Growth Hormone Action in Somatostatin Neurons Regulates Anxiety and Fear Memory.

Dysfunctions in growth hormone (GH) secretion increase the prevalence of anxiety and other neuropsychiatric diseases. GH receptor (GHR) signaling in the amygdala has been associated with fear memory, a key feature of posttraumatic stress disorder. However, it is currently unknown which neuronal population is targeted by GH action to influence the development of neuropsychiatric diseases. Here, we showed that approximately 60% of somatostatin (SST)-expressing neurons in the extended amygdala are directly responsive to GH. GHR ablation in SST-expressing cells (SSTΔGHR mice) caused no alterations in energy or glucose metabolism. Notably, SSTΔGHR male mice exhibited increased anxiety-like behavior in the light-dark box and elevated plus maze tests, whereas SSTΔGHR females showed no changes in anxiety. Using auditory Pavlovian fear conditioning, both male and female SSTΔGHR mice exhibited a significant reduction in fear memory. Conversely, GHR ablation in SST neurons did not affect memory in the novel object recognition test. Gene expression was analyzed in a micro punch comprising the central nucleus of the amygdala (CEA) and basolateral (BLA) complex. GHR ablation in SST neurons caused sex-dependent changes in the expression of factors involved in synaptic plasticity and function. In conclusion, GHR expression in SST neurons is necessary to regulate anxiety in males, but not female mice. GHR ablation in SST neurons also decreases fear memory and affects gene expression in the amygdala, although marked sex differences were observed. Our findings identified for the first time a neurochemically-defined neuronal population responsible for mediating the effects of GH on behavioral aspects associated with neuropsychiatric diseases.SIGNIFICANCE STATEMENT Hormone action in the brain regulates different neurological aspects, affecting the predisposition to neuropsychiatric disorders, like depression, anxiety, and posttraumatic stress disorder. Growth hormone (GH) receptor is widely expressed in the brain, but the exact function of neuronal GH action is not fully understood. Here, we showed that mice lacking the GH receptor in a group of neurons that express the neuropeptide somatostatin exhibit increased anxiety. However, this effect is only observed in male mice. In contrast, the absence of the GH receptor in somatostatin-expressing neurons decreases fear memory, a key feature of posttraumatic stress disorder, in males and females. Thus, our study identified a specific group of neurons in which GH acts to affect the predisposition to neuropsychiatric diseases.

Gap junctions regulate the activity of AgRP neurons and diet-induced obesity in male mice.

Recent studies indicated an important role of connexins, gap junction proteins, in the regulation of metabolism. However, most of these studies focused on the glial expression of connexins, whereas the actions of connexins in neurons are still poorly investigated. Thus, the present study had the objective to investigate the possible involvement of gap junctions, and in particular connexin 43 (CX43), for the central regulation of energy homeostasis. Initially, we demonstrated that hypothalamic CX43 expression was suppressed in fasted mice. Using whole-cell patch-clamp recordings, we showed that pharmacological blockade of gap junctions induced hyperpolarization and decreased the frequency of action potentials in 50-70% of agouti-related protein (AgRP)-expressing neurons, depending on the blocker used (carbenoxolone disodium, TAT-Gap19 or Gap 26). When recordings were performed with a biocytin-filled pipette, this intercellular tracer was detected in surrounding cells. Then, an AgRP-specific CX43 knockout (AgRPΔCX43) mouse was generated. AgRPΔCX43 mice exhibited no differences in body weight, adiposity, food intake, energy expenditure and glucose homeostasis. Metabolic responses to 24 h fasting or during refeeding were also not altered in AgRPΔCX43 mice. However, AgRPΔCX43 male, but not female mice, exhibited a partial protection against high-fat diet-induced obesity, even though no significant changes in energy intake or expenditure were detected. In summary, our findings indicate that gap junctions regulate the activity of AgRP neurons, and AgRP-specific CX43 ablation is sufficient to mildly prevent diet-induced obesity specifically in males.

Effects of the Isolated and Combined Ablation of Growth Hormone and IGF-1 Receptors in Somatostatin Neurons.

Hypophysiotropic somatostatin (SST) neurons in the periventricular hypothalamic area express growth hormone (GH) receptor (GHR) and are frequently considered as the key neuronal population that mediates the negative feedback loop controlling the hypothalamic-GH axis. Additionally, insulin-like growth factor-1 (IGF-1) may also act at the hypothalamic level to control pituitary GH secretion via long-loop negative feedback. However, to the best of our knowledge, no study so far has tested whether GHR or IGF-1 receptor (IGF1R) signaling specifically in SST neurons is required for the homeostatic control of GH secretion. Here we show that GHR ablation in SST neurons did not impact the negative feedback mechanisms that control pulsatile GH secretion or body growth in male and female mice. The sex difference in hepatic gene expression profile was only mildly affected by GHR ablation in SST neurons. Similarly, IGF1R ablation in SST neurons did not affect pulsatile GH secretion, body growth, or hepatic gene expression. In contrast, simultaneous ablation of both GHR and IGF1R in SST-expressing cells increased mean GH levels and pulse amplitude in male and female mice, and partially disrupted the sex differences in hepatic gene expression. Despite the increased GH secretion in double knockout mice, no alterations in body growth and serum or liver IGF-1 levels were observed. In summary, GHR and IGF1R signaling in SST neurons play a redundant role in the control of GH secretion. Furthermore, our results reveal the importance of GH/IGF-1 negative feedback mechanisms on SST neurons for the establishment of sex differences in hepatic gene expression profile.

Growth hormone receptor in dopaminergic neurones regulates stress-induced prolactin release in male mice.

Arcuate nucleus (ARH) dopaminergic neurones regulate several biological functions, including prolactin secretion and metabolism. These cells are responsive to growth hormone (GH), although it is still unknown whether GH action on ARH dopaminergic neurones is required to regulate different physiological aspects. Mice carrying specific deletion of GH receptor (GHR) in tyrosine hydroxylase (TH)- or dopamine transporter (DAT)-expressing cells were produced. We investigated possible changes in energy balance, glucose homeostasis, fertility, pup survival and restraint stress-induced prolactin release. GHR deletion in DAT- or TH-expressing cells did not cause changes in food intake, energy expenditure, ambulatory activity, nutrient oxidation, glucose tolerance, insulin sensitivity and counter-regulatory response to hypoglycaemia in male and female mice. In addition, GHR deletion in dopaminergic cells caused no gross effects on reproduction and pup survival. However, restraint stress-induced prolactin release was significantly impaired in DAT- and TH-specific GHR knockout male mice, as well as in pegvisomant-treated wild-type males, whereas an intact response was observed in females. Patch clamp recordings were performed in ARH DAT neurones and, in contrast to prolactin, GH did not cause acute changes in the electrical activity of DAT neurones. Furthermore, TH phosphorylation at Ser40 in ARH neurones and median eminence axonal terminals was not altered in DAT-specific GHR knockout male mice during restraint stress. In conclusion, GH action in dopaminergic neurones is required for stress-induced prolactin release in male mice, suggesting the existence of sex differences in the capacity of GHR signalling to affect prolactin secretion. The mechanism behind this regulation still needs to be identified.

Tumor Necrosis Factor α and Interleukin-1ß Acutely Inhibit AgRP Neurons in the Arcuate Nucleus of the Hypothalamus.

Obesity-associated low-grade inflammation favors weight gain, whereas systemic infection frequently leads to anorexia. Thus, inflammatory signals can either induce positive or negative energy balance. In this study, we used whole-cell patch-clamp to investigate the acute effects of three important proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-6, and interleukin-1ß (IL-1ß) on the membrane excitability of agouti-related peptide (AgRP)- or proopiomelanocortin (POMC)-producing neurons. We found that both TNF-α and IL-1ß acutely inhibited the activity of 35-42% of AgRP-producing neurons, whereas very few POMC neurons were depolarized by TNF-α. Interleukin-6 induced no acute changes in the activity of AgRP or POMC neurons. Our findings indicate that the effect of TNF-α and IL-1ß, especially on the activity of AgRP-producing neurons, may contribute to inflammation-induced anorexia observed during acute inflammatory conditions.

High-intensity-exercise-induced intestinal damage is protected by fermented milk supplemented with whey protein, probiotic and pomegranate (Punica granatum L.).

Here we evaluated the effect of fermented milk supplemented with whey protein (approximately 80 % protein), probiotic (Bifidobacterium animalis subsp. lactis BB12) and pomegranate juice (Punica granatum L.) on the physical performance, intestinal motility and villi structure, inflammatory markers and intestinal microbiota of rats under high-intensity acute exercise. In all, twenty-four Wistar rats were separated into groups: control (Ctrl), supplemented (Supp), exercised (Exe) and exercised and supplemented (Exe+Supp). Rats in the Supp groups received fermented milk during 6 weeks by oral administration. At the end of the supplementation period, the Exe groups were submitted to high-intensity acute exercise on a treadmill. We found that intense acute exercise caused changes in the intestinal villi interspace, changes in the proportion of Lactobacillus species and an increase in Clostridium species, as well as a decrease in intestinal motility. Supplementation increased intestinal motility, and maintained the intestinal villi interspace and the natural microbiota proportions of the exercised rats. Physical performance was not improved by fermented milk supplementation. We conclude that the fermented milk containing whey protein, B. animalis (BB12) and pomegranate juice can re-establish intestinal microbiota and protect the animals from the undesirable effects of intense acute exercise.