Ethology and pharmacology of hypothalamic aggression in the rat

Stimulation of a restricted area of the rat's hypothalamus elicits unprovoked violent attacks of a species-specific and strain-specific nature. Serotonergic drugs affecting 5HT1 receptors, propranolol, the 5HT re-uptake inhibitor fluvoxamine, and the anxiolytic oxazepam, inhibit hypothalamic attack selectively. However, hypothalamic attack is extremely unsensitive for many drugs that do affect attack provoked by natural stimuli. The pharmacology, the form, the impulsive nature, the absence of preliminaries, the insensitivity for contexts and ultimate aims of aggressive behaviour, suggest that a mechanism with the limited function of damaging adversaries of any kind is activated in the hypothalamus. This hypothalamic attack release mechanism (harm) requires specific sensory input for the expression of specific motor components, such as biting and kicking. The back and dorsal part of the opponent's head are the important attack releasing and directing stimuli. Attacks of this nature are part of the "aggressive" repertoire of the rat in natural settings. "Lateral" or "sideways" postures, specific for intermale fighting cannot be induced by hypothalamic stimulation. Drug, lesion, and stimulation studies suggest that attack and "sideways" postures are under the control of different central mechanisms. These results suggest new ways to describe the patterning of aggressive behaviour. There are interesting ethopharmacological similarities between hypothalamic responses and obsessive compulsive disorders (OCD) in man. It is suggested that further study of the ethopharmacology of hypothalamic responses may shed light on the pathophysiology of impulsive behavioural symptoms which in man seem to be beyond the control of appraisal or context.     

Learning abilities, NGF and BDNF brain levels in two lines of TNF-alpha transgenic mice, one characterized by neurological disorders, the other phenotypically normal.

In this study we used two lines of transgenic mice overexpressing tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS), one characterized by reactive gliosis, inflammatory demyelination and neurological deficits (Tg6074) the other showing no neurological or phenotypical alterations (TgK3) to investigate the effect of TNF-alpha on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) levels and learning abilities. The results showed that the amount of NGF in the brain of Tg6074 and TgK3 transgenic mice is low in the hippocampus and in the spinal cord, increases in the hypothalamus of Tg6074 and showed no significant changes in the cortex. BDNF levels were low in the hippocampus and spinal cord of TgK3. BDNF increased in the hypothalamus of TgK3 and Tg6074 while in the cortex, BDNF increased only in Tg6074 mice. Transgenic mice also had memory impairments as revealed by the Morris Water Maze test. These findings indicate that TNF-alpha significantly influences BDNF and NGF synthesis, most probably in a dose-dependent manner. Learning abilities were also differently affected by overexpression of TNF-alpha, but were not associated with inflammatory activity. The possible functional implications of our findings are discussed.     

Differential regulation of neurotrophins and serotonergic function in mice with genetically reduced glucocorticoid receptor expression

The neurotrophin and serotonin (5-HT) hypotheses of depression were studied in a mouse model of reduced glucocorticoid receptor (GR) function (GR(+/-) mice), which recently has been proven as a murine model of predisposition for depressive behaviour under stressful conditions. In this model we studied diurnal changes in neurotrophins and serotonergic function in candidate brain regions mediating depressive behaviour. Morning and evening levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were analyzed in representative brain regions of GR(+/-) and wildtype mice. The diurnal variation of hippocampal BDNF in wildtypes with higher levels in the morning was absent in GR(+/-) mice. Hypothalamus and parietal cortex displayed enhanced BDNF levels in GR(+/-) mice. In the frontal cortex, striatum and hypothalamus NGF increased from morning to evening in both genotypes, with an exaggeration in GR(+/-) mice. The diurnal variation of 5-HT levels and turnover did not differ significantly between genotypes. It was only in the hypothalamus that the evening level of 5-HIAA was lower in GR(+/-) mice than in wildtype mice. In conclusion, the present data indicate a contribution of altered BDNF and NGF protein levels to the predisposition for depressive behaviour in the GR(+/-) mouse model of depression, but argue against an eminent role of the serotonergic system.     

Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry

The expression of nerve growth factor (NGF) receptor mRNA and NGF receptor protein was examined in the adult rat basal forebrain using in situ hybridization and immunohistochemical techniques. NGF receptor mRNA and protein were detected within cells in the medial septum, diagonal band of Broca, and nucleus basalis of Meynert. Controls showed that the hybridization signal was not due to nonspecific binding of the probe to heterologous RNAs or other molecules. As expected, the distribution of NGF receptor mRNA-containing cells correlated nicely with the distribution of NGF receptor immunoreactive cells in each of these areas. These data extend previous work which suggests that neurons in these areas express the NGF receptor mRNA and manufacture functional NGF receptors. NGF receptor immunoreactivity was also detected in the arcuate nucleus of the hypothalamus, in the leptomeninges at the base of the brain and overlying the tectum, and within ependymal regions along the lateral walls of the cerebral ventricles. A few weakly stained neurons in the lateral hypothalamus and ventrolateral striatum were also consistently observed. In contrast, NGF receptor mRNA was not detected within any meningial, ependymal, or hypothalamic tissues using in situ hybridization. A cross-linking/immunoprecipitation assay demonstrated normal, membrane-bound NGF receptors within extracts of dorsal superior colliculus, ventromedial hypothalamic, and overlying meningial tissues, proving that the staining observed in these areas was not a non-specific artifact associated with the immunohistochemistry. The lack of hybridization in these areas may reflect levels of NGF receptor mRNA which are too low to be detected by the in situ hybridization methods being used. Alternatively, the staining may represent innervation of these areas by afferents whose cell bodies are located elsewhere, and whose terminals contain the NGF receptor protein.     

Nerve growth factor released into the bloodstream following intraspecific fighting induces mast cell degranulation in adult male mice

Nerve growth factor (NGF) is stored in the salivary glands of adult male mice. Recent evidence has shown that NGF is active on cell lines of the immune system. Following isolation-induced fighting (20 min session) NGF is released into the bloodstream, causing disruption of cytoplasmic membranes and concomitant degranulation of peritoneal mast cells (MCs). Fluorometric determinations showed an increase in histamine levels in the peritoneal fluid of fighting mice. Administration of NGF antibodies or sialoadenectomy prior to the fighting session blocks MC degranulation. These results, together with other observations, suggest that endogenous NGF plays a physiological role in MC activation.     

Brain derived neurotrophic factor and neurotrophin-4 employ different intracellular pathways to modulate norepinephrine uptake and release in rat hypothalamus

Classical actions of the neurotrophin family are related to cellular survival and differentiation. Moreover, acute effects of neurotrophins have been reported. Although neurotrophins effects on synaptic transmission at central nervous system level have been largely studied, acute effects of neurotrophins on hypothalamic noradrenergic transmission are still poorly understood. Thus, we have studied the effects of the neurotrophin family members nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) on norepinephrine (NE) neuronal uptake and its evoked release, as well as the receptor and the intracellular pathways involved in these processes in rat hypothalamus.Present results indicate that BDNF increased NE uptake and decreased its evoked release through a mechanism that involve Trk B receptor and phospholipase C. Moreover, NT-4, also through the Trk B receptor, decreased NE uptake and its evoked release by activating phosphatidylinositol 3-OH-kinase. These effects were observed in whole hypothalamus as well as in the anterior hypothalamic zone. On the other hand, NGF did not modify noradrenergic transmission.In conclusion, we showed for the first time that BDNF and NT-4 activate two different intracellular signalling pathways through a Trk B receptor dependent mechanism. Furthermore, present findings support the hypothesis that BDNF and NT-4 acutely applied, could be considered as modulators of noradrenergic transmission and thus may regulate hypothalamic physiological as well as pathophysiological responses.     

Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain

Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double-labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.     

Learning abilities, NGF and BDNF brain levels in two lines of TNF-α transgenic mice, one characterized by neurological disorders, the other phenotypically normal

In this study we used two lines of transgenic mice overexpressing tumor necrosis factor alpha (TNF-α) in the central nervous system (CNS), one characterized by reactive gliosis, inflammatory demyelination and neurological deficits (Tg6074) the other showing no neurological or phenotypical alterations (TgK3) to investigate the effect of TNF-α on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) levels and learning abilities. The results showed that the amount of NGF in the brain of Tg6074 and TgK3 transgenic mice is low in the hippocampus and in the spinal cord, increases in the hypothalamus of Tg6074 and showed no significant changes in the cortex. BDNF levels were low in the hippocampus and spinal cord of TgK3. BDNF increased in the hypothalamus of TgK3 and Tg6074 while in the cortex, BDNF increased only in Tg6074 mice. Transgenic mice also had memory impairments as revealed by the Morris Water Maze test. These findings indicate that TNF-α significantly influences BDNF and NGF synthesis, most probably in a dose-dependent manner. Learning abilities were also differently affected by overexpression of TNF-α, but were not associated with inflammatory activity. The possible functional implications of our findings are discussed.     

Tanycytes and a differential fatty acid metabolism in the hypothalamus

Although the brain controls all main metabolic pathways in the whole organism, its lipid metabolism is partially separated from the rest of the body. Circulating lipids and other metabolites are taken up into brain areas like the hypothalamus and are locally metabolized and sensed involving several hypothalamic cell types. In this study we show that saturated and unsaturated fatty acids are differentially processed in the murine hypothalamus. The observed differences involve both lipid distribution and metabolism. Key findings were: (i) hypothalamic astrocytes are targeted by unsaturated, but not saturated lipids in lean mice; (ii) in obese mice labeling of these astrocytes by unsaturated oleic acid cannot be detected unless β‐oxidation or ketogenesis is inhibited; (iii) the hypothalamus of obese animals increases ketone body and neutral lipid synthesis while tanycytes, hypothalamic cells facing the ventricle, increase their lipid droplet content; and (iv) tanycytes show different labeling for saturated or unsaturated lipids. Our data support a metabolic connection between tanycytes and astrocytes likely to impact hypothalamic lipid sensing. GLIA 2017;65:231–249     

Behavioural responses to hypothalamic cooling and heating in the rat

Rats with hypothalamic thermodes had their hypothalamus cooled or warmed for short sessions. In a first series of experiments, rats could bar-press to obtain fanning with cool air. Cooling the hypothalamus did not suppress or inhibit this behaviour although rectal temperature was markedly increased. In a second series of experiments, bar-pressing would warm the water flowing in the thermode. The rats thus self suppressed the cooling of their hypothalamus. This behaviour was absent at 10 degrees C ambient temperature, and increased with increasing ambient temperature up to 35 degrees C. The result of this behaviour was a small hyperthermia in warm and hot environment compared to control when the rats could not self-suppress the cooling of hypothalamus. The results of both experiments suggest that no conscious direct sensation is aroused by hypothalamic cooling. Hypothalamic heating increased the rat's bar-pressing for cool air and decreased the rat's rectal temperature. When pressure on the lever would suppress a warm hypothalamic stimulus rats self-cooled their hypothalamus, especially in warm environments. Such behaviour resulted in an increased somatic hyperthermia due to the warm environment and hypothalamic cooling. These results are compatible with the hypothesis of a direct conscious sensation from a warm hypothalamus.     

Increased thirst and drinking in Huntington's disease and the R6/2 mouse

While Huntington's disease (HD) is a condition that primarily involves the basal ganglia, there is evidence to suggest that the hypothalamus is also affected. Because the osmoreceptors regulating thirst are situated in the circumventricular region of the hypothalamus, we were interested in whether altered thirst is a part of the HD phenotype. We used the LABORAS behavioural monitoring system and water consumption to show that drinking behaviour was abnormal in R6/2 mice. By 10 weeks of age, R6/2 mice spent significantly more time drinking and drank a greater volume than their wild-type (WT) littermates. The numbers of immunoreactive vasopressin neurons in the paraventricular nucleus (PVN) of the hypothalamus in R6/2 mice were significantly decreased from 8 weeks of age, suggesting that the change in drinking behaviour may be the result of hypothalamic dysfunction. We gave a xerostomia (dry mouth) questionnaire to HD patients and control subjects, and also measured their urine osmolality and serum vasopressin. The mean total xerostomia score was significantly higher in HD patients than in controls, indicating greater thirst in HD patients. Urine osmolality was unaffected in HD patients up to clinical stage III, and none of the patients had diabetes. However, serum vasopressin was increased, suggesting a dysregulation in the control of hypothalamic vasopressin release. A dry mouth can affect taste, mastication and swallowing, all of which may contribute to the significant weight loss seen in both HD patients and R6/2 mice, as can dehydration. We suggest that increased thirst may be an important and clinically relevant biomarker for the study of disease progression in HD.     

Prolonged prenatal exposure to low-level ozone affects aggressive behaviour as well as NGF and BDNF levels in the central nervous system of CD-1 mice

The long-term effects on isolation-induced aggressive behaviour and central NGF and BDNF levels of gestational exposures to ozone (O(3)) were evaluated in adult CD-1 mice. Females were exposed to O(3), at the dose of 0.0, 0.3 or 0.6 ppm from 30 days prior the formation of breeding pairs until gestational day 17. Litters were fostered at birth to untreated dams and, at adulthood, male offspring underwent five successive daily encounters (15 min each) with a standard opponent of the same strain, sex, weight and age. The encounters on day 1, 3 and 5 were videotaped and agonistic and non-agonistic behavioural items finely scored. O(3)-exposed mice showed a significant increase in freezing and defensive postures, a decrease in nose-sniffing behaviour and reduced progressively the aggressive behavioural profile displayed on day 1. Reduced NGF levels in the hippocampus and increased BDNF in the striatum were also found upon O(3) exposure.     

Levels of nerve growth factor and neurotrophin-3 are affected differentially by the presence of p75 in sympathetic neurons in vivo

The development and survival of sympathetic neurons is critically dependent on the related neurotrophic factors nerve growth factor (NGF) and neurotrophin-3 (NT3), the actions of which must be executed appropriately despite spatial and temporal overlaps in their activities. The tyrosine receptor kinases, trkA and trkC, are the cognate receptors for NGF and NT3, respectively. The p75 neurotrophin receptor has been implicated in neurotrophin binding and signaling for both NGF and NT3. In this study, the authors used mice that overexpressed NGF (NGF-OE) or NT3 (NT3-OE) in skin and mice that lacked p75 (p75(-/-)) to understand the dynamics of sympathetic neuron response to each neurotrophin and to address the role of p75. NGF and NT3 were measured in sympathetic ganglia and skin (a major target of sympathetic neurons) by using the enzyme-linked immunosorbent assay (ELISA) technique. A three- to four-fold increase in skin NT3 was seen in both NT3-OE and p75(-/-) mice. Moreover, both mouse lines exhibited a three-fold increase in ganglionic NT3. However, the increase in ganglionic NT3 was accompanied by a decrease in ganglionic NGF in p75(-/-) mice but not in NT3-OE mice. This indicated that p75 plays an important role in determining the level of NGF within sympathetic neurons. In NGF-OE mice, the overexpression of NGF was correlated with increased ganglionic NGF and increased ganglionic expression of p75 mRNA. In addition, in NGF-OE mice, ganglionic trkC expression was decreased, as was the amount of NT3 present within sympathetic ganglia. These results indicate that the level of p75 is integral in determining the level of sympathetic NGF and that NGF competes with NT3 by increasing the expression of p75 and decreasing the expression of trkC.     

Altered hypothalamic c-Fos-like immunoreactivity in diet-induced obese mice

High-fat diet can induce obesity. However, it is not known if the neural activity of the hypothalamus is altered under high-fat diet. The aim of the present study is to search for the altered hypothalamic neuronal activity in C57BI/6J mice fed a high-fat diet for 15 weeks. Hypothalamic c-Fos-like immunoreactivity (FLI) and serum leptin were measured after mice were fed a high-fat diet for 15 weeks. Our results demonstrate that increased body weight and serum leptin are accompanied by an elevated neuronal c-Fos-like immunoreactivity in the lateral hypothalamus, the lateral part of the dorsomedial hypothalamic and perifornical nuclei of diet-induced obese mice. Fasting increases FLI neurons in the arcuate hypothalamic nucleus and decreases FLI neurons in the lateral hypothalamic area and dorsomedial hypothalamic nucleus of both diet-induced obese and lean mice. The current data suggest that constantly activated status of these neurons in the hypothalamus may be responsible for differences in body weight and serum leptin between obese and lean mice.     

The effect of increased levels of endogenous nerve growth factor on mouse sympathetic ganglia

The present experiments showed that treatment of mice with a range of doses of testosterone propionate led to a significant increase in weight of the superior cervical ganglia (SCG). In all cases this was accompanied by an elevation of the level of nerve growth factor (NGF) in the submandibular glands (SMG) and, with the exception of animals treated with the lowest dose of testosterone, by an increase in weight of the SMG.Hypertrophy of the SCG was also found in mice treated with 5α-dihydrotestosterone which therefore excludes the possibility of the hypertrophy being due to a general metabolic effect of testosterone propionate.Treatment of mice with isoproterenol and of rats with testosterone propionate resulted in hypertrophy of the SMG but had no effect on the levels of NGF in the glands or on the weights of the SCG. These findings exclude the possibility that the hypertrophy of the SCG in testosterone-treated mice is a consequence of an increase in size of the SMG.It is concluded that the hypertrophy of the SCG in testosterone-treated mice results from the action of increased levels of endogenous NGF. The route of access of NGF responsible for the hypertrophy is discussed and a comparison is made of the effects produced by systemically administered NGF and increased levels of endogenous NGF.     

Repeated restraint and nerve growth factor administration in male and female mice: effect on sympathetic and cardiovascular mediators of the stress response

Chronic stress and increased sympathetic nerve activity have been associated with cardiovascular disorders such as hypertension, myocardial infarction and stroke. The aim of this study was to investigate the role of nerve growth factor (NGF) on the expression of tyrosine hydroxylase (TH), vascular-endothelial growth factor (VEGF) and leptin receptor (OB-R) in brain, adrenal and cardiovascular tissues of adult male and female mice following a chronic stress procedure. It was found that daily restraint for 10 consecutive days alters TH levels in hypothalamic and brainstem areas related to sympathetic activation, in both male and female mice. Chronic stress procedure also modifies heart and aorta VEGF levels in male mice, and adrenal glands TH in female mice. The NGF administration in stressed mice reverted the stress-induced up-regulation of TH levels in male and female mice hypothalamic nuclei and in male locus coeruleus. Administration of NGF in stressed animals also down-regulated OB-R levels in the hypothalamus of both male and female mice and in the female aorta. Our findings indicate that repeated restraint in mice has an effect on TH and VEGF protein content at different brain and peripheral sites involved in the sympathetic and cardio-vascular response to stressful stimuli. NGF administration is able to counteract some of these stress-induced changes. Since NGF is known to be up-regulated during stress, a possible functional significance of our observations is that the circulating NGF released during and following stress may serve to prevent possible deficits and/or damage linked to stress-induced sympathetic and cardiovascular activation.     

Early social enrichment shapes social behavior and nerve growth factor and brain-derived neurotrophic factor levels in the adult mouse brain.

BACKGROUND: Early experiences produce persistent changes in brain and behavioral function. We investigate whether being reared in a communal nest (CN), a form of early social enrichment that characterizes the natural ecological niche of many rodent species including the mouse, has effects on adult social/aggressive behavior and nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) levels in mice. METHODS: The CN consisted of a single nest where three mothers kept their pups together and shared care-giving behavior from birth to weaning (postnatal day 25). RESULTS: Compared to standard laboratory conditions, in CN condition, mouse mothers displayed higher levels of maternal care. At adulthood, CN mice displayed higher propensity to interact socially and achieved more promptly the behavioral profile of either dominant or subordinate male. Furthermore, CN adult mice showed higher NGF levels, which were further affected by social status, and higher BDNF levels in the brain. CONCLUSIONS: Our findings indicate that CN, a highly stimulating early social environment, produces differences in social behavior later in life associated with marked changes of neurotrophin levels in selected brain areas, including hippocampus and hypothalamus.