5212 • The Journal of Neuroscience, June 29, 2022 • 42(26):5212–5228

Systems/Circuits

Interactions between Brainstem Neurons That Regulate the

Motility to the Stomach
Lorenza Bellusci,1 Selena N. Garcia DuBar,1 Michelle Kuah,1 David Castellano,1 Vinona Muralidaran,2
Elizabeth Jones,2 Aaron M. Rozeboom,2 Richard A. Gillis,1 Stefano Vicini,1 and Niaz Sahibzada1
1
Departments of Pharmacology and Physiology and 2Pathology, Georgetown University Medical Center, Washington, DC 20007

Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown
that this activity is greatly influenced by local GABAergic signaling, primarily because of somatostatin (SST)-expressing
GABAergic neurons. To further understand the network dynamics associated with gastric motility control in the DVC, we
focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of
these neurons on gastric motility remains unknown. Here, we investigate the anatomic and functional characteristics of the
NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both
sexes. We sought to determine whether NPY neurons influence the activity of gastric-projecting neurons, synaptically interact
with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in
vivo show that NPY neurons are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from
the gastric antrum, are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum-projec-
ting neurons, are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a
potent inhibitory effect on the action potential firing of the NPY neurons, and affect gastric tone and motility as reflected by
their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the
network that controls vagal transmission to the stomach.
Key words: DMV: gastric motility; hindbrain; NTS; optogenetics; vagus

Significance Statement
The brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach
via tone and motility. Two distinct nonoverlapping populations of predominantly excitatory NPY neurons and predominantly
inhibitory SST neurons form reciprocal connections with each other in the NTS and with premotor neurons in the dorsal
motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS NPY neurons increased and
decreased gastric motility, respectively, whereas both activation and inhibition of NTS SST neurons enhanced gastric motility

Introduction nuclei essential to the regulation of vagovagal gastric activity.

Gastric function is dependent on the complex interplay between
visceral sensory input to the brain, activities in brain circuitries,
autonomic outflow, and function in the enteric nervous system.
In the hindbrain, the dorsal vagal complex (DVC) contains
Received Mar. 1, 2022; revised Apr. 4, 2022; accepted May 16, 2022.
Author contributions: L.B., S.N.G.-D., E.J., A.M.R., R.A.G., S.V., and N.S. designed research; L.B., S.N.G.-D.,
D.C., V.M., E.J., A.M.R., S.V., and N.S. performed research; S.V. contributed unpublished reagents/analytic tools; L.B.,
S.N.G.-D., M.K., D.C., S.V., and N.S. analyzed data; L.B., S.N.G.-D., R.A.G., S.V., and N.S. wrote the paper.
This work was supported by the National Institutes of Health–National Institute of Diabetes and Digestive
and Kidney Diseases Grant R01-DK117508. The Histopathology and Tissue Shared Resource at Georgetown
University Medical Center is partially supported by National Institutes of Health–National Cancer Institute
Grant P30 CA051008.
The authors declare no competing financial interests.
Correspondence should be addressed to Stefano Vicini at svicin01@georgetown.edu or Niaz Sahibzada at
sahibzan@georgetown.edu.
https://doi.org/10.1523/JNEUROSCI.0419-22.2022
Copyright © 2022 the authors
These are the nucleus tract of solitarius, specifically the medial
subnucleus [referred to as the nucleus tractus solitarius (NTS)]
and the dorsal motor nucleus of the vagus (DMV; Gillis et al.,
1989; Rogers et al., 1996; Berthoud, 2004; De Jonghe et al., 2011;
Gillis et al., 2022).
In a series of studies, we found a distinct projection that con-
trols the gastric tone and motility by the local g -aminobutyric
acid (GABA) signaling in the DVC (albeit into the NTS; Herman
et al., 2009, 2010, 2012). Pharmacological in vivo studies with
GABA and glutamate antagonists (Herman et al., 2009) showed
the evidence for NTS-DMV-projecting neurons to be both exci-
tatory and inhibitory (Glatzer et al., 2003; Davis et al., 2004).
Blockade of the GABAA receptors in NTS is accompanied by ro-
bust decreases in gastric mechanical activity (Herman et al.,
2009a). Although glutamatergic signaling is essential, its block-
ade in the NTS per se does not affect gastric mechanical activity
(Herman et al., 2009).
Bellusci et al. · Brainstem Neurons Influence Gastric Function
This GABAergic drive was further supported by the advent of
transgenic mice with GFP-expressing inhibitory neurons (GIN)
mice (Oliva et al., 2000), showing its presence in the DVC as
essential to the inhibition of the brainstem network dynamics
system (Glatzer et al., 2007; Gao et al., 2009). Furthermore, stud-
ies using sst-Cre transgenic mice revealed the abundant presence
of somatostatin (SST) neurons in the NTS (Lewin et al., 2016;
Thek et al., 2019) and in the DMV (Lewin et al., 2016) that likely
correspond at least in part to neurons identified in GIN mice
(Oliva et al., 2000). Optogenetic activation of SST neurons results
in IPSCs and robust inhibition of the action potentials of retro-
gradely labeled gastric antrum DMV neurons. Retrograde poly-
synaptic tracing revealed that SST neurons are part of the
vagovagal system (Wang and Bradley, 2010; Lewin et al., 2016).
Moreover, the DVC afferent vagus directly activates SST neurons
in NTS (Thek et al., 2019) as well as neurons in GIN mice in this
nucleus (Glatzer et al., 2007), thus, mediating the visceral sensory
motility input to the hindbrain.
In addition to SST neurons, neuropeptide-Y (NPY) neurons
are conspicuously present in the DVC and receive direct input
from sensory afferents (van den Pol et al., 2009; Chen et al.,
2020), but their role in the gastric vagal reflex circuitry is
unknown. NPY neurons are involved in different physiological
and homeostatic processes that include, among others, feeding
(Kamiji and Inui, 2007; Zhang et al., 2019), blood pressure
(Lettgen et al., 1994), and stress and depression (Heilig, 2004;
Morales-Medina et al., 2010; Reichmann and Holzer, 2016).
To assess the involvement of the NPY neuron in the gastric
circuitry of the DVC and, in particular, its relation to SST signal-
ing, we took advantage of distinct transgenic mice, including
npy-Cre;rosa26-tdTomato (Milstein et al., 2015) and sst-Cre;
rosa26-tdTomato (Taniguchi et al., 2011) mice expressing fluo-
rescence protein and the optogenetic effectors of inhibitory and
excitatory opsins. Initially, we showed the connection between
SST and NPY neurons and their neural function using Cre/Flox
and Flp/Frt combinations of drivers and reporter mice. Later, we
used sst- or npy-Cre;rosa26-ChR2-YFP mice to assess the impact
of SST or NPY neuron activation based on their synaptic connec-
tivity and electrophysiological characteristics on gastric activity
in vivo. Our goals were to (1) characterize neurons in the dorso-
vagal complex of in the npy-Cre;rosa26-tdTomato, (2) profile
their electrophysiological characteristics, (3) assess the synaptic
action of NPY and SST neurons on DMV motoneurons and
their reciprocal synaptic connectivity in the vagovagal circuitry,
and (4) determine their influence on end-organ function (that is,
gastric mechanical function). We found that brainstem NPY and
SST neurons interact and control stomach motility and tone.
Materials and Methods
Animal models. All animals were housed in a climate-controlled ani-
mal facility (22 6 2°C) and maintained on a 12 h light/dark cycle with
ad libitum access to food and water. Animal housing rooms were main-
tained at MP14 barrier (pathogen and opportunistic free) in the animal
facility prior to experiments.
Transgenic mice examined in this study were all The Jackson
Laboratory reporter strains NPY-Cre (RRID:IMSR_JAX:027851), a
gift from Boris V. Zemelman, Center for Learning and Memory,
University of Texas at Austin (Milstein et al., 2015); Rosa-ChR2-
EYFP reporter (RRID:IMSR_JAX:012569); Bac transgenic NPY-eGFP
mouse (RRID:IMSR_JAX:006417); Sst-ires-Flp (RRID:IMSR_JAX:028579);
mCherry-Frt reporter (RRID:IMSR_JAX:029040); Sst-Cre (RRID:IMSR_
JAX:013044); rosa26-tdTom (RRID:IMSR_JAX:007905); ChR2-tdTomato
(RRID:IMSR_JAX:012567); and ArchT-EGFP (RRID:IMSR_JAX:021188).
Based on the study in NPY or Sst neuron vagal gastric circuity, the driver
J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5213
and reporter strains were bred individually and were used to create a colony
by mating differentially with each other to develop the distinct mouse lines
used in the study. Mice of either sex 1–2 months old were anesthetized
with isofluorane and killed to prepare brain slice by decapitation after
cardiac perfusion following the National Institutes of Health guide-
lines, United Kingdom regulations for the ethical use of animals in
research (Drummond, 2009), and the approval of the Georgetown
University Animal Care and Use Committee. SST and NPY trans-
genic mice examined in this study have been described previously
(Milstein et al., 2015; Lewin et al., 2016).
RNA scope for NPY, SLC7A6, GAD67, and tdTomato mRNA. Mice
were anesthetized with isoflurane, followed by transcardiac perfusion
with ice-cold PBS solution. Brainstems were extracted and fixed over-
night in 10% neutral buffered formalin and transferred to 70% EtOH,
sectioned at 5 mm on a rotary microtome and stored at room tempera-
ture until use in RNAScope assay. Slides were baked at 60°C for 1 h,
deparaffinized in xylene twice for 5 minutes at room temperature and
dehydrated with 100% EtOH twice for 2 minutes and dried for 59 at 60°
C. The RNAScope assay proceeded according to the protocol of the
manufacturer, following the guidelines for standard tissue processing of
formalin-fixed paraffin-embedded tissue. Sequential brainstem sections
were hybridized with one of two three-plex probe mixtures, (1) NPY
(catalog #313321, ACD Bio), tdTomato (catalog #317041-C2, ACD Bio),
and Gad1 (catalog #400951-C3, ACD Bio), or (2) NPY, tdTomato, and
SLC17A6 (catalog #319171-C3, ACD Bio). Signal amplification was
achieved through multiple amplification steps using the RNAscope mul-
tiplex fluorescence kit (catalog #323100, ACD Bio) according to the pro-
tocol of the manufacturer. The following fluorophores were used: OPAL
570, 620, 690 (catalog #FP1488001KT, FP1495001KT, FP1497001KT,
Akoya Biosciences). At the end of the procedure, slides were cover-
slipped using ProLong Gold Antifade Mountant (Thermo Fisher
Scientific) and left to dry at RT before storing at 4°C and imaging.
Immunolocalization image acquisition and analysis. Brainstem sec-
tions were obtained from ;3 week postnatal day transgenic mice.
Following anesthesia with isoflurane, mice were initially perfused trans-
cardially with PBS (0.1 M, pH 7.3), followed by 4% buffered paraformal-
dehyde fixative (PFA). The brains were removed and stored overnight in
4% PFA. Free-floating coronal brainstem sections (50 mm) were obtained
using a vibratome (VT1000S, Leica). Sections were blocked with 4%
donkey serum in PBS for 1 h at room temperature and washed three
times for 10 min each in PBS containing 0.1% Triton X-100 (Tx). They
were then incubated overnight (minimum 12 h in a cold room at 4°C).
They were then incubated overnight (minimum 12 h in a cold room at
4°C) with a primary anti-NPY antibody (polyclonal rabbit, 1:400; catalog
#ab30914, Abcam; RRID:AB_1566510) or anti-vGlut2 (polyclonal
guinea pig, 1:500; catalog #AB2251-I, Millipore; RRID:AB_2665454)
that were diluted in PBS/Tx/1% BSA. Following this treatment, the
brainstem sections were rewashed three times for 10 min each in PBS/
Tx and then further incubated (2–4 h) at room temperature with a
secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (cata-
log #A11094, Thermo Fisher Scientific; RRID:AB_221544) or Alexa
Fluor 564 (catalog #A21312, Thermo Fisher Scientific; RRID:AB_
221478) at 1:500 dilution, which were constituted in PBS/Tx/BSA. At
the end of the incubation, the brainstem slices were washed three
times for 10 min each with PBS/Tx. All slices were mounted in
Vectashield Mounting Medium (Vector Laboratories).
Imaging. Slides were scanned at 10 magnification using the Vectra
3.0 Automated Quantitative Pathology Imaging System (PerkinElmer/
Akoya Biosciences). Whole slide scans were viewed with Phenochart
(PerkinElmer/Akoya Biosciences), which also allows for the selection of
high-powered images at 40 (resolution of 0.25 mm per pixel) for multi-
spectral image capture. Multispectral images were unmixed using
inForm Advanced Image Analysis software (inForm 2.4.6; PerkinElmer/
Akoya Biosciences) and exported as component image TIFFs for analysis
in QuPath 0.3.0 (Bankhead et al., 2017). Cell segmentation was done
using the StarDist extension (Schmidt et al., 2018).
Confocal microscope and colocalization analysis. To obtain the
DVC images shown, brain slices were transferred into the viewing
chamber of a resonant scanning confocal (Thorlabs) mounted on a
5214 • J. Neurosci., June 29, 2022 • 42(26):5212–5228
Nikon Eclipse F1 microscope. Acquired z-stack images (0.5 mm) using
20 or 40 immersion objective were processed with ImageJ soft-
ware (National Institutes of Health) to illustrate the distribution of
cell bodies for green or red fluorescent neurons. For the analysis of
colocalization z-stacks of images spanning the whole 50 mm thickness
of stained brainstem slices were acquired using a 20 or 40 immer-
sion objective and projected on a 2D image. Each channel was thresh-
olded to generate a binary image following manual clearing of the
background. The two colors images were merged, converted to 32-
bit, and individual cells counted. One randomly chosen slice per ani-
mal was acquired.
Anatomical tracing. To record from identified stomach projecting
DVC neurons, mice were labeled by monosynaptic tracer DiI18 or injected
with the polysynaptic tracers pseudorabies virus (PRV)-152EFGP in
the stomach (PRV-152EGFP was a gift from Lynn Enquist, Princeton
University Center for Neuroanatomy with Neurotropic Viruses, National
Institutes of Health Grant P40RR018604.). The tracer was applied to the
gastric antrum of transgenic mice expressing -tdTomato in a manner previ-
ously described by us (Lewin et al., 2016). To retrograde uptake of DiI in
the gastric-antrum-projecting DMV neurons, animals were allowed to
recover for 7–10 d after surgery, whereas PRV-injected mice were killed 2–
3 d after inoculation.
Brainstem slices. Slices were prepared from male and female mice at
least 4 weeks of age. Briefly, after isoflurane anesthesia, followed by
transcardiac perfusion with ice-cold N-Methyl-D-glucamine (NMDG)
solution (Ting et al., 2018), brains were quickly removed into oxygenated
solution (95% O2 plus 5% CO2, 4°C, pH 7.4, 296 mOsm) containing the
following (in mM): 93 NMDG, 93 HCl, 2.5 KCL, 1.2 NaH2PO4, 30
NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 so-
dium pyruvate, 10 MgSO4.7H2O, and 0.5 CaCl2.2H2O. Coronal brain
sections (250 mm) containing the brainstem DVC were cut in NMDG so-
lution using a vibratome (VT1000S, Leica) and incubated at 37°C in oxy-
genated HEPES holding artificial CSF (ACSF pH 7.4, 296 mOsm) with
the following composition (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30
NaHCO3, 20 HEPES, 2.5 glucose, 5 sodium ascorbate, 2 thiourea, 3 so-
dium pyruvate, and 2 MgSO4.7H2O. Slices were variably exposed to
NaCl (Ting et al., 2018). They were then allowed to be equilibrated for
an additional 3 h at room temperature (21°C). Subsequently, the slices
were transferred to a recording chamber (500 ml volume) attached to a
microscope stage (E600-FN, Nikon). There, they were continuously per-
fused with oxygenated ACSF (pH 7.4, 296 mOsm) composed of the fol-
lowing (in mM): 121 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2
CaCl2, 2 MgSO4, 5 HEPES, and 2.5 glucose.
Electrophysiology. Neurons were identified visually by infrared-dif-
ferential interference contrast or episcopic fluorescence optics and a
CMOS camera (Thorlabs). All recordings from NPY and SST neurons
were in the NTS. A 60 water immersion objective was used for identi-
fying and approaching neurons. Recordings were made with patch elec-
trodes (5–6 MV; Warner Instruments) with internal pipette solution
(pH 7.2, 285 mOsm) that was composed of the following (in mM): 145
K-gluconate, 5 EGTA, 5 MgCl2, 10 HEPES, 5 ATP.Na, and 0.2 GTP-Na.
In studies in which IPSCs were specifically studied, 145 mM KCl
was substituted for potassium gluconate in the pipette solution. Cell-
attached (loose seal ,40 MV), whole-cell voltage-clamp mode at a
holding potential (Vhold) = 60 mV or 30 mV) whole-cell volt-
age-clamp mode at a holding potential (Vhold) = 60mV or
30mV or current clamp or current-clamp recordings were performed
using a MultiClamp 700B amplifier (Molecular Devices). (Note: Action
potential firing frequency was not significantly different in parallel cell-
attached recordings with ACSF pipette solution.) A 5 mV hyperpolariza-
tion pulse monitored input and access resistances; series resistance was
typically ,10 MX and was not compensated. Resting membrane poten-
tials were corrected for liquid junction potential, which for the intracellu-
lar solutions, K-gluconate was 15 mV, and for the KCl was 3 mV.
Signals were low-pass filtered at 2 kHz and acquired with a Digidata
1440A Digitizer (Molecular Devices).
In vitro optogenetic control. Light delivery in coronal brainstem slices
was accomplished using filter cubes of the microscope [channelrhodop-
sin (ChR2), l = 450–490 nM; ArchT-EGFP l = 510–560 nM] via white
Bellusci et al. · Brainstem Neurons Influence Gastric Function
light from an X-Cite 120LED (Excelitas Technologies). Slices were
excited with a maximal light intensity adjusted to prevent loss of voltage
clamp (Vhold = 70 mV). The diameter of the area exposed to optoge-
netic control under the 60 objective was ,100 mm and encompassed
the whole DVC area.
Drugs. Stock solutions of the following drugs were prepared in water:
D( ) 2-Amino-5-phosphonopentanoic acid (catalog #3693, Tocris
Bioscience), bicuculline methobromide (BMR; catalog #HB0894, Hello
Bio), 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfon-
amide disodium salt (NBQX, catalog #ab120046, Abcam), tetrodo-
toxin (TTX, catalog #HB1035, Hello Bio), and 4-Aminopyridine
(catalog #A-0152, Sigma-Aldrich). Drug-containing stock solutions
were diluted to desired concentrations in ACSF. All drugs were applied
via the Y-tube application adapted to brain slices (Murase et al., 1989;
Hevers and Lüddens, 2002).
In vitro data analysis. Electrophysiological data were analyzed off-
line using pClamp 11 (Molecular Devices). Data were acquired from
neurons that had a stable baseline membrane potential. Threshold, rheo-
base, and rate and characteristic of action potentials were assessed
from a baseline membrane potential of 60 mV by injecting current.
To analyze postsynaptic currents, semiautomated threshold pClamp
11 software was used with 5–10 times the baseline noise depending
on the voltage clamp (Vhold = 60 mV or 30 mV) and the internal
pipette solution (K-gluconate or KCl). The control and treatment
data were acquired from two consecutive 500 ms segments. For
studies where optogenetic activation was used to excite NPY-Cre;
ChR2 or Sst-Cre;ChR2 neurons while recording from the NPY or
Sst neuron or gastric-antrum-projecting DMV neurons, the voltage-
clamp was set at 30 mV. This procedure allowed us to separate
IPSCs and EPSCs, which were displayed as upward deflections (out-
ward currents) and downward deflections (inward currents). Both
IPSCs and EPSCs were measured together as there was minimal, if
any, overlap between the two types of currents. Moreover, it enabled
us to clearly visualize a one-to-one correlation between the onset of
the optogenetic activation and its effect on the identity of the postsy-
naptic current (i.e., IPSC or EPSC).
Gastric physiology. Experiments were performed on NPY-Cre;ChR2
and Sst-Cre;ChR2/or Cre;ArchT-EGFP mice.
Surgical preparation. Before all experiments, food was withheld for
4 h, whereas water was provided ad libitum. Animals were anesthetized
with an intraperitoneal injection containing a mixture of urethane
(1000 mg/kg) and a-chloralose (60 mg/kg) dissolved in 0.9% saline.
Body temperature was maintained at 37 6 1°C with an infrared heat
lamp.
After the depth of anesthesia was confirmed by lack of pedal and cor-
neal reflexes, mice were intubated via the trachea following a tracheot-
omy to maintain an open airway and to institute artificial respiration
when necessary. Both cervical vagi were carefully isolated from each ca-
rotid artery on either side and looped with a 5–0 silk thread for later
avulsion during the experiment.
Subsequent, to the cervical vagi loop, a laparotomy was performed to
expose the stomach. An intragastric balloon (made from the tip of a latex
condom ;0.3 cm long) was inserted into the stomach via the fundus
and positioned in the distal region of the antrum, secured in place by a
purse-string suture. The balloon was inflated (with warm water ;100–
150 ml) to produce a baseline pressure of 3–6 mmHg, which was
measured by a pressure transducer (sensitivity 5 mV/V/mmHg) that was
connected to it as described by (Richardson et al., 2013) for monitoring
blood pressure in rats. At the end of the procedure, the abdominal cavity
was closed using a gut suture.
In vivo optogenetic. To gain access to the dorsal medulla, mice were
positioned in a stereotaxic apparatus (David Kopf Instruments). A lim-
ited dorsal craniotomy was performed to expose the medulla, and the
underlying dura and pia were cut and reflected. The caudal tip of the
area postrema, the calamus scriptorius (CS), was viewed as a reference
point for determining the coordinates with optic fiber cannula (50–
70 mm, Doric Lenses) directly connected to a laser output for blue light
(;473 nM, 1.20 mW) or green light (;532 nM, 1.36 mW; Shanghai Laser
& Optics Century). The laser output was controlled by a Master-8
Bellusci et al. · Brainstem Neurons Influence Gastric Function
stimulator (AMPI), and the intragastric pressure data were acquired
using the PowerLab data acquisition system (ADInstruments).
Optogenetic control with the optic cannula was given either into the
NTS or DMV at a 30° angle from the perpendicular. Responses evoked
from the NTS were induced by a 90 or 60 s envelope of photograph exci-
tation or inhibition by trains of light pulses (frequency 15 Hz, pulse
width 10 or 40 ms, duty cycle of 15 or 60%). Duty cycles of 60% for a 90
s duration of light activation were used in early experiments, but a duty
cycle of 15% for 60 s was more reliable and used for the rest of the study.
These optogenetic control parameters are compatible with those used in
respiration studies (Abbott et al., 2013). Stereotaxic coordinates for the
optic fiber cannula into the NTS were area postrema (AP) = 10.3–0.5
mm rostral to CS, ML = 0.1–0.3 mm lateral to the midline, and DV =
0.0–0.1 mm ventral to the dorsal surface of the medulla. Coordinates for
the DMV were 10.3–0.5 mm rostral to CS, 0.1–0.3 mm lateral to the
midline, and 0.2–0.3 mm ventral to the dorsal surface of the medulla.
Optogenetic activation tags were also acquired using the PowerLab data
acquisition system.
Histologic verification of pipette tracks. At the end of each experi-
ment, the mice were killed with an overdose of urethane. The brain was
removed and placed in a fixative-cryoprotectant solution composed of
4% phosphate-buffered paraformaldehyde and 10% sucrose (0.1 M, pH
7.4) for at least 48 h. The brainstem was dissected and cut on a cryostat
into 50 mm coronal sections, which were mounted serially onto gelati-
nized slides and stained with neutral red staining solution. The locations
of the cannula tracks were identified using the mouse brain atlas
(Paxinos and Franklin, 2007). To document cannula sites in the brain,
microphotographs and camera lucida drawings were made of each pip-
ette track.
Experimental design and statistical analysis. Analysis of the intra-
gastric pressure recordings was completed off-line using Chart
(ADInstruments) and Prism (GraphPad) software packages. All the ex-
perimental recordings were initially filtered using a root mean square
(RMS; 2 s moving window) algorithm to account for respiration and vari-
ous other signal artifacts. All peaks were selected within the 90 or 60 s of
light activation and compared with the immediately prior peaks, selected
for the same amount of time (baseline).
An algorithm for calculating average peak-to-peak values was used
to determine a change in amplitude of gastric phasic contractions. This
algorithm was used because gastric phasic contractions have a low fre-
quency of occurrence. Because of the natural variance in the amplitude
of gastric contractions, the average peak-to-peak values proved to be a
uniform method to compare all datasets. However, following analysis of
peak-to-peak parameters of baseline values and those measured during
light activation, it was found that differences in intrinsic activity of an
animal could introduce considerable variance in the results. An optoge-
netic-induced response in each animal was normalized to its baseline
value to solve this problem, which was displayed as a percentage change in
the gastric contractions. The noise level on all completed peak-to-peak
analysis was set to zero as an RMS algorithm had already filtered the data.
Baseline and peak values during light activation were taken as a per-
centage difference using the following formula: ((light activation-base-
line)/(baseline))  100). The same formula has been used to calculate
the area under the trace (tone).
Quantification and statistical analysis . Statistical analyses were per-
formed using Prism 9 (GraphPad). All data indicated in the text and fig-
ures were checked for normal distribution, and the results expressed as
means 6 SEM unless otherwise stated. The statistical analysis compari-
sons between two groups was performed by a paired Student’s t test or
unpaired t test with Welch’s correction. Comparisons among three or
more groups was performed with one-way ANOVA followed by the
Holm–Sídák’s post hoc test, Brown–Forsythe and Welch test followed by
Dunnett’s T3 multiple comparisons test, or mixed-effects model fol-
lowed by Tukey’s multiple comparisons test.
All percentage from in vivo data were analyzed for the significance of
normality using the D’Agostino–Pearson omnibus and or Shapiro–Wilk
normality test. A one-sample t test was performed on the data with the
hypothetical value set at zero. For ipsilateral vagotomy data, a two-sam-
ple paired t test was performed.
J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5215
Animal number and cell numbers are indicated in the text. The crite-
rion used to determine statistical significance was p , 0.05.
Results
It is well documented that the DVC serves as an autonomic gate-
way that comprises many neuronal subtypes, which are essential
to the homeostatic regulation of autonomic functions such as
those related to the gastrointestinal, cardiovascular, and respi-
ratory systems (Feldman and Ellenberger, 1988; Rinaman et al.,
1989; Lettgen et al., 1994; Lawrence and Jarrott, 1996; Gillis et
al., 2022). We focused on the NPY neurons in the DVC to
delineate their role in gastric motility. To accomplish this, we
examined their (1) distribution in the DVC, (2) presence in the
vagovagal circuitry, (3) electrophysiological characteristics, (4)
synaptic connectivity, and (5) influence on end-organ function
(i.e., stomach).
Distribution of NPY neurons in the DVC and their
phenotype
We first established distribution of the NPY neurons in the
DVC of the npy-Cre knock-in mouse (catalog #027851, The
Jackson Laboratory), crossed with the Rosa-tdTomato Flox re-
porter mouse (catalog #007905B6, The Jackson Laboratory)
that expresses robust tdTomato fluorescence following Cre-
mediated recombination directed by the mouse npy promoter
(Milstein et al., 2015). This strain was also reported in a recent
study (Chen et al., 2020). A prominent diagonal band of
TdTomato-expressing neuron was observed in NTS (Fig. 1H),
which extended mediolaterally from the central canal encom-
passing subnuclei, including the medial, central, dorsal (medial
and lateral), with a few scattered neurons and their dendrites in
the commissural and intermediate subnuclei. Distinct pockets
of neurons were also observed in the ventrolateral NTS. In the
underlying DMV nucleus, although relatively few neurons were
scattered along the mediolateral axis, neurons favored a more
lateral location and were interspersed with other DMV cells at
its border with the NTS. This appearance was in contrast with
NPY terminals in the DMV that showed distinct punctations
that were more or less evenly spread throughout the nucleus as
they were in the overlying NTS. Also, labeled neurons were
present in the dorsally located AP (Fig. 1H).
Using the Cre/Lox technology, transgenic breeding can
potentially lead to recombination during development, inducing
expression in off-target neurons (Taniguchi et al., 2011). Using
in situ hybridization with RNAscope and probes targeting NPY
and TdTomato (Fig. 1A–E) revealed that NPY mRNA expression
is present in 84 6 10% of a cell expressing mRNA for TdTomato
(mean 6 SD, three mice, 27 sections analyzed). We also per-
formed immunohistochemistry for NPY (n = 2 mice, six slices)
to visualize tdTomato neurons expressing NPY (Fig. 1I,J) using a
polyclonal antibody, which was visualized with confocal micros-
copy using a far-red secondary Alexa 647 antibody. TdTomato
neurons were partially labeled with anti-NPY antibodies (48 6
8%; mean 6 SD) whereas 74 6 14% (mean 6 SD) of NPY
positive neurons expressed TdTomato. NPY antibody stain-
ing appeared to be restricted to the perikaryal cytoplasm
and surrounding neuropil, as evident from the labeled
fibers and puncta (Fig. 1J). The discrepancy between NPY
mRNA and antibodies detection could be because, as in
most peptides, NPY expression fluctuates because of altera-
tions in physiological activity and development.
5216 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 1. A subset of NPY neurons in the NTS and DMV of a transgenic npy-Cre;tdTomato animal is glutamatergic. A, B, Representative micrograph of the DVC (A) that was used for
RNAscope in situ hybridization probes for NPY, Slc17A6 (VGlut2), tdTomato mRNA (B), and GAD1 (GAD67 not shown) in distinct adjacent brainstem paraffin sections. C–G, Graph shows the per-
centage RNA target expression of GAD1, VGlut2, and NPY in the NTS (C). White box indicates region imaged at higher magnification in D and E; F and G are from adjacent 5 mm section.
Colocalization with the NPY and Slc17A6 (E) or NPY and GAD1 (G) with tdTomato mRNA (TdTom, D, G). H, Distribution of td-Tomato neurons in the DVC of an npy-Cre;tdTomato mouse.
I, J, Colocalization of td-Tomato neurons (I) with anti-NPY immunostaining in the NTS (J, NPY-Ab). K–M, Coexpression of transgenically td-Tomato labeled NPY neurons with YFP expression
induced by unilateral virus injection in the DVC and their overlap in the NTS. N–P, Example of immunoreactivity to the VGlut2 transporter antibody as visualized by the secondary Alexa 647
antibody in the NTS of a npy-Cre;tdTomato mouse. (Note: Double arrowheads in all micrographs identify neurons that show respective colocalization.) 12N, Hypoglossal nucleus; CC, central
canal. Calibration: A, B, 100 mm; D–G, 50 mm; H, 100 mm; I–M, 40 mm; N–O, 60 mm.

To further ascertain that cre-expression in DVC neurons the dorsomedial NTS region to target neurons in npy-Cre;
was not because of developmental off-target recombination, tdTomato mice (n = 2 mice). The virus-labeled NTS neurons
unilateral microinjection of the rAAV5/EF1a-DIO-EYFP virus also exhibited npy-Cre mediated recombinatorial tdTomato
(University of North Carolina Virus Vector Core) was made in expression (Fig. 1K–M; 93 6 4% colabeling, n = 5 slices). This
Bellusci et al. · Brainstem Neurons Influence Gastric Function
indicates that the cre transcript is similar between neurons of
adult mice. In general, when the transcriptional activity of NPY
goes down, Cre expression should also decrease, although at a
slower pace, which results in decreased reporter expression.
Because the regulation of gastric motility is intimately de-
pendent on the nature of the ongoing neural activity in the DVC
(Rogers et al., 1996), we next examined the potential neurotrans-
mitter associated with the NPY neurons. We focused on gluta-
mate as its VGlut2 transporter has been reported to be present,
among others, in the medial and dorsomedial NTS subnuclei,
which is central to the vagovagal circuitry of the DVC controlling
gastric tone and motility (Lin et al., 2004, 2008; Okada et al.,
2008).
To further characterize the phenotype of npy-cre neurons,
RNAscope studies extended to probes targeting, in addition to
NPY and TdTomato, VGlut2 (Slc17A6) or for comparison
GAD67 (GAD1) in distinct adjacent brainstem paraffine sections
(14 sections, three mice) analyzed at the intermediate rostrocau-
dal level, which is where the majority of subsequent studies were
performed (Fig. 1B,C,F,G). Our experiments were similar to
those performed to validate the Sst-Cre line in the DVC (Thek et
al., 2019, their Fig. 1). These results revealed that Slc17A6 mRNA
expression is present in 53 6 24% (mean 6 SD) of a cell express-
ing mRNA for TdTomato. In contrast, GAD1 mRNA expression
was present in 22 6 16% (mean 6 SD) of a cell expressing
mRNA for TdTomato.
We also assessed the VGlut2 transporter antibody with im-
munoreactivity as visualized by the secondary Alexa 647 anti-
body as shown in Figure 1N–P. In 19 hindbrain sections from
three npy-Cre;tdTomato mice 52 6 14% of TdTomato neurons
were positive for the VGlut2 antibody.
Altogether, these studies demonstrate that NPY neurons in
npy-Cre;tdTomato mice are distinctly distributed throughout
the extent of the NTS and DMV, with their density especially
notable in the NTS. Furthermore, a larger subset of the NPY
neurons in the DVC is glutamatergic, whereas a smaller subset
is GABAergic.
NPY neurons are part of the vagal reflex circuit that
regulates gastric motility
To establish that the NPY neurons of the DVC are part of the au-
tonomic circuitry that controls gastric motility, we trans-synapti-
cally traced their connectivity from the stomach with PRV (Card
et al., 1993; Smith et al., 2000; Lewin et al., 2016). The PRV iso-
variant 152 was injected into the gastric antrum of npy-Cre;
tdTomato transgenic mice. Our reason for tracing NPY neurons
from the antrum was the important role of this gastric region in
gastric motility (el-Sharkawy and Szurszewski, 1978; Gillis et al.,
1989; Lüdtke et al., 1991; Rogers et al., 1996; Gillis et al., 2022).
PRV retrogradely labeled NPY neurons in the DMV and NTS
(Fig. 2A–F). Notably, particularly in the DMV, premotor
antrum-projecting neurons were observed to be surrounded by
NPY terminals (Fig. 2E,F), suggesting that DMV neurons may
be the recipients of regulatory modulation by these neurons.
This observation led us to explore what other neurons may like-
wise be in close apposition with these terminals. Of particular in-
terest were the SST neurons in the DVC, which we previously
reported to have a strong influence on the activity of antrum-
projecting DMV neurons and, by extension, on gastric motility
(Lewin et al., 2016). To examine whether SST neurons received
NPY terminals, as both SST and NPY transgenic mice are cre
drivers, we have bred sst-Cre;tdTomato mice with npy-hrGFP-
BAC (van den Pol et al., 2009; Fig. 2G–J), and their brains were
J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5217
processed for imaging. Inspection of the DVC under confocal
microscopy showed that both NPY and SST neurons in this com-
plex represented distinct populations of neurons with no overlap
(n = 4 mice, eight slices) whose terminals were in apposition to
each other (Fig. 2H–J). Moreover, each neuron population dis-
played terminals from the other population and suggested recip-
rocal connectivity within the same population (Fig. 2J, arrows).
These results show that the NPY neurons are (1) part of the
vagovagal circuitry that controls the stomach and (2) reciprocally
connected to themselves and SST neurons in the DVC.
NPY neurons are functionally interconnected by an
excitatory network
To study the functional characteristics of NPY neurons, we com-
bined optogenetics with electrophysiology and pharmacological
techniques in brainstem slices from transgenic mice expressing
Chr2. Excitation of NPY neurons in the NTS of npy-Cre;ChR-
EYFP mice produced consistent action potentials with a train or
persistent (2 s) light activation in extracellular or intracellular
recordings (Fig. 3A,B). Depolarization current steps produced
action potentials that displayed little spike-frequency adaptation
(Fig. 3C,D), whereas injection of hyperpolarizing currents pro-
duced a noticeable depolarization sag (2.1 6 1.5 mV, n = 11 cells
in six npy-Cre;tdTomato mice, mean 6 SD). Cell-attached
recordings (loose seal ,40 MV) showed that NPY neurons were
spontaneously active with a mean firing rate of 3.6 6 3.6 Hz (n =
11 cells in five npy-Cre;tdTomato mice, mean 6 SD; Fig. 3E).
The intracellular current-clamp recording showed the mean
peak amplitude, rise time, and half width of the action potential
to be 76.5 6 4.8 mV, 0.8 6 0.04 ms, and 2.3 6 0.2 ms in 11 neu-
rons from 6 npy-Cre;tdTomato mice, respectively (Fig. 3E). The
average resting membrane potential and input resistance were
61.4 6 1.6 mV and 1.2 6 0.2 GV in 11 neurons from 6 npy-
Cre;tdTomato mice (Fig. 3E). Moreover, optogenetic inhibition
of tonically active NPY neurons via activation of the proton-
pump opsin ArchT in npy-Cre;ArchT-EGFP mice showed that
they exhibit robust rebound activity on hyperpolarization (Fig.
3F) from a mean baseline of 3.3 6 1.2 Hz before light activation
to 5.9 6 1.1 Hz afterward.
We next examined the effect of the NPY neurons in response
to short (5 ms) and long (2 s) pulses of light in neurons in the
DVC of npy-Cre;ChR2-EYFP mice. Light excitation evoked large
inward currents with superimposed postsynaptic currents (PSCs;
Fig. 3G). Spontaneous and light-evoked PSCs but not the large
inward currents were reversely blocked by local exposure, via a
y-tube, to the glutamatergic AMPA receptor antagonist NBQX
(5 mM; Fig. 3H,I). Subtraction of the average trace in Figure 3H
from all traces in Figure 3G revealed light-evoked EPSC from re-
ciprocal connections between NPY neurons that were blocked by
NBQX (Fig. 3J; control 3K plus NBQX) as shown in the super-
imposed traces in Figure 3, J and K, and the summary of results
in Figure 3L (n = 9 cells in nine mice) comparing the peak
EPSCs with the peak of the direct ChR2 currents. These studies
show that NPY neurons of the DVC of npy-Cre mice (1) are toni-
cally active, (2) display action potentials with little spike-frequency
adaptation in response to depolarizing current injection, (3) exhibit
hyperpolarization-induced depolarization sags, and (4) are function-
ally connected via an excitatory synaptic network.
Gastric-projecting DMV neurons are excited by optogenetic
activation of NPY neurons
The DMV forms the parasympathetic efferent arm of the vagova-
gal gastric reflex circuitry, which, among other gastric functions,
5218 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 2. NPY neurons in the DVC are part of the vagal circuit that regulates gastric motility and tone. A–C, Micrographs showing, after an ;72 h survival period, the result of the injection
of the PRV (isovariants 152) into the gastric antrum polysynaptically labels NPY neurons in DMV and NTS of a npy-Cre;tdTomato. D, Quantification of the percentage of neurons labeled with
PRV-152 throughout the rostrocaudal extent of the DVC of npy-Cre;tdTomato mouse (n = 14 sections, .15 PRV1 cell count per section). AP, area postrema; 4V, 4th ventricle. E, F,
Representative micrographs of PRV-614-labeled gastric-antrum-projecting neurons in the DMV displaying apposition with tdTomato positive terminals. (Note: Double arrowheads in all micro-
graphs identify neurons showing colocalization.) G, Breeding strategy for transgenic mice labeling both NPY and SST neurons. H–I, Confocal micrographs showing digitally rendered images of
NPY (H) and SST (J) neurons showing the apposition of NPY terminals onto SST neurons (white/green arrows) and SST terminal onto NPY neurons (white/red arrows). Calibration: A–C, 60 mm;
E, 20 mm; F, 4 mm; H–J, 20 mm.

controls motility of the upper gastrointestinal (GI) tract (Gillis et
al., 1989; Ferreira et al., 2002; Niedringhaus et al., 2007; Cruz et
al., 2007; Gillis et al., 2022). As gastric-projecting DMV neurons
in this nucleus display close apposition of NPY terminals on
their cell bodies (Fig. 2F), we wondered about the functional con-
sequence of stimulating these terminals on the activity of the
DMV neurons. To identify these gastric-projecting neurons,
crystals of the cell-viable monosynaptic retrograde tracer DiI
were applied to the gastric antrum and isolated from the sur-
rounding tissue by a silicone glue as previously described by us
(Lewin et al., 2016). Optogenetic excitation of NPY neurons
increased the firing frequency of the antrum-projecting neurons
from a mean baseline of 1.9 1 0.3–2.9 1 0.4 Hz (Fig. 4A–C; p =
0.0024, t = 3.300, two-tailed paired t test, n = 33). Similarly, base-
line synaptic activity was also increased on long-lasting light
exposure from a baseline of 4.4 1 0.7–7.9 1 1.2 Hz (Fig. 4D–F;
p = 0.0132, t = 3.170, two-tailed paired t test, n = 9). Light excita-
tion evoked synaptic currents with both excitatory and inhibitory
components. This feature was particularly evident from the
increase in the amplitude of the EPSCs as opposed to IPSCs, an
observation that was made possible by voltage clamping each
DMV neuron at 40 mV or by exposing the neurons to the
GABAA or glutamate antagonists, respectively (Fig. 4G–J).
Figure 4K summarized the percentage of neurons responsive to
light on action potential firing and their synaptic activation. In
cells displaying light-induced EPSCs or EPSC-IPSC sequence, we
Bellusci et al. · Brainstem Neurons Influence Gastric Function J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5219

Figure 3. NPY neurons are functionally interconnected by an excitatory network. A, B, Response to light stimulation of NPY neurons in the cell-attached (A) and current-
clamp modes (B) in a npy-Cre;ChR2-EYFP transgenic mouse. C, D, Representative voltage responses (C) and phase plot (D) of an NPY neuron to 10 pA current steps injections.
[Note: hyperpolarization-induced depolarization sag in C (arrow)]. E, Graphs showing the spontaneous action potentials (AP) firing rate (5 npy-Cre;tdTomato mice), parame-
ters of the action potential, resting potential, and input resistance (RM) in NPY neurons in the DVC (6 npy-Cre;tdTomato mice). F, Representative light-induced (train)
suppression of action potentials in an NPY neuron in a npy-Cre;ArchT-EYFP mouse. [Note: the robust rebound response on cessation of the light train (red stippled arrows)].
G–I, Representative recordings of light-evoked excitatory currents in an NPY neuron before and after exposure to NBQX. J, K, Subtraction of the average trace of I from all
traces in J exposure to NBQX. L, Graph of the peak of light-evoked EPSC from reciprocal connections between NPY neurons that were blocked by NBQX (EPSC) compared with
peak of the direct-light-evoked ChR2 current (n = 9 cells in 9 mice).

measured the latency of peak EPSC and IPSCs from the begin-
ning of the light pulse (lag time) and their relative lag time as
time difference between the peak EPSC minus peak IPSC (Fig.
4L). The EPSCs latency (11.2 6 0.8 ms, n = 43) was shorter than
that for IPSC (19.4 6 2.8 ms, n = 17). The lag time between IPSC
and EPSCs was 6.4 6 2.8 ms (n = 17). These studies may
indicate that the action of NPY neurons on gastric-antrum-
projecting neurons is mainly excitation but can also trigger
inhibition via the polysynaptic activation of GABAergic
neurons. However, evoked PSC latency has been shown to
5220 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 4. Gastric-projecting DMV neurons are excited by optogenetic stimulation of NPY neurons. A, B, Light-induced responses from retrogradely labeled DMV neurons (configuration as
depicted in the diagram; star denotes the gastric site of the retrograde dye application). B, Consistent responses are elicited by a single pulse followed by a train of pulses (15 Hz; overlay of 5
sweeps shown). C, Graph showing light-evoked changes in the firing rate of action potentials in DMV neurons (n = 33 neurons from 9 mice). D–F, Change in excitatory synaptic currents in
response to light stimulation in DMV neurons (n = 9 neurons from 4 mice). G, Overlay (top) and distribution (bottom) of traces illustrating light-induced EPSC-IPSC sequences at 40 mV hold-
ing voltage (Vh). H, Graph showing light-evoked changes in postsynaptic currents in DMV gastric-projecting neurons at 70 or 40 mV Vh. I, J, Representative recordings of light-evoked exci-
tatory currents in at DMV neurons at Vh 70 mV and two additional DMV neurons at Vh 40 mV that are abolished by NBQX (5 mM) but not by BMR (25 mM). K, Summary graphs showing
Bellusci et al. · Brainstem Neurons Influence Gastric Function
be a poor predictor of monosynaptic versus polysynaptic
connections in the DVC (Doyle and Andresen, 2001;
Appleyard et al., 2007; Neyens et al., 2020), and light-
evoked IPSCs could be because of the small portion of
GAD1-expressing NPY neurons.
Synaptic interactions of NPY and SST neurons and their
effect on premotor DMV neurons in the DVC
Previously, we reported that SST neurons are connected to pre-
motor DMV gastric neurons, whose activity is robustly affected
by their excitation (Lewin et al., 2016). Our present imaging data
show that in addition to the DMV neurons, SST neurons appear
to be anatomically connected to the NPY neurons (Fig. 2J). This
observation compelled us to assess the interaction between these
two neurons and gastric premotor neurons in the DVC. To ac-
complish this, a similar type of breeding strategy as mentioned
before was used (i.e., crossing npy-Cre;Chr2-YFP transgenic
mice with sst-Flp;;mCherry mice or crossing sst-Cre;Chr2-YFP
mice with npy- hrGFP-BAC mice).
As with the DMV premotor gastric neurons (Fig. 4), we first
determined whether the excitation of NPY neurons would influ-
ence the synaptic activity of the SST neurons. Optogenetic stimu-
lation of NPY neurons in 4 npy-Cre;ChR2-YFP mice induced
light-activated EPSC-IPSC timing sequences that varied between
the different SST neurons; in some neurons, light-evoked EPSCs
occurred before the IPSCs, whereas in others, the opposite was
true (Fig. 5A,B). Overall, as seen in the percentage of light-re-
sponsive SST neurons, the EPSCs evoked were more than the
IPSCs. The amplitudes and occurrence of both were enhanced
by exposure to 4-AP (100 mM; Fig. 5C) to increase synaptic
release, suggesting polysynaptic activation of inhibitory neurons.
Light activation of SST neurons, as was reported for the
DMV, antrum projection neurons (Lewin et al., 2016) pro-
foundly suppressed (88 6 5%, 21 cells, six mice) the activity of
NPY neurons from a mean baseline of 2.5 6 0.5 Hz (Fig. 5D),
which was associated with robust light-evoked IPSCs (Fig. 5E,F;
n = 10 cells from 2 npy-Cre;ChR2-YFP mice; n = 12 cells from 5
sst-Cre;ChR2-YFP mice). Furthermore, as noted in our experi-
ments with npy-Cre;ArchT-EGFP mice (Fig. 3F), recovery of
NPY neurons from inhibition (in this case by SST neuron excita-
tion) was accompanied by rebound spiking (Fig. 5G). Altogether,
the striking inhibitory effect of SST stimulation on NPY neurons
is best illustrated by the dual-cell attached recording example
shown in Figure 5H (n = 5).
To determine whether the light-induced effects were direct
from presynaptic neuron terminals, we used subcellular channel
rhodopsin-assisted circuit mapping (Petreanu et al., 2009). We
used TTX (1 mM and 4-AP, 50 mM) to prevent action potentials
with ChR2 activation, thus demonstrating that the synaptic activ-
ity onto a neuron is because of the presence of synaptic terminal
on the recorded neuron (Petreanu et al., 2009). Studies of light-
evoked synaptic activity using this technique revealed that the
DMV premotor gastric neurons received differential functional
input from SST and NPY neurons. Whereas the SST neuron
more reliably influenced DMV gastric neurons via a direct
/ by a noticeable quiescent period marked by an initial drop in
the percentage of cells displaying the light-evoked changes in action potentials (spike),
EPSCs, or EPSC-IPSC sequence in DMV-gastric projecting neurons (n = 16 mice). L, Latency of
peak EPSC and IPSCs from the beginning of the light pulse and the differences in their lag
time as time difference between the peak EPSC minus peak IPSC (n = 43 and 17 neurons
from 16 mice).
J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5221
monosynaptic input (five of five cells responding), the NPY neu-
ron was seen to elicit its effect with optogenetic excitation in the
presence of both TTX and 4-AP in only two of six DMV neurons
tested (Figure 5I–L; n = 5 cells from two sst-Cre;ChR2-YFP mice;
n = 6 cells from three npy-Cre;ChR2-YFP mice).
In assessing the interaction between the different neurons in
the DVC, we were also interested to know whether, similar to
the NPY, light activation of SST neurons was capable of eliciting
changes in the synaptic activity of other SST neurons in the
DMV, as has been previously reported in the NTS (Thek et al.,
2019). Light excitation of SST neurons evoked PSCs blocked by
the GABAA antagonist gabazine (10 mM; n = 4 of 6 neurons, two
mice; Fig. 5M,N). Altogether, these studies demonstrate that
NPY neurons are connected to SST neurons in the DVC, which
in turn have a powerful influence on their tonic activity.
Furthermore, they show that whereas NPY neurons may indi-
rectly influence DMV gastric neurons and SST neurons, they
also directly influence premotor neurons.
In vivo optogenetic manipulation of NPY and SST neuronal
activity in the DVC affects gastric motility and is site
dependent
The DVC of the hindbrain is critical to the vagovagal control
of gastric motility. In particular, its nuclei, NTS and DMV, differ-
entially regulate the activity of the upper GI tract. Microinjection
of L-glutamate in the NTS inhibits GI activity, whereas its applica-
tion in the DMV excites it (Ferreira et al., 2002; Cruz et al., 2007;
Niedringhaus et al., 2007; Herman et al., 2009, 2010; Richardson
et al., 2013). As both NPY and SST neurons are found in these
nuclei and strongly affect the activity of gastric neurons (Fig. 1;
Gao et al., 2009; Lewin et al., 2016; Thek et al., 2019), optogenetic
studies were undertaken to parse out the role of these neurons in
regulating gastric motility. Responses evoked from the NTS were
induced by a 60 s envelope of photograph excitation or inhibition
using trains of light pulses (frequency 15 Hz, pulse width 10 ms,
duty cycle of 15%). These optogenetic parameters are derived
empirically from preliminary in vitro studies (Fig. 6A), which
showed that they robustly activate SST or NPY neurons in the
DVC. The parameters above were similar to those used by
Guyenet and colleagues to study control of respiration (Abbott et
al., 2013).
To differentiate responses induced from the NTS from those
deriving from the underlying DMV, we used ipsilateral vagot-
omy, which we have shown to inhibit responses evoked only
from the DMV as this nucleus projects ipsilaterally to postgan-
glionic gastric neurons (Ferreira et al., 2002; Cruz et al., 2007;
Herman et al., 2009; Richardson et al., 2013). This procedure is
in contrast to the NTS, which projects bilaterally to the DMV
(Norgren, 1978); hence, responses evoked from this nucleus are
not blocked by ipsilateral vagotomy (Fig. 6B; Ferreira et al., 2002;
Cruz et al., 2007; Herman et al., 2009; Richardson et al., 2013).
To target the NTS or DMV, a direct visual approach was adopted
in anesthetized mice (Fig. 6C,D) using calamus scriptorius as a
reference point (Ferreira et al., 2002; Cruz et al., 2007).
In npy-Cre;ChR2-YFP mice, light activation of the NTS (n =
9) induced a robust increase in gastric motility and tone followed
tone. (Fig. 6E). The increase in motility and tone to optogenetic
activation was not affected by ipsilateral vagotomy (Fig. 6F; n =
7). This result is in contrast to light activation of Chr2 in the
DMV (n = 5 mice), where suppression of gastric motility induced
by light (Fig. 6G) was blocked by ipsilateral vagotomy (Fig. 6H),
5222 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 5. Synaptic interactions and connectivity of neurons in the DVC. A, B, Optogenetic stimulation of NPY neurons evokes EPSCs and IPSCs in SST neurons that vary in their timing
sequence. C, % neurons displaying light evoked EPSCs or IPSCs (top) and graphs showing differential changes in ChR2 light-evoked synaptic activity at 70 and 40 mV holding potential in
two Sst neurons in a npy-Cre;ChR2-YFP mouse crossed with a sst-Flp;mCherry mouse in the absence (left) or the presence (right) of 4-AP. D, Representative waterfall plot displaying the robust
suppression of action potentials in a NPY neuron in response to a short and long light-pulse stimulation in a sst-Cre;ChR2 neuron. E, Overlay (top) and distribution (bottom) of light-evoked
IPSCs at 40 mV holding potential in an NPY-GFP neuron in a sst-Cre-Chr2-YFP; npy-hrGFP BAC mouse. F, Summary graph showing a side-by-side comparison of evoked postsynaptic currents
by light in SST and NPY neurons in npy- or sst-Cre;ChR2-YFP mice crossed with the respective reporter n = 10 cells from 2 npy-Cre;ChR2-YFP mice; n = 12 cells from 5 sst-Cre;ChR2-YFP mice.
Bellusci et al. · Brainstem Neurons Influence Gastric Function
indicating an effective placement of the optic cannula in this
nucleus.
During light activation of Chr2 neurons in the NTS, both ampli-
tude of phasic contractions and gastric tone significantly increased
compared with the baseline (44.2 1 5.8% and 54.3 1 8.5%, respec-
tively; Fig. 6I, Table 1; p = 0.0001, t = 7.586 and p = 0.0002, t =
6.402, two-tailed, one sample t test). In contrast, light activation of
ChR2 in the underlying DMV significantly decreased the amplitude
of phasic contractions that averaged 26.5 1 8.1% relative to baseline,
whereas gastric tone was not significantly affected (14.7 1 13.1%
decrease from the baseline; Fig. 6I, Table 1; p = 0.0304, t = 3.283
and p = 0.3225, t = 1.128, two-tailed, one sample t test).
A group of npy-Cre;ArchT-EGFP mice that had been simi-
larly tested showed opposite results on gastric activity compared
with those described in npy-Cre;ChR2-YFP mice. Light activa-
tion of ArchT in the NTS in these mice (n = 7) produced inhibi-
tory effects on gastric motility that exhibited a robust rebound
activity at the end of the inhibition but not tone (Fig. 6J; average
data are 27.6 6 7.6 and 9.4 6 5.8, respectively; p = 0.0112, t =
3.615 and p = 0.1561, t = 1.621, two-tailed, one sample t test;
Table 1). Conversely, light activation of ArchT in the DMV (n =
5) in the of npy-Cre;ArchT-EGFP mice significantly increased
gastric motility and tone (18.8 6 4.2% and 17.5 6 4.4%, respec-
tively; p = 0.0116, t = 4.411 and p = 0.017, t = 3.939 and, two-
tailed, one sample t test; Table 1).
In sst-Cre;ChR2-YFP transgenic mice, light-activated gastric
motility was similar to that of npy-Cre;ChR2-YFP. However, de-
spite the analogy with NPY-ChR2, the response elicited in the
NTS produced short-duration contraction(s) that sometimes
quickly proceeded to a longer duration of depression of the am-
plitude of motility (Fig. 6K,L, examples in different mice). In
NTS of sst-Cre;ChR2-YFP transgenic mice (n = 9), light activa-
tion of ChR2 produced a significant increase in the amplitude
of phasic contractions and gastric tone (35.6 1 6.8% and
26.2 1 7.7%, respectively; Fig. 6N, Table 1; p = 0.0008, t = 5.225
and 0.0112, t = 3.415, two-tailed, one sample t test), which was
not affected by ipsilateral vagotomy (n = 7).
In contrast, in the DMV (n = 5) as shown in the example in
Figure 6M, light activation of ChR2 suppressed the amplitude of
phasic contractions by 25.4 1 8.4% (Fig. 6N, Table 1; p = 0.0396,
t = 3.009, two-tailed, one sample t test), and this effect was
blocked by ipsilateral vagotomy (Fig. 6M). Instead, the gastric
tone was not significantly affected (16.0 1 11.5% decrease from
the baseline, Fig. 6N, Table 1, p = 0.2365, t = 1.391, two-tailed,
one sample t test). Similar to Sst-ChR2 mice, in sst-Cre;ArchT-
EGFP transgenic mice, excitatory effects on gastric tone and mo-
tility increased during NTS light stimulation (n = 7). Gastric mo-
tility was significantly affected (27.3 6 3.5%, p = 0.0002, t =
7.759, two-tailed, one sample t test), whereas tone was not (10.0
6 5.6%, p = 0.1269, t = 1.772, two-tailed, one sample t test).
/
G, Representative recording of light-induced suppression of action potentials in a NPY neuron
in npy-Cre;ArchT-EGFP mouse is accompanied by an increase in the rebound firing of action
potentials that is potentiated by closely spaced light pulses (green overlays). H, Dual record-
ings from SST and NPY neurons in response to light activation in an sst-Cre;ChR2-YFP mouse.
I, J, Subcellular channelrhodopsin-assisted circuit mapping of light-evoked responses from
DMV neurons in two sst-Cre;ChR2-YFP (IPSCs, I) and 3 npy-Cre;ChR2-YFP (EPSCs, J) mice. K,
L, Summary of results. M, Micrograph showing red fluorescence expression in DVC neurons
of sst-Cre;tdTomato mice (arrows, calibration: 20 mm). N, Light stimulation elicits postsynap-
tic currents in these SST neurons (left trace) that are inhibited by gabazine (10 mM), a GABAA
antagonist, (right trace). (Note: The remaining light-induced current in the right trace is
ChR2-induced direct current.)
J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5223
Stimulation of the DMV (n = 6), conversely, induced significant
inhibitory effects on the amplitude of phasic contractions but
not on gastric tone (17.6 6 4.5%, p = 0.0112 t = 3.921 and 6.9 6
4.3%, p = 0.1724, t = 1.591, two-tailed, one sample t test, respec-
tively), which were subsequently abolished by ipsilateral vagot-
omy (Fig. 6O, Table 1).
To document cannula placement sites in the hindbrain, pho-
tomicrographs and camera lucida drawings were made.
A representative photomicrograph illustrating an optic
cannula track in the DVC (brightfield and darkfield images) is
displayed in Figure 6P. The camera lucida drawings of the
hindbrain sections denote the localization of cannula tracts in
npy-Cre;ChR2-YFP and sst-Cre;ChR2-YFP transgenic animals
(Fig. 6Q).
These in vivo studies (summarized in Table 1) show that in
vivo optogenetic control in the DVC of NPY and SST transgenic
mice affects gastric motility. Although light activation of ChR2
in NTS significantly increases the amplitude of phasic contrac-
tions and gastric tone in both mice, ChR2 activation in the DMV
induces an opposite effect.
Altogether, the present study demonstrates that (1) NPY
neurons in DVC, like SST neurons (i.e., Lewin et al., 2016), are
important components of the gastric vagovagal reflex, are excita-
tory, and prevalently release glutamate, which is in contrast to
other regions of the brain where NPY neurons are described as
prevalently inhibitory (Pelkey et al., 2017); (2) NPY neurons may
function in the gastric vagovagal reflex by mediating excitation
from the NTS to the DMV and homeostatically regulate the vagal
circuitry and gastric motility by the interaction of excitatory
NPY neurons with inhibitory SST neurons; and (3) this is the
first time to our knowledge that optogenetics has been used in
vivo to excite (and inhibit) brainstem nuclei composing the main
components of the gastric vagovagal reflex while monitoring this
influence on gastric contractility and tone.
Discussion
This study observed the conspicuous presence of NPY neurons
in the DVC that regulate vagal transmission to the stomach.
Distribution of NPY neurons and their connection to SST
and gastric DMV neurons
Our results reveal abundant labeled cell distribution in the DVC
of npy-Cre;tdTom mice. NPY mRNA expression in tdTomato
neurons in the DVC was high, although the immunoreactivity
for the neuropeptide was considerably lower. The discrepancy
between NPY mRNA and antibodies detection could be because,
as with most peptides, NPY expression fluctuates because of
alterations in physiological activity and development. mRNA
expression for both the VGlut2 and GAD67 transporter was
detected in TdTomato neurons in the DVC of npy-Cre;tdTom
mice. Excitatory Vglut2 mRNA-expressing neurons were more
abundant than inhibitory GAD67 mRNA-expressing neurons,
consistent with the distribution of light-evoked postsynaptic
responses in target neurons of NPY neurons in npy-Cre;ChR2-
EYFP mice. The immunoreactivity for Vglut2 is exceptional, as
the NPY neurons are predominantly GABAergic in most brain
areas (Horvath et al., 1997).
NPY neuron characteristics and functional interconnectivity
in the DVC
In determining the synaptic interactions and connectivity of
NPY neurons to SST neurons or DMV gastric output neurons,
5224 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 6. In vivo optogenetic stimulation of NPY and SST neurons in the DVC influences gastric motility and tone. A, Stimulation parameters used in vivo reliably elicit action potentials in vitro in both
NPY and SST neurons in the DMV of npy- and sst-Cre;ChR2-YFP mice. B, A diagram of the ipsilateral vagotomy strategy used to separate light-evoked effects on gastric motility in the NTS from those in
the underlying DMV (see text). C, D, Stereotaxic location and hindbrain approach of optogenetic stimulation in the DVC. E, F, Chart recordings demonstrating that optogenetic stimulation of the NTS in a
npy-Cre;ChR2-YFP mouse robustly excites intragastric pressure (IGP) effect that is not blocked by ipsilateral vagotomy (E). G, H, Representative recordings showing that DMV stimulation leads to consistent
inhibition of gastric motility (H) that is blocked by ipsilateral vagotomy. I, Graph displaying the extent of changes in percentage of motor index of the amplitude of phasic contractions (Amplitude) and
gastric tone (Area Under Trace) evoked by light from baseline activity in npy-Cre;ChR2-YFP transgenic animals. pppp p , 0.0001, p p = 0.0304, ppp p = 0.0002. J, Representative recording showing
inhibition of gastric tone and motility in the NTS in an npy-Cre;ArchT-EGFP. K, L, Two representative chart recordings showing how stimulation of the NTS in distinct sst-Cre;ChR2-YFP mice induce an initial
excitatory phase followed by an inhibitory effect. M, Representative chart recording showing the light-induced inhibitory effect from the DMV of a sst-Cre;ChR2-YFP mouse that was blocked by ipsilateral
vagotomy. N, Graph depicting the changes in the amplitude and gastric motility index evoked by light from baseline activity in sst-Cre;ChR2-YFP mice. O, Representative chart recording showing light-
induced inhibition of gastric tone and motility in the DMV in a sst-Cre;ArchT-EGFP mouse that was blocked by ipsilateral vagotomy. ppp p = 0.0008, p p = 0.0396(amplitude), p p = 0.0112 (AUT).
P, Light (top) and phase-contrast (bottom) micrographs showing a representative optic cannula track in the DVC. Q, Camera lucida drawings of hindbrain sections denoting examples of the localization of
cannula tracts (NTS, squares; DMV, circles) in npy- and sst-Cre;ChR2-YFP mice transgenic animals (blue and red circles, respectively) along the rostrocaudal axis of the DVC.

we initially optogenetically excited or inhibited these, profiling
them in npy -Cre transgene crossed with ChR2 or ArchT re-
porter mice.
In NPY neurons of NPY-ChR2-eyfp mice, we observed evi-
dence for reciprocal excitatory coupling to each other as seen
using subtraction of direct ChR2 current recorded in NQBX.
Silencing these neurons with ArchT opsin or with direct hyper-
polarizations produced a large rebound increase in firing on re-
covery similar to that observed in cortical Camk2a kinase-
expressing glutamatergic neurons (Han et al., 2011; Madisen et al.,
2012). Our in vitro results with Vglut2 staining match the electro-
physiological findings of light-induced EPSC in DMV neurons in
Bellusci et al. · Brainstem Neurons Influence Gastric Function J. Neurosci., June 29, 2022 • 42(26):5212–5228 • 5225

Table 1. Optogenetic control of change of gastric motility (MMI%) and tone (AUT%) in the NTS and DMV nuclei
Mouse Stim area N Mean 6 SEM t df p Test
npy:Chr2 NTS Motility 9 44.2 6 5.8 7.586 8 ,0.0001 Two-tailed, one sample t test
Tone 9 54.3 6 8.5 6.402 8 0.0002 Two-tailed, one sample t test
DMV Motility 9 26.5 6 8.1 3.283 4 0.0304 Two-tailed, one sample t test
Tone 9 14.7 6 13.1 1.128 4 0.3225 Two-tailed, one sample t test
npy:Archt NTS Motility 7 27.6 6 7.6 3.615 6 0.0112 Two-tailed, one sample t test
Tone 7 9.4 6 5.8 1.621 6 0.1561 Two-tailed, one sample t test
DMV Motility 5 18.8 6 4.3 4.411 4 0.0116 Two-tailed, one sample t test
Tone 5 17.5 6 4.5 3.939 4 0.017 Two-tailed, one sample t test
Sst:Chr2 NTS Motility 9 35.6 6 6.8 5.225 8 0.0008 Two-tailed, one sample t test
Tone 8 26.2 6 7.7 3.415 7 0.0112 Two-tailed, one sample t test
DMV Motility 5 25.4 6 8.4 3.009 4 0.0396 Two-tailed, one sample t test
Tone 5 16.0 6 11.5 1.391 4 0.2365 Two-tailed, one sample t test
Sst:ArchT NTS Motility 7 27.2 6 3.2 7.759 6 0.0002 Two-tailed, one sample t test
Tone 7 9.9 6 5.6 1.772 6 0.1269 Two-tailed, one sample t test
DMV Motility 6 17.6 6 4.5 3.921 5 0.0112 two-tailed, one sample t test
Tone 6 6.9 6 4.3 1.591 5 0.1724 Two-tailed, one sample t test
A summary of in vivo results obtained with optogenetic control in SST and NPY cre driver mice crossed with different opsin reporters. AUT = area under the trace, MMI = mean motility index.

NPY ChR2 mice. This excitatory action was observed between
NPY and SST neurons, albeit much weaker. Thus, the most plausi-
ble hypothesis is that NPY neurons are prevalently excitatory. The
coexpression between tdTomato and NPY mRNA argues against
the possibility of misexpression in excitatory neurons using Cre-
lox breeding (Madisen et al., 2012; Hu et al., 2013).
Light activation of an NPY neuron allows EPSC and IPSC
sequences in the SST neuron, albeit with lower synaptic strength,
and IPSCs probably because of the subpopulation of GAD67-
NPY neurons or to excitatory polysynaptic activation of GABA
neurons. Together, these results reveal a complexity of the inter-
action between the inhibitory SST neurons and the excitatory
NPY neurons that is further increased by the reciprocal connec-
tivity of both SST (Fig. 7N; Thek et al., 2019) and NPY neurons
(Fig. 3J) and may play a substantial role as dynamic inhibitors of
gastric vagal circuitry (Lewin et al., 2016).
In vivo optogenetic control of NPY and SST neurons in the DVC
influences gastric motility and tone
After obtaining in vitro data on the synaptic interactions between
NPY and SST neurons, we examined in vivo the end organ of the
whole animal, namely, gastric stomach function, in NPY and
SST mice (also, refer to a model of the signaling shown in Fig. 7).
Optogenetic stimulation of SST neurons may predominantly
underlie the differential effects on gastric tone and motility seen
with the blockade of the local GABA signaling in the DVC
(Ferreira et al., 2002; Herman et al., 2009, 2010; Richardson et
al., 2013). Moreover, the reciprocal inhibition between SST neu-
rons is a vital synchronicity mechanism (Fig. 7A; Thek et al.,
2019) that can be a target of suppression by both excitatory and
inhibitory opsins. We suggest this as an explanation for the simi-
lar action of ChR2 activation and ArchT silencing, excitatory in
the NTS and inhibitory in the DMV, and for the finding that
light activation in the NTS and DMV of sst-Cre;ChR2-YFP
transgenic mice elicited the same motility responses observed in
npy-Cre;ChR2-YFP mice (see below).
Light activation of ArchT in the NTS of sst-Cre;ArchT-YFP
mice increases contraction. SST neurons inhibit each other
(Lewin et al., 2016; Thek et al., 2019), and light decreases their
firing. However, as their GABAergic synapses are not blocked,
the remaining spontaneous activity of SST neurons increases in-
hibition of GABA output neurons. Light activation of ArchT in
the DMV of sst-Cre; ArchT-YFP mice reduces contraction via a
similar inhibitory action on the SST-SST reciprocal inhibition
that has the result to increase inhibition of DMV neurons
directly. These results suggest that the SST-SST reciprocal inhibi-
tion acts as a brake of SST neurons and mediates inhibitory
action on DMV neurons.
In the NTS, in vivo optogenetic control in NPY-ChR2-eyfp
mice also showed activation of gastric motility, a robust effect
that was not affected by ipsilateral vagotomy (Fig. 6). Previous
reports showed that microinjection of glutamate, along with
GABA blockade in the NTS, decreases gastric motility (Ferreira
et al., 2002; Cruz et al., 2007; Herman et al., 2009; Richardson et
al., 2013). Because NTS mainly inhibits DMV neurons projec-
ting to the stomach, it is reasonable that glutamatergic and
GABAergic exposure strikingly affects motility behaviors. We
propose that NPY neurons may directly excite glutamatergic
NTS-DMV-projecting neurons, overwhelming the dominant
GABAergic-projecting neurons from the NTS to the DMV (Fig.
7B). In addition, the gastric motility induced by light activation
of ChR2 was strong and durable during all the stimulation pe-
riod, probably because of the monosynaptic excitatory network
that NPY neurons create with each other (Fig. 7B). In fact, gas-
tric motility is blocked in npy-Cre;ArchT-EGFP mice with light
in the NTS (Fig. 6J). These results could also be explained with
an activation of a direct NPY glutamatergic output to the
DMV. However, this hypothesis is not consistent with the
increased contractility produced by light in the DMV of ArchT
mice and the unreliable light-evoked synaptic excitation seen
with intracellular recording from DMV neurons in the presence
of TTX and 4-AP when NPY-ChR2-YFP afferents were optoge-
netically activated. What should also be considered is the possi-
bility of light-off rebound responses and the differential impact
of ArchT activation at soma versus the synapse. (Mahn et al.,
2016).
Light activation of ArchT in the DMV in npy-Cre;ArchT-
EGFP mice increases baseline contraction because it decreases
the ability of NPY neurons to activate the SST neurons. To
explain the contradictory action of inhibition of contractility
seen with the light on the DMV of sst-Cre;ChR2-YFP mice, we
propose a significant role of activation of GABAB receptors that
inhibits GABAergic terminals on DMV neurons and stimulates
contraction (Cruz et al., 2019).
In the DVC, SST/GIN neurons of the vagovagal circuitry in
the NTS receive direct vagus afferents (Glatzer et al., 2007;
5226 • J. Neurosci., June 29, 2022 • 42(26):5212–5228 Bellusci et al. · Brainstem Neurons Influence Gastric Function

Figure 7. A schematic illustration of a possible model to explain the results of the optogenetic stimulation in vivo of SST and NPY neurons in the DVC on gastric motility. A, In sst-Cre;ChR2-
YFP mice light stimulation of the SST neurons of the NTS inhibited the GABA projection output neuron to the DMV, thus, exciting gastric motility. In the DMV, stimulation of SST neurons inhib-
its the acetylcholine (ACh) projection to the stomach, thereby, decreasing gastric motility. Light stimulation of NTS in SST ArchT mice increases contraction. SST neurons inhibit each other, and
light decreases their firing. However, as their GABAergic synapses are not blocked, the remaining spontaneous activity of SST neurons increases inhibition of GABA output neurons. Light stimu-
lation of DMV in SST-ArchT mice reduces contraction via a similar inhibitory action on the SST-SST reciprocal inhibition, which, however, has the end result of directly increasing inhibition of
DMV neurons. NPY neurons are no longer inhibited by the SST neurons, thus allowing glutamate projection neurons on the NTS to become unopposed to enhance contraction motility. In the
DMV, the ACh output projection neuron to the stomach has extensive GABA tone from local (SST) and extrinsic (GABA projection) neurons. Although ArchT proton pump activation silences SST
neurons, extensive GABA tone still remains, which produces inhibition of the motility of the stomach. B, In npy-Cre;ChR2-YFP mice, light in the NTS excites NPY neurons, which in turn stimulate
glutamate output neuron and ultimately activate ACh neuron in the DMV, increasing gastric motility. In the DMV, activation of the NPY neurons decreases motility by exciting SST neurons,
which inhibit the ACh output projecting neuron to the stomach. In npy-Cre;ArchT-EGFP mice the light-induced gastric tone and motility in the two nuclei is opposite. In the NTS, silencing the
NYP neurons via its proton pump stops the excitatory drive of the glutamatergic NTS-DMV projection neuron, inhibiting ACh neuron and in turn contractions.

Lewin et al., 2016; Thek et al., 2019). In the DMV, these neu-
rons inhibit projecting neurons to the stomach (Lewin et al.,
2016). This inhibitory effect of SST is attenuated by a-melano-
cyte-stimulating hormone (a-MSH), which is a proopiomelano-
cortin (POMC)-derived peptide agonist melanocortin 4R, and
DAMGO, m-opioid agonist in the DMV (Lewin et al., 2016).
However, the administration of these agonists in the DVC has
contrasting effects on gastric motility. By suppressing local
GABAergic signaling, a-MSH and DAMGO inhibit gastric mo-
tility in the NTS (Herman et al., 2010; Richardson et al., 2013)
and excites it in the DMV (Richardson et al., 2013).
Like SST neurons, POMC neurons are also present in the
DVC (Joseph et al., 1983; Appleyard et al., 2005) and may be
directly activated by glutamatergic vagus afferents (Appleyard et
al., 2005) and CCK neuron (Moran et al., 2001) via MC4-R sig-
naling depending (Fan et al., 2004). These neurons are depressed
by opioids (Appleyard et al., 2005; Glatzer et al., 2007).
Furthermore, GLP-1 agonists in DVC (Ludwig et al., 2021;
Zhang et al., 2021) that contribute to anorexic effects (Zhang et
al., 2021) may do so via the POMC neurons releasing a-MSH in
the NTS. This peptide could decouple GABA projection NTS-
DMV neurons from local GABAergic SST neurons in the NTS.
Although GABA projection NTS-DMV neurons receive vagus
afferents, these neurons of gastric motility are subservient to local
GABA signaling (Herman et al., 2009) mediated SST neurons
(Lewin et al., 2016; Gillis et al., 2022). Altogether, we believe that
the effects of SST and POMC neurons on gastric motility (among
others) are influenced by vagus afferents and local neurons to
regulate energy homeostasis. Of the local neurons, NPY neurons
may play an essential role in this instance to stimulate SST neu-
rons directly or indirectly. For example, orexigenic peptide NPY
in NTS reduces the c-fos activation produced by peripheral CCK
Bellusci et al. · Brainstem Neurons Influence Gastric Function
(McMinn et al., 2000) that positively influences POMC neurons.
Activation of POMC neurons reduces feeding, partly by decreas-
ing gastric motility by inhibiting local GABA SST neurons via
releasing a-MSH. Moreover, microinjections of NPY in the NTS
induced c-fos neuronal activity in DVC (Yang et al., 1995) and a
significantly decreased gastric load-sensitive NTS cell response to
distending gastric loads (Schwartz and Moran, 2002). An alterna-
tive explanation for our contrasting results may refer to nonca-
nonical signaling (Chen et al., 2020) and the possibility of
contrasting effects of secretion of NPY or either glutamate or
both (Nectow et al., 2017). In summary, our finding show that
the interaction of SST and NPY neurons in NTS has a potent
effect on gastric contractions. This finding provides a model for
studying other premotor systems in the nucleus, including cardi-
orespiratory systems, which among others, involve the monitor-
ing of arterial baroreceptors and adapting pulmonary stretch
receptors reflexes (Andresen and Kunze, 1994; Lawrence and
Jarrott, 1996; Sapru, 1996; Kubin et al., 2006; Zoccal et al., 2014;
Bai et al., 2019; Kupari et al., 2019).
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