Forebrain projections to brainstem nuclei involved in the control of mandibular movements in rats

Mandibular movements occur through the triggering of trigeminal motoneurons. Aberrant movements by orofacial muscles are characteristic of orofacial motor disorders, such as nocturnal bruxism (clenching or grinding of the dentition during sleep). Previous studies have suggested that autonomic changes occur during bruxism episodes. Although it is known that emotional responses increase jaw movement, the brain pathways linking forebrain limbic nuclei and the trigeminal motor nucleus remain unclear. Here we show that neurons in the lateral hypothalamic area, in the central nucleus of the amygdala, and in the parasubthalamic nucleus, project to the trigeminal motor nucleus or to reticular regions around the motor nucleus (Regio h) and in the mesencephalic trigeminal nucleus. We observed orexin co-expression in neurons projecting from the lateral hypothalamic area to the trigeminal motor nucleus. In the central nucleus of the amygdala, neurons projecting to the trigeminal motor nucleus are innervated by corticotrophin-releasing factor immunoreactive fibers. We also observed that the mesencephalic trigeminal nucleus receives dense innervation from orexin and corticotrophin-releasing factor immunoreactive fibers. Therefore, forebrain nuclei related to autonomic control and stress responses might influence the activity of trigeminal motor neurons and consequently play a role in the physiopathology of nocturnal bruxism.

Leptin Targets in the Mouse Brain

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin`s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRbI-RES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518-532, 2009. (C) 2009 Wiley-Liss, Inc.

Investigation of the hypothalamic defensive system in the mouse

The hypothalamus plays especially important roles in various endocrine, autonomic, and behavioral responses that guarantee the survival of both the individual and the species. In the rat, a distinct hypothalamic defensive circuit has been defined as critical for integrating predatory threats, raising an important question as to whether this concept could be applied to other prey species. To start addressing this matter, in the present study, we investigated, in another prey species (the mouse), the pattern of hypothalamic Fos immunoreactivity in response to exposure to a predator (a rat, using the Rat Exposure Test). During rat exposure, mice remained concealed in the home chamber for a longer period of time and increased freezing and risk assessment activity. We were able to show that the mouse and the rat present a similar pattern of hypothalamic activation in response to a predator. Of particular note, similar to what has been described for the rat, we observed in the mouse that predator exposure induces a striking activation in the elements of the medial hypothalamic defensive system, namely, the anterior hypothalamic nucleus, the dorsomedial part of the ventromedial hypothalamic nucleus and the dorsal premammillary nucleus. Moreover, as described for the rat, predator-exposed mice also presented increased Fos levels in the autonomic and parvicellular parts of the paraventricular hypothalamic nucleus, lateral preoptic area and subfornical region of the lateral hypothalamic area. In conclusion, the present data give further support to the concept that a specific hypothalamic defensive circuit should be preserved across different prey species. (C) 2008 Elsevier B.V. All rights reserved.

Sensing danger through the olfactory system: The role of the hypothalamic dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment. (C) 2008 Elsevier Ltd. All rights reserved.

Hypo- and hyperthyroidism affect NEI concentration in discrete brain areas of adult male rats

To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic-pituitary-thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and L-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of L-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis. (C) 2011 Elsevier Inc. All rights reserved.

Central mechanisms involved in pilocarpine-induced pressor response

Pilocarpine (cholinergic muscarinic agonist) injected peripherally may act centrally to produce pressor responses; in the present study, using c-fos immunoreactive expression, we investigated the forebrain and brainstem areas activated by pressor doses of intravenous (i.v.) pilocarpine. In addition, the importance of vasopressin secretion and/or sympathetic activation and the effects of lesions in the anteroventral third ventricle (AV3V) region in awake rats were also investigated. In male Holtzman rats, pilocarpine (0.04 to 4 mu mol/kg b.w.) i.v. induced transitory hypotension followed by long lasting hypertension. Sympathetic blockade with prazosin (1 mg/kg b.w.) i.v. or AV3V lesions (1 day) almost abolished the pressor response to i. v. pilocarpine (2 mu mol/kg b.w.), whereas the vasopressin antagonist (10 mu g/kg b.w.) i.v. reduced the response to pilocarpine. Pilocarpine (2 and 4 mu mol/kg b.w.) i.v. increased the number of c-fos immunoreactive cells in the subfornical organ, paraventricular and supraoptic nuclei of the hypothalamus, organ vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and caudal and rostral ventrolateral medulla. These data suggest that i.v. pilocarpine activates specific forebrain and brainstem mechanisms increasing sympathetic activity and vasopressin secretion to induce pressor response. (C) 2011 Elsevier B.V. All rights reserved.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

Hypothalamic sites responding to predator threats - the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.

Distinct subsets of hypothalamic genes are modulated by two different thermogenesis-inducing stimuli

Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-D-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t) SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e. g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EHrats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

Urinary 6-Sulphatoxymelatonin Levels Are Depressed in Chronic Migraine and Several Comorbidities

Objective.- To assess urinary 6-sulphatoxymelatonin levels in a large consecutive series of patients with migraine and several comorbidities (chronic fatigue, fibromyalgia, insomnia, anxiety, and depression) as compared with controls. Background.- Urine analysis is widely used as a measure of melatonin secretion, as it is correlated with the nocturnal profile of plasma melatonin secretion. Melatonin has critical functions in human physiology and substantial evidence points to its importance in the regulation of circadian rhythms, sleep, and headache disorders. Methods.- Urine samples were collected into a single plastic container over a 12-hour period from 8:00 pm to 8:00 am of the next day, and 6-sulphatoxymelatonin was measured by quantitative ELISA. All of the patients were given a detailed questionnaire about headaches and additionally answered the following questionnaires: Chalder fatigue questionnaire, Epworth somnolence questionnaire, State-Trait Anxiety Inventory, and the Beck Depression Inventory. Results.- A total of 220 subjects were evaluated - 73 (33%) had episodic migraine, 73 (33%) had chronic migraine, and 74 (34%) were enrolled as control subjects. There was a strong correlation between the concentration of 6-sulphatoxymelatonin detected and chronic migraine. Regarding the comorbidities, this study objectively demonstrates an inverse relationship between 6-sulphatoxymelatonin levels and depression, anxiety, and fatigue. Conclusions.- To our knowledge, this is the first study to evaluate the relationship between the urinary concentration of melatonin and migraine comorbidities. These results support hypothalamic involvement in migraine pathophysiology.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

Deletion of Tumor Necrosis Factor-alpha Receptor 1 (TNFR1) Protects against Diet-induced Obesity by Means of Increased Thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org