Andrei V. Derbenev

Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve

Cannabinoids bind central type 1 receptors (CB1R) and modify autonomic functions, including feeding and anti‐emetic behaviours, when administered peripherally or into the dorsal vagal complex. Western blots and immunohistochemistry indicated the expression of CB1R in the rat dorsal vagal complex, and tissue polymerase chain reaction confirmed that CB1R message was made within the region. To identify a cellular substrate for the central autonomic effects of cannabinoids, whole‐cell patch‐clamp recordings were made in brainstem slices to determine the effects of CB1R activation on synaptic transmission to neurones of the dorsal motor nucleus of the vagus (DMV). A subset of these neurones was identified as gastric related after being labelled retrogradely from the stomach. The CB1R agonists WIN55,212‐2 and anandamide decreased the frequency of spontaneous excitatory or inhibitory postsynaptic currents in a concentration‐related fashion, an effect that persisted in the presence of tetrodotoxin. Paired pulse ratios of electrically evoked postsynaptic currents were also increased by WIN55,212‐2. The effects of  WIN55,212‐2 were sensitive to the selective CB1R antagonist AM251. Cannabinoid agonist effects on synaptic input originating from neurones in the nucleus tractus solitarius (NTS) were determined by evoking activity in the NTS with local glutamate application. Excitatory and inhibitory synaptic inputs arising from the NTS were attenuated by WIN55,212‐2. Our results indicate that cannabinoids inhibit transfer of synaptic information to the DMV, including that arising from the NTS, in part by acting at receptors located on presynaptic terminals contacting DMV neurones. Inhibition of synaptic input to DMV neurones is likely to contribute to the suppression of visceral motor responses by cannabinoids.

Excitatory and inhibitory local circuit input to the rat dorsal motor nucleus of the vagus originating from the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV) constitute sensory and motor nuclei of the dorsal vagal complex, respectively. We used whole-cell patch-clamp recordings from DMV neurons in rat brain slices and three methods of stimulation (electrical, glutamate microdrop, glutamate photostimulation) to test the hypothesis that convergent excitatory and inhibitory inputs to DMV neurons originate from intact neurons in multiple NTS areas. Electrical stimulation of the NTS resulted in evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) in DMV neurons. Stimulation of the dorsal NTS with glutamate microdrops, which selectively stimulates the soma and dendrites of intact neurons, resulted in 31% of DMV neurons receiving eEPSCs, 44% receiving eIPSCs, and 6% receiving convergent excitatory and inhibitory inputs. Glutamate photostimulation allowed selective activation of intact neurons in multiple, discrete areas of the NTS and resulted in 36% of DMV neurons receiving eEPSCs, 65% receiving eIPSCs and 20% receiving both inputs. Data obtained by stimulation of multiple NTS areas support the hypothesis that there are anatomically convergent inputs to DMV neurons originating from intact neurons within the NTS. These data support the hypothesis that there is transfer of convergent information from the NTS to the DMV, implying that significant sensory-motor processing occurs within the brainstem.

Leptin receptor neurons in the dorsomedial hypothalamus are key regulators of energy expenditure and body weight, but not food intake

Objective Leptin responsive neurons play an important role in energy homeostasis, controlling specific autonomic, behavioral, and neuroendocrine functions. We have previously identified a population of leptin receptor (LepRb) expressing neurons within the dorsomedial hypothalamus/dorsal hypothalamic area (DMH/DHA) which are related to neuronal circuits that control brown adipose tissue (BAT) thermogenesis. Intra-DMH leptin injections also activate sympathetic outflow to BAT, but whether such effects are mediated directly via DMH/DHA LepRb neurons and whether this is physiologically relevant for whole body energy expenditure and body weight regulation has yet to be determined. Methods We used pharmacosynthetic receptors (DREADDs) to selectively activate DMH/DHA LepRb neurons. We further deleted LepRb with virally driven cre-recombinase from DMH/DHA neurons and determined the physiological importance of DMH/DHA LepRb neurons in whole body energy homeostasis. Results Neuronal activation of DMH/DHA LepRb neurons with DREADDs promoted BAT thermogenesis and locomotor activity, which robustly induced energy expenditure (p < 0.001) and decreases body weight (p < 0.001). Similarly, intra-DMH/DHA leptin injections normalized hypothermia and attenuated body weight gain in leptin-deficient ob/ob mice. Conversely, ablation of LepRb from DMH/DHA neurons remarkably drives weight gain (p < 0.001) by reducing energy expenditure (p < 0.001) and locomotor activity (p < 0.001). The observed changes in body weight were largely independent of food intake. Conclusion Taken together, our data highlight that DMH/DHA LepRb neurons are sufficient and necessary to regulate energy expenditure and body weight.

Vanilloid-Mediated Heterosynaptic Facilitation of Inhibitory Synaptic Input to Neurons of the Rat Dorsal Motor Nucleus of the Vagus

Vanilloid type-1 receptors (VR1) are abundant in the dorsal vagal complex, where their function is mostly unknown. We examined the role of VR1 in regulating synaptic inputs to neurons of the dorsal motor nucleus of the vagus (DMV). Using patch-clamp recordings from DMV neurons in brainstem slices, capsaicin was found to increase action potential-independent inhibitory input onto DMV neurons. This rapid effect was mimicked by application of the endogenous cannabinoid, anandamide and blocked by VR1 antagonists. The VR1-mediated facilitation of synaptic inhibition was reduced by ionotropic and metabotropic glutamate receptor antagonists, suggesting an indirect, heterosynaptic enhancement of GABA release caused by a VR1-mediated increase in glutamate release from presynaptic terminals of excitatory neurons. Application of l-glutamate also increased GABA release. The paired-pulse ratio was increased for IPSCs evoked after electrical stimulation of the nucleus tractus solitarius, but the effect was slower than for the enhancement of spontaneous and miniature IPSCs. Capsaicin also increased the frequency of glutamatergic postsynaptic currents in a VR1-mediated manner. Results of these studies suggest that VR1-containing glutamatergic terminals contact DMV neurons. Activation of VR1 potently enhances glutamate release onto GABAergic terminals, facilitating GABA release. Endogenous cannabinoids can thereby rapidly enhance inhibitory input to DMV neurons via VR1-mediated presynaptic mechanisms.

Functional plasticity of central TRPV1 receptors in brainstem dorsal vagal complex circuits of streptozotocin-treated hyperglycemic mice

Emerging data indicate that central neurons participate in diabetic processes by modulating autonomic output from neurons in the dorsal motor nucleus of the vagus (DMV). We tested the hypothesis that synaptic modulation by transient receptor potential vanilloid type 1 (TRPV1) receptors is reduced in the DMV in slices from a murine model of type 1 diabetes. The TRPV1 agonist capsaicin robustly enhanced glutamate release onto DMV neurons by acting at preterminal receptors in slices from intact mice, but failed to do so in slices from diabetic mice. TRPV1 receptor protein expression in the vagal complex was unaltered. Brief insulin preapplication restored TRPV1-dependent modulation of glutamate release in a PKC- and PI3K-dependent manner. The restorative effect of insulin was prevented by brefeldin A, suggesting that insulin induced TRPV1 receptor trafficking to the terminal membrane. Central vagal circuits critical to the autonomic regulation of metabolism undergo insulin-dependent synaptic plasticity involving TRPV1 receptor modulation in diabetic mice after several days of chronic hyperglycemia.

CNS Inflammation and Bone Marrow Neuropathy in Type 1 Diabetes

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

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

The paraventricular nucleus (PVN) of the hypothalamus controls the autonomic neural output to the liver, thereby participating in the regulation of hepatic glucose production (HGP); nevertheless, mechanisms controlling the activity of liver-related PVN neurons are not known. Transient receptor potential vanilloid type 1 (TRPV1) is involved in glucose homeostasis and colocalizes with liver-related PVN neurons; however, the functional role of TRPV1 regarding liver-related PVN neurons has to be elucidated. A retrograde viral tracer was used to identify liver-related neurons within the brain-liver circuit in control, type 1 diabetic, and insulin-treated mice. Our data indicate that TRPV1 regulates liver-related PVN neurons. This TRPV1-dependent excitation diminished in type 1 diabetic mice. In vivo and in vitro insulin restored TRPV1 activity in a phosphatidylinositol 3-kinase/protein kinase C–dependent manner and stimulated TRPV1 receptor trafficking to the plasma membrane. There was no difference in total TRPV1 protein expression; however, increased phosphorylation of TRPV1 receptors was observed in type 1 diabetic mice. Our data demonstrate that TRPV1 plays a pivotal role in the regulation of liver-related PVN neurons. Moreover, TRPV1-dependent excitation of liver-related PVN neurons diminishes in type 1 diabetes, thus indicating that the brain-liver autonomic circuitry is altered in type 1 diabetes and may contribute to the autonomic dysfunction of HGP.

Glutamatergic Preoptic Area Neurons That Express Leptin Receptors Drive Temperature-Dependent Body Weight Homeostasis

The preoptic area (POA) regulates body temperature, but is not considered a site for body weight control. A subpopulation of POA neurons express leptin receptors (LepRbPOA neurons) and modulate reproductive function. However, LepRbPOA neurons project to sympathetic premotor neurons that control brown adipose tissue (BAT) thermogenesis, suggesting an additional role in energy homeostasis and body weight regulation. We determined the role of LepRbPOA neurons in energy homeostasis using cre-dependent viral vectors to selectively activate these neurons and analyzed functional outcomes in mice. We show that LepRbPOA neurons mediate homeostatic adaptations to ambient temperature changes, and their pharmacogenetic activation drives robust suppression of energy expenditure and food intake, which lowers body temperature and body weight. Surprisingly, our data show that hypothermia-inducing LepRbPOA neurons are glutamatergic, while GABAergic POA neurons, originally thought to mediate warm-induced inhibition of sympathetic premotor neurons, have no effect on energy expenditure. Our data suggest a new view into the neurochemical and functional properties of BAT-related POA circuits and highlight their additional role in modulating food intake and body weight. SIGNIFICANCE STATEMENT Brown adipose tissue (BAT)-induced thermogenesis is a promising therapeutic target to treat obesity and metabolic diseases. The preoptic area (POA) controls body temperature by modulating BAT activity, but its role in body weight homeostasis has not been addressed. LepRbPOA neurons are BAT-related neurons and we show that they are sufficient to inhibit energy expenditure. We further show that LepRbPOA neurons modulate food intake and body weight, which is mediated by temperature-dependent homeostatic responses. We further found that LepRbPOA neurons are stimulatory glutamatergic neurons, contrary to prevalent models, providing a new view on thermoregulatory neural circuits. In summary, our study significantly expands our current understanding of central circuits and mechanisms that modulate energy homeostasis.

Morphological and electrophysiological features of motor neurons and putative interneurons in the dorsal vagal complex of rats and mice

The dorsal motor nucleus of the vagus (DMV) contains preganglionic motor neurons that control viscera along the subdiaphragmatic digestive tract, but may also contain neurons that do not project to the viscera. Neurons that expressed EGFP 60-72 h subsequent to PRV-152 inoculation of vagal terminals in the stomach wall were targeted for whole-cell patch-clamp recording and biocytin filling in transverse brainstem slices from rats and their quantitative morphological and electrophysiological characteristics were compared with uninfected cells. Over 90% of PRV-152 labeled neurons were also labeled subsequent to intraperitoneal injection of FluoroGold, indicating that most were preganglionic motor neurons. In reconstructed neurons with an identifiable axon trajectory, two cellular subtypes were distinguished. The axon projected ventrolaterally from the DMV in 44 of 49 cells and these were likely to be vagal motor neurons. Axons of other neurons ramified within the nucleus tractus solitarius (NTS) or DMV. These cells were smaller and otherwise morphologically distinct from putative motor neurons. Transgenic mice with GFP-expressing inhibitory neurons (i.e., GIN mice) were used to identify a GABAergic subset of DMV neurons. These neurons had locally ramifying axons and formed a morphologically distinct subset of DMV cells, which were similar in size and axon trajectory to GABAergic neurons in the NTS. Most neurons in the DMV therefore possess morphological features of motor neurons, but locally projecting cells and inhibitory neurons with distinct morphological features are also found within the DMV. These cells likely contribute to regulation of vagal function.

Potential therapeutic value of TRPV1 and TRPA1 in diabetes mellitus and obesity

Diabetes mellitus and obesity, which is a major risk factor in the development of type 2 diabetes mellitus, have reached epidemic proportions worldwide including the USA. The current statistics and forecasts, both short- and long-term, are alarming and predict severe problems in the near future. Therefore, there is a race for developing new compounds, discovering new receptors, or finding alternative solutions to prevent and/or treat the symptoms and complications related to obesity and diabetes mellitus. It is well demonstrated that members of the transient receptor potential (TRP) superfamily play a crucial role in a variety of biological functions both in health and disease. In the recent years, transient receptor potential vanilloid type 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) were shown to have beneficial effects on whole body metabolism including glucose homeostasis. TRPV1 and TRPA1 have been associated with control of weight, pancreatic function, hormone secretion, thermogenesis, and neuronal function, which suggest a potential therapeutic value of these channels. This review summarizes recent findings regarding TRPV1 and TRPA1 in association with whole body metabolism with emphasis on obese and diabetic conditions.