Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.

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.

The Ventral Premammillary Nucleus Links Fasting-Induced Changes in Leptin Levels and Coordinated Luteinizing Hormone Secretion

Physiological conditions of low leptin levels like those observed during negative energy balance are usually characterized by the suppression of luteinizing hormone (LH) secretion and fertility. Leptin administration restores LH levels and reproductive function. Leptin action on LH secretion is thought to be mediated by the brain. However, the neuronal population that mediates this effect is still undefined. The hypothalamic ventral premammillary nucleus (PMV) neurons express a dense concentration of leptin receptors and project to brain areas related to reproductive control. Therefore, we hypothesized that the PMV is well located to mediate leptin action on LH secretion. To test our hypothesis, we performed bilateral excitotoxic lesions of the PMV in adult female rats. PMV-lesioned animals displayed a clear disruption of the estrous cycle, remaining in anestrus for 15-20 d. After apparent recovery of cyclicity, animals perfused in the afternoon of proestrus showed decreased Fos immunoreactivity in the anteroventral periventricular nucleus and in gonadotropin releasing hormone neurons. PMV-lesioned animals also displayed decreased estrogen and LH secretion on proestrus. Lesions caused no changes in mean food intake and body weight up to 7 weeks after surgery. We further tested the ability of leptin to induce LH secretion in PMV-lesioned fasted animals. We found that complete lesions of the PMV precluded leptin stimulation of LH secretion on fasting. Our findings demonstrate that the PMV is a key site linking changing levels of leptin and coordinated control of reproduction.

A comparative study on the effects of the benzodiazepine midazolam and the dopamine agents, apomorphine and sulpiride, on rat behavior in the two-way avoidance test

In recent years. studies in behavioral pharmacology have shown the involvement of dopaminergic mechanisms in avoidance behavior as assessed by the two-way active avoidance test (CAR). Changes in dopaminergic transmission also occur in response to particularly threatening challenges. However, studies on the effects of benzodiazepine (BZD) drugs ill this test are still unclear. Given the interplay of dopamine and other neurotransmitters in the neurobiology of anxiety and schizophrenia the aim of this work was to evaluate the effects of systemic administration of midazolam, the dopaminergic agonist apomorphine, and the D(2) receptor antagonist sulpiride using the CAR, a test that shows good sensitivity to typical neuroleptic drugs. Whereas midazolam did not alter the avoidance response. apomorphine increased and sulpiride reduced them in this test. Escape was not affected by any drug treatments. Heightened avoidance was not associated with the increased motor activity caused by apomorphine. In contrast with the benzodiazepine midazolam, activation of post-synaptic D(2) receptors with apomorphine facilitates, whereas the D(2) receptor antagonism with sulpiride inhibited the acquisition of the avoidance behavior. Together, these results bring additional evidence for a role of D(2) mechanisms in the acquisition of the active avoidance. (C) 2009 Elsevier Inc. All rights reserved.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 mu m(2) to 805.89 mu m(2). Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers. (C) 2008 Elsevier B.V. All rights reserved.

New Perspectives on beta-Adrenergic Mediation of Innate and Learned Fear Responses to Predator Odor

In the present study, we investigated the role of noradrenergic transmission in unconditioned and conditioned responses to predatory threats. First, we examined the effects of systemically injected beta-blockers on unconditioned and contextual conditioned response to cat odor. The centrally acting beta-blocker (propranolol) was able to impair unconditioned responses, as well as the acquisition of the contextual fear to cat odor; however, the peripherally acting (nadolol) was not effective. Next, we examined the neural substrate underlying the noradrenergic modulation of the defensive response to cat odor and focused on the dorsal premammillary nucleus (PMd), because it represents the hypothalamic site most responsive to predatory threats and, at the same time, presents a dense plexus of noradrenergic fibers. We were able to see that propranolol significantly reduced PMd-Fos expression in response to cat odor and that beta-adrenoceptor blockade in the PMd, before cat odor exposure, reduced defensive responses to the cat odor and to the cat odor-related environment. We have also shown that beta-adrenoceptor blockade in the PMd, before the exposure to cat odor-related context, impaired the contextual conditioned responses. Overall, the present results provide convincing evidence suggesting that central noradrenergic mediation is critical for the expression of unconditioned and contextual conditioned antipredatory responses. We have further shown that the PMd appears to be an important locus to mediate these beta-adrenoceptor effects.

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.

Exercise reduces inhibitory neuroactivity and protects myenteric neurons from age-related neurodegeneration

The practice of regular exercise is indicated to prevent some motility disturbances in the gastrointestinal tract, such as constipation, during aging. The motility alterations are intimately linked with its innervations. The goal of this study is to determine whether a program of exercise (running on the treadmill), during 6 months, has effects in the myenteric neurons (NADH- and NADPH-diaphorase stained neurons) in the colon of rats during aging. Male Wister rats 6 months (adult) and 12 months (middle-aged) old were divided into 3 different groups: AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged submitted to physical activity). The aging did not cause a decline significant (p > 0.05) of the number of NADH-diaphorase stained neurons in sedentary rats (AS vs. MS group). In contrast, a decline of 3 1% was observed to NADPH-diaphorase stained neurons. Thus, animals that underwent physical activity (AS vs. MT group) rescued neurons from degeneration caused by aging (total number, density and profile of neurons did not change with age - NADH-diaphorase method). On the other hand, physical activity augmented the decline of NADPH-diaphorase positive neurons (total number, density and profile of neurons decreased). Collectively, the results show that exercise inhibits age-related decline of myenteric neurons however, exercise augments the decline of neurons with inhibitory activity (nitric oxide) in the colon of the rats. (c) 2008 Elsevier B.V. All rights reserved.

Hemiparkinsonian rats rotate toward the side with the weaker dopaminergic neurotransmission

Rats with unilateral lesion of the substantia nigra pars compacta (SNpc) have been used as a model of Parkinson`s disease. Depending on the lesion protocol and on the drug challenge, these rats rotate in opposite directions. The aim of the present study was to propose a model to explain how critical factors determine the direction of these turns. Unilateral lesion of the SNpc was induced with 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Separate analysis showed that neither the type of neurotoxin nor the site of lesion along the nigrostriatal. pathway was able to predict the direction of the turns these rats made after they were challenged with apomorphine. However, the combination of these two factors determined the magnitude of the lesion estimated by tyrosine-hydroxylase immunohistochemistry and HPLC-ED measurement of striatal dopamine. Very small lesions did Dot cause turns, medium-size lesions caused ipsiversive turns, and large lesions caused contraversive turns. Large-size SNpc lesions resulted in an increased binding of [H-3] raclopride to D2 receptors, while medium-size lesions reduced the binding of [H-3]SCH-23390 D1 receptors in the ipsilateral striatum. These results are coherent with the model proposing that after challenged with a dopamine receptor agonist, unilaterally SNpc-lesioned rats rotate toward the side with the weaker activation of dopamine receptors. This activation is weaker on the lesioned side in animals with small SNpc lesions due to the loss of dopamine, but stronger in animals with large lesions due to dopamine receptor supersensitivity. (C) 2008 Elsevier B.V. All rights reserved.

Architectonic subdivisions of the amygdalar complex of a primitive marsupial (Didelphis aurita)

The architecture of the amygdaloid complex of a marsupial, the opossum Didelphis aurita, was analyzed using classical stains like Nissl staining and myelin (Gallyas) staining, and enzyme histochemistry for acetylcholinesterase and NADPH-diaphorase. Most of the subdivisions of the amygdaloid complex described in eutherian mammals were identified in the opossum brain. NADPH-diaphorase revealed reactivity in the neuropil of nearly all amygdaloid subdivisions with different intensities, allowing the identification of the medial and lateral subdivisions of the cortical posterior nucleus and the lateral subdivision of the lateral nucleus. The lateral, central, basolateral and basomedial nuclei exhibited acetylcholinesterase positivity, which provided a useful chemoarchitectural criterion for the identification of the anterior basolateral nucleus. Myelin stain allowed the identification of the medial subdivision of the lateral nucleus, and resulted in intense staining of the medial subdivisions of the central nucleus. The medial, posterior, and cortical nuclei, as well as the amygdalopiriform area did not exhibit positivity for myelin staining. On the basis of cyto- and chemoarchitectural criteria, the present study highlights that the opossum amygdaloid complex shares similarities with that of other species, thus supporting the idea that the organization of the amygdala is part of a basic plan conserved through mammalian evolution. (C) 2008 Elsevier Inc. All rights reserved.

Patterns of fos activation in rat raphe nuclei during feeding behavior

To analyze the differential recruitment of the raphe nuclei during different phases of feeding behavior, rats were subjected to a food restriction schedule (food for 2 h/day, during 15 days). The animals were submitted to different feeding conditions, constituting the experimental groups: search for food (MFS), food ingestion (MFI), satiety (MFSa) and food restriction control (MFC). A baseline condition (BC) group was included as further control. The MFI and MFC groups, which presented greater autonomic and somatic activation, had more FOS-immunoreactive (FOS-IR) neurons. The MFI group presented more labeled cells in the linear (LRN) and dorsal (DRN) nuclei; the MFC group showed more labeling in the median (MRN), pontine (PRN), magnus (NRM) and obscurus (NRO) nuclei; and the MFSa group had more labeled cells in the pallidus (NRP). The BC exhibited the lowest number of reactive cells. The PRN presented the highest percentage of activation in the raphe while the DRN the lowest. Additional experiments revealed few double-labeled (FOS-IR+ 5-HT-IR) cells within the raphe nuclei in the MFI group, suggesting little serotonergic activation in the raphe during food ingestion. These findings suggest a differential recruitment of raphe nuclei during various phases of feeding behavior. Such findings may reflect changes in behavioral state (e.g., food-induced arousal versus sleep) that lead to greater motor activation, and consequently increased FOS expression. While these data are consistent with the idea that the raphe system acts as gain setter for autonomic and somatic activities, the functional complexity of the raphe is not completely understood. (c) 2008 Elsevier B.V. All rights reserved.

Melanin-concentrating hormone projections to areas involved in somatomotor responses

The lateral hypothalamic area (LHA) participates in the integration of sensory information and somatomotor responses associated with hunger and thirst. Although the LHA is neurochemically heterogeneous, a particularly high number of cells express melanin-concentrating hormone (MCH), which has been reported to play a role in energy homeostasis. Treatment with MCH increases food intake, and MCH mRNA is overexpressed in leptin-deficient (ob/ob) mice. Mice lacking both MCH and leptin present reduced body fat, mainly due to increased resting energy expenditure and locomotor activity. Dense MCH innervation of the cerebral motor cortex (MCx) and the pedunculopontine tegmental nucleus (PPT), both related to motor function, has been reported. Therefore, we postulated that a specific group of MCH neurons project to these areas. To investigate our hypothesis, we injected retrograde tracers into the MCx and the PPT of rats, combined with immunohistochemistry. We found that 25% of the LHA neurons projecting to the PPT were immunoreactive for MCH, and that 75% of the LHA neurons projecting to the MCx also contained MCH. Few MCH neurons were found to send collaterals to both areas. We also found that 15% of the incerto-hypothalamic neurons projecting to the PPT expressed MCH immunoreactivity. Those neurons preferentially innervated the rostral PPT. In addition, we observed that the MCH neurons express glutamic acid decarboxylase mRNA, a gamma-aminobutyric acid (GABA) synthesizing enzyme. We postulate that MCH/GABA neurons are involved in the inhibitory modulation of the innervated areas, decreasing motor activity in states of negative energy balance. (C) 2007 Published by Elsevier B.V.

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats (Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 +/- 1 degrees C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia. respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem. (C) 2011 Elsevier B.V. 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.