Abstract
Oxytocin receptors (OTR) have been found in the paraventricular thalamus (PVT) for the regulation of feeding and maternal behaviors. However, the functional projections of OTR-expressing PVT neurons remain largely unknown. Here, we used chemogenetic and optogenetic tools to test the role of OTR-expressing PVT neurons and their projections in the regulation of food intake in both male and female OTR-Cre mice. We found chemogenetic activation of OTR-expressing PVT neurons promoted food seeking under trials with a progressive ratio schedule of reinforcement. Using Feeding Experimentation Devices for real-time meal measurements, we found chemogenetic activation of OTR-expressing PVT neurons increased meal frequency but not cumulative food intake because of a compensatory decrease in meal sizes. In combination with anterograde neural tracing and slice patch-clamp recordings, we found optogenetic stimulation of PVT OTR terminals excited neurons in the posterior basolateral amygdala (pBLA) and nucleus accumbens core (NAcC) as well as local PVT neurons through monosynaptic glutamatergic transmissions. Photostimulation of OTR-expressing PVT-NAcC projections promoted food-seeking, whereas selective activation of PVT-pBLA projections produced little effect on feeding. In contrast to selective activation of OTR terminals, photostimulation of a broader population of glutamatergic PVT terminals exerted direct excitation followed by indirect lateral inhibition on neurons in both NAcC and anterior basolateral amygdala. Together, these results suggest that OTR-expressing PVT neurons are a distinct population of PVT glutamate neurons that regulate feeding motivation through projections to NAcC.
SIGNIFICANCE STATEMENT The paraventricular thalamus plays an important role in the regulation of feeding motivation. However, because of the diversity of paraventricular thalamic neurons, the specific neuron types promoting food motivation remain elusive. In this study, we provide evidence that oxytocin receptor-expressing neurons are a specific group of glutamate neurons that primarily project to the nucleus accumbens core and posterior amygdala. We found that activation of these neurons promotes the motivation for food reward and increases meal frequency through projections to the nucleus accumbens core but not the posterior amygdala. As a result, we postulate that oxytocin receptor-expressing neurons in the paraventricular thalamus and their projections to the nucleus accumbens core mainly regulate feeding motivation but not food consumption.
- basolateral amygdala
- food intake
- food motivation
- nucleus accumbens
- oxytocin receptor
- paraventricular thalamus
Introduction
Oxytocin (OT), a neuropeptide that is synthesized by neurons exclusively located in the paraventricular nucleus (PVN) and supraoptic nucleus of the hypothalamus, plays crucial roles in the regulation of social bonding, stress response, motivated behaviors, and food intake through OT projections widely throughout the brain. Although the majority of previous studies support the idea that OT decreases food intake by targeting multiple brain areas for homeostatic and hedonic regulations (Mullis et al., 2013; Ong et al., 2015; Liu et al., 2020; Wald et al., 2020), recent findings from rodent and clinical studies raise the possibility that activation of specific OT signaling promotes feeding motivation when food intake is inhibited by anxiety and other stress-associated conditions (Douglas et al., 2007; Olszewski et al., 2014, 2015, 2016). For instance, intraperitoneal injection of an OT receptor agonist alleviated anxiety-induced hypophagia in mice (Olszewski et al., 2014). In pregnant rats, OT administration increased food intake in 12 h after drug application (Douglas et al., 2007). Clinical studies also reported that OT only reduced food intake in the fasted state in obese but not in normal-weight men (Thienel et al., 2016). OT dysfunction is reported to be involved in anorexia nervosa (AN), a common eating disorder characterized by severe hypophagia, high anxiety, and social deficits (Oldershaw et al., 2011). In women with AN, OT levels of both cerebrospinal fluid and blood are lower than those in control subjects (Demitrack et al., 1990; Lawson et al., 2011), suggesting that central OT system dysfunction might underline elevated anxiety and hypophagia in AN (Fetissov et al., 2005). In addition, the potential treatment of OT on AN has been reported by previous studies and some clinical trials are still under investigation (Maguire et al., 2013; Kim et al., 2014a, b; Russell et al., 2018). Given the divergent projections of OT neurons and the wide expression of OT receptors in many brain areas, it is important to know whether specific OT signaling promotes feeding motivation based on neural circuit connections.
Paraventricular thalamus (PVT) is a midline nucleus that spans the anteroposterior dorsomedial thalamus with extensive connections throughout the brain for the regulation of arousal, emotion, and motivated behaviors (Li and Kirouac, 2012; Hsu et al., 2014; Li et al., 2014; Kirouac, 2015; Ye and Zhang, 2021). Both OT fibers and OT receptors were found in the PVT by previous studies (Knobloch et al., 2012; Hammock and Levitt, 2013; Horie et al., 2020; Newmaster et al., 2020; Watarai et al., 2020). Our latest findings indicate that OT excited PVT neurons through activating OT receptors (Barrett et al., 2021). OT activation of PVT neurons promoted feeding motivation to attenuate stress-induced hypophagia but produced little effect on normal food intake (Barrett et al., 2021). Although a recent study using retrograde viruses in OTR-ires-Cre knock-in prairie voles reported that OTR-expressing PVT neurons project to nucleus accumbens (Horie et al., 2020), the functional projections of OTR-expressing PVT neurons remain largely unknown. According to previous reports, the diversity of PVT neurons contributes to the complexity of the PVT in the regulation of emotion and motivated behaviors including feeding because of the distinct connections of PVT neuronal subpopulations with other brain regions (Gao et al., 2020). Therefore, identifying the functional projections of OTR-expressing PVT neurons will help us understand how PVT diversely regulates emotion, motivation, and behavior.
In the present study, we used OTR-Cre mice to test the role of OTR-expressing neurons in feeding regulation. In combination with slice electrophysiology, optogenetics, chemogenetics, and neural circuit tracing based on viral vectors and tracers, we identified the functional neural circuits originating from OTR-expressing PVT neurons in the control of feeding motivation.
Materials and Methods
Animals
C57BL/6J and vGlut2-Cre (B6J.129S6(FVB)-Slc17a6tm2(cre)Lowl/MwarJ) mice were purchased from The Jackson Laboratory. and BAC-transgenic OTR-Cre mice (Tg(Oxtr-cre)ON66Gsat; was originally obtained from the Mutant Mouse Resource and Research Center (MMRRC) at University of California at Davis, and was donated to the MMRRC by Nathaniel Heintz of Rockefeller University and Charles Gerfen of National Institute of Mental Health) were generously provided by the Elizabeth Hammock lab at Florida State University (Gong et al., 2007). All mice of both sexes were housed in a climate-controlled vivarium on a 12/12 h light/dark cycle with ad libitum access to food and water. All animals and experimental procedures in this study were approved by the Florida State University Institutional Animal Care and Use Committee. Ketamine (100 mg/kg) plus xylazine (10 mg/kg) were used for anesthesia, and meloxicam (5 mg/kg) was used for reducing pain or discomfort at the time of survival surgery and at least 2–3 d following the surgery. For brain slice preparation, mice were killed with an overdose of ketamine (300 mg/kg) and xylazine (30 mg/kg).
Drugs
D-2-amino-5-phosphonovalerate (AP5; catalog #0106), bicuculline methiodide (Bic; catalog #2503), 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; catalog #0190), tetrodotoxin (TTX; catalog #1069), and 4-aminopyridine (4-AP, catalog #0940) were purchased from Tocris Bioscience. Oxytocin (OT, catalog #051-01) and [Thr4, Gly7]-Oxytocin (TGOT, catalog #051-04) were obtained from Phoenix Pharmaceuticals. All drugs were dissolved in water or DMSO and aliquoted as stock solutions that were stored at −80°C. On the day of recording, the stock solutions were diluted to final concentrations in artificial (A)CSF (at least 1:1000 dilution) for the experimental test. During the recording, drugs were delivered locally via a multichannel drug application system (Warner Instruments) with a flow pipette of 250 μm diameter so that the drug solutions were quickly washed out after the drug channel was switched to normal ACSF.
Stereotactic injection of viral vectors and CTB-555
Naive male and female mice 6–8 weeks old were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a stereotaxic apparatus (David Kopf Instruments). Meloxicam (5 mg/kg) was also given for reducing pain or discomfort before the surgery and for the following 2–3 d. After exposing the skull via a small incision and drilling a hole in the skull, a pulled-glass pipette with beveled tip of 20–40 µm diameter was inserted into the brain to target PVT (coordinates relative to bregma, AP, −1.00–1.70 mm; DV, −3.00–3.20 mm; ML, ±0.05 mm). AAV1-pCAG-FLEX-EGFP-WPRE (300 nl; catalog #51502-AAV1, Addgene), AAV5-hSyn-DIO-mCherry (300–500 nl; catalog #50549-AAV5, Addgene) AAV5-hSyn-DIO-hM3D(Gq)-mCherry (300-500 nl; catalog #44361-AAV5, Addgene) or AAV1-EF1a-DIO-ChR2(H134R)-EYFP-WPRE-HGHpA (300–500 nl; catalog #20298-AAV1, Addgene) was injected into PVT with a speed of 100 nl per min using a pressure pump. The pipette was slowly withdrawn 10 min after injection. Mice were returned to their home cages for recovery. At least 4 weeks after surgery to allow Cre-dependent protein expression, mice were used for behavioral tests or slice patch-clamp recording. For optogenetic behavioral experiments, mice received a second surgery to implant fiber optics that target PVT (AP, −1.50 mm; DV, −2.80 mm; ML, ±0.05 mm) nucleus accumbens core (AP, +1.00 mm; DV, −4.20 mm; ML, ±1.00 mm) or posterior basolateral amygdala (pBLA; AP, −2.50 mm; DV, −4.30 mm; ML, ±3.00 mm).
For nonspecific retrograde labeling, cholera toxin subunit B conjugated with Alexa Fluor 555 (CTB-555, 200 nl; catalog #C34776, Thermo Fisher Scientific) was injected into NAcC (coordinates relative to bregma, AP, +1.00 mm; DV, −4.50 mm; ML, −0.80 mm); 3–5 d after injection, mice were ready for patch-clamp recording and anatomic imaging. For retrograde tracing of OTR-expressing PVT neurons that project to pBLA and NAcC, AAVrg-CAG-FLEX-EGFP-WPRE (250 nl; catalog #51502-AAVrg, Addgene) was injected into the pBLA (AP, −2.50 mm; DV, −4.50 mm; ML, ±3.00 mm) and AAVrg-CAG-FLEX-tdTomato-WPRE (250 nl; catalog #28306-AAVrg, Addgene) was injected into the NAcC (AP, +1.00 mm; DV, −4.50 mm; ML, ±0.80 mm) of the same OTR-Cre mice. Four weeks after injections, mice were perfused for anatomic imaging.
Slice preparation and patch-clamp recording
Coronal brain slices (300 µm thick) containing the PVT, nucleus accumbens core (NAcC), or amygdala were prepared for patch-clamp recordings. After recovery of over 1 h from slicing, brain sections were transferred to a recording chamber mounted on a Zeiss upright microscope and perfused with a continuous flow of gassed ACSF solution containing the following (in mm): 124 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.23 NaH2PO4, 26 NaHCO3, and 10 glucose (gassed with 95% O2/5%CO2; 300–305 mOsm). Pipettes used for whole-cell recording had resistances ranging from 4 to 7 MΩ when filled with K-gluconate pipette solution containing the following (in mm): 145 potassium gluconate, 1 MgCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, and 5 disodium phosphocreatine, pH 7.3, with KOH; 290–295 mOsm). The recording was performed at 33 ± 1°C using a dual-channel heat controller (Warner Instruments). EPC-10 patch-clamp amplifier (HEKA Instruments) and PatchMaster 2 × 90.5 software (HEKA Elektronik) were used to acquire and analyze the data. For voltage-clamp recording, the membrane potentials were held at −70 mV or −45 mV for recording photostimulation-evoked currents. Traces were processed using Igor Pro 6.37 (WaveMetrics). The postsynaptic currents were analyzed with MiniAnalysis 6.03 (Synaptosoft).
Optogenetic stimulation
To photostimulate ChR2-positive axons, blue light (470 nm) was delivered to target the recorded neurons through an optical fiber from a laser. The regular laser pulses (10 ms) at different frequencies were controlled with an optogenetics TTL Pulse Generator (Doric Lenses) to generate photostimulation-evoked currents.
Progressive ratio schedule of reinforcement
Before operant conditioning training in mouse operant chambers (Med Associates) all mice were food restricted (70% of their daily food intake) to facilitate the acquisition of lever-press responding until they learned to press the lever to obtain the food pellet in 3–5 d. Mice were provided with their daily quota of food in the home cage after termination of the training session. During the training, mice were initially trained under fixed-ratio 1 (FR1) sessions for 45 min daily during light cycles. Animals had a choice between the following levers: an active lever press associated with a 3 s light cue with concomitant delivery of high-fat high-sucrose (HFHS) pellets (20 mg, 48.9% Kcal as fat, Bio-Serv), and an inactive lever press that remained inoperative and served as a control for general activity. Each active lever press triggered the delivery of one pellet during FR1 sessions. The active lever had a 5 s refractory period after each food delivery so that mice could retrieve the single pellet but not drive supplementary food delivery. After a training period of ∼7–10 d when three successive sessions of obtaining ≥20 pellets during the FR1 session of 45 min, mice were then engaged in consecutive 45 min progressive ratio (PR) sessions during light cycles (11:00 A.M.–5:00 P.M.). For the PR session, the number of lever presses required for one food pellet delivery followed the following order (calculated by the formula [5e (R*0.2)]−5, where R is equal to the number of food rewards already earned plus 1): 1, 2, 4, 6, 9, 12, 15, 20, and so on (Barrett et al., 2021). The maximal number of active lever presses performed to reach the final ratio was defined as the break point, a value reflecting motivation of the animals to obtain the food reward. When a relatively stable break point was reached, mice were ready for PR tasks with drug treatments (intraperitoneal injection of saline or 2.0 mg/kg clozapine-N-oxide (CNO) 30 min before the tests) or photostimulation (20 Hz during the tests).
Meal pattern and food intake test
An open-source Feeding Experimentation Device (FED) was used for food intake test and meal pattern analysis (Nguyen et al., 2016; Matikainen-Ankney et al., 2021). The FED is designed with an Arduino microcontroller and container to store and dispense food pellets (20 mg). When active, the device releases a single pellet into a retrieval well, where its presence is monitored by an infrared beam. On pellet retrieval, FED time stamps the event to a secure digital card and dispenses a new pellet into the well. Feeding data are stored in individual comma-separated value files that can be analyzed. Briefly, mice were individually housed in the cages containing water bottles and FED that fit well within the cages, allowing monitoring of feeding behavior and meal pattern in a home environment without any special equipment. Dustless food pellets (size, 20 mg per pellet, 3.35 kcal/g, Bio-Serv) were used for the tests over 5–7 d. For experimental tests with chemogenetic stimulation, saline or CNO (2.0 mg/kg) was injected at the onset of dark phase.
HFHS food intake
HFHS food intake was measured over 30 min daily when mice were tethered to a laser for photostimulation. After mice were habituated to tethering for 3–5 d and obtained a relatively stable baseline food intake, they were ready for the tests with photostimulation (20 Hz) delivered by a laser with an optogenetics TTL Pulse Generator (Doric Lenses).
c-fos Immunocytochemistry
Mice with hM3D(Gq)-mCherry received an injection of saline or CNO (2.0 mg/kg,, i.p.) and were then killed for perfusion and paraformaldehyde (PFA) fixation 2 h after CNO injection. Briefly, mice were deeply anesthetized with ketamine (300 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.), and then perfused transcardially with saline followed by 4% paraformaldehyde in PBS. Brains were fixed overnight in 4% PFA and then in 30% sucrose for 2 d. The 30-μm-thick coronal sections were cut on a cryostat and collected in PBS. Free-floating slices were washed three times for 10 min each time in PBS and incubated in rabbit anti-c-fos antibody (catalog #2250, Cell Signaling Technology) 1:2000 in PBS with 2% normal donkey serum at 4°C overnight. After being washed three times in PBS for 10 min each time, sections were incubated in donkey anti-rabbit Alexa Fluor 488 antibody (catalog #711-545-152, Jackson ImmunoResearch) at a dilution of 1:500 for 4 h at room temperature. Sections were then washed in PBS for three times and mounted on glass slides for imaging under a microscope. ImageJ was used to count c-fos-positive and mCherry-positive neurons from 12 coronal sections including all PVT regions for quantification.
Statistical analysis
Data are expressed as mean ± SEM. Statistical significance was assessed using a two-sided Student's t test and χ2 test for comparison of two groups and one-way or two-way ANOVA followed by a Bonferroni post hoc test for three or more groups. Prism 9 (GraphPad) was used for statistical analysis and figure making.
Results
Activation of OTR-expressing PVT neurons promotes motivation for food rewards
According to our latest findings (Barrett et al., 2021), PVT OT administration promoted feeding motivation to attenuate stress-induced hypophagia. Here, we first used a PR schedule of reinforcement in operant chambers to test whether activation of OTR-expressing PVT neurons regulates the motivation for food rewards (Fig. 1A). To selectively manipulate the activity of OTR-expressing PVT neurons, we injected AAV5-hSyn-DIO-hM3D(Gq)-mCherry into the PVT to express hM3D(Gq) selectively in OTR neurons of OTR-Cre mice for chemogenetic activation using intraperitoneal injection of CNO (Fig. 1B). Our electrophysiological data indicate that application of CNO (5 μm) markedly increased the firing rate of hM3D(Gq)-mCherry-positive PVT neurons in slices (Fig. 1C), confirming a functional expression of hM3D(Gq) for the chemogenetic activation. Furthermore, we found that CNO (2.0 mg/kg, i.p.) injection increased active lever presses from 189.4 ± 20.9 times to 277.7 ± 35.4 times (t(20) = 3.593, p = 0.0009, paired t test, Fig. 1D), break points from 39.0 ± 3.8 to 57.2 ± 6.8 (t(20) = 3.657, p = 0.0008, paired t test; Fig. 1E), and food rewards earned from 10.4 ± 0.5 pellets to 12.0 ± 0.5 pellets (t(20) = 3.442, p = 0.0013, paired t test; Fig. 1F) during the operant PR tests. However, no difference in inactive lever presses between CNO and saline-treated mice was observed (t(20) = 0.283, p = 0.3900, paired t test; Fig. 1G). In control mice with only mCherry expression in OTR-expressing PVT neurons, CNO intraperitoneal injection produced no obvious effect on active lever presses, break points, reward earned, or inactive lever presses (Fig. 1H–K). Together, these results demonstrate that activation of OTR-expressing PVT neurons promotes the motivation for food rewards.
Chemogenetic activation of OTR-expressing PVT neurons promotes the motivation for food rewards. A, Diagram shows an operant chamber that was used for measuring the motivation for HFHS food pellets under PR schedule of reinforcement. B, Diagram shows AAV5-hSyn-DIO-hM3D(Gq)-mCherry injection in PVT of OTR-Cre mice, and a representative image shows hM3D(Gq)-mCherry expression in PVT neurons D3V, the third ventricle. C, Representative trace showing CNO (5.0 μm) excited a hM3D(Gq)-mCherry-positive neuron in PVT of slice. D, Bar graph with data plots shows active levers pressed by PVT-OTR-hM3D(Gq)-mCherry mice (n = 21) during PR sessions following saline or CNO (2.0 mg/kg) injection. Paired t test, t(20) = 3.593, p = 0.0009, ***p < 0.001. E, Break points reached by PVT-OTR-hM3D(Gq)-mCherry (n = 21) during PR sessions. Paired t test, t(20) = 3.657, p = 0.0008, ***p < 0.001. F, HFHS pellet reward earned by PVT-OTR-hM3D(Gq)-mice (n = 21) during PR sessions. Paired t test, t(20) = 3.442, p = 0.0012, **p < 0.01. G, Inactive levers pressed by PVT-OTR-hM3D(Gq)-mCherry mice (n = 21) during PR tests. Paired t test, t(20) = 0.283, p = 0.3900. ns, Not significant. H, Bar graph with data plots shows active levers pressed by control PVT-OTR-mCherry mice (n = 7) during PR sessions following saline or CNO injection. Paired t test, t(6) = 0.1620, p = 8766. ns, Not significant. I, Break points reached by control PVT-OTR-mCherry mice (n = 7) during PR sessions. Paired t test, t(60) = 0.0336, p = 0.9743, ns, Not significant. J, HFHS pellet reward earned by control PVT-OTR-mCherry mice (n = 7) during PR sessions. Paired t test, t(6) = 0.5477, p = 0.6036. ns, Not significant. K, Inactive levers pressed by control PVT-OTR-mCherry mice (n = 7) during PR tests. Paired t test, t(6) = 0.4970, p = 0.6369. ns, Not significant.
Activation of OTR-expressing PVT neurons regulates daily meal pattern
To further test whether OTR-expressing PVT neurons regulate daily food intake because of an increased feeding motivation, we used an FED device with an open-source program to measure real-time food intake of mice continuously for multiple days. Similarly, we injected AAV5-hSyn-DIO-hM3D(Gq)-mCherry into PVT to express hM3D(Gq) selectively in OTR neurons of OTR-Cre mice. Using c-fos immunocytochemistry, we first tested c-fos expression in PVT-OTR-hM3D(Gq)-mCherry neurons used for food intake tests and found CNO intraperitoneal injection increased c-fos immunoreactivity in hM3D(Gq)-positive neurons (Fig. 2A–C). These results further confirm the functional expression of hM3D(Gq) in OTR-expressing PVT neurons, which can be activated by CNO. Furthermore, we tested the real-time daily food intake in OTR-Cre mice with hM3D(Gq)-mCherry expression in PVT OTR-expressing neurons. We found CNO intraperitoneal injection significantly increased meal frequency of 0–6, 6–12, 12–18, and 18–24 h after drug injection (p = 0.0006, two-way repeated-measures ANOVA; Fig. 2F). The meal frequency of 0–6 h after drug injection was increased from 4.6 ± 0.6 meals per 6 h to 6.0 ± 0.6 meals per 6 h (p = 0.0287, post hoc Bonferroni test; Fig. 2F). However, no obvious effect was found on the cumulative food intake measured at 6, 12, 18, and 24 h following drug injection (p = 0.3183, two-way repeated-measures ANOVA; Fig. 2G). We also compared average meal sizes during the 24 h test after drug injection. Surprisingly, we found CNO intraperitoneal injection decreased the meal size from 0.44 ± 0.05 g to 0.34 ± 0.05 g (p = 0.028, paired t test; Fig. 2H). These data thus indicate that chemogenetic activation of OTR-expressing PVT neurons increases meal frequency but decreases meal sizes, which leads to no obvious effect on cumulative food intake.
Chemogenetic activation of OTR-expressing PVT neurons increases meal frequency but decreases meal size. A1–A3, Representative images show mCherry expression (A1) in OTR-expressing PVT neurons, c-fos immunoreactivity (A2) in PVT, and lack of c-fos expression in PVT OTR-hM3D(Gq)-mCherry neurons (A3) following intraperitoneal injection of saline in PVT-OTR-hM3D(Gq)-mCherry mice. B1–B3, Representative images show mCherry expression (B1) in OTR-expressing PVT neurons, c-fos immunoreactivity (B2) in PVT, and c-fos expression in the majority of PVT OTR-hM3D(Gq)-mCherry neurons (B3) following intraperitoneal injection of CNO (2.0 mg/kg) in PVT-OTR-hM3D(Gq)-mCherry mice. C, Bar graph with data plots showing percentages of c-fos-positive PVT OTR neurons following intraperitoneal injection of saline and CNO (2.0 mg/kg) in PVT-OTR-hM3D(Gq)-mCherry mice. ****p < 0.0001. D, Bar graph shows the real-time meal size of a PVT-OTR-hM3D(Gq)-mCherry mouse following intraperitoneal injection of saline. E, Real-time meal size in the same mouse following intraperitoneal injection of CNO (2.0 mg/kg). F, Meal frequency of 0–6, 6–12, 12–18, and 18–24 h in mice (n = 7) after saline or CNO injection. Two-way repeated-measures ANOVA, CNO effect, F(1,24) = 15.73, p = 0.0006, ***p < 0.001; post hoc Bonferroni's test, CNO versus saline for 0–6 h, p = 0.0287, #p < 0.05. G, Cumulative food intake in mice (n = 7) following saline or CNO (2.0 mg/kg) injection. Two-way repeated-measures ANOVA, CNO effect, F(1,24) = 1.039, p = 0.3183. ns, Not significant. H, Bar graph with data plots shows meal sizes of mice (n = 7) in 24 h following saline or CNO injection. Paired t test, t(6) = 2.877, p = 0.0282, *p < 0.05.
OT activates OTR-expressing PVT neurons that excite neighboring neurons through excitatory local circuits
To confirm that OTR-expressing PVT neurons are activated by OT, we injected AAV1-EF1a-DIO-ChR2-EYFP in the PVT of OTR-Cre mice to express ChR2-EYFP on OTR-expressing PVT neurons and tested the effect of OT on ChR2-EYFP-positive PVT neurons in slices (Fig. 3A). To characterize the response of ChR2-EYFP-positive neurons to photostimulation, we recorded these neurons in both current-clamp and voltage-clamp modes and found each light pulse (10 ms, 1 Hz) activated high-amplitude inward currents (Fig. 3C) to evoke action potentials (Fig. 3B). Thus, these data indicate that ChR2 was functionally expressed in EYFP-positive PVT neurons. We further tested the effect of OT on ChR2-EYFP-positive PVT neurons. As a standard used for our previous studies (Barrett et al., 2021), we considered drug-induced responses as excitatory effects when the depolarization was >1.5 mV and/or the firing rate was increased at least 20% by the drug treatment. OT (1 μm) application of 1 min depolarized all recorded neurons from −54.0 ± 1.4 mV to −48.5 ± 1.5 mV (p < 0.0001, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 3D,E) and increased the firing rate from 0.24 ± 0.05 Hz to 1.78 ± 0.50 Hz (p = 0.0315, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 3D,F, n = 8 neurons). In the presence of AP5 (50 μm) and CNQX (10 μm), selective OTR agonist TGOT (0.3 μm) depolarized the recorded neurons from −50.6 ± 1.5 mV to −46.4 ± 1.3 mV (p = 0.0004, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 3D,E) and increased the firing rate from 0.08 ± 0.07 Hz to 2.21 ± 0.59 Hz (p = 0.0024, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 3D,F). These data thus indicate that OT and TGOT excite all recorded ChR2-EYFP-positive neurons in the presence of AP5 and CNQX, confirming that these neurons express OTR for OT activation.
OTR-expressing PVT neurons are excited by OTR agonists and send excitatory projections to neighboring neurons. A, Diagram shows the experimental design that AAV2-EF1a-DIO-ChR2-EYFP was injected into PVT of OTR-Cre mice and patch-clamp recording with photostimulation was performed in PVT neurons with ChR2-EYFP expression. B, Representative trace shows action potentials evoked by photostimulation of 1 Hz under patch-clamp configuration. C, Representative trace shows photostimulation (1 Hz) evoked inward currents mediated by ChR2-formed channels when the membrane potential of the recorded neuron was held at −70 mV. D, Representative traces show both OT (1.0 μm) and TGOT (0.3 μm in the presence of AP5 and CNQX) increased the firing rate of ChR2-EYFP-positive PVT neurons. E, Bar graph with data plots showing the membrane potentials of ChR2-EYFP-positive PVT neurons in control and in the presence of OT (n = 8 cells) or TGOT (n = 9 cells). Two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons, Control (Ctrl) versus OT, ****p < 0.0001; Ctrl versus TGOT, ***p = 0.0004. F, The firing rates of Chr2-EYFP-positive PVT neurons in control and in the presence of OT (n = 8 cells) or TGOT (n = 9 cells). Two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons, Ctrl versus OT, *p = 0.0315; Ctrl versus TGOT, **p = 0.0024. G, Diagram shows the experimental design that AAV2-EF1a-DIO-ChR2-EYFP was injected into PVT of OTR-Cre mice and patch-clamp recording with photostimulation was performed in PVT neurons without ChR2-EYFP expression. H, Representative trace shows that photostimulation (0.5 Hz) evoked excitatory postsynaptic currents (oEPSCs) in a PVT neuron without ChR2 expression when membrane potential was held at −45 mV. I, oEPSCs evoked by laser pulses (10 ms) in control and in the presence of TTX (1 μm), TTX plus 4-AP (100 μm), or AP5 (50 μm) and CNQX (10 μm) when the membrane potentials were held at −70 mV. J, Bar graph with data plots showing oEPSC amplitude in control and in the presence of different blockers (n = 6 cells). One-way ANOVA followed by Bonferroni's multiple comparisons, ****p < 0.0001.
Our latest study reported that OT excited PVT neurons not only through direct depolarization but also by increasing glutamatergic transmission onto these neurons (Barrett et al., 2021). Here, we asked whether OTR-expressing PVT neurons send glutamatergic projections to excite local neurons. To test this hypothesis, we performed voltage clamp to record EYFP-negative PVT neurons in slices of mice with ChR2-EYFP expression in PVT OTR neurons (Fig. 3G). Photostimulation (10 ms, 0.5 Hz) evoked optical (o)EPSCs on ChR2-EYFP-negative PVT neurons, which were blocked by TTX (1 μm) and abolished by the application of AP5 (50 μm) and CNQX (10 μm; Fig. 3I,J). However, oEPSCs were recovered when 4-AP (100 μm) was added to a TTX-containing solution (Fig. 3I,J). Together these data suggest that OTR neurons regulate local PVT neurons through monosynaptic glutamatergic projections.
PVT OTR neurons predominantly project to posterior amygdala and nucleus accumbens
To identify brain areas outside PVT that are innervated by OTR-expressing PVT neurons, we injected AAV1-hSyn-DIO-EGFP into the PVT of OTR-Cre mice for EGFP expression to mark PVT OTR-positive soma and axons. In PVT, we found a high density of EGFP-labeled OTR neurons (Fig. 4A,B). We scanned major areas that were reported to be innervated by PVT neurons, and dense EGFP-labeled axons were found in posterior basolateral amygdala (pBLA; Fig. 4C,D), posterior basomedial amygdala (BMP; Fig. 4E,F), and NAcC (Fig. 4G). However, we didn't find obvious EGFP-labeled axons in anterior basolateral amygdala (aBLA) or central amygdala (CeA), which were major targets of PVT glutamate neurons. In contrast to a wide projection from a general population of PVT neurons to amygdala and NAc, OTR-expressing PVT neurons selectively send axons to pBLA, BMP, and NAcC, suggested by these data. In addition to NAcC and posterior amygdala, PVT OTR neurons also send their projections to the ventral part of lateral septum (LSV), bed nucleus of stria terminalis (BNST), rostral zona incerta (ZIR), and PVN with a lower density.
OTR-expressing PVT neurons send axons to NAc, pBLA, and BMP. A, EGFP was expressed in OTR-expressing PVT neurons in OTR-Cre mice after AAV1-hSyn-DIO-EGFP injection in PVT. D3V, Third ventricle. B, Zoomed-in image shows EGFP expression in the soma of OTR-expressing PVT neurons. C, EGFP-positive axons were found in bilateral pBLA. LV, Lateral ventricle. D, Zoomed-in image shows high density of EGFP-positive axons in pBLA. E, EGFP-positive axons in BMP. F, Zoomed-in image shows dense EGFP-positive axons in BMP. G, EGFP-positive axons in NAcC. H, Table showing EGFP-positive axons were found in LSV, NAcC, BNST, ZIR, PVN, pBLA, and BMP.
OTR-expressing PVT neurons regulate feeding motivation through monosynaptic glutamatergic transmission to NAcC
Our anatomic data showed that OTR-expressing PVT neurons send projections to NAcC (Fig. 4G). Here, we used both retrograde tracers and optogenetics to identify the functional property of OTR-expressing neural projections from PVT to NAc. We first injected retrograde tracer CTB (conjugated with Alexa 555) into NAcC to label NAc-projecting neurons in PVT and tested the response of NAc-projecting PVT neurons to OT using patch-clamp recordings in slices (Fig. 5A,B). We found that TGOT (0.3 μm) depolarized the membrane potential of NAc-projecting PVT neurons (50%, 7/14) from −51.1 ± 2.0 mV to −46.6 ± 1.8 mV (p = 0.0003, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 5D) and increased the firing rate of these neurons from 0.30 ± 0.07 to 1.26 ± 0.53 Hz (p = 0.0057, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 5E). In the presence of AP5 (50 μm) and CNQX (10 μm), TGOT (0.3 μm) still depolarized the membrane potential of NAc-projecting PVT neurons (40%, 6/15) from −56.7 ± 2.4 mV to −53.3 ± 2.1 mV (p = 0.0067, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 5D) and increased the firing rate of these neurons from 0.45 ± 0.11 to 2.18 ± 0.37 Hz (p = 0.038, two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons; Fig. 5E). These data indicate that only part of NAc-projecting PVT neurons express OTR that mediate OT modulation on NAc neurons through direct projections to NAc.
OTR-expressing PVT neurons innervate NAcC neurons to regulate food seeking through glutamatergic transmission. A, Diagram shows that retrograde tracer CTB-555 was injected into NAcC of mice and patch-clamp recordings were performed on PVT neurons of slices labeled with CTB-555. B, Two images show CTB-labeled neurons in both NAcC (left) and PVT (right). C, Representative traces show that TGOT excited CTB-labeled PVT neurons in the absence and presence of AP5 (50 μm) and CNQX (10 μm). D, Membrane potentials of CTB-labeled PVT neurons were depolarized by TGOT in the absence (n = 7 cells) and presence (n = 6 cells) of AP5 (50 μm) and CNQX (10 μm). Two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons, Control (Ctrl) versus TGOT in the absence of AP5/CNQX, ***p = 0.0003; Ctrl versus TGOT in the presence of AP5/CNQX, **p = 0.0067. E, TGOT increased the firing rates of CTB-labeled PVT neurons in the absence (n = 7 cells) and presence (n = 6 cells) of AP5 (50 μm) and CNQX (10 μm). Two-way repeated-measures ANOVA followed by Bonferroni's multiple comparisons, Ctrl versus TGOT in the absence of AP5/CNQX, **p = 0.0057; Ctrl versus TGOT in the presence of AP5/CNQX, *p = 0.0377. F, Diagram shows that AAV2-EF1a-DIO-ChR2-EYFP was injected into PVT of OTR-Cre mice and patch-clamp recording with photostimulation was performed in NAc neurons surrounded by ChR2-EYFP-positive axons. G, Representative trace shows that photostimulation (1 Hz) evoked oEPSCs in a NAcC neuron when membrane potentials were held at −45 mV. H, oEPSCs evoked by laser pulses (10 ms) in control and in the presence of TTX (1 μm), TTX plus 4-AP (100 μm), or AP5 (50 μm) and CNQX (10 μm) when the membrane potentials were held at −70 mV. I, A bar graph with data plots showing oEPSC amplitude in control and in the presence of different blockers (n = 7 cells). One-way ANOVA followed by Bonferroni's multiple comparisons, ****p < 0.0001. J, Representative image showing EYFP-positive terminals were found in NAcC of OTR-Cre mice with AAV-induced ChR2-EYFP expression in PVT OTR neurons. K, Bar graph with data plots shows active levers pressed during PR sessions with or without intra-NAcC photostimulation (20 Hz) in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 4.253, **p = 0.0021. L, Break points reached during PR sessions in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 5.037, ***p = 0.0007. M, HFHS pellet reward during PR sessions in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 5.547, ***p = 0.0004. N, Break points reached by mice during PR sessions in control PVT-OTR-mCherry mice (n = 7). Paired t test, t(6) = 0.1682, p = 0.8720. ns, Not significant. O, HFHS pellet reward during PR sessions in control PVT-OTR-mCherry mice (n = 7). Paired t test, t(6) = 0.000, p > 0.9999. ns, Not significant. P, HFHS food intake over 30 min with or without intra-NAcC photostimulation (20 Hz) in PVT-OTR-ChR2-EYFP mice (n = 12). Paired t test, t(11) = 0.4519, p = 0.6601. ns, Not significant.
To further test the functional neural circuits that link PVT OTR-expressing neurons to NAc, we used slice voltage-clamp recordings to detect synaptic currents on NAc neurons surrounded by ChR2-positive axons in OTR-Cre mice with ChR2-EYFP expression selectively in PVT OTR neurons (Fig. 5F). We found photostimulation (10 ms, 1 Hz) evoked oEPSCs on NAcC neurons (Fig. 5G). oEPSCs evoked by laser pulses (10 ms) were blocked by TTX (1 μm) and abolished by AP5 (50 μm) plus CNQX (10 μm; Fig. 5H,I), suggesting OTR-expressing PVT neurons send excitatory projections to NAcC. Similarly, oEPSCs were recovered by 4-AP (100 μm) application in the presence of TTX (Fig. 5H,I). These data thus indicate that OTR-expressing PVT neurons excite NAcC neurons through monosynaptic excitatory projections.
To examine whether PVT-NAc projections regulate food motivation to control food intake mediated by OTR-expressing PVT neurons, we injected viral vectors to induce ChR2 expression on PVT OTR neurons and implanted fiber optics to target NAcC for selective optogenetic activation of PVT-NAc pathways (Fig. 5J). We found photostimulation (20 Hz) increased active lever presses from 87.7 ± 14.3 times to 162.5 ± 22.4 times (t(9) = 4.253, p = 0.0021, paired t test; Fig. 5K), the break points from 21.3 ± 3.0 to 34.8 ± 3.8 (t(9) = 5.037, p = 0.0007, paired t test; Fig. 5L), and food rewards earned from 8.0 ± 0.6 pellets to 10.1 ± 0.6 pellets (t(9) = 5.547, p = 0.0004, paired t test; Fig. 5M) during the operant PR tests. However, we didn't observe any difference in break points and food reward earned by control mice with only mCherry expression in PVT OTR neurons (Fig. 5N,O). Furthermore, photostimulation of PVT OTR-expressing terminals in NAcC produced no obvious effect on cumulative HFHS food intake over 30 min (Fig. 5P). These data together indicate that activation of PVT-NAcC OTR-expressing projections promotes food seeking, suggesting a role of this pathway in mediating the control of PVT OTR neurons on feeding.
OTR-expressing PVT neurons send monosynaptic glutamatergic transmissions to pBLA without effect on food intake
We next examined how PVT OTR neurons regulate the activity of pBLA neurons using patch-clamp recordings combined with optogenetics in slices of mice with ChR2 expression specifically in PVT OTR neurons and ChR2-positive terminals in pBLA (Fig. 6A,B). We found photostimulation (10 ms, 1 Hz) of PVT OTR axonal terminals in pBLA evoked oEPSCs on pBLA neurons (Fig. 6C). The oEPSCs evoked by laser pulses (10 ms) were blocked by TTX (1 μm) and abolished by AP5 (50 μm) plus CNQX (10 μm; Fig. 6D,E), suggesting OTR-expressing PVT neurons release glutamate to produce excitatory transmissions onto pBLA neurons. However, oEPSCs were recovered by 4-AP (100 μm) application in the presence of TTX (Fig. 6D,E), suggesting OTR-expressing PVT neurons regulate pBLA neurons through monosynaptic excitatory projections.
OTR-expressing PVT neurons send monosynaptic glutamatergic transmission to excite pBLA neurons. A, Diagram shows the experimental design that AAV2-EF1a-DIO-ChR2-EYFP was injected into PVT of OTR-Cre mice and slice patch-clamp recording with photostimulation was performed in pBLA neurons surrounded by ChR2-EYFP-positive terminals. B, Representative image showing EYFP-positive terminals were found in pBLA of OTR-Cre mice with AAV-induced ChR2-EYFP expression in PVT OTR neurons. C, Representative trace shows that photostimulation (1 Hz) evoked oEPSCs in a pBLA neuron when membrane potential was held at −45 mV. D, oEPSCs evoked by laser pulses (10 ms) in control and in the presence of TTX (1 μm), TTX plus 4-AP (100 μm), or AP5 (50 μm) and CNQX (10 μm) when the membrane potentials were held at −70 mV. E, Bar graph with data plots showing oEPSC amplitude in control and in the presence of different blockers (n = 7 cells). One-way ANOVA followed by Bonferroni's multiple comparisons, ****p < 0.0001. F, Bar graph with data plots shows active levers pressed during PR sessions with or without intra-pBLA photostimulation (20 Hz) in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 1.127, p = 2889. G, Break points reached during PR sessions in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 0.8591, p = 0.4126. H, HFHS pellet reward earned during PR sessions in PVT-OTR-ChR2-EYFP mice (n = 10). Paired t test, t(9) = 1.000, p = 0.3434. I, HFHS food intake over 1 h with or without intra-pBLA photostimulation (20 Hz) in PVT-OTR-ChR2-EYFP mice (n = 13). Paired t test, t(12) = 0.8843, p = 0.3939. ns, Not significant. J, Representative image showing tdTomato-positive terminals were found in NAcC in an OTR-Cre mouse with AAVrg-CAG-FLEX-tdTomato injection into NAcC and AAVrg-CAG-FLEX-EGFP-WPRE injection into pBLA. K, Representative image showing EGFP-positive terminals were found in pBLA in the same OTR-Cre mouse with AAVrg-CAG-FLEX-tdTomato injection into NAcC and AAVrg-CAG-FLEX-EGFP-WPRE injection into pBLA. L1, L2, Representative images showing both tdTomato-positive and EGFP-positive PVT neurons without colocation in one section. L2 is a zoomed-in image from L1 showing clear tdTomato-positive PVT neurons that project to NAcC. M1, M2, Representative images showing both tdTomato-positive and EGFP-positive PVT neurons without colocation in another section. M2 is a zoomed-in image from M1 showing clear EGFP-positive PVT neurons that project to pBLA.
To further test whether pBLA mediates the effect of OTR-expressing PVT neurons on food seeking, we similarly injected viral vectors to induce ChR2 expression on PVT OTR neurons and implanted fiber optics to target pBLA for selective optogenetic activation of PVT-pBLA pathways. We found that photostimulation (20 Hz) produced no obvious effect on active lever presses or break points to obtain food rewards (Fig. 6F–H). In addition, photostimulation of PVT OTR-expressing terminals in pBLA had little effect on HFHS food intake (Fig. 6I). Together, these data indicate that PVT-pBLA projections play no functional role in feeding regulation mediated by PVT OTR-expressing neurons.
To examine whether the same PVT OTR neurons bifurcately project to both the NAcC and the pBLA for behavioral regulation, we injected retrograde pAAV-CAG-FLEX-tdTomato into the NAcC and AAV-pCAG-FLEX-EGFP-WPRE into the pBLA of the same OTR-Cre mice for tracing PVT OTR neurons that project to these two regions (Fig. 6J,K). We found both tdTomato-labeled and EGFP-labeled neurons in PVT, further confirming PVT OTR-expressing neurons project to both NAcC and pBLA. However, we did not observe any colocalization of tdTomato and EGFP in the same PVT neurons (Fig. 6L1–M2). Together, these data suggest that no PVT OTR neurons bifurcately project to both NAcC and pBLA for functional regulation of food intake.
PVT glutamate neurons differentially modulate aBLA and pBLA neurons
According to our data above, OTR-expressing PVT neurons are one group of glutamate neurons that participate in the functional regulation of the PVT. Anatomically, OTR-expressing PVT neurons selectively project to pBLA but not aBLA or CeA. To determine whether PVT glutamate neurons differentially regulate aBLA and pBLA, we tested the effect of optogenetic activation of PVT glutamatergic projections on the activity of both aBLA and pBLA neurons using vGlut2-Cre mice with Cre-dependent expression of ChR2-EYFP on PVT glutamate neurons. When the membrane potentials of aBLA neurons were held at −70 mV, laser pulses (0.5, 5, 10, and 20 Hz) evoked inward synaptic currents, and high frequency of photostimulation (10 and 20 Hz) also activated obvious tonic inward currents, which may be mediated by extrasynaptic glutamate receptors including both ionotropic and metabotropic types (Fig. 7A). However, when the membrane potentials of these aBLA neurons were held at −45 mV, laser pulses of 10 ms evoked rapidly decaying inward currents followed by slowly decaying outward currents (Fig. 7B). In the presence of Bic (30 μm), the amplitude of the inward currents was increased, but the amplitude of outward currents was decreased, suggesting that activation of GABAA receptors contributed to the outward currents. When a cocktail of AP5 and CNQX was applied in the recording solution, laser pulses evoked no current on aBLA neurons surrounded by ChR2-positive PVT glutamatergic axons. These data thus indicate that PVT glutamate neurons not only directly excite aBLA neurons but also activate certain GABA neurons that form local neural circuits for a secondary lateral inhibition.
PVT glutamate neurons differentially regulate aBLA and pBLA neurons. A, Representative traces show phasic (synaptic) and tonic inward currents evoked by photostimulation (0.5, 5, 10, 20 Hz) on an aBLA neuron surrounded by ChR2-EYFP-positive PVT glutamatergic terminals in voltage-clamp configuration when membrane potentials were held at −70 mV. B, Representative traces show that every light pulse (10 ms) evoked a rapid-decay inward current followed by a slow-decay outward current in aBLA neurons in normal ACSF solution when the membrane potential was held at −45 mV. Bath application of Bic decreased the amplitudes of the outward component but increased those of the inward component of the currents. Photostimulation-evoked currents were abolished by the application of AP5 (50 μm) and CNQX (10 μm). C, Representative traces show both phasic and tonic inward currents evoked by photostimulation (1, 5, 10, 20 Hz) on a pBLA neuron surrounded by ChR2-EYFP-positve PVT glutamatergic terminals when membrane potentials were held at −70 mV. D, Light pulses (10 ms, 1 Hz) evoked only inward currents but not outward currents on pBLA neurons when membrane potentials were held at −45 mV.
We then tested the effect of photostimulation of PVT glutamatergic terminals on the activity of pBLA neurons. Similarly, laser pulses (1, 5, 10, and 20 Hz) evoked phasic inward currents, and high-frequency photostimulation (5, 10 and 20 Hz) also activated obvious tonic inward currents when the membrane potentials of the recorded neurons were held at −70 mV (Fig. 7C). However, when the membrane potentials were held at −45 mV, photostimulation with 1 Hz evoked inward synaptic currents only but not delayed outward currents (Fig. 7D). These data indicate that PVT glutamatergic projections only exert direct excitation on pBLA neurons through releasing glutamate. Together, these data confirm that PVT glutamate neurons differentially regulate aBLA and pBLA neurons.
PVT glutamate neurons excite NAc GABA neurons that form local inhibitory circuits
To determine whether OTR-expressing neurons are a small population of PVT neurons that innervate NAc differentially from major glutamatergic projections, we tested the effect of photostimulation of the broader population of PVT glutamatergic projections on the activity of NAc neurons in vGlut2-Cre mice with ChR2-EYFP expression selectively in PVT glutamate neurons. When the membrane potentials of recorded NAc neurons were held at −70 mV, photostimulation of ChR2-positive glutamatergic axons in NAc evoked both phasic and tonic inward currents in neurons surrounded by ChR2-positive terminals (Fig. 8A). When the membrane potentials of the recorded neurons were held at −45 mV, laser pulses evoked rapidly decaying inward currents followed by slowly decaying outward currents (Fig. 8B). Both inward and outward currents were abolished by the application of 50 μm AP5 and 10 μm CNQX (Fig. 8B), suggesting glutamate released from PVT glutamatergic terminals initiated the currents on recorded neurons. Furthermore, we found that laser pulses evoked only inward currents in the presence of Bic (Fig. 8B), indicating the contribution of GABAA receptor activation on the outward currents. Thus, these data indicate that PVT glutamatergic projections not only directly excite NAc neurons but also inhibit these neurons by activating local inhibitory circuits. Together, these results suggest that OTR neurons are a special subpopulation of PVT neurons, which are different from other PVT glutamate neurons in the regulation of NAc.
PVT glutamate neurons directly excite NAc neurons but indirectly inhibit them through local inhibitory circuits. A, Photostimulation (1, 5, 10, 20 Hz) evoked both fast EPSCs and tonic inward currents on NAc neurons surrounded by ChR2-EYFP-positve axons when membrane potentials were held at −70 mV. B, Light pulses (10 ms) evoked rapid-decay inward currents followed by slow-decay outward currents on NAc neurons when membrane potentials were held at −45 mV (top). Application of AP5 (50 μm) and CNQX (10 μm) abolished photostimulation-evoked currents (middle), whereas Bic (30 μm) treatment only blocked the outward currents (bottom).
Discussion
In this study, we confirmed that activation of OTR-expressing PVT neurons promotes the motivation for food reward and increases meal frequency without an effect on cumulative food consumption. In contrast to PVT glutamatergic transmissions that target wide areas in both NAc and amygdala, OTR-expressing PVT neurons mainly project to the NAcC, the pBLA, and the BMP. In addition, activation of PVT-NAc OTR-positive terminals but not PVT-pBLA projections promotes food seeking, indicating the NAcC contributes to feeding regulation mediated by PVT OTR neurons (Fig. 9).
Diagram shows major projections of OTR-expressing PVT neurons and their role in feeding regulation. OTR-expressing PVT neurons send dense axons to both NAc and pBLA together with relatively weaker projections to other areas including BMP, BNST, LSV, PVN, and ZIR. Selective activation of OTR-expressing PVT-NAc glutamatergic axons promotes feeding motivation, whereas selective stimulation of OTR-expressing PVT-pBLA glutamatergic terminals has no obvious effect on feeding.
Growing evidence supports that PVT plays important roles in the regulation of arousal, valence, emotion, and motivated behaviors by receiving inputs from many regions such as locus ceruleus, dorsal raphe, hippocampus, and prefrontal cortex (Li and Kirouac, 2012; Hsu et al., 2014; Li et al., 2014; Kirouac, 2015; Ye and Zhang, 2021). The convergent signals from distinct presynaptic areas are integrated by the PVT that sends projections divergently to other regions including the NAc and the amygdala for motivational and behavioral regulation (Li and Kirouac, 2012; Dong et al., 2017; Li et al., 2021). Specifically, PVT receives both homeostatic and cognitive signals to control feeding motivation and food consumption. GABA neurons from zona incerta and AgRP neurons from arcuate nucleus both send inhibitory projections to PVT for a promoting effect on food consumption when they are activated (Livneh et al., 2017; Zhang and van den Pol, 2017). In contrast, activation of both excitatory projections from parasubthalamic nucleus and ventromedial hypothalamus suppresses food intake (Zhang and van den Pol, 2017; Zhang et al., 2020). Together, these studies indicate that activation of PVT neurons exerts inhibitory control on food consumption. In addition to the effect on food consumption, activation of specific PVT neurons and their projections by OT, orexin, glucose deprivation, and optogenetics has been reported to produce a promoting effect on motivated food seeking (Choi et al., 2012; Labouèbe et al., 2016; Sofia Beas et al., 2020; Barrett et al., 2021). Based on these previous studies, it is highly possible that food seeking and food consumption are differentially regulated by distinct PVT neuronal types that are activated by selective neural projections and signals. Although PVT neurons are primarily glutamatergic, a recent study has revealed the diversity of these neurons in neural circuit connections and potential behavioral regulation (Gao et al., 2020). Therefore, it is important to understand specific neuronal types in the PVT for the differential regulation of food seeking (motivation) and food intake (consumption), the two aspects of feeding behaviors.
In the present study, we found that chemogenetic activation of OTR-expressing PVT neurons increased operant lever presses for food rewards, suggesting that activation of OTR-expressing PVT neurons promotes feeding motivation. However, activation of OTR-expressing neurons increased meal frequency but decreased the average meal size, which counteracted the promoting effect of increased meal frequency on cumulative food intake. This is consistent with previous studies that activation of dopamine neurons in the ventral tegmental area and their projections to the NAc promoted motivation to increase meal frequency but decreased meal size (Boekhoudt et al., 2017; 2018). In addition, our recent findings reported that intra-PVT OT infusion promoted feeding motivation to attenuate stress-induced hypophagia but not regular food consumption (Barrett et al., 2021). Together, these findings suggest that OTR-expressing PVT neurons could be activated to increase food intake in special circumstances such as stress conditions when normal food consumption is inhibited. However, the present study is limited in revealing when PVT OTR-expressing neurons are physiologically regulated for feeding control. Therefore, future studies are needed to specifically examine whether OTR-expressing PVT neurons are activated for feeding behavior using tools such as fiber photometry. Mice under both normal and stressed conditions should be considered for the study. In addition, chemogenetic inhibition of PVT OTR-expressing neurons and their projections provides another strategy to test whether and how these neurons are physiologically activated for promoting feeding motivation.
As we discussed above, PVT sends dense projections to multiple brain areas, especially the NAc, the amygdala, and the bed nucleus of the stria terminalis for the regulation of emotion and motivated behaviors (Li and Kirouac, 2012; Dong et al., 2017; Li et al., 2021). The diverse projections by distinct PVT neurons contribute to the differential control of behaviors including feeding. Our present study indicates that OTR-expressing PVT neurons respond to OT and release glutamate to regulate postsynaptic neurons both inside and outside the PVT. Our results show that OTR-expressing PVT neurons project to the NAc. In contrast to PVT glutamate neurons that send axons to target wide areas in both the shell of NAc (NAcSh) and NAcC, OTR-expressing PVT neurons mainly innervate the NAcC using glutamatergic transmission. Although both NAcC and NAcSh receive dopaminergic projections for the regulation of reward and motivation, previous studies indicate that these two NAc subregions are functionally different in the control of drug-seeking behaviors. NAcSh mainly responds to spatial/contextual information, whereas NAcC more likely regulates drug-seeking behavior mediated by discrete cues (Bossert et al., 2007; Ito et al., 2008; Chaudhri et al., 2010; Ito and Hayen, 2011). In addition to drug reward, natural food reward processing is also regulated by NAc that affects feeding motivation and food consumption (Wise, 2006; Krause et al., 2010; O'Connor et al., 2015; Aitken et al., 2016). Intra-NAcC injection of glucagon-like peptide 1 (GLP-1) activated NAcC neurons and reduced food intake (Dossat et al., 2011), whereas injection of GLP-1 receptor antagonist exendin-(9–39) increased the meal size but decreased meal numbers in 2 h when sweetened condensed milk was tested (Dossat et al., 2013). In addition, PVT GLP-1 receptor activation reduced food motivation and consumption by inhibiting NAcC-projecting PVT neurons (Ong et al., 2017). Together, these studies suggested activation of NAcC neurons promotes food motivation but causes an inconsistent effect on food consumption. The promoting effects of activating PVT-NAc projections on food motivation are also supported by other pharmacological and optogenetic studies (Labouèbe et al., 2016; Meffre et al., 2019; Barrett et al., 2021). Consistent with these studies, our results indicate that activation of OTR-expressing PVT projections promoted food seeking but not cumulative food consumption. These findings thus confirm that the NAcC is a postsynaptic target for OTR-expressing PVT to sense OT signaling and regulate food motivation.
Both OTR-expressing and glutamate neurons in the PVT also project to the amygdala. However, the projection patterns of the two neuronal types are distinct in two aspects. First, the two neuronal types project to different amygdala nuclei. PVT glutamate neurons send axons to both anterior and posterior amygdala including aBLA, pBLA, and CeA, whereas OTR-expressing PVT neurons predominantly innervate posterior amygdala including pBLA and BMP. Second, pBLA is differentially innervated by PVT glutamate neurons and specific OTR-expressing PVT neurons although they all release glutamate for neurotransmission. Photostimulation of ChR2-positive PVT glutamatergic terminals evoked a rapidly decaying glutamate current (inward component) followed by a slowly decaying GABA current (outward component) on aBLA neurons but only inward currents on pBLA neurons. A cocktail of AP5 and CNQX abolished both inward and outward components of photostimulation-evoked currents, indicating that direct glutamate release from PVT glutamatergic terminals initiates the currents. The outward but not the inward component of the currents was blocked by Bic, suggesting glutamate release from PVT glutamate neurons activated GABA neurons that further inhibited neighboring aBLA neurons. Therefore, the general population of PVT glutamate neurons not only excite pBLA neurons but also activate aBLA neurons that form GABAergic local neural circuits for a lateral inhibition. However, OTR-expressing PVT neurons only modulate pBLA neurons through monosynaptic glutamatergic projections. Together, these results suggest that OTR-expressing neurons are a specific type of PVT glutamate neurons.
So far, the function of pBLA remains largely unknown. Some studies have indicated that aBLA contributes more to contextual fear conditioning, whereas pBLA has a greater effect on reward conditioning because of specific gene expression (Kantak et al., 2002; Kim et al., 2016). Additionally, a potential role of pBLA in feeding regulation was reported by pBLA lesion (Parker and Coscina, 2001). Therefore, pBLA neurons may be involved in feeding regulation. However, the present study indicates that selective activation of OTR-expressing PVT projections to pBLA produced little effect on food seeking and consumption. These results thus rule out the possibility that OTR-expressing PVT neurons target pBLA for feeding control. The function of this pathway especially in stress-related conditions should be examined in future studies.
Local neural circuits are important for lateral inhibition or recurrent excitation that causes amplification in the cortex (Douglas et al., 1995; Mateo et al., 2011). In the PVT, somato-dendritic secretion of neuropeptides from local neurons was proposed to be involved in the regulation of PVT in emotional and behavioral control (Hartmann and Pleil, 2021). Our present study reports optogenetic activation of OTR-expressing PVT neurons evoked glutamatergic EPSC on local PVT neurons. Therefore, OTR-expressing PVT neurons form monosynaptic neural circuits for recurrent excitation to further amplify PVT efferents to downstream brain areas such as the NAc and the amygdala for the control of emotion and behavior.
Footnotes
This work was supported by Florida State University Startup funding, First Year Assistant Professor Award, and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK131441 to X.Z. We thank Dr. Elizabeth Hammock for comments about the manuscript and Dr. Diana Williams for providing microscope services.
The authors declare no competing financial interests.
- Correspondence should be addressed to Xiaobing Zhang at xzhang{at}psy.fsu.edu
