller (P-97, Sutter-
instruments). All subep-ChAT+ neurons used in the electrophysiology recordings were identified by their distinct morphology, loca-
tion (within 15–20 mM from LV surface), their non-spontaneous firing activity, and their depolarized resting membrane potentials
(-45 mV). For measuring EPSCs when holding at – 60 mV, the following internal solution was used: 130 mM potassium gluconate,
2 mM NaCl, 4 mM MgCl2, 20 mM HEPES, 4 mM Na2ATP, 0.4 mM NaGTP and 0.5 mM EGTA, pH adjusted to 7.2 with KOH. A 40 mM
picrotoxin ((124-87-8), Millipore Sigma) was added in the bath solution in voltage-clamp mode. For measuring inward IPSCs when
holding at – 60 mV, we used internal solution containing K-Cl (high Chloride) KCl (135 Mm), HEPES (10 mM), Na2ATP (2mM), NaGTP
(0.2 mM), MgCl2 (2 mM), and EGTA (0.1 mM), pH adjusted to 7.2 with KOH. To block glutamatergic transmission, 50 mM DL-AP5
(NMDA antagonist, R&D Systems) and 50 mM CNQX (AMPA antagonist, Tocris) were added in the bath solution in voltage-clamp
mode. To block action potentials in the patched cell, the sodium channel blocker QX-314 (10 mM, 552233, Millipore Sigma) was
also added. Signals were amplified with Multiclamp 700B (filtered at 10 kHz), digitized with Digidata 1440A (20 kHz), and recorded
using pClamp 10 software (Axon). Light-activation of channelrhodopsin was achieved using a 473-nm laser (X-Cite exacte) through a
40x objective (Nikon). Action potentials, evoked EPSCs and evoked IPSCs were analyzed using AxoGrapth (AxoGrapth). One or two
subep-ChAT+ neurons were recorded per wholemount SVZ. Up to three neurons from each animal were recorded. The mean of at
least five responses was used as a data point.
For focal (targeted) photo-stimulation of local CR+-IPSCs from subep-ChAT+ neurons, we used Polygon 400 Digital Micromirror
Device (Mightex; multiwavelength pattered illuminator) to control the temporal dynamics (the size, shape, intensity, and position)
of light inputs. The illumination consisted of a 100 ms light pulse (470-nm) at 50% intensity in the selected area (50 mm), triggered
using a TTL pulse from the Digidata to synchronize the stimulation with electrophysiology. Photo-stimulation targeted one fluorescent
subep-CR+ neuron in the field of view, which resulted in postsynaptic effects in the recorded subep-ChAT+ neuron.

Immunofluorescence staining and imaging

For the preparation of brain tissue for IHC staining, we utilized primary antibodies to GFP (#GFP-1020, 1:400, AVES lab), Choline Ace-
tyltransferase Antibody (#AB144P, 1:250, Millipore sigma), tdTomato [16D7] (#EST203, 1:200, Kerafast), RFP (#600-401-379, 1:250,
Rockland), Calretinin (#ab702, 1:200, Abcam), calretinin (#6B3, 1:250, Swant), Calretinin (#MCA-3G9, 1:250, EnCor Biotechnology),
RFP (#ab62341, 1:200, Abcam), Doublecortin (#AB2253, 1:250, Millipore), Doublecortin (#4604S, 1:200, Cell Signaling Technology),

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Doublecortin (#MA5-17066, 1:200, ThermoFisher Scientific), Ki67 (#14-5698-95, 1:25, Invitrogen), Ki67 (## MA5-14520, 1:200, Invi-
trogen), GFAP (#GFAP, 1:500, Aves Labs). In brief, for circuit tracing and in vivo optogenetic experiments, after experiments mice
were deeply anesthetized with isoflurane, perfused transcardially with phosphate buffered saline (PBS), followed by 4% PFA in
PBS. The perfused brains were removed and postfixed overnight at 4 C in 4% PFA. The fixed brains were either cut into 50 mm cor-
onal sections by Precisionary Instruments VF-500-0Z vibrating microtome or the SVZ wholemounts were dissected. The coronal sli-
ces or SVZ wholemounts were incubated in a blocking solution containing 5% donkey serum and TBST for 100 min at room temper-
ature. The sections were then incubated at room temperature overnight in PBS containing 1% donkey serum and antibodies. They
were then washed with PBS and incubated with secondary antibodies, Alexa 594 (1:1000, LifeTech) or Alexa 488 (1:1000, LifeTech) or
Alexa 647 (1:1000, LifeTech) for 2 h at room temperature, before washing with PBS. The stained sections were counterstained with a
40 ,6-diamidino-2-phenylindole solution (DAPI; (D9542) Sigma-Aldrich). After washing four times with TBST, the sections were cover-
slipped with Fluoromount (Sigma) aqueous mounting medium. Images were taken using Leica SP8 upright confocal microscope
(Zeiss) with 10 3, 203 and 403 objectives under the control of Zen software (Zeiss). In order to study the number of EdU+/
GFAP+ and EdU+/Ki67+ cells that are adjacent to subep-ChAT+ neurons in V-SVZ and SVZ layers, 40mm 3 40mm square images
were taken where the subep-ChAT+ neuron is in center of each image.
All antibodies used were validated as in previous publications25,41 or by publications available on vendor websites specific to each
antibody.

In vivo optogenetic stimulation and inhibition

Cannula targeting the ACC region was implanted (AP: +0.9, ML ± 0.25, DV: 0.4 from brain surface) after injecting pAAV-Ef1a-DIO
hChR2(E123T/T159C)-mCherry and pAAV_hSyn1-SIO-stGtACR2-FusionRed viruses as described in Figures 4A, 5A, 6A, and 7A, us-
ing implantable mono fiber-optic fiber (200 mm, 0.22 NA, Doric). The protruding ferrule end of cannula was then connected via fiber
cord and a rotary coupling joint (Doric) was used to permit free movement. Three to four weeks after viral infection, light-stimulation
was delivered by TTL control (Master 8, AMPI) of a 473-nm laser (IkeCool). For In vivo optogenetic stimulation, the ipsilateral ACC
region was stimulated for 10 ms pulses (7 mW laser power) at 10 Hz, lasting 10 s, given once every 1 min. For In vivo optogenetic
inhibition, the ACC regions in ipsilateral and contralateral sides were continuously stimulated using 2 mW laser power only to study
and avoid overheating in the local cortical areas.
Cannula targeting the LV regions was implanted (AP: +0.9, ML ± 0.65, DV: 2.1 from brain surface) of P30 Ai40D; ChAT-Cre mice as
illustrated in Figure 6A. After three to four weeks, light-stimulation was delivered by TTL control (Master 8, AMPI) of a 556-nm laser
(IkeCool). The LV region in ipsilateral side was continuously stimulated using 2 mW laser power only to study and avoid overheating in
the local areas.
For ChAT, DCX, Ki67, GFAP, CR and RFP analyses, 50-mm brain coronal sections were cut and collected by Precisionary Instru-
ments VF-500-0Z vibrating microtome or SVZ wholemounts were dissected. The sections or areas surrounding the activated/in-
hibited subep-ChAT+ neurons were selected for analyses.

In vivo chemogenetic
The Compound 21 (Cpd21; DREADD Agonist 21 dihydrochloride (SML2392)) (Millipore Sigma) was Dissolved in 0.9% saline and
stored at
20 C until used as shown in Figures 7 and S9. All the injections were given intraperitoneally, at a volume of 1 mg/kg daily
for one- or two-days. For DCX and P-S6 analysis, 50-mm brain coronal sections were cut and collected on Leica VT1000S vibratome
or Precisionary Instruments VF-500-0Z vibrating microtome. Coronal sections of slices at AP: +0.75–0.40 mm relative to bregma
were selected for analyses, comparing ipsilateral sides (activation) versus contralateral sides (Control). For EdU, GFAP and Ki67 anal-
ysis, SVZ wholemounts were dissected and the number of EdU+/GFAP+ and EdU+/Ki67+ cells surrounding subep-ChAT+ neurons in
ipsilateral sides (activation) were compared versus contralateral sides (Control).

In vivo EdU labeling

The 5-ethynyl-20 -deoxyuridine (EdU) staining was conducted using EdU Cell Proliferation Kit for Imaging (EdU in vivo Kits) (baseclick
GmbH, Germany) according to the manufacturer’s protocol. EdU was prepared at 50mg/20mL in sterile PBS and used for pulse la-
beling of adult mice by performing an intraperitoneal (IP) injection of 500 ml dissolved EdU (50 mg/kg). The mice were harvested as
described in Figures 4, 5, 6, and 7. The intended SVZ wholemounts for EdU labeling were first stained for other antibodies, and then
counterstained with DAPI as described in the immunofluorescence staining section. Subsequently, the sections were washed three
times with 3% BSA in PBS. Then, incubated for 30 min in a reaction cocktail containing Deionized water, Reaction buffer, Catalyst
solution, Dye Azide and Buffer additive while protected from light. After the reaction cocktail was removed, sections were washed
three times with 3% BSA in PBS. They were then mounted in vectashield mounting media (vector laboratories Inc, Burlingame,
CA) and imaged by using Leica SP8 upright confocal microscope (Zeiss) as above. All steps were carried out at room temperature.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data are expressed as difference between means ± standard error of the means (SEM) and all statistical analyses were performed
using GraphPad Prism (version 8) program. One-sample t-tests were used for the analysis of action potential frequency, evoked

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EPSC amplitude, and evoked EPSC latency of excitatory and inhibitory postsynaptic currents recorded from subep-ChAT+ neurons
in Figures 1, 2, and 3. Paired t-tests were used for analysis of in vivo optogenetics and chemogenetic stimulation and inhibition
studies. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
For quantification analysis of DCX+ neuroblast in brain slices, all the coronal sections where subep-ChAT+ neurons are found were
included in analysis. This area in SVZ is around 300–350 mm in length (Figure 6C; yellow circle (upper image)). Therefore, 6 coronal
sections (50 mm) from each mouse were included in analysis. Prior to statistical analysis, the quantified DCX+ neuroblasts in sections
from the same mouse were averaged. Similarly, the intensity quantifications of subep-ChAT+ neuronal activity were also averaged
within the same mouse prior to statistical analysis.
For quantifying the number of EdU+/GFAP+ and EdU+/Ki67+ cells in SVZ wholemount, the 40mm 3 40mm square images of V-SVZ
and SVZ layers were made where the subep-ChAT+ neuron is in the center of each image. This allows for studying the number of
EdU+/GFAP+ and EdU+/Ki67+ cells that are adjacent to subep-ChAT+ neurons.

22 Cell Reports 42, 112783, July 25, 2023