High-Fat Diet Modulates the Excitability of Neurons within the Brain-Liver Pathway.

Stimulation of hepatic sympathetic nerves increases glucose production and glycogenolysis. Activity of pre-sympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the ventrolateral and ventromedial medulla (VLM/VMM) largely influence the sympathetic output. Increased activity of the sympathetic nervous system (SNS) plays a role in the development and progression of metabolic diseases; however, despite the importance of the central circuits, the excitability of pre-sympathetic liver-related neurons remains to be determined. Here, we tested the hypothesis that the activity of liver-related neurons in the PVN and VLM/VMM is altered in diet-induced obese mice, as well as their response to insulin. Patch-clamp recordings were conducted from liver-related PVN neurons, VLM-projecting PVN neurons, and pre-sympathetic liver-related neurons in the ventral brainstem. Our data demonstrate that the excitability of liver-related PVN neurons increased in high-fat diet (HFD)-fed mice compared to mice fed with control diet. Insulin receptor expression was detected in a population of liver-related neurons, and insulin suppressed the firing activity of liver-related PVN and pre-sympathetic VLM/VMM neurons in HFD mice; however, it did not affect VLM-projecting liver-related PVN neurons. These findings further suggest that HFD alters the excitability of pre-autonomic neurons as well as their response to insulin.

Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia.

The sympathetic nervous system (SNS) plays a crucial role in the regulation of renal and hepatic functions. Although sympathetic nerves to the kidney and liver have been identified in many species, specific details are lacking in the mouse. In the absence of detailed information of sympathetic prevertebral innervation of specific organs, selective manipulation of a specific function will remain challenging. Despite providing major postganglionic inputs to abdominal organs, limited data are available about the mouse celiac-superior mesenteric complex. We used tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DbH) reporter mice to visualize abdominal prevertebral ganglia. We found that both the TH and DbH reporter mice are useful models for identification of ganglia and nerve bundles. We further tested if the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver. The retrograde viral tracer, pseudorabies virus (PRV)-152 was injected into the cortex of the left kidney or the main lobe of the liver to identify kidney-projecting and liver-projecting neurons in the celiac-superior mesenteric complex. iDISCO immunostaining and tissue clearing were used to visualize unprecedented anatomical detail of kidney-related and liver-related postganglionic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia compared with TH-positive neurons. Kidney-projecting neurons were restricted to the suprarenal and aorticorenal ganglia, whereas only sparse labeling was observed in the celiac-superior mesenteric complex. In contrast, liver-projecting postganglionic neurons were observed in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia, suggesting spatial separation between the sympathetic innervation of the mouse kidney and liver.

Activation of ADAM17 (A Disintegrin and Metalloprotease 17) on Glutamatergic Neurons Selectively Promotes Sympathoexcitation.

Chronic activation of the brain renin-angiotensin system contributes to the development of hypertension by altering autonomic balance. Beyond the essential role of Ang II (angiotensin II) type 1 receptors, ADAM17 (A disintegrin and metalloprotease 17) is also found to promote brain renin-angiotensin system overactivation. ADAM17 is robustly expressed in various cell types within the central nervous system. The aim of this study was to determine whether ADAM17 modulates presympathetic neuronal activity to promote autonomic dysregulation in salt-sensitive hypertension. To test our hypothesis, ADAM17 was selectively knocked down in glutamatergic neurons using Cre-loxP technology. In mice lacking ADAM17 in glutamatergic neurons, the blood pressure increase induced by deoxycorticosterone acetate-salt treatment was blunted. Deoxycorticosterone acetate-salt significantly elevated cardiac and vascular sympathetic drive in control mice, while such effects were reduced in mice with ADAM17 knockdown. This blunted sympathoexcitation was extended to the spleen, with a lesser activation of the peripheral immune system, translating into a sequestration of circulating T cells within this organ, compared with controls. Within the paraventricular nucleus, Ang II-induced activation of kidney-related presympathetic glutamatergic neurons was reduced in ADAM17 knockdown mice, with the majority of cells no longer responding to Ang II stimulation, confirming the supportive role of ADAM17 in increasing presympathetic neuronal activity. Overall, our data highlight the pivotal role of neuronal ADAM17 in regulating sympathetic activity and demonstrate that activation of ADAM17 in glutamatergic neurons leads to a selective increase of sympathetic output, but not vagal tone, to specific organs, ultimately contributing to dysautonomia and salt-sensitive hypertension.

Interaction between TRPV1-expressing neurons in the hypothalamus.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel expressed in the peripheral and central nervous systems. TRPV1-dependent mechanisms take part in a wide range of physiological and pathophysiological pathways including the regulation of homeostatic functions. TRPV1 expression in the hypothalamus has been described as well as evidence that TRPV1-dependent excitatory inputs to hypothalamic preautonomic neurons are diminished in diabetic conditions. Here we aimed to determine the functional expression of TRPV1 in two hypothalamic nuclei known to be involved in the central control of metabolism and to test the hypothesis that TRPV1-expressing neurons receive TRPV1-expressing inputs. A mouse model (TRPV1Cre/tdTom) was generated to identify TRPV1-expressing cells and determine the cellular properties of TRPV1-expressing neurons in adult mice. Our study demonstrated the functional expression of TRPV1 in the dorsomedial hypothalamic nucleus and paraventricular nucleus in adult mice. Our findings revealed that a subset of TRPV1Cre/tdTom neurons receive TRPV1-expressing excitatory inputs, indicating direct interaction between TRPV1-expressing neurons. In addition, astrocytes likely play a role in the modulation of TRPV1-expressing neurons. In summary, this study identified specific hypothalamic regions where TRPV1 is expressed and functional in adult mice and the existence of direct connections between TRPV1Cre/tdTom neurons. NEW & NOTEWORTHY Transient receptor potential vanilloid type 1 (TRPV1) is expressed in the hypothalamus, and TRPV1-dependent regulation of preautonomic neurons is decreased in hyperglycemic conditions. Our study demonstrated functional expression of TRPV1 in two hypothalamic nuclei involved in the control of energy homeostasis. Our results also revealed that a subset of TRPV1-expressing neurons receive TRPV1-expressing excitatory inputs. These findings suggest direct interaction between TRPV1-expressing neurons.

Androgen excess in pancreatic ß cells and neurons predisposes female mice to type 2 diabetes.

Androgen excess predisposes women to type 2 diabetes (T2D), but the mechanism of this is poorly understood. We report that female mice fed a Western diet and exposed to chronic androgen excess using dihydrotestosterone (DHT) exhibit hyperinsulinemia and insulin resistance associated with secondary pancreatic ß cell failure, leading to hyperglycemia. These abnormalities are not observed in mice lacking the androgen receptor (AR) in ß cells and partially in neurons of the mediobasal hypothalamus (MBH) as well as in mice lacking AR selectively in neurons. Accordingly, i.c.v. infusion of DHT produces hyperinsulinemia and insulin resistance in female WT mice. We observe that acute DHT produces insulin hypersecretion in response to glucose in cultured female mouse and human pancreatic islets in an AR-dependent manner via a cAMP- and mTOR-dependent pathway. Acute DHT exposure increases mitochondrial respiration and oxygen consumption in female cultured islets. As a result, chronic DHT exposure in vivo promotes islet oxidative damage and susceptibility to additional stress induced by streptozotocin via AR in ß cells. This study suggests that excess androgen predisposes female mice to T2D following AR activation in neurons, producing peripheral insulin resistance, and in pancreatic ß cells, promoting insulin hypersecretion, oxidative injury, and secondary ß cell failure.

Overactivity of Liver-Related Neurons in the Paraventricular Nucleus of the Hypothalamus: Electrophysiological Findings in db/db Mice.

Preautonomic neurons in the paraventricular nucleus (PVN) of the hypothalamus play a large role in the regulation of hepatic functions via the autonomic nervous system. Activation of hepatic sympathetic nerves increases glucose and lipid metabolism and contributes to the elevated hepatic glucose production observed in the type 2 diabetic condition. This augmented sympathetic output could originate from altered activity of liver-related PVN neurons. Remarkably, despite the importance of the brain-liver pathway, the cellular properties of liver-related neurons are not known. In this study, we provide the first evidence of overall activity of liver-related PVN neurons. Liver-related PVN neurons were identified with a retrograde, trans-synaptic, viral tracer in male lean and db/db mice and whole-cell patch-clamp recordings were conducted. In db/db mice, the majority of liver-related PVN neurons fired spontaneously; whereas, in lean mice the majority of liver-related PVN neurons were silent, indicating that liver-related PVN neurons are more active in db/db mice. Persistent, tonic inhibition was identified in liver-related PVN neurons; although, the magnitude of tonic inhibitory control was not different between lean and db/db mice. In addition, our study revealed that the transient receptor potential vanilloid type 1-dependent increase of excitatory neurotransmission was reduced in liver-related PVN neurons of db/db mice. These findings demonstrate plasticity of liver-related PVN neurons and a shift toward excitation in a diabetic mouse model. Our study suggests altered autonomic circuits at the level of the PVN, which can contribute to autonomic dysfunction and dysregulation of neural control of hepatic functions including glucose metabolism.SIGNIFICANCE STATEMENT A growing body of evidence suggests the importance of the autonomic control in the regulation of hepatic metabolism, which plays a major role in the development and progression of type 2 diabetes mellitus. Despite the importance of the brain-liver pathway, the overall activity of liver-related neurons in control and diabetic conditions is not known. This is a significant gap in knowledge, which prevents developing strategies to improve glucose homeostasis via altering the brain-liver pathway. One of the key findings of our study is the overall shift toward excitation in liver-related hypothalamic neurons in the diabetic condition. This overactivity may be one of the underlying mechanisms of elevated sympathetic activity known in metabolically compromised patients and animal models.

High Fructose Diet inducing diabetes rapidly impacts olfactory epithelium and behavior in mice.

Type 2 Diabetes (T2D), a major public health issue reaching worldwide epidemic, has been correlated with lower olfactory abilities in humans. As olfaction represents a major component of feeding behavior, its alteration may have drastic consequences on feeding behaviors that may in turn aggravates T2D. In order to decipher the impact of T2D on the olfactory epithelium, we fed mice with a high fructose diet (HFruD) inducing early diabetic state in 4 to 8 weeks. After only 4 weeks of this diet, mice exhibited a dramatic decrease in olfactory behavioral capacities. Consistently, this decline in olfactory behavior was correlated to decreased electrophysiological responses of olfactory neurons recorded as a population and individually. Our results demonstrate that, in rodents, olfaction is modified by HFruD-induced diabetes. Functional, anatomical and behavioral changes occurred in the olfactory system at a very early stage of the disease.

Anatomical and molecular consequences of Unilateral Naris Closure on two populations of olfactory sensory neurons expressing defined odorant receptors.

Mammalian olfactory sensory neurons (OSNs), the primary elements of the olfactory system, are located in the olfactory epithelium lining the nasal cavity. Exposed to the environment, their lifespan is short. Consequently, OSNs are regularly regenerated and several reports show that activity strongly modulates their development and regeneration: the peripheral olfactory system can adjust to the amount of stimulus through compensatory mechanisms. Unilateral naris occlusion (UNO) was frequently used to investigate this mechanism at the entire epithelium level. However, there is little data regarding the effects of UNO at the cellular level, especially on individual neuronal populations expressing a defined odorant receptor. Here, using UNO during the first three postnatal weeks, we analyzed the anatomical and molecular consequences of sensory deprivation in OSNs populations expressing the MOR23 and M71 receptors. The density of MOR23-expressing neurons is decreased in the closed side while UNO does not affect the density of M71-expressing neurons. Using Real Time qPCR on isolated neurons, we observed that UNO modulates the transcript levels for transduction pathway proteins (odorant receptors, CNGA2, PDE1c). The transcripts modulated by UNO will differ between populations depending on the receptor expressed. These results suggest that sensory deprivation will have different effects on different OSNs' populations. As a consequence, early experience will shape the functional properties of OSNs differently depending on the type of odorant receptor they express.

Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.

Mammalian olfactory sensory neurons (OSNs) form the primary elements of the olfactory system. Inserted in the olfactory mucosa lining of the nasal cavity, they are exposed to the environment and their lifespan is brief. Several reports say that OSNs are regularly regenerated during the entire life and that odorant environment affects the olfactory epithelium. However, little is known about the impact of the odorant environment on OSNs at the cellular level and more precisely in the context of early postnatal olfactory exposure. Here we exposed MOR23-green fluorescent protein (GFP) and M71-GFP mice to lyral or acetophenone, ligands for MOR23 or M71, respectively. Daily postnatal exposure to lyral induces plasticity in the population of OSNs expressing MOR23. Their density decreases after odorant exposure, whereas the amount of MOR23 mRNA and protein remain stable in the whole epithelium. Meanwhile, quantitative PCR indicates that each MOR23 neuron has higher levels of olfactory receptor transcripts and also expresses more CNGA2 and phosphodiesterase 1C, fundamental olfactory transduction pathway proteins. Transcript levels return to baseline after 4 weeks recovery. Patch-clamp recordings reveal that exposed MOR23 neurons respond to lyral with higher sensitivity and broader dynamic range while the responses' kinetics were faster. These effects are specific to the odorant-receptor pair lyral-MOR23: there was no effect of acetophenone on MOR23 neurons and no effect of acetophenone and lyral on the M71 population. Together, our results clearly demonstrate that OSNs undergo specific anatomical, molecular, and functional adaptation when chronically exposed to odorants in the early stage of life.

Functional evidence of multidrug resistance transporters (MDR) in rodent olfactory epithelium.

BACKGROUND: P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated. PRINCIPAL FINDINGS: Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of species-specific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species, MRPs inhibitors induced a marked reduction of the EOG magnitude, while Pgp inhibitors had only a minor or no measurable effect. CONCLUSIONS: The findings suggest that both Pgp and MRP transporters are functional in the olfactory mucosa and in olfactory receptor neurons. Pgp and MRPs may be cellular constituents of olfactory receptor neurons and represent potential mechanisms for modulation of the olfactory response.