Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Mar 1.
Published in final edited form as: Auton Neurosci. 2022 Dec 14;245:103058. doi: 10.1016/j.autneu.2022.103058

GABAergic leptin receptor-expressing neurons in the dorsomedial hypothalamus project to brown adipose tissue-related neurons in the paraventricular nucleus of mice

Yanyan Jiang 1,2, Kavon Rezai-Zadeh 3, Lucie D Desmoulins 1, Heike Muenzberg 3, Andrei V Derbenev 1,2, Andrea Zsombok 1,2
PMCID: PMC9899324  NIHMSID: NIHMS1859622  PMID: 36538864

Abstract

Brown adipose tissue (BAT) contributes to energy homeostasis via nonshivering thermogenesis. The BAT is densely innervated by the sympathetic nervous system (SNS) and activity of pre-autonomic neurons modulates the sympathetic outflow. Leptin, an adipocyte hormone, alters energy homeostasis and thermogenesis of BAT via several neuronal circuits; however, the cellular effects of leptin on interscapular BAT (iBAT)-related neurons in the hypothalamus remain to be determined. In this study, we used pseudorabies virus (PRV) to identify iBAT-related neurons in the paraventricular nucleus (PVN) of the hypothalamus and test the hypothesis that iBAT-related PVN neurons are modulated by leptin. Inoculation of iBAT with PRV in leptin receptor reporter mice (Lepr:EGFP) demonstrated that a population of iBAT-related PVN neurons expresses Lepr receptors. Our electrophysiological findings revealed that leptin application caused hyperpolarization in some of iBAT-related PVN neurons. Bath application of leptin also modulated excitatory and inhibitory neurotransmission to most of iBAT-related PVN neurons. Using channel rhodopsin assisted circuit mapping we found that GABAergic and glutamatergic Lepr-expressing neurons in the dorsomedial hypothalamus/dorsal hypothalamic area (dDMH/DHA) project to PVN neurons; however, connected iBAT-related PVN neurons receive exclusively inhibitory signals from Lepr-expressing dDMH/DHA neurons.

Keywords: iBAT, hypothalamus, patch-clamp electrophysiology, PRV, leptin

Introduction

The autonomic nervous system (ANS) plays an important role in the regulation of homeostatic functions including energy and glucose homeostasis (Schlaich et al. 2015; Thorp and Schlaich 2015). Brown adipose tissue (BAT) contributes to energy homeostasis via nonshivering thermogenesis. In rodents, the largest BAT is the interscapular BAT (iBAT), which is densely innervated by the sympathetic nervous system (SNS) (Bamshad et al. 1999; Bartness et al. 2001; Foster et al. 1982; Huesing et al. 2021). In the brainstem, neurons involved in the control of iBAT (iBAT-related neurons) were identified with trans-synaptic viral labeling in the reticular area, raphe, rostroventrolateral medulla (RVLM), the nucleus of the solitary tract, and the locus ceruleus (Bamshad et al. 1999; Cano et al. 2003; Oldfield et al. 2002; Zhang et al. 2011). In the forebrain, BAT-related neurons are in the paraventricular nucleus (PVN) of the hypothalamus, preoptic area, lateral hypothalamus (LH), dorsomedial hypothalamus (DMH) and arcuate nucleus (Bamshad et al. 1999; Bartness et al. 2001; Bartness et al. 2010; Cano et al. 2003; Oldfield et al. 2002). Pre-autonomic PVN neurons, neurons with long descending projections to spinal cord and brainstem areas involved in autonomic regulation, are key to the regulation of the SNS, and accumulating evidence suggests that the PVN plays a vital role in energy homeostasis (Cano et al. 2003; Kalsbeek et al. 2010; O’Hare and Zsombok 2016).

Studies linking hypothalamic neurons and iBAT function also suggested an important role for the hormone leptin (Enriori et al. 2011; Haynes et al. 1997b; Pandit et al. 2017; Zhang et al. 2011). Leptin is an adipocyte hormone and best recognized for its anorexigenic action; however, leptin is also known to increase SNS and iBAT function when injected directly into the DMH (Enriori et al. 2011; Marsh et al. 2003) and ventromedial hypothalamus (Marsh et al. 2003; Minokoshi et al. 1999). Increased sympathetic activity resulted in increased iBAT thermogenesis (Enriori et al. 2011; Minokoshi et al. 1999; Rezai-Zadeh et al. 2014), and retrograde labeling from iBAT has identified iBAT-related neurons in the hypothalamus including the PVN (Bamshad et al. 1999; Oldfield et al. 2002; Voss-Andreae et al. 2007). Moreover, a recent study demonstrated that leptin receptors are expressed on glutamatergic PVN neurons, some of which project to sympathetic ventral brainstem nuclei and can modulate sympathetic nerve activity to muscle and BAT (Shi et al. 2020). While whole animal studies are available, the cellular properties of the iBAT-related PVN neurons and their regulation remain to be determined.

In this study, we used a retrograde viral tracer, pseudorabies virus (PRV) to identify iBAT-related neurons in the mouse PVN and determine their basic cellular properties and synaptic regulation. We also tested the hypothesis that iBAT-related PVN neurons receive projections from leptin receptor expressing neurons of the DMH/dorsal hypothalamic area (dDMH/DHA). In addition, PRV injection was performed in leptin receptor reporter mice (Lepr:EGFP) to determine Lepr expression in iBAT-related PVN neurons. Indeed, inoculation of iBAT in Lepr:EGFP mice demonstrated that only few iBAT-related PVN neurons express Lepr receptors. Bath application of leptin modulated GABAergic and glutamatergic neurotransmission to iBAT-related PVN neurons. In striking contrast, channel rhodopsin assisted circuit mapping revealed only GABAergic connections between dDMH/DHA Lepr neurons and iBAT-related PVN neurons.

Materials and Methods

Animals

Male (age 8–16 weeks) C57BL6/J mice (Stock No. 000664, Jackson Laboratories) were used for viral inoculations and electrophysiological experiments. Male and female LeprCre mice (JAX_032457 RRID; original breeding pairs were provided by Dr. Martin G. Myers, Jr., University of Michigan) were used for the light stimulation experiments. In addition, LeprCre mice were crossed with Rosa26-EGFP mice (RRID:IMSR_JAX:004077) to generate Lepr:EGFP reporter mice. All breeding was performed at Pennington Biomedical Research Center.

The animals were housed in 12-hour dark-light cycle rooms at room temperature and had free access to water and standard rodent chow. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Tulane University and Pennington Biomedical Research Center (Louisiana State University System).

Inoculation of the iBAT with pseudorabies virus

Isogenic Bartha strains of pseudorabies virus, PRV-152 (GFP) or PRV-614 (RFP) (1×109 pfu/ml) was used to identify iBAT-related neurons. PRVs were obtained from the Center for Neuroanatomy with Neurotropic Viruses (University of Pittsburgh, supported by NIH P40 OD010996). The PRV injections were performed as described previously (Francois et al. 2019; Gao et al. 2012; Gao et al. 2017; Jiang et al. 2013; Zhang et al. 2011) and a detailed protocol of PRV injections into the iBAT is available at protocols.io (https://dx.doi.org/10.17504/protocols.io.w2vfge6).

Briefly, under isoflurane or ketamine/xylazine anesthesia the iBAT was exposed and PRV (5 × 100 nl) was injected with a pulled glass pipette attached to a 0.5 μl Hamilton syringe. After injection the glass pipette was left in place for an additional 30 s before withdrawal. A drop of adhesive “liquid bandage” was used to seal each injection sites to prevent leakage. The animals were maintained in a biosafety level 2 facility for up to 4 days (~96 hours) following inoculation to ensure labeling of hypothalamic neurons.

Stereotaxic surgery

Stereotaxic viral injections were performed as described previously (Rezai-Zadeh et al. 2014). Briefly, mice were deeply anesthetized with isoflurane (1–5 % isoflurane/0.8 L O2/min), the scalp was shaved, and they were placed in a stereotaxic head frame (#1900, David Kopf Instruments, Tujunga, CA). Ophthalmic ointment was applied to the eyes and a subcutaneous injection of carprofen (5–10 mg/kg) was given to each mouse prior surgery and daily over 48 hours post-surgery. The scalp was cleaned with Novalsan and 70 % Ethanol before making the skin incision. A craniotomy was made to insert a guide cannula (Model C253, Plastics One) to the dDMH/DHA (1.7 mm posterior, +/−0.25 mm lateral and 5.5 mm ventral to Bregma according to the Paxinos Mouse Brain Atlas; Paxinos and Franklin, 2004). Bilateral viral injections were done with AAV5-hSyn-DIO-ChR2-mCherry (AAV-DIO-ChR2, 200 nl per site, made available by Dr. Karl Deisseroth, UNC, Chapel Hill, NC) using injectors from Plastic One (Model C235IS or Model C317IS, Plastics One) attached to a Hamilton syringe to infuse at a rate of 20 nL/30 s. Cannula and injector remained in place for 5 min to prevent backflow, the skull access was then sealed with bone wax (Lukens #901, Medline Industries), and the incision closed with wound clips (#203–1000, CellPoint Scientific). Local anesthetics were applied to the incision site (bupivacaine/lidocaine, 5 mg/kg). After the surgery, mice were single housed and allowed for recovery for at least 2 weeks to ensure sufficient viral expression, then were transported to Tulane University. After arrival to Tulane University, the mice were given a minimum of one week for acclimatization. In a second surgery mice received PRV-152 (GFP) injection in the iBAT as described above to allow identification of iBAT-projecting PVN neurons. Animals were euthanized ~ 96 h after PRV injection to prepare acute brain slices for electrophysiological recordings.

Brain slice preparation and whole-cell patch-clamp recordings

Acute brain slices were prepared as described previously (Gao et al. 2012; Gao et al. 2017; Jiang et al. 2013; Molinas et al. 2019; Rezai-Zadeh et al. 2014). Briefly, under isoflurane anesthesia mice were decapitated, the brains were removed and immersed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 124 NaCl, 26 NaHCO3, 1.4 NaH2PO4, 11 glucose, 3 KCl, 1.3 MgCl, 1.5 CaCl2, pH: 7.3–7.4. Coronal brain slices containing the hypothalamus (300 μm) were prepared with a vibrating microtome (Leica VT 1200S). The slices were transferred to a holding chamber containing aCSF (for ~1 hour) before recordings.

Whole-cell patch-clamp recordings were performed at 34–36 °C from PVN neurons identified under a 40x water-immersion objective (N.A.=0.8). Epifluorescence was used to identify PRV-containing neurons and infrared illumination and differential interference contrast optics (IR-DIC) to target specific cells. The recording electrodes (2–5 MΩ) were filled with a solution containing the following (in mM): 130 K+ or Cs+-gluconate, 10 HEPES, 5 EGTA, 1 NaCl, 1 MgCl2, 1 CaCl2, 3 KOH or CsOH, 2–3 Mg-ATP, 0.2 % biocytin, pH 7.3–7.4. Liquid junction potential was corrected before determining membrane potential. The resting membrane potential was examined at rest in current-clamp mode.

To determine the excitability of iBAT-related PVN neurons, recordings were conducted in current clamp mode. During a step protocol, the recorded neurons were hyperpolarized to ~ −90 mV and then depolarizing current steps (duration of 1s) were applied to reveal the firing activity of iBAT-related PVN neurons before and after application of leptin (300 nM). The effect of bath application of leptin on firing rate was assessed 5 min after leptin reached the chamber.

Spontaneous excitatory postsynaptic currents (sEPSCs) were examined at a holding potential of −60 mV. Spontaneous inhibitory postsynaptic current (sIPSC) or evoked inhibitory postsynaptic current (eIPSCs) were examined at a holding potential of −10 mV. This experimental condition allowed us to exclude excitatory glutamatergic transmission without using glutamate receptor antagonists which are known to decrease the presynaptic release of GABA (Boychuk and Smith 2016; Xu and Smith 2015). Tetrodotoxin (TTX, 1 μM) was bath applied to record miniature excitatory and inhibitory post-synaptic currents (mEPSCs, mIPSCs). Electrophysiological signals were recorded using an Axoclamp 700B amplifier (Molecular Devices) and acquired by pClamp 10 software (Molecular Devices). Synaptic currents were analyzed offline using pClamp 10 and MiniAnalysis (Synaptosoft). Murine leptin (300 nM, PeproTech Inc) was dissolved in aCSF and bath applied in specific experiments.

For light-stimulation of channel rhodopsin (ChR2)-positive fibers a wide-spectrum UV light was used. The light was focused on the back aperture of the microscope, producing a wide-field exposure around the recorded cell. The light output was controlled by a programmable pulse stimulator, Master-8 (AMPI Co., Israel) and pClamp 10 software. The glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM, Tocris Bioscience) and D-(−)-2-amino-5-phosphonopentanoic acid (AP5, 50 μM; Tocris Bioscience), the GABAA receptor antagonist bicuculline methiodide (30 μM; Tocris Bioscience), and 4-aminopyridine (4-AP, 100 μM, Tocris Bioscience) were included in the aCSF and bath applied for specific experiments. After recordings the brain slices were fixed in 4 % paraformaldehyde in 0.15 M sodium phosphate buffer overnight at 4 °C. After several rinses in 0.01 M phosphate-buffered saline (PBS), slices were immersed in AMCA Avidin D (1:200, Vector Labs) in 0.01M PBS containing 1 % Triton X-100 (Acros organics) for 72 h at 4 °C to visualize the recorded neurons.

Statistical analysis

Continuous recordings of EPSCs and IPSCs were conducted before and after drug application. The effects of drugs across the neuron groups were analyzed using paired t-test. The effects of drugs on PSC frequency and amplitude were analyzed within individual cells using the Kolmogorov-Smirnov (K-S) test by comparing 2 min epochs before and after drug application. Neurons were grouped (decrease, increase, or no change) based on K-S test. Significance was set at p<0.05 and numbers are reported as mean ± standard error (SEM).

Results

Lepr expression on iBAT-related PVN neurons

Inoculation of iBAT with PRV in C57BL6/J mice was used to verify expression in previously described brain areas (Cano et al. 2003; Voss-Andreae et al. 2007; Zhang et al. 2011). Labeling of iBAT-related neurons at 96 h post-inoculation with PRV was observed in the ventral brainstem, particularly in the raphe, gigantocellular reticular nucleus, reticular, and the ventrolateral medulla and in the PVN.

Then, expression of Lepr in the PVN was confirmed in the Lepr:EGFP reporter mouse (Fig. 1). Adult Lepr:EGFP male (n=2) and female (n=2) mice (age 8–9 month) were used for viral inoculation of the iBAT. Lepr expressing neurons were sparse, but consistently observed throughout the PVN including in subnuclei involved in autonomic regulation (Fig. 1). PRV-614 (RFP) injection into the iBAT of Lepr:EGFP mice showed that few PRV labeled neurons co-localized with Lepr expressing neurons in the mouse PVN (Fig. 1B).

Figure 1: A subset of iBAT-related neurons expresses Lepr in the mouse PVN.

Figure 1:

Open in a new tab

A: Image of the paraventricular nucleus (PVN) showing Lepr-expressing neurons. B: Pseudorabies virus (PRV-614, RFP) injection into the iBAT showed robust PRV infection 96 hours post-injections and revealed occasional co-localization with Lepr expressing PVN neurons (insert, open arrowhead), even though the majority of PVN Lepr neurons (insert, filled arrowheads) were not PRV labeled. C: Lepr-expressing PVN neurons in the same section. Scale bar represents 200 μm (A) and 100 μm (C).

Leptin hyperpolarized a subset of iBAT-related neurons in the PVN

Electrophysiological recordings were conducted approximately 96 h post-inoculation from PRV-EGFP labeled iBAT-related PVN neurons to determine their cellular properties. The average resting membrane potential of iBAT-related PVN neurons was −46.08 ± 1.94 mV (range −59.87–−32.74 mV, n=14 from 5 mice). To determine the excitability of iBAT-related PVN neurons, recordings were conducted in current clamp mode. After a hyperpolarizing step, depolarizing current steps were applied to reveal the firing activity of iBAT-related PVN neurons. At baseline 5 out of 14 iBAT-related PVN neurons had spontaneous firing (0.8 ± 0.26 Hz, n=5) and the input resistance was 442 ± 29 MΩ (range 240–582 MΩ, n=14). Then, leptin was bath applied to determine its effect on the firing rate of iBAT-related PVN neurons (Fig. 2). Application of leptin hyperpolarized approximately 1/3 of iBAT-related PVN neurons (5 out of 14 cells), depolarized one neuron, while the remaining neurons (n=8) did not respond to leptin. The average resting membrane potential of the hyperpolarizing subset of iBAT-related PVN neurons was −43.60 ± 2.85 mV before and −49.69 ± 4.00 mV after leptin application (n=5 cells from 4 mice) (paired t-test, p=0.02). Two of these neurons had spontaneous firing, which was reduced after leptin application (0.75 Hz and 0.94 Hz before, 0.09 Hz and 0.12 Hz after leptin) (Fig. 2B).

Figure 2. Leptin modulates excitability of a subset of iBAT-related PVN neurons in mice.

Figure 2.

Open in a new tab

A: Fluorescence image shows iBAT-related neurons in the PVN. Arrow points to a recorded neuron filled with biocytin (blue). Insert is enlarged boxed area. B: Representative traces illustrate that leptin decreased the excitability of an iBAT-related PVN neurons. During a step protocol the recorded neuron was hyperpolarized to ~ −90 mV, then depolarizing current steps were applied (duration 1s) to reveal the firing activity before (upper trace) and after (lower trace) bath application of leptin (300 nM). The effect of leptin was assessed 5 min after it reached the chamber. While multiple current steps were applied, only one trace is shown for better visibility. Scale bar represents 10 μm.

Leptin modulates the inhibitory neurotransmission to iBAT-related PVN neurons

Leptin was shown to alter thermogenesis and modulate both excitatory and inhibitory neurotransmission in multiple brain areas (Ghamari-Langroudi 2012; Rezai-Zadeh et al. 2014; Williams et al. 2007; Yu et al. 2018); therefore, we examined the effect of leptin on spontaneous and miniature inhibitory neurotransmission to iBAT-related PVN neurons.

To determine the effect of leptin on spontaneous inhibitory PSCs, iBAT-related PVN neurons were clamped at −10 mV. To reveal the effect of leptin on the frequency of synaptic events within a single neuron Kolmogorov-Smirnov (K-S) test was used. Based on the results of the K-S test, the neurons were grouped to cells with significant increase, decrease or cells with no changes. Bath application of leptin resulted in a decreased frequency of sIPSCs in 50% of recorded iBAT-related PVN neurons (10 out of 20). The average frequency of sIPSCs decreased from 2.07 ±0.58 (range 0.15–5.72 Hz) to 1.04 ±0.35 Hz (range 0.08–3.10 Hz, n=10 from 7 mice) (paired t-test, p=0.009) (Fig. 3A, D). This decrease in frequency was accompanied with a decrease of the amplitude of sIPSCs following leptin application (21.97 ± 1.93 pA vs. 16.23 ± 1.20 pA) (paired t-test, p=0.019). The rest of the neurons either did not respond to leptin (n=6 from 6 mice) (p>0.05) or showed an increase 0.76 ± 0.22 Hz (range 0.30–1.28 Hz) to 1.34 ± 0.23 Hz (range 0.84–1.89 Hz, n=4 from 3 mice) (p=0.0001) (Fig. 3). These data suggest that the effect of leptin on spontaneous inhibitory neurotransmission of iBAT-related PVN neurons is heterogenous.

Figure 3. Leptin modulates inhibitory neurotransmission to iBAT-related neurons in the mouse PVN.

Figure 3.

Open in a new tab

A-C: Representative recordings of sIPSCs show decrease (A), increase (B), and no change (C) to application of leptin (300 nM). D-G: Bar graphs demonstrate that leptin application resulted in decrease (n=10 from 7 mice) (p=0.019) (D), increase (n=4 from 3 mice) (p=0.0001) (E), or no change (n=6 from 6 mice) (p>0.05) (F) of sIPSC frequency in iBAT-related PVN neurons. G: Percent change of sIPSC frequency after leptin application. Data shown as mean ±SEM. sI: spontaneous inhibitory postsynaptic currents, ctrl: control.

Similarly, application of leptin resulted in a decreased frequency of miniature IPSCs. The overall frequency from all recorded neurons was 0.63 ± 0.12 Hz (range 0.17–1.63 Hz, n=16 from 4 mice) before and 0.36 ± 0.06 Hz after leptin application (range 0.01–0.66 Hz) (p=0.025). Analysis of each individual cell with K-S test revealed that approximately half of the recorded iBAT-PVN neurons responded with a decrease of mIPSC frequency following leptin application. The average frequency was 0.86 ± 0.24 Hz (range 0.21–1.70 Hz, n=7 from 3 mice) before and 0.29 ± 0.08 Hz (range 0.10–0.71 Hz) (p=0.014) after bath application of leptin. The frequency of one neuron showed an increase (0.50 vs 0.89 Hz), while the rest of the recorded cells did not respond to application of leptin (0.44 vs 0.36 Hz, n=8 from 4 mice). The amplitude of mIPSCs did not change after leptin application in any of the groups. The average amplitude was 15.3 ± 1.5 pA before and 13.3 ± 1.4 pA after leptin application in neurons with decreased frequency (p=0.06), while it was 15.9 ± 2.7 pA vs 14.8 ± 2.0 pA in neurons without change (p=0.29). These data demonstrate that in mice bath applied leptin can modulate mIPSC frequency to half of iBAT-related PVN neurons.

Most of iBAT-related PVN neurons receive direct inputs from GABAergic dDMH/DHALepr neurons

Lepr neurons in the dDMH/DHA regulate energy expenditure likely via glutamatergic projections to iBAT-related premotor raphe pallidus neurons (Rezai-Zadeh et al. 2014). However, the vast majority of DMH Lepr neurons are GABAergic neurons (Vong et al. 2011), suggesting that GABAergic projections could contribute to modulation of energy expenditure. Thus, we tested whether dDMH/DHALepr neurons also send projections to iBAT-related PVN neurons. ChR2 was expressed in dDMH/DHALepr neurons and recordings were conducted from PVN neurons (Fig. 4). First, light stimulation of dDMH/DHALepr fibers in the PVN showed that dDMH/DHALepr neurons project to randomly selected PVN neurons (non-PRV labeled) via excitatory and inhibitory connections (Fig. 4A, B). These evoked IPSCs and EPSCs were diminished in the presence of bicuculline or AP-5 and CNQX, respectively. Then, recordings were conducted from iBAT-related PVN neurons, while ChR2-expressing inputs from dDMH/DHALepr neurons were stimulated (Fig. 4C–F). In this case, we found that approximately 70 % of iBAT-related PVN neurons (13/19) responded to light stimulation and received only inhibitory projections from dDMH/DHALepr neurons (Fig. 4F). No evoked EPSCs were observed in iBAT-PVN neurons (not shown). Bath application of TTX abolished light-induced IPSCs in iBAT-related PVN neurons, while application of 4-AP reinstated the evoked IPSCs (Fig. 4F), suggesting that the projections from dDMH/DHALepr neurons to iBAT-PVN neurons are monosynaptic. These data demonstrate that in mice even though dDMH/DHALepr send both excitatory and inhibitory projections to PVN neurons, pre-autonomic PVN neurons involved in the regulation of iBAT receive exclusively inhibitory inputs from dDMH/DHALepr neurons.

Figure 4. GABAergic dDMH/DHALepr neurons project to iBAT-related PVN neurons in mice.

Figure 4.

Open in a new tab

A: Schematic illustration of the experimental design. AAV-DIO-ChR2 was injected into the dDMH/DHA of LeprCre mice. Light-evoked post-synaptic currents were recorded from random (non-iBAT) PVN neurons to identify glutamatergic and/or GABA projections from Lepr-expressing dDMH/DHA neurons. B: Light-evoked IPSCs were observed in approximately 1/3 of all recorded non-iBAT-PVN neurons. An example of evoked IPSCs in a non-iBAT-PVN neuron before and after application of the GABA receptor antagonist bicuculline (30 μM). Bicuculline blocked light evoked IPSCs. Blue rectangle indicates light stimulation (2 ms). Light stimulation triggered evoked EPSCs in approximately half of non-iBAT PVN neurons. These evoked EPSCs were blocked by bath application of the glutamate receptor antagonists CNQX (10 μM) and AP5 (50 μM), demonstrating glutamatergic transmission. C: Schematic illustration of the experimental design. AAV-DIO-ChR2 was injected into the dDMH/DHA of LeprCre mice, then PRV-152 (GFP) into the interscapular brown adipose tissue (iBAT). Light-evoked post-synaptic currents were recorded from iBAT-related PVN neurons. D: Image illustrates the injection site and tracks (asterisks) of AAV-DIO-ChR2 into the dDMH/DHA. E: Images illustrate a recorded iBAT-related PVN neuron (green) filled with biocytin (middle). The images were generated post-recording from fixed tissue. F: Light stimulation (2 ms) in the PVN evoked IPSCs in most of iBAT-PVN neurons (13/19). Bath application of TTX (1 μM) abolished, while application of 4-AP (100 μM) reinstated the light-induced IPSCs in iBAT-related PVN neurons. G: The average amplitude of eIPSCs was not different in random and iBAT-related PVN neurons. Scale bar represents 500 μm (D) 10 μm (E).

The effects of leptin on excitatory neurotransmission to iBAT-related PVN neurons

Leptin application did not alter the overall EPSC frequency in iBAT-related PVN neurons. The average sEPSC frequency from all recorded cells was 2.85 ± 0.34 Hz (range 0.78–6.06 Hz) before and 2.94 ± 0.34 Hz (range 0.45–7.51 Hz, n=25 from 11 mice) after leptin application (p>0.05). Examination of each individual neuron with K-S test revealed that leptin application decreased sEPSC frequency in a subset of neurons (7 out of 25) from 3.81 ± 0.66 Hz (range 0.87–5.74 Hz, n=7) to 2.75 ± 0.48 Hz (range 0.45–4.33 Hz, n=7 from 4 mice) (paired t-test, p=0.01) (Fig. 5A, B). The amplitude was not affected by leptin application (11.27 ± 1.15 pA vs 10.83 ± 1.48 pA) (p>0.05). In another subset of iBAT-related PVN neurons leptin resulted in an increase in frequency of sEPSCs (10 out of 25). The average frequency of sEPSCs increased from 2.64 ± 0.52 (range 0.78–6.06 Hz) to 3.44 ± 0.62 Hz (range 1.20–7.52 Hz, n=10 from 7 mice) (paired t-test, p=0.0003) (Fig. 5A, C). The amplitude of sEPSCs did not change following leptin application (12.89 ± 1.05 pA vs 13.24 ± 2.15 pA, n=10) (p>0.05). Leptin did not affect the frequency and amplitude of the remaining 8 cells.

Figure 5: Leptin has heterogenous effect on the excitatory neurotransmission in a subset of iBAT-related neurons in the mouse PVN.

Figure 5:

Open in a new tab

A: Representative recordings of sEPSCs from iBAT-related PVN neurons illustrate that leptin (300 nM) decreased (top), increased (middle) or had no effect (bottom) on the frequency of sEPSCs. B-D: Bar graphs demonstrate decrease (n=7 from 4 mice) (p=0.01) (B), increase (n=10 from 7 mice) (p=0.0003) (C), and no change (n=10 from 7 mice) (p>0.05) (D) of sEPSC frequency to application of leptin. E: Illustration of percent change of sEPSC frequency after bath application of leptin. Data shown as mean ±SEM. sE: spontaneous excitatory postsynaptic currents, ctrl: control.

Similarly, the overall frequency and amplitude of miniature EPSCs did not change after application of leptin (2.26 ± 0.5 Hz vs 2.45 ± 0.41 Hz (n=15 from 5 mice) (p>0.05). Although a subset of individually tested iBAT-related PVN neurons showed increase of mEPSC frequency (5 out of 15 cells from 4 mice using K-S test as statistical test), the overall response was not significant (1.75 ± 0.36 Hz vs 2.53 ± 0.30 Hz, p=0.07). The rest of the recorded neurons did not respond to leptin.

Discussion

Our data confirmed the distribution of a broad population of iBAT-related neurons in the mouse forebrain and demonstrated that a subset of Lepr-expressing PVN neurons is part of the PVN-iBAT pathway. Bath application of leptin hyperpolarized a subset of iBAT-related PVN neurons and had heterogenous effects on postsynaptic currents. We also found that Lepr-expressing neurons in the dDMH/DHA modulate excitatory and inhibitory neurotransmission to PVN neurons; however, dDMH/DHALepr neurons provide exclusively inhibitory inputs to most of iBAT-related PVN neurons (Figure 6).

Figure 6. Schematic illustration of the connections between dDMH/DHALepr and PVN neurons in the mouse.

Figure 6.

Open in a new tab

Non-iBAT PVN neurons receive both excitatory (Glut, blue) and inhibitory (GABA, red) inputs from Lepr-expressing neurons in the dDMH/DHA. In contrast, the connections between dDMH/DHA Lepr-expressing neurons and iBAT-related PVN neurons (green) are exclusively inhibitory. 3rd v: 3rd ventricle.

Effects of leptin on iBAT-related neurons

Leptin, an adipocyte-derived hormone, acts via leptin receptors in the central nervous system to decrease food intake and increase energy expenditure, and thus contributes to the regulation of energy homeostasis (Allison and Myers 2014; Munzberg and Morrison 2015). Leptin was shown to play a role in the regulation of iBAT thermogenesis via increased norepinephrine turnover, and a subsequent study demonstrated that leptin infusion results in increased efferent sympathetic nerve traffic to iBAT in a dose-dependent manner (Haynes et al. 1997a; Haynes et al. 1997b). In contrast, Zucker obese rats showed blunted BAT sympathetic response to leptin (Haynes et al. 1997a), which provides further evidence for leptin-dependent regulation of iBAT.

Leptin affects a variety of brain nuclei that are essential for thermoregulation. In addition to the DMH and preoptic area (Wada et al. 2014; Zhang et al. 2011), the role of the PVN in the regulation of thermogenesis is acknowledged (Cowley et al. 1999; Morrison and Madden 2014). Cold exposure increases c-fos expression in PVN neurons, and lesions of the PVN attenuate fever, both of which suggest that PVN neurons are involved in thermoregulation (Caldeira et al. 1998; Cano et al. 2003), even though melanocortin-induced thermogenesis is specifically not mediated via the PVN (Balthasar et al. 2005). Interestingly, NMDA-activation of the rat PVN did not increase BAT sympathetic nerve activity and BAT thermogenesis, indeed it reversed cold-induced BAT sympathetic nerve activity, suggesting that PVN neurons exert an inhibitory influence on the sympathetic outflow, and thereby BAT energy expenditure and thermogenesis (Madden and Morrison 2009). This observation is in contrast with other studies showing that glutamate-activation of the PVN increased iBAT temperature in a dose-dependent manner, which was prevented by systemic injection of a sympathetic ganglionic blocker or a β-adrenergic receptor antagonist (Amir 1990). Deletion of Lepr from Sim1 expressing neurons, which are mainly located in the PVN, resulted in decreased surface and core body temperature as well as energy expenditure (Cakir et al. 2019). In addition, nonshivering thermogenesis was disrupted in these mice due to defective regulation of uncoupling protein 1 in BAT and an altered hypothalamic-pituitary-thyroid axis. A more recent study demonstrated that bilateral leptin injection into the PVN of rats held at ~35 °C increased sympathetic nerve activity and temperature of BAT (Shi et al. 2020). Shi et al also showed that leptin-induced elevation of sympathetic nerve activity, mean arterial blood pressure, and heart rate was partially mediated by ionotropic glutamate receptors. Some Lepr-expressing glutamatergic neurons are pre-sympathetic neurons that project to the rostral ventrolateral medulla (Shi et al. 2020), which is in line with our data from mice showing iBAT-related neurons in nuclei involved in sympathetic regulation. Overall, despite the somewhat conflicting observations, the findings demonstrate that in rodents the PVN can modulate iBAT temperature through the sympathetic nervous system.

At the cellular levels, studies revealed that PVN neurons express leptin receptors and/or receive inputs from leptin receptor expressing neurons (Elmquist et al. 1998; Mercer et al. 1996; Wada et al. 2014), and thus may influence energy expenditure directly or indirectly as part of multisynaptic pathways originating from other hypothalamic regions (Pandit et al. 2017). Although the PVN contains heterogenous population of neurons (Luther et al. 2002; Luther and Tasker 2000; Swanson and Kuypers 1980; Swanson and Sawchenko 1980; Swanson et al. 1980), in case of pre-autonomic neurons having common features like low-threshold spikes (LTS) and strong inward rectification (Luther and Tasker 2000; Stern 2001; Tasker and Dudek 1991), only limited information is available about the properties of organ-related neurons including iBAT-related neurons.

Our findings showed a sparse but consistent expression of Lepr in the PVN and revealed that a subset of iBAT-related PVN neurons expresses Lepr. It is known, that leptin has the potential to modulate the excitability of a variety of PVN neurons (Powis et al. 1998) including CRF and thyrotropin-releasing hormone-expressing neurons (Ghamari-Langroudi et al. 2010; Huang et al. 1998). Our electrophysiological study demonstrate that application of leptin modulates a population of iBAT-related PVN neurons. Leptin hyperpolarized a subset of iBAT-related PVN neurons that may suggest expression of Lepr on PRV labeled PVN neurons, which is supported by the observation that some of the PRV labeled neurons co-localized with Lepr:EGFP neurons. The hyperpolarization caused by leptin is in contrast with Shi and colleagues recent work (Shi et al. 2020) revealing that PVN glutamatergic neurons are excited by leptin measured with calcium imaging. The difference could be due to investigating specific groups of neurons (iBAT-related vs vGlut2-expressing neurons) or the method (patch-clamp vs calcium measurements).

Neuronal excitability is largely determined by the activity of both excitatory and inhibitory inputs and the PVN receives inputs from several brain areas including the arcuate nucleus, the lateral, and dorsomedial hypothalamus, where abundant expression of leptin receptors was shown (Elmquist et al. 1998; Leshan et al. 2006; Myers et al. 2009). Leptin regulates synaptic inputs of neurons in other brain areas (Williams et al. 2007), which is consistent with our findings. In electrophysiological studies leptin is used in a wide range of concentrations. While some of the studies demonstrated that leptin has an effect in pM range (Nagamori et al. 2003), others showed reliable effect only in nM range (Otgon-Uul et al. 2016; Yu et al. 2016; Zsombok et al. 2014). Recordings from PVN neurons showed that leptin effects excitability in nM range (Ghamari-Langroudi et al. 2011; Ghamari-Langroudi et al. 2010) and GABAergic DMH neurons also respond to leptin in nM range. These GABAergic neurons send projections to the PVN (Otgon-Uul et al. 2016); therefore, in our recordings leptin was applied in nM range.

Our data demonstrated that leptin has a heterogenous effect on neurotransmission, as subsets of neurons responded with either a decrease or increase of PSC frequency, and another subset did not respond to leptin. Several subsets of neurons in the DMH/DHA are involved in the regulation of energy expenditure via projections to a variety of nuclei. Leptin alters the excitability of DMH neurons including GABAergic DMH neurons (Otgon-Uul et al. 2016), which leads to altered neurotransmission at the projection sites. Specifically, a population of DMH GABAergic neurons responded to leptin application, and light stimulation of DMH GABAergic axon terminals in the PVN increased IPSCs and promoted food intake (Otgon-Uul et al. 2016). Our data provide further information about this DMH – PVN circuit by demonstrating that GABAergic Lepr-expressing dDMH/DHA neurons project to iBAT-related PVN neurons. Moreover, our data provide functional evidence for direct connections between Lepr-expressing GABAergic neurons and iBAT-related neurons, which suggest the involvement of this circuit in the central control of thermoregulation. Activation of Lepr neurons selectively in the dDMH/DHA is sufficient to increase BAT thermogenesis, and even though this circuit involve glutamatergic dDMH/DHALepr projections to premotor raphe pallidus neurons (Rezai-Zadeh et al. 2014), the vast majority of DMH neurons are GABAergic neurons. Here we demonstrate connections between dDMH/DHALepr and PVN neurons, including iBAT-related PVN neurons using channel rhodopsin assisted circuit mapping. Our study revealed that GABAergic and glutamatergic dDMH/DHALepr neurons send projections to PVN neurons; however, connected iBAT-related PVN neurons receive exclusively inhibitory inputs from dDMH/DHALepr neurons (Fig. 6).

Bicuculline injection into the DMH activated BAT SNA, whereas subsequent injection of bicuculline into the PVN prevented this activation (Madden and Morrison 2009). Our data also suggest that Lepr-expressing neurons in the dDMH/DHA provide inhibitory inputs to PVN neurons. On the other hand, it has been shown that arcuate nucleus rat insulin promoter (RIP) expressing neurons with projections to the PVN are excited by leptin, and GABA transporter deficiency in these neurons results in an impaired thermogenic response to leptin treatment (Kong et al. 2012). These findings suggest that the arcuate nucleus is also an important source of GABAergic inputs (Kong et al. 2012). Although, the relative impact of the GABAergic DMH>PVN circuits in body weight homeostasis remains unclear, our data show the existence of GABAergic connections between dDMH/DHALepr and iBAT-related PVN neurons. Ultimately, further work would be necessary to identify which Lepr populations contribute to the excitatory neuromodulations of iBAT-related PVN neurons, and their potential role in the regulation of feeding or energy expenditure (e.g. phenotype- or circuits-dependent manner) as well as their response to leptin.

Technical considerations

By using PRV, we identified iBAT-related neurons in the mouse brain. PRVs are used to identify neurons in the nervous system with projections to peripheral organs including but not limited to the liver, stomach, kidney, iBAT and pancreas (Buijs et al. 2003; Cano et al. 2004; Doslikova et al. 2019; Gao et al. 2019; Gao et al. 2012; Gao et al. 2017; Glatzer et al. 2003; Huesing et al. 2022; Torres et al. 2021; Zhang et al. 2011). Previous studies demonstrated the specificity of the labeling and extensive research has been completed to reveal many aspects of the virus (Card 1998; Card et al. 1993).

Sympathetic circuits identified with PRV to the BAT were shown in rodents including in Siberian hamsters, rats, and mice (Bamshad et al. 1999; Cano et al. 2003; Oldfield et al. 2002; Song et al. 2008; Voss-Andreae et al. 2007; Zhang et al. 2011). In rats, 72 hours after iBAT inoculation sympathetic preganglionic neurons were identified in a variety of brain regions including but not limited to the RVLM, raphe and PVN (Oldfield et al. 2002). 96 hours after inoculation, more widespread labeling of iBAT-related neurons was observed in the ventrolateral medulla and in the PVN, indicating neurons projecting to the spinal cord via the brainstem (Oldfield et al. 2002). In our study, inoculation with PRV resulted in labeling in the described brain regions and was consistent with previous findings in mice (Voss-Andreae et al. 2007; Zhang et al. 2011). Our patch-clamp recordings were conducted approximately 96 hours after iBAT inoculation; therefore, the recorded neurons are likely representing a heterogenous population since they may have a variety of potential projection targets or could be interneurons within the PVN. Although distinguishing iBAT-related neurons based on their projection sites and determining their cellular properties would be intriguing, it would require future detailed studies. Previous studies investigated the cellular properties of PRV infected neurons in slices and dissociated primary culture cells (Derbenev et al. 2004; McCarthy et al. 2009; Smith et al. 2000). In slices, electrophysiological studies showed that the cellular properties of PRV-labeled neurons were indistinguishable from neurons identified with traditional tracers (Davis et al. 2003; Derbenev et al. 2004; Gao et al. 2012; Irnaten et al. 2001). In general, there was no difference in the membrane properties or the synaptic currents between uninfected and PRV-labeled rat and mouse neurons (Davis et al. 2003; Derbenev et al. 2004; Gao et al. 2012; Irnaten et al. 2001; Smith et al. 2000). Consistently, our recordings from non-labeled PVN neurons identified as pre-autonomic based on the presence of low-threshold spikes (LTS) (Luther et al. 2002; Stern 2001) did not reveal significant difference between the average resting membrane potential of random pre-autonomic PVN neurons and iBAT-related PVN neurons (random: −49.4 ± 2.4 mV (n=14) vs. iBAT-related: −46.0 ± 1.9 mV (n=14). The input resistance of pre-autonomic neurons was also similar (440 ± 30 MΩ (range 180 – 560 MΩ) vs. 442 ± 29 MΩ (range 240 – 582 MΩ), suggesting that PRV did not alter basic properties of PVN neurons at the time point of our recordings.

While in general there was no difference when recordings were performed from slices, patch-clamp recordings from rat cultured sympathetic neurons showed increased action potential firing and became electrically coupled after infection with the virulent Becker strain of PRV (McCarthy et al. 2009). In case of the attenuated PRV Bartha, like PRV-152, the infected neurons did not show elevated action potential firing until 18 hours of the 24-hour infection cycle (McCarthy et al. 2009). It is important to note the difference between ex vivo and in vitro recordings. When recordings are conducted from slices, due to the multi-synaptic circuits, it is difficult to determine the exact time of infection; therefore, the measurements in slices were likely performed in an early stage of the PRV infection as no differences were detected between infected and uninfected neurons (Derbenev et al. 2004; Gao et al. 2012; Irnaten et al. 2001; Smith et al. 2000). Additionally, we must keep in mind that in ex vivo conditions the neurons are surrounded with different cells and have variable degree of inter-connectivity compared with cell culture. In our studies, we controlled time to obtain reproducible infection rate and pattern, and our data are consistent with previous observations in mice demonstrating existence of iBAT-related neurons in the brainstem and forebrain (Voss-Andreae et al. 2007; Zhang et al. 2011).

Acknowledgements

This work was supported by research grants from the National Institutes of Health (NIH) (DK-099598 to A. Zsombok; DK-092587 to H. Muenzberg, DK-122842 to A. Zsombok and A. Derbenev), and Marko Spark Innovation Research Fund to A. Zsombok and A. Derbenev. We also thank the Tulane Brain Institute Cell and Tissue Imaging Core and NIH Center for Neuroanatomy and Neurotropic viruses for the PRVs (P40 OD010996).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of interest: none.

Reference list

  1. Allison MB, and Myers MG Jr. 20 years of leptin: connecting leptin signaling to biological function. J Endocrinol 223: T25–35, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amir S Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats. Brain Res 508: 152–155, 1990. [DOI] [PubMed] [Google Scholar]
  3. Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T, Ferreira M, Tang V, McGovern RA, Kenny CD, Christiansen LM, Edelstein E, Choi B, Boss O, Aschkenasi C, Zhang CY, Mountjoy K, Kishi T, Elmquist JK, and Lowell BB. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123: 493–505, 2005. [DOI] [PubMed] [Google Scholar]
  4. Bamshad M, Song CK, and Bartness TJ. CNS origins of the sympathetic nervous system outflow to brown adipose tissue. Am J Physiol 276: R1569–1578, 1999. [DOI] [PubMed] [Google Scholar]
  5. Bartness TJ, Song CK, and Demas GE. SCN efferents to peripheral tissues: implications for biological rhythms. J Biol Rhythms 16: 196–204, 2001. [DOI] [PubMed] [Google Scholar]
  6. Bartness TJ, Vaughan CH, and Song CK. Sympathetic and sensory innervation of brown adipose tissue. Int J Obes (Lond) 34 Suppl 1: S36–42, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boychuk CR, and Smith BN. Glutamatergic drive facilitates synaptic inhibition of dorsal vagal motor neurons after experimentally induced diabetes in mice. J Neurophysiol 116: 1498–1506, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buijs RM, la Fleur SE, Wortel J, Van Heyningen C, Zuiddam L, Mettenleiter TC, Kalsbeek A, Nagai K, and Niijima A. The suprachiasmatic nucleus balances sympathetic and parasympathetic output to peripheral organs through separate preautonomic neurons. J Comp Neurol 464: 36–48, 2003. [DOI] [PubMed] [Google Scholar]
  9. Cakir I, Diaz-Martinez M, Lining Pan P, Welch EB, Patel S, and Ghamari-Langroudi M. Leptin Receptor Signaling in Sim1-Expressing Neurons Regulates Body Temperature and Adaptive Thermogenesis. Endocrinology 160: 863–879, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caldeira JC, Franci CR, and Pela IR. Bilateral lesion of hypothalamic paraventricular nucleus abolishes fever induced by endotoxin and bradykinin in rats. Ann N Y Acad Sci 856: 294–297, 1998. [DOI] [PubMed] [Google Scholar]
  11. Cano G, Card JP, and Sved AF. Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in rat. J Comp Neurol 471: 462–481, 2004. [DOI] [PubMed] [Google Scholar]
  12. Cano G, Passerin AM, Schiltz JC, Card JP, Morrison SF, and Sved AF. Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure. J Comp Neurol 460: 303–326, 2003. [DOI] [PubMed] [Google Scholar]
  13. Card JP. Practical considerations for the use of pseudorabies virus in transneuronal studies of neural circuitry. Neurosci Biobehav Rev 22: 685–694, 1998. [DOI] [PubMed] [Google Scholar]
  14. Card JP, Rinaman L, Lynn RB, Lee BH, Meade RP, Miselis RR, and Enquist LW. Pseudorabies virus infection of the rat central nervous system: ultrastructural characterization of viral replication, transport, and pathogenesis. J Neurosci 13: 2515–2539, 1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cowley MA, Pronchuk N, Fan W, Dinulescu DM, Colmers WF, and Cone RD. Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat. Neuron 24: 155–163, 1999. [DOI] [PubMed] [Google Scholar]
  16. Davis SF, Williams KW, Xu W, Glatzer NR, and Smith BN. Selective enhancement of synaptic inhibition by hypocretin (orexin) in rat vagal motor neurons: implications for autonomic regulation. J Neurosci 23: 3844–3854, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Derbenev AV, Stuart TC, and Smith BN. Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve. J Physiol 559: 923–938, 2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Doslikova B, Tchir D, McKinty A, Zhu X, Marks DL, Baracos VE, and Colmers WF. Convergent neuronal projections from paraventricular nucleus, parabrachial nucleus, and brainstem onto gastrocnemius muscle, white and brown adipose tissue in male rats. J Comp Neurol 527: 2826–2842, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, and Saper CB. Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395: 535–547, 1998. [PubMed] [Google Scholar]
  20. Enriori PJ, Sinnayah P, Simonds SE, Garcia Rudaz C, and Cowley MA. Leptin action in the dorsomedial hypothalamus increases sympathetic tone to brown adipose tissue in spite of systemic leptin resistance. J Neurosci 31: 12189–12197, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Foster DO, Depocas F, and Zaror-Behrens G. Unilaterality of the sympathetic innervation of each pad of rat interscapular brown adipose tissue. Can J Physiol Pharmacol 60: 107–113, 1982. [DOI] [PubMed] [Google Scholar]
  22. Francois M, Torres H, Huesing C, Zhang R, Saurage C, Lee N, Qualls-Creekmore E, Yu S, Morrison CD, Burk D, Berthoud HR, and Munzberg H. Sympathetic innervation of the interscapular brown adipose tissue in mouse. Ann N Y Acad Sci 1454: 3–13, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gao H, Korim WS, Yao ST, Heesch CM, and Derbenev AV. Glycinergic neurotransmission in the rostral ventrolateral medulla controls the time course of baroreflex-mediated sympathoinhibition. J Physiol 597: 283–301, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gao H, Miyata K, Bhaskaran MD, Derbenev AV, and Zsombok A. Transient receptor potential vanilloid type 1-dependent regulation of liver-related neurons in the paraventricular nucleus of the hypothalamus diminished in the type 1 diabetic mouse. Diabetes 61: 1381–1390, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gao H, Molinas AJR, Miyata K, Qiao X, and Zsombok A. Overactivity of Liver-Related Neurons in the Paraventricular Nucleus of the Hypothalamus: Electrophysiological Findings in db/db Mice. J Neurosci 37: 11140–11150, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ghamari-Langroudi M Electrophysiological analysis of circuits controlling energy homeostasis. Mol Neurobiol 45: 258–278, 2012. [DOI] [PubMed] [Google Scholar]
  27. Ghamari-Langroudi M, Srisai D, and Cone RD. Multinodal regulation of the arcuate/paraventricular nucleus circuit by leptin. Proc Natl Acad Sci U S A 108: 355–360, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ghamari-Langroudi M, Vella KR, Srisai D, Sugrue ML, Hollenberg AN, and Cone RD. Regulation of thyrotropin-releasing hormone-expressing neurons in paraventricular nucleus of the hypothalamus by signals of adiposity. Mol Endocrinol 24: 2366–2381, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Glatzer NR, Hasney CP, Bhaskaran MD, and Smith BN. Synaptic and morphologic properties in vitro of premotor rat nucleus tractus solitarius neurons labeled transneuronally from the stomach. J Comp Neurol 464: 525–539, 2003. [DOI] [PubMed] [Google Scholar]
  30. Haynes WG, Morgan DA, Walsh SA, Mark AL, and Sivitz WI. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 100: 270–278, 1997a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Haynes WG, Sivitz WI, Morgan DA, Walsh SA, and Mark AL. Sympathetic and cardiorenal actions of leptin. Hypertension 30: 619–623, 1997b. [DOI] [PubMed] [Google Scholar]
  32. Huang Q, Rivest R, and Richard D. Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice. Endocrinology 139: 1524–1532, 1998. [DOI] [PubMed] [Google Scholar]
  33. Huesing C, Zhang R, Gummadi S, Lee N, Qualls-Creekmore E, Yu S, Morrison CD, Burk D, Berthoud HR, Neuhuber W, and Munzberg H. Organization of sympathetic innervation of interscapular brown adipose tissue in the mouse. J Comp Neurol 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Huesing C, Zhang R, Gummadi S, Lee N, Qualls-Creekmore E, Yu S, Morrison CD, Burk D, Berthoud HR, Neuhuber W, and Munzberg H. Organization of sympathetic innervation of interscapular brown adipose tissue in the mouse. J Comp Neurol 530: 1363–1378, 2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Irnaten M, Neff RA, Wang J, Loewy AD, Mettenleiter TC, and Mendelowitz D. Activity of cardiorespiratory networks revealed by transsynaptic virus expressing GFP. J Neurophysiol 85: 435–438, 2001. [DOI] [PubMed] [Google Scholar]
  36. Jiang Y, Gao H, Krantz AM, Derbenev AV, and Zsombok A. Reduced GABAergic inhibition of kidney-related PVN neurons in streptozotocin-treated type 1 diabetic mouse. J Neurophysiol 110: 2192–2202, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kalsbeek A, Bruinstroop E, Yi CX, Klieverik LP, La Fleur SE, and Fliers E. Hypothalamic control of energy metabolism via the autonomic nervous system. Ann N Y Acad Sci 1212: 114–129, 2010. [DOI] [PubMed] [Google Scholar]
  38. Kong D, Tong Q, Ye C, Koda S, Fuller PM, Krashes MJ, Vong L, Ray RS, Olson DP, and Lowell BB. GABAergic RIP-Cre neurons in the arcuate nucleus selectively regulate energy expenditure. Cell 151: 645–657, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Leshan RL, Bjornholm M, Munzberg H, and Myers MG Jr. Leptin receptor signaling and action in the central nervous system. Obesity (Silver Spring) 14 Suppl 5: 208S–212S, 2006. [DOI] [PubMed] [Google Scholar]
  40. Luther JA, Daftary SS, Boudaba C, Gould GC, Halmos KC, and Tasker JG. Neurosecretory and non-neurosecretory parvocellular neurones of the hypothalamic paraventricular nucleus express distinct electrophysiological properties. J Neuroendocrinol 14: 929–932, 2002. [DOI] [PubMed] [Google Scholar]
  41. Luther JA, and Tasker JG. Voltage-gated currents distinguish parvocellular from magnocellular neurones in the rat hypothalamic paraventricular nucleus. J Physiol 523 Pt 1: 193–209, 2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Madden CJ, and Morrison SF. Neurons in the paraventricular nucleus of the hypothalamus inhibit sympathetic outflow to brown adipose tissue. Am J Physiol Regul Integr Comp Physiol 296: R831–843, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Marsh AJ, Fontes MA, Killinger S, Pawlak DB, Polson JW, and Dampney RA. Cardiovascular responses evoked by leptin acting on neurons in the ventromedial and dorsomedial hypothalamus. Hypertension 42: 488–493, 2003. [DOI] [PubMed] [Google Scholar]
  44. McCarthy KM, Tank DW, and Enquist LW. Pseudorabies virus infection alters neuronal activity and connectivity in vitro. PLoS Pathog 5: e1000640, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, and Trayhurn P. Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 387: 113–116, 1996. [DOI] [PubMed] [Google Scholar]
  46. Minokoshi Y, Haque MS, and Shimazu T. Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats. Diabetes 48: 287–291, 1999. [DOI] [PubMed] [Google Scholar]
  47. Molinas AJR, Desmoulins LD, Hamling BV, Butcher SM, Anwar IJ, Miyata K, Enix CL, Dugas CM, Satou R, Derbenev AV, and Zsombok A. Interaction between TRPV1-expressing neurons in the hypothalamus. J Neurophysiol 121: 140–151, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Morrison SF, and Madden CJ. Central nervous system regulation of brown adipose tissue. Compr Physiol 4: 1677–1713, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Munzberg H, and Morrison CD. Structure, production and signaling of leptin. Metabolism 64: 13–23, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Myers MG Jr., Munzberg H, Leinninger GM, and Leshan RL. The geometry of leptin action in the brain: more complicated than a simple ARC. Cell Metab 9: 117–123, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Nagamori K, Ishibashi M, Shiraishi T, Oomura Y, and Sasaki K. Effects of leptin on hypothalamic arcuate neurons in Wistar and Zucker rats: an in vitro study. Exp Biol Med (Maywood) 228: 1162–1167, 2003. [DOI] [PubMed] [Google Scholar]
  52. O’Hare JD, and Zsombok A. Brain-liver connections: role of the preautonomic PVN neurons. Am J Physiol Endocrinol Metab 310: E183–189, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Oldfield BJ, Giles ME, Watson A, Anderson C, Colvill LM, and McKinley MJ. The neurochemical characterisation of hypothalamic pathways projecting polysynaptically to brown adipose tissue in the rat. Neuroscience 110: 515–526, 2002. [DOI] [PubMed] [Google Scholar]
  54. Otgon-Uul Z, Suyama S, Onodera H, and Yada T. Optogenetic activation of leptin- and glucose-regulated GABAergic neurons in dorsomedial hypothalamus promotes food intake via inhibitory synaptic transmission to paraventricular nucleus of hypothalamus. Mol Metab 5: 709–715, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Pandit R, Beerens S, and Adan RAH. Role of leptin in energy expenditure: the hypothalamic perspective. Am J Physiol Regul Integr Comp Physiol 312: R938–r947, 2017. [DOI] [PubMed] [Google Scholar]
  56. Powis JE, Bains JS, and Ferguson AV. Leptin depolarizes rat hypothalamic paraventricular nucleus neurons. Am J Physiol 274: R1468–1472, 1998. [DOI] [PubMed] [Google Scholar]
  57. Rezai-Zadeh K, Yu S, Jiang Y, Laque A, Schwartzenburg C, Morrison CD, Derbenev AV, Zsombok A, and Munzberg H. Leptin receptor neurons in the dorsomedial hypothalamus are key regulators of energy expenditure and body weight, but not food intake. Mol Metab 3: 681–693, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Schlaich M, Straznicky N, Lambert E, and Lambert G. Metabolic syndrome: a sympathetic disease? Lancet Diabetes Endocrinol 3: 148–157, 2015. [DOI] [PubMed] [Google Scholar]
  59. Shi Z, Pelletier NE, Wong J, Li B, Sdrulla AD, Madden CJ, Marks DL, and Brooks VL. Leptin increases sympathetic nerve activity via induction of its own receptor in the paraventricular nucleus. Elife 9: 2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Smith BN, Banfield BW, Smeraski CA, Wilcox CL, Dudek FE, Enquist LW, and Pickard GE. Pseudorabies virus expressing enhanced green fluorescent protein: A tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits. Proc Natl Acad Sci U S A 97: 9264–9269, 2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Song CK, Vaughan CH, Keen-Rhinehart E, Harris RB, Richard D, and Bartness TJ. Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence. Am J Physiol Regul Integr Comp Physiol 295: R417–428, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Stern JE. Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus. J Physiol 537: 161–177, 2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Swanson LW, and Kuypers HG. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194: 555–570, 1980. [DOI] [PubMed] [Google Scholar]
  64. Swanson LW, and Sawchenko PE. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31: 410–417, 1980. [DOI] [PubMed] [Google Scholar]
  65. Swanson LW, Sawchenko PE, Wiegand SJ, and Price JL. Separate neurons in the paraventricular nucleus project to the median eminence and to the medulla or spinal cord. Brain Res 198: 190–195, 1980. [DOI] [PubMed] [Google Scholar]
  66. Tasker JG, and Dudek FE. Electrophysiological properties of neurones in the region of the paraventricular nucleus in slices of rat hypothalamus. J Physiol 434: 271–293, 1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Thorp AA, and Schlaich MP. Relevance of Sympathetic Nervous System Activation in Obesity and Metabolic Syndrome. J Diabetes Res 2015: 341583, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Torres H, Huesing C, Burk DH, Molinas AJR, Neuhuber WL, Berthoud HR, Munzberg H, Derbenev AV, and Zsombok A. Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia. Am J Physiol Regul Integr Comp Physiol 321: R328–R337, 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Vong L, Ye C, Yang Z, Choi B, Chua S Jr., and Lowell BB. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71: 142–154, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Voss-Andreae A, Murphy JG, Ellacott KL, Stuart RC, Nillni EA, Cone RD, and Fan W. Role of the central melanocortin circuitry in adaptive thermogenesis of brown adipose tissue. Endocrinology 148: 1550–1560, 2007. [DOI] [PubMed] [Google Scholar]
  71. Wada N, Hirako S, Takenoya F, Kageyama H, Okabe M, and Shioda S. Leptin and its receptors. J Chem Neuroanat 61-62: 191–199, 2014. [DOI] [PubMed] [Google Scholar]
  72. Williams KW, Zsombok A, and Smith BN. Rapid inhibition of neurons in the dorsal motor nucleus of the vagus by leptin. Endocrinology 148: 1868–1881, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Xu H, and Smith BN. Presynaptic ionotropic glutamate receptors modulate GABA release in the mouse dorsal motor nucleus of the vagus. Neuroscience 308: 95–105, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Yu S, Cheng H, Francois M, Qualls-Creekmore E, Huesing C, He Y, Jiang Y, Gao H, Xu Y, Zsombok A, Derbenev AV, Nillni EA, Burk DH, Morrison CD, Berthoud HR, and Munzberg H. Preoptic leptin signaling modulates energy balance independent of body temperature regulation. Elife 7: 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Yu S, Qualls-Creekmore E, Rezai-Zadeh K, Jiang Y, Berthoud HR, Morrison CD, Derbenev AV, Zsombok A, and Munzberg H. Glutamatergic Preoptic Area Neurons That Express Leptin Receptors Drive Temperature-Dependent Body Weight Homeostasis. J Neurosci 36: 5034–5046, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Zhang Y, Kerman IA, Laque A, Nguyen P, Faouzi M, Louis GW, Jones JC, Rhodes C, and Munzberg H. Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits. J Neurosci 31: 1873–1884, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Zsombok A, Jiang Y, Gao H, Anwar IJ, Rezai-Zadeh K, Enix CL, Munzberg H, and Derbenev AV. Regulation of leptin receptor-expressing neurons in the brainstem by TRPV1. Physiol Rep 2: 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES