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 and morphology of calbindin neurons in the Amygdaloid Complex of the marmoset monkey (callithrix jacchus).

The location and distribution of the calcium-binding protein calbindin-D28k (CB) has been considered to be of great value as a neuronal marker for identifying distinct brain regions and discrete neuronal populations. In the amygdaloid complex (AC), the balance of excitatory and inhibitory inputs is controlled by CB immunoreactive interneurons. Alterations of inhibitory mechanisms in the AC may play a role in the emotional symptomatology of neurological diseases like Alzheimer's and psychiatric disorders like posttraumatic stress disorder. The present investigation examined the distribution and morphology of CB-containing neurons, neuropils and fibers in marmoset monkey ACs by using immunohistochemical and morphometrical methods. We recognized four types of CB cells in the AC: type 1 (multipolar), type 2 (spherical or bipolar), type 3 (pyramidal) and type 4 (halo cells), a cell type specific to the marmoset located in the basal and central nuclei. We detected CB cells in all nuclei and areas of the AC, where most of the cells were present in the deep nuclei (lateral, basal, accessory basal and paralaminar). In the superficial nuclei (the nucleus of the lateral olfactory tract, medial nucleus, periamygdaloid cortex and cortical nuclei), the CB cells were abundant in layers 2 and 3. The intercalated nuclei contained small densely packed cells. The CB neuropils were particularly dense in layer 1 of the superficial nuclei, in the deep nuclei and in the amygdalohippocampal area. Large CB immunoreactive neurons in the white matter and fibers with varicosities were found in the myelin tracts that surrounded the AC. These findings are the first step in determining whether some of these cells are specifically disrupted in pathological states.

Cyto- and Myelo-Architecture of the Amygdaloid Complex of the Common Marmoset Monkey (Callithrix jacchus).

The amygdaloid complex (AC) is a heterogeneous aggregate of nuclei located in the rostromedial region of the temporal lobe. In addition to being partly connected among themselves, the AC nuclei are strongly interconnected with the cerebral cortex, striatum, basal forebrain, hypothalamus and brainstem. Animal and human functional studies have established that the AC is a central hub of the neuronal networks supporting emotional responsivity, particularly its negative/aversive components. Dysfunction of AC circuits in humans has been implicated in anxiety, depression, schizophrenia and bipolar disorder. The small New-World marmoset monkey (Callithrix jacchus) has recently become a key model for neuroscience research. However, the nuclear and fiber tract organization of marmoset AC has not been examined in detail. Thus, the extent to which it can be compared to the AC of Old-World (human and macaque) primates is yet unclear. Here, using Nissl and acetylcholinesterase (AChE) histochemical stains as a reference, we analyzed the cytoarchitecture and nuclear parcellation of the marmoset AC. In addition, given the increasing relevance of tractographic localization for high-resolution in vivo imaging studies in non-human primates, we also identified the myelin fiber tracts present within and around the AC as revealed by the Gallyas method. The present study provides a detailed atlas of marmoset AC. Moreover, it reveals that, despite phylogenetic distance and brain size differences, every nucleus and myelinated axon bundle described in human and macaque studies can be confidently recognized in marmosets.

Retinofugal Projections Into Visual Brain Structures in the Bat Artibeus planirostris: A CTb Study.

A well-developed visual system can provide significant sensory information to guide motor behavior, especially in fruit-eating bats, which usually use echolocation to navigate at high speed through cluttered environments during foraging. Relatively few studies have been performed to elucidate the organization of the visual system in bats. The present work provides an extensive morphological description of the retinal projections in the subcortical visual nuclei in the flat-faced fruit-eating bat (Artibeus planirostris) using anterograde transport of the eye-injected cholera toxin B subunit (CTb), followed by morphometrical and stereological analyses. Regarding the cytoarchitecture, the dorsal lateral geniculate nucleus (dLGN) was homogeneous, with no evident lamination. However, the retinal projection contained two layers that had significantly different marking intensities and a massive contralateral input. The superior colliculus (SC) was identified as a laminar structure composed of seven layers, and the retinal input was only observed on the contralateral side, targeting two most superficial layers. The medial pretectal nucleus (MPT), olivary pretectal nucleus (OPT), anterior pretectal nucleus (APT), posterior pretectal nucleus (PPT) and nucleus of the optic tract (NOT) were comprised the pretectal nuclear complex (PNT). Only the APT lacked a retinal input, which was predominantly contralateral in all other nuclei. Our results showed the morphometrical and stereological features of a bat species for the first time.

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.

Retinal, NPY- and 5ht- inputs to the aged suprachiasmatic nucleus in common marmosets (Callithrix jacchus).

The circadian timing system (CTS) anticipates optimal physiological patterns in response to environmental fluctuations, such as light-dark cycle. Since age-related disruption of circadian synchronization is linked to several pathological conditions, we characterized alterations of neurochemical constituents and retinal projections to the major pacemaker of CTS, the suprachiasmatic nucleus (SCN), in adult and aged marmosets. We used intraocular injections of neural tracer Cholera toxin b (CTb) to report age-related reductions in CTb, neuropeptide Y and serotonin immunoreactivities. Considering these projections arise in SCN from nuclei that relay environmental information to entrain the circadian clock, we provide important anatomical correlates to age-associated physiological deficits.

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.

Effect of FGF-2 and sciatic nerve grafting on ChAT expression in dorsal root ganglia neurons of spinal cord transected rats.

Neurotrophic factors and peripheral nerves are known to be good substrates for bridging CNS trauma. The involvement of fibroblast growth factor-2 (FGF-2) activation in the dorsal root ganglion (DRG) was examined following spinal cord injury in the rat. We evaluated whether FGF-2 increases the ability of a sciatic nerve graft to enhance neuronal plasticity, in a gap promoted by complete transection of the spinal cord. The rats were subjected to a 4mm-long gap at low thoracic level and were repaired with saline (Saline or control group, n=10), or fragment of the sciatic nerve (Nerve group, n=10), or fragment of the sciatic nerve to which FGF-2 (Nerve+FGF-2 group, n=10) had been added immediately after lesion. The effects of the FGF-2 and fragment of the sciatic nerve grafts on neuronal plasticity were investigated using choline acetyl transferase (ChAT)-immunoreactivity of neurons in the dorsal root ganglion after 8 weeks. Preservation of the area and diameter of neuronal cell bodies in dorsal root ganglion (DRG) was seen in animals treated with the sciatic nerve, an effect enhanced by the addition of FGF-2. Thus, the addition of exogenous FGF-2 to a sciatic nerve fragment grafted in a gap of the rat spinal cord submitted to complete transection was able to improve neuroprotection in the DRG. The results emphasized that the manipulation of the microenvironment in the wound might amplify the regenerative capacity of peripheral neurons.

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.

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.

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.