Distribution of nitric oxide in the rock cavy (Kerodon rupestris) brain II: The brainstem.

In a recent paper, we described the distribution of Nitric oxide (NO) in the diencephalon of the rock cavy (Kerodon rupestris). This present paper follows this work, showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For this, we used immunohistochemistry against the neuronal form of nitric oxide synthase (NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the rock cavy, where the NOS neurons were seen to be limited to some nuclei in the thalamus and hypothalamus, the distribution of NOS in the brainstem is widespread. Neurons immunoreactive to NOS (NOS-ir) were seen as rostral as the precommissural nuclei and as caudal as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex, periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus, pedunculopontine tegmental, and other nuclei of the reticular formation are among the locations with the most NOS-ir neurons. This distribution is similar, but with some differences, to those described for other rodents, indicating that NO also has an important role in rock cavy's physiology.

Distribution of nitric oxide synthase in the rock cavy (Kerodon rupestris) brain I: The diencephalon.

Nitric oxide (NO) is a highly soluble and membrane-permeable neurotransmitter, so it does not need to be packed in vesicles or have a membrane receptor. In the nervous system, NO is synthesized by the neuronal form of the nitric oxide synthase (NOS) enzyme and has been considered as a local neurotransmitter. NOS distribution is widespread in the nervous system of various vertebrate species, which may explain its participation in many functions such as memory, blood pressure regulation and sexual behavior. Here we used immunohistochemistry against NOS and NADPH diaphorase histochemistry to map the distribution of NO in the diencephalon of the rock cavy (Kerodon rupestris), a rodent endemic to the Brazilian Northeast. Rock cavy has crepuscular habits and is adapted to ecological conditions such as heat and scarcity of water and food. This study found that NOS distribution was more concentrated in the hypothalamus of this animal. Among the hypothalamic nuclei, the median preoptic, supraoptic, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, ventral and dorsal premammillary nucleus, supramammillary nucleus, lateral mammillary nucleus and dorsal hypothalamic nucleus had the largest collections of NOS immunoreactive (NOS-ir) neurons. Some nuclei of the thalamus and epithalamus such as the paraventricular nucleus of the thalamus, the ventral lateral geniculate nucleus, the medial geniculate nucleus and the lateral habenula showed NOS-ir neurons. This distribution is similar to that described in other rodents, indicating that NO also has an important role in rock cavy's physiology.

Topographic specializations of catecholaminergic cells and ganglion cells and distribution of calcium binding proteins in the crepuscular rock cavy (Kerodon rupestris) retina.

The rock cavy (Kerodon rupestris) is a crepuscular Hystricomorpha rodent that has been used in comparative analysis of retinal targets, but its retinal organization remains to be investigated. In order to better characterize its visual system, the present study analyzed neurochemical features related to the topographic organization of catecholaminergic cells and ganglion cells, as well the distribution of calcium-binding proteins in the outer and inner retina. Retinal sections and/or wholemounts were processed using tyrosine hydroxylase (TH), GABA, calbindin, parvalbumin and calretinin immunohistochemistry or Nissl staining. Two types of TH-immunoreactive (TH-IR) cells were found which differ in soma size, dendritic arborization, intensity of TH immunoreactivity and stratification pattern in the inner plexiform layer. The topographic distribution of all TH-IR cells defines a visual streak along the horizontal meridian in the superior retina. The ganglion cells are also distributed in a visual streak and the visual acuity estimated considering their peak density is 4.13 cycles/degree. A subset of TH-IR cells express GABA or calbindin. Calretinin is abundant in most of retinal layers and coexists with calbindin in horizontal cells. Parvalbumin is less abundant and expressed by presumed amacrine cells in the INL and some ganglion cells in the GCL. The topographic distribution of TH-IR cells and ganglion cells in the rock cavy retina indicate a suitable adaptation for using a broad extension of its inferior visual field in aspects that involve resolution, adjustment to ambient light intensity and movement detection without specialized eye movements.

Distribution of urocortin 3 neurons innervating the ventral premammillary nucleus in the rat brain.

Urocortin 3 (Ucn 3) is a recently described peptide of the corticotropin-releasing factor family. Neurons expressing Ucn 3 mRNA and peptide are distributed in specific brain areas, including the median preoptic nucleus, the perifornical area (PFx), and the medial nucleus of the amygdala (MEA). Fibers immunoreactive to Ucn 3 are confined to certain brain nuclei, being particularly dense in the ventral premammillary nucleus (PMV). In studies involving electrolytic lesions and analysis of Fos distribution according to behavioral paradigms, the PMV has been potentially implicated in conspecific aggression and sexual behavior. However, the role that Ucn 3 plays in this pathway has not been explored. Therefore, we investigated the origins of the urocortinergic innervation of the PMV of Wistar rat in an attempt to map the brain circuitry and identify likely related functions. We injected the retrograde tracer cholera toxin b subunit into the PMV and found that 88% of the Ucn 3-immunoreactive fibers in the PMV originate in the dorsal MEA, and that few originate in the PFx. As a control, we injected the anterograde tracer biotin dextran amine into both regions. We observed that the PMV is densely innervated by the MEA, and scarcely innervated by the PFx. The MEA is a secondary relay of the vomeronasal system and projects amply to hypothalamic nuclei related to hormonal and behavioral adjustments, including the PMV. Although physiological studies should also be performed, we hypothesize that Ucn 3 participates in such pathways, conveying sensory information to the PMV, which in turn modulates behavioral and neuroendocrine responses.

The cytoarchitectonic and TH-immunohistochemical characterization of the dopamine cell groups in the substantia nigra, ventral tegmental area and retrorubral field in a bat (Artibeus planirostris).

The dopamine (DA) neurons of the retrorubral field (RRF - A8), the substantia nigra (SN - A9), and the ventral tegmental area (VTA - A10) have been implicated in motor regulation, reward, aversion, cognition, and several neuropsychiatric disorders. A series of studies have identified subdivisions of these cell groups in rodents, but these cell groups have not been well described in bats. An understanding of the motor system organization in bats would provide a context for comparing motor systems across rodent, primate, and bat phylogenies. The aim of this work was to determine whether typical subdivisions of RRF, SN, and VTA are present in Artibeus planirostris, a common frugivorous bat species found throughout South America. Coronal and sagittal sections of bat brain were subjected to Nissl staining and TH immunohistochemistry. The organizational pattern of the nuclei in A. planirostris showed a conspicuous tail in the SN, which has been not described in bats to date, and also contained a well-defined substantia nigra reticulata (SNR) not previously reported in microbats. This work provides for the first time a morphometric analysis of DA neurons in a microchiropteran species, enabling a comparative investigation of vertebrates. Our analysis revealed an apparent phylogenetic stability in these structures, although the SN tail might represent a functional specialization in this species.

The differential mice response to cat and snake odor.

Studies from the last two decades have pointed to multiple mechanisms of fear. For responding to predators, there is a group of highly interconnected hypothalamic nuclei formed by the anterior hypothalamic nucleus, the ventromedial hypothalamic nucleus and the dorsal premammillary nucleus­the predator-responsive hypothalamic circuit. This circuit expresses Fos in response to predator presence or its odor. Lesion of any component of this system blocks or reduces the expression of fear and consequently defensive behavior when faced with a predator or its cue. However, most of the knowledge about that circuit has been obtained using the rat as a model of prey and the cat as a source of predator cues. In the present study, we exposed mice to strong cat or snake odors, two known mice predators, and then we used the rat exposure test (RET) to study their behavior when confronted with the same predator's odor. Our data point to a differential response of mice exposed to these odors. When Swiss mice were exposed to the cat odor, they show defensive behavior and the predator-responsive hypothalamic circuit expressed Fos. The opposite was seen when they faced snake's odor. The acute odor exposure was not sufficient to activate the mouse predator-responsive hypothalamic circuit and the mice acted like they were not in a stressful situation, showing almost no sign of fear or defensive posture. This leads us to the conclusion that not all the predator cues are sufficient to activate the predator-responsive hypothalamic circuit of mice and that their response depends on the danger that these predators represent in the natural history of the prey.

Distribution of the neuronal inputs to the ventral premammillary nucleus of male and female rats.

The ventral premammillary nucleus (PMV) expresses dense collections of sex steroid receptors and receptors for metabolic cues, including leptin, insulin and ghrelin. The PMV responds to opposite sex odor stimulation and projects to areas involved in reproductive control, including direct innervation of gonadotropin releasing hormone neurons. Thus, the PMV is well positioned to integrate metabolic and reproductive cues, and control downstream targets that mediate reproductive function. In fact, lesions of PMV neurons blunt female reproductive function and maternal aggression. However, although the projections of PMV neurons have been well documented, little is known about the neuronal inputs received by PMV neurons. To fill this gap, we performed a systematic evaluation of the brain sites innervating the PMV neurons of male and female rats using the retrograde tracer subunit B of the cholera toxin (CTb). In general, we observed that males and females show a similar pattern of afferents. We also noticed that the PMV is preferentially innervated by neurons located in the forebrain, with very few projections coming from brainstem nuclei. The majority of inputs originated from the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic nucleus. A moderate to high density of afferents was also observed in the ventral subiculum, the arcuate nucleus and the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus. Our findings strengthen the concept that the PMV is part of the vomeronasal system and integrates the brain circuitry controlling reproductive functions.

Retinal projections into the Zona Incerta of the rock cavy (Kerodon rupestris): a CTb study.

The Zona Incerta is a key neural substrate of higher brain functions. A neural population in the caudal ZI projects into the superior colliculus. This recently has been identified as an important structure for the saccades. Applying CTb, we describe a retinal projection into the caudal ZI and the distribution of its terminal varicosities in the rock cavy, a Brazilian rodent, which has been used as an anatomical model to enhance the comprehension about the phylogeny of the nervous system. Contrary to other investigated rodents, the retinal fibers in the rock cavy lie in the caudal Zona Incerta (ZIc), suggesting a functional specialization in the rock cavy. The high resolution and qualitative analysis of retinal fibers in the present work provide a substrate to interpretation of the visual system, and its phylogenetic pathways among species.

Serotonergic fibers distribution in the midline and intralaminar thalamic nuclei in the rock cavy (Kerodon rupestris).

The thalamic midline/intralaminar complex is part of the higher-order thalamus, which receives little sensory input, and instead forms extensive cortico-thalamo-cortical pathways. The midline thalamic nuclei connect with the medial prefrontal cortex and the medial temporal lobe. On the other hand, the intralaminar nuclei connect with the fronto-parietal cortex. Taking into account this connectivity pattern, it is not surprising that the midline/intralaminar complex has been implicated in a broad variety of cognitive functions, including memory process, attention and orientation, and also reward-based behavior. Serotonin (5-HT) is a neurotransmitter that exerts different post-synaptic roles. Serotonergic neurons are almost entirely restricted to the raphe nuclei and the 5-HT fibers are distributed widely throughout the brain, including the midline/intralaminar complex. The present study comprises a detailed description of the morphologic features and semiquantitative analysis of 5-HT fibers distribution in the midline/intralaminar complex in the rock cavy, a typical rodent of the Northeast region of Brazil, which has been used by our group as an anatomical model to expand the comprehension about phylogeny on the nervous system. The 5-HT fibers in the midline/intralaminar nuclei of the rock cavy were classified into three distinct categories: (1) beaded fibers, which are relatively fine and endowed with large varicosities; (2) fine fibers, with thin axons and small varicosities uniformly distributed in whole axon; and (3) stem axons, showing thick non-varicose axons. Moreover, the density of 5-HT fibers is variable among the analyzed nuclei. On the basis of this diversity of the morphological fibers and the differential profile of optical density among the midline/intralaminar nuclei of the rock cavy, we conclude that the serotonergic system uses a diverse morphologic apparatus to exert a large functional repertory in the midline/intralaminar thalamic nuclei.

Mediodorsal thalamic nucleus receives a direct retinal input in marmoset monkey (Callithrix jacchus): a subunit B cholera toxin study.

The mediodorsal thalamic nucleus is a prominent nucleus in the thalamus, positioned lateral to the midline nuclei and medial to the intralaminar thalamic complex in the dorsal thalamus. Several studies identify the mediodorsal thalamic nucleus as a key structure in learning and memory, as well as in emotional mechanisms and alertness due to reciprocal connections with the limbic system and prefrontal cortex. Fibers from the retina to the mediodorsal thalamic nucleus have recently been described for the first time in a crepuscular rodent, suggesting a possible regulation of the mediodorsal thalamic nucleus by visual activity. The present study shows retinal afferents in the mediodorsal thalamic nucleus of a new world primate, the marmoset (Callithrix jacchus), using B subunit of cholera toxin (CTb) as an anterograde tracer. A small population of labeled retinofugal axonal arborizations is consistently labeled in small domains of the medial and lateral periphery of the caudal half of the mediodorsal nucleus. Retinal projections in the mediodorsal thalamic nucleus are exclusively contralateral and the morphology of the afferent endings was examined. Although the functional significance of this projection remains unknown, this retina-mediodorsal thalamic nucleus pathway may be involved in a wide possibility of functional implications.

Sciatic nerve grafting and inoculation of FGF-2 promotes improvement of motor behavior and fiber regrowth in rats with spinal cord transection.

PURPOSE: Failure of severed adult central nervous system (CNS) axons to regenerate could be attributed with a reduced intrinsic growing capacity. Severe spinal cord injury is frequently associated with a permanent loss of function because the surviving neurons are impaired to regrow their fibers and to reestablish functional contacts. Peripheral nerves are known as good substrate for bridging CNS trauma with neurotrophic factor addition. We evaluated whether fibroblastic growth factor 2 (FGF-2) placed in a gap promoted by complete transection of the spinal cord may increase the ability of sciatic nerve graft to enhance motor recovery and fibers regrow. METHODS: We used a complete spinal cord transection model. Rats received a 4 mm-long gap at low thoracic level and were repaired with saline (control) or fragment of the sciatic nerve (Nerve) or FGF-2 was added to nerve fragment (Nerve+FGF-2) to the grafts immediately after complete transection. The hind limbs performance was evaluated weekly for 8 weeks by using motor behavior score (BBB) and sensorimotor tests-linked to the combined behavior score (CBS), which indicate the degree of the motor improvement and the percentage of functional deficit, respectively. Neuronal plasticity were evaluated at the epicenter of the injury using MAP-2 and GAP-43 expression. RESULTS: Spinal cord treatment with sciatic nerve and sciatic nerve plus FGF-2 allowed recovery of hind limb movements compared to control, manifested by significantly higher behavioral scores. Higher amounts of MAP-2 and GAP-43 immunoreactive fibers were found in the epicenter of the graft when FGF-2 was added. CONCLUSIONS: FGF-2 added to the nerve graft favored the motor recovery and fiber regrowth. Thus, these results encourage us to explore autologous transplantation as a novel and promising cell therapy for treatment of spinal cord lesion.

Comparative distribution of cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus of the capuchin monkey (Cebus apella) and the common marmoset (Callithrix jacchus).

Cocaine- and amphetamine-regulated transcript (CART) is widely distributed in the brain of many species. In the hypothalamus, CART neurotransmission has been implicated in diverse functions including energy balance, stress response, and temperature and endocrine regulation. Although some studies have been performed in primates, very little is known about the distribution of CART neurons in New World monkeys. New World monkeys are good models for systems neuroscience, as some species have evolved several behavioral and anatomical characteristics shared with humans, including diurnal and social habits, intense maternal care, complex manipulative abilities and well-developed frontal cortices. In the present study, we assessed the distribution of CART mRNA and peptide in the hypothalamus of the capuchin monkey (Cebus apella) and the common marmoset (Callithrix jacchus). We found that the distribution of hypothalamic CART neurons in these monkeys is similar to what has been described for rodents and humans, but some relevant differences were noticed. Only in capuchin monkeys CART neurons were observed in the suprachiasmatic and the intercalatus nuclei, whereas only in marmoset CART neurons were observed in the dorsal anterior nucleus. We also found that the only in marmoset displayed CART neurons in the periventricular preoptic nucleus and in an area seemingly comprising the premammillary nucleus. These hypothalamic sites are both well defined in rodents but poorly defined in humans. Our findings indicate that CART expression in hypothalamic neurons is conserved across species but the identified differences suggest that CART is also involved in the control of species-specific related functions.

Male and female odors induce Fos expression in chemically defined neuronal population.

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide urocortin 3, or glutamic acid decarboxylase mRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir in the MEA and PMV comparing fed and fasted animals. In the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 mRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine- and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues.

Retinal afferents to the thalamic mediodorsal nucleus in the rock cavy (Kerodon rupestris).

The MD has reciprocal connections with the ventromedial prefrontal cortex (PFC) and with limbic cortices and appears to participate in learning and memory-related processes. In this study, we report the identification of a hitherto not reported direct retinal projection to the MD of the rock cavy, a typical rodent species of the Northeast region of Brazil. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-imunoreactive fibers and presumptive terminals were seen in the MD. A few labeled retinal fibers/terminals detected in the MD of the rock cavy brain show clear varicosities, suggesting terminal fields. The present work is the first to show a direct retinal projection to the MD of rodents and may contribute for elucidating the anatomical substrate of the functional involvement of this thalamic nucleus in the modulation of the visual recognition, emotional learning and object-reward association memory.

Serotonin and circadian rhythms

All mammal behaviors and functions exhibit synchronization with environmental rhythms. This is accomplished through an internal mechanism that generates and modulates biological rhythms. The circadian timing system, responsible for this process, is formed by connected neural structures. Pathways receive and transmit environmental cues to the central oscillator, the hypothalamic suprachiasmatic nucleus, which mediates physiological and behavioral alterations. The suprachiasmatic nucleus has three major inputs: the retinohypothalamic tract (a direct projection from the retina), the geniculohypothalamic tract (an indirect photic projection originating in the intergeniculate leaflet), and a dense serotonergic plexus from the raphe nuclei. The serotonergic pathway, a source of non-photic cues to the suprachiasmatic nucleus, modulates its activity. The importance of raphe nuclei in circadian rhythms, especially in photic responses, has been demonstrated in many studies. Serotonin is the raphe neurotransmitter that triggers phase shifts, inhibits light-induced phase-shifts, and plays a role in controlling the sleep-wake cycle. All data to date have demonstrated the importance of the raphe, through serotonergic afferents, in adjusting circadian rhythms and must therefore be considered a component of the circadian timing system. The aim of this paper is to review the literature addressing the involvement of serotonin in the modulation of circadian rhythm.(AU)

Serotonin and circadian rhythms

All mammal behaviors and functions exhibit synchronization with environmental rhythms. This is accomplished through an internal mechanism that generates and modulates biological rhythms. The circadian timing system, responsible for this process, is formed by connected neural structures. Pathways receive and transmit environmental cues to the central oscillator, the hypothalamic suprachiasmatic nucleus, which mediates physiological and behavioral alterations. The suprachiasmatic nucleus has three major inputs: the retinohypothalamic tract (a direct projection from the retina), the geniculohypothalamic tract (an indirect photic projection originating in the intergeniculate leaflet), and a dense serotonergic plexus from the raphe nuclei. The serotonergic pathway, a source of non-photic cues to the suprachiasmatic nucleus, modulates its activity. The importance of raphe nuclei in circadian rhythms, especially in photic responses, has been demonstrated in many studies. Serotonin is the raphe neurotransmitter that triggers phase shifts, inhibits light-induced phase-shifts, and plays a role in controlling the sleep-wake cycle. All data to date have demonstrated the importance of the raphe, through serotonergic afferents, in adjusting circadian rhythms and must therefore be considered a component of the circadian timing system. The aim of this paper is to review the literature addressing the involvement of serotonin in the modulation of circadian rhythm