Distribution of neurogranin-like immunoreactivity in the brain and sensory organs of the adult zebrafish.

We studied the expression of neurogranin in the brain and some sensory organs (barbel taste buds, olfactory organs, and retina) of adult zebrafish. Database analysis shows zebrafish has two paralog neurogranin genes (nrgna and nrgnb) that translate into three peptides with a conserved IQ domain, as in mammals. Western blots of zebrafish brain extracts using an anti-neurogranin antiserum revealed three separate bands, confirming the presence of three neurogranin peptides. Immunohistochemistry shows neurogranin-like expression in the brain and sensory organs (taste buds, neuromasts and olfactory epithelium), not being able to discern its three different peptides. In the retina, the most conspicuous positive cells were bipolar neurons. In the brain, immunopositive neurons were observed in all major regions (pallium, subpallium, preoptic area, hypothalamus, diencephalon, mesencephalon and rhombencephalon, including the cerebellum), a more extended distribution than in mammals. Interestingly, dendrites, cell bodies and axon terminals of some neurons were immunopositive, thus zebrafish neurogranins may play presynaptic and postsynaptic roles. Most positive neurons were found in primary sensory centers (viscerosensory column and medial octavolateral nucleus) and integrative centers (pallium, subpallium, optic tectum and cerebellum), which have complex synaptic circuitry. However, we also observed expression in areas not related to sensory or integrative functions, such as in cerebrospinal fluid-contacting cells associated with the hypothalamic recesses, which exhibited high neurogranin-like immunoreactivity. Together, these results reveal important differences with the patterns reported in mammals, suggesting divergent evolution from the common ancestor.

Nkx2.9 Contributes to Mid-Hindbrain Patterning by Regulation of mdDA Neuronal Cell-Fate and Repression of a Hindbrain-Specific Cell-Fate.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2-high versus r2-Pet1Vglut3-high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1Tph2-high and r2-Pet1Vglut3-high neurons likely regulate distinct brain regions and behaviors. Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+/5-HT+ identity; this suggests that some r2-Pet1Vglut3-high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.SIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1+ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1 boutons-those that are VGLUT3+ or VGLUT3+/5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.

Connections of the laterodorsal tegmental nucleus with the habenular-interpeduncular-raphe system.

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb-IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state-setting modulatory projection systems.

Zebrafish and medaka offer insights into the neurobehavioral correlates of vertebrate magnetoreception.

An impediment to a mechanistic understanding of how some species sense the geomagnetic field ("magnetoreception") is the lack of vertebrate genetic models that exhibit well-characterized magnetoreceptive behavior and are amenable to whole-brain analysis. We investigated the genetic model organisms zebrafish and medaka, whose young stages are transparent and optically accessible. In an unfamiliar environment, adult fish orient according to the directional change of a magnetic field even in darkness. To enable experiments also in juveniles, we applied slowly oscillating magnetic fields, aimed at generating conflicting sensory inputs during exploratory behavior. Medaka (but not zebrafish) increase their locomotor activity in this assay. Complementary brain  activity mapping reveals neuronal activation in the lateral hindbrain during magnetic stimulation. These comparative data support magnetoreception in teleosts, provide evidence for a light-independent mechanism, and demonstrate the usefulness of zebrafish and medaka as genetic vertebrate models for studying the biophysical and neuronal mechanisms underlying magnetoreception.

sept8a and sept8b mRNA expression in the developing and adult zebrafish.

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1-4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.

Increased complexity of circRNA expression during species evolution.

Circular RNAs (circRNAs) are broadly identified from precursor mRNA (pre-mRNA) back-splicing across various species. Recent studies have suggested a cell-/tissue- specific manner of circRNA expression. However, the distinct expression pattern of circRNAs among species and its underlying mechanism still remain to be explored. Here, we systematically compared circRNA expression from human and mouse, and found that only a small portion of human circRNAs could be determined in parallel mouse samples. The conserved circRNA expression between human and mouse is correlated with the existence of orientation-opposite complementary sequences in introns that flank back-spliced exons in both species, but not the circRNA sequences themselves. Quantification of RNA pairing capacity of orientation-opposite complementary sequences across circRNA-flanking introns by Complementary Sequence Index (CSI) identifies that among all types of complementary sequences, SINEs, especially Alu elements in human, contribute the most for circRNA formation and that their diverse distribution across species leads to the increased complexity of circRNA expression during species evolution. Together, our integrated and comparative reference catalog of circRNAs in different species reveals a species-specific pattern of circRNA expression and suggests a previously under-appreciated impact of fast-evolved SINEs on the regulation of (circRNA) gene expression.

Early life versus lifelong oral manganese exposure differently impairs skilled forelimb performance in adult rats.

Recent studies of children suggest that exposure to elevated manganese (Mn) levels disrupts aspects of motor, cognitive and behavioral functions that are dependent on dopamine brain systems. Although basal ganglia motor functions are well-known targets of adult occupational Mn exposure, the extent of motor function deficits in adults as a result of early life Mn exposure is unknown. Here we used a rodent model early life versus lifelong oral Mn exposure and the Montoya staircase test to determine whether developmental Mn exposure produces long-lasting deficits in sensorimotor performance in adulthood. Long-Evans male neonate rats (n=11/treatment) were exposed daily to oral Mn at levels of 0, 25, or 50mg Mn/kg/d from postnatal day (PND) 1-21 (early life only), or from PND 1-throughout life. Staircase testing began at age PND 120 and lasted 1month to objectively quantify measures of skilled forelimb use in reaching and pellet grasping/retrieval performance. Behavioral reactivity also was rated on each trial. Results revealed that (1) behavioral reactivity scores were significantly greater in the Mn-exposed groups, compared to controls, during the staircase acclimation/training stage, but not the latter testing stages, (2) early life Mn exposure alone caused long-lasting impairments in fine motor control of reaching skills at the higher, but not lower Mn dose, (3) lifelong Mn exposure from drinking water led to widespread impairment in reaching and grasping/retrieval performance in adult rats, with the lower Mn dose group showing the greatest impairment, and (4) lifelong Mn exposure produced similar (higher Mn group) or more severe (lower Mn group) impairments compared to their early life-only Mn exposed counterparts. Collectively, these results substantiate the emerging clinical evidence in children showing associations between environmental Mn exposure and deficits in fine sensorimotor function. They also show that the objective quantification of skilled motor performance using the staircase test can serve as a sensitive measure of early life insults from environmental agents. Supported by NIEHS R01ES018990.

Osteocalcin- and osteopontin-containing neurons in the rat hind brain.

Immunohistochemistry for osteocalcin (OC) and osteopontin (OPN) was performed to know their distributions in the hind brain of adult rats. OC- and OPN-immunoreactivity (-ir) were detected in neuronal cell bodies, including perikarya and proximal dendrites and the neuropil. In the cranial nerve motor nuclei, numerous OC- and OPN-immunoreactive (-ir) neurons were detected. The neuropil in the cranial motor nuclei mostly showed strong OC- and OPN-staining intensity. The cranial nerve sensory nuclei and other relay and modulating structures in the lower brain stem also contained various numbers of OC- and OPN-ir neurons. The staining intensities in the neuropil were varied among these regions. In the cerebellar cortex, Purkinje cells and granule cells showed OPN-ir but not OC-ir. However, OC- and OPN-ir neurons were abundantly distributed throughout the cerebellar nuclei. The neuropil in the cerebellar nuclei showed moderate OC-ir and strong OPN-ir staining intensities. These findings indicate that the distribution patterns of OC- and OPN-ir neurons were similar in many structures within the hind brain. OC may play a role in modulating neuroprotective function of OPN.

Leptin amplifies the action of thyrotropin-releasing hormone in the solitary nucleus: an in vitro calcium imaging study.

Leptin exerts a powerful permissive influence on neurogenic thermogenesis. During starvation and an absence of leptin, animals cannot produce thermogenic reactions to cold stress. However, thermogenesis is rescued by restoring leptin. We have previously observed a highly cooperative interaction between leptin and thyrotropin-releasing hormone [TRH] to activate hindbrain-generated thermogenic responses (Hermann et al., 2006). In vivo physiological studies (Rogers et al., 2009) suggested that the thermogenic impact of TRH in the hindbrain is amplified by the action of leptin through a leptin receptor-mediated production of phosphoinositol-trisphosphate [PIP3]. In turn, PIP3 can activate a tyrosine kinase whose target is the Src-SH2 regulatory site on the phospholipase C [PLC] complex. The TRH receptor signals through the PLC complex. Our immunohistochemical studies (Barnes et al., 2010) suggest that this transduction interaction between leptin and TRH occurs within neurons of the solitary nucleus [NST], though this interaction had not been verified. The present in vitro live cell calcium imaging study shows that while medial NST neurons are rarely activated by leptin alone, leptin pre-treatment significantly augments NST neurons' responsiveness to TRH. This leptin-mediated priming of NST neurons was uncoupled by pre-treatment with the phosphoinositide 3-kinase [PI3K] inhibitor [wortmannin], the phospholipase C inhibitor [U73122] and the Src-SH2 antagonist [PP2]. TTX did not eliminate the synergistic response of the agonists, thus the sensitization cannot be attributed to pre-synaptic mechanisms. It seems likely that NST neurons are involved in the leptin-mediated increase in BAT temperature by sensitizing the TRH-PLC-IP3-calcium release mechanism.

Essential role of Phox2b-expressing ventrolateral brainstem neurons in the chemosensory control of inspiration and expiration.

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO(2)/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem-spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO(2)-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO(2)-evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO(2) in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO(2)-evoked increases of inspiratory activity.

Co-localization of TRHR1 and LepRb receptors on neurons in the hindbrain of the rat.

We have reported a highly cooperative interaction between leptin and thyrotropin releasing hormone (TRH) in the hindbrain to generate thermogenic responses (Hermann et al., 2006) (Rogers et al., 2009). Identifying the locus in the hindbrain where leptin and TRH act synergistically to increase thermogenesis will be necessary before we can determine the mechanism(s) by which this interaction occurs. Here, we performed heat-induced epitope recovery techniques and in situ hybridization to determine if neurons or afferent fibers in the hindbrain possess both TRH type 1 receptor and long-form leptin receptor [TRHR1; LepRb, respectively]. LepRb receptors were highly expressed in the solitary nucleus [NST], dorsal motor nucleus of the vagus [DMN] and catecholaminergic neurons of the ventrolateral medulla [VLM]. All neurons that contained LepRb also contained TRHR1. Fibers in the NST and the raphe pallidus [RP] and obscurrus [RO] that possess LepRb receptors were phenotypically identified as glutamatergic type 2 fibers (vglut2). Fibers in the NST and RP that possess TRHR1 receptors were phenotypically identified as serotonergic [i.e., immunopositive for the serotonin transporter; SERT]. Co-localization of LepRb and TRHR1 was not observed on individual fibers in the hindbrain but these two fiber types co-mingle in these nuclei. These anatomical arrangements may provide a basis for the synergy between leptin and TRH to increase thermogenesis.

Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro.

We have cultivated highly uniform populations of neural precursor cells, which retain their region-specific identities, from various rat embryonic brain regions. The roles of the proneural basic-helix-loop-helix (bHLH) factors neurogenin2 (Ngn2) and Mash1 in gamma-aminobutyric acid (GABA) neuron differentiation were explored in the region-specific cultures. Consistent with previous in vivo studies, forced expression of Mash1 promoted GABA neuron formation from the precursors derived from the developing forebrains, whereas Ngn2 displayed an inhibitory role in forebrain GABA neuron differentiation. Functional analyses of mutant bHLH proteins indicated that the helix-loop-helix domains of Mash1 and Ngn2, known as the structures for protein-protein interactions, impart the distinct activities. Intriguingly, the regulatory activities of Mash1 and Ngn2 in GABA neuron differentiation from the hindbrain- and spinal cord-derived precursor cells were completely opposite of those observed in the forebrain-derived cultures: increased GABA neuron yield by Ngn2 and decreased yield by Mash1 were shown in the precursors of those posterior brain regions. No clear difference that depended on dorsal-ventral brain regions was observed in the bHLH-mediated activities. Finally, we demonstrated that Otx2, the expression of which is developmentally confined to the regions anterior to the isthmus, is a factor responsible for the anterior-posterior region-dependent opposite effects of the bHLH proteins.

Neurochemical organization of the nucleus paramedianus dorsalis in the human.

We have characterized the neurochemical organization of a small brainstem nucleus in the human brain, the nucleus paramedianus dorsalis (PMD). PMD is located adjacent and medial to the nucleus prepositus hypoglossi (PH) in the dorsal medulla and is distinguished by the pattern of immunoreactivity of cells and fibers to several markers including calcium-binding proteins, a synthetic enzyme for nitric oxide (neuronal nitric oxide synthase, nNOS) and a nonphosphorylated neurofilament protein (antibody SMI-32). In transverse sections, PMD is oval with its long axis aligned with the dorsal border of the brainstem. We identified PMD in eight human brainstems, but found some variability both in its cross-sectional area and in its A-P extent among cases. It includes calretinin immunoreactive large cells with oval or polygonal cell bodies. Cells in PMD are not immunoreactive for either calbindin or parvalbumin, but a few fibers immunoreactive to each protein are found within its central region. Cells in PMD are also immunoreactive to nNOS, and immunoreactivity to a neurofilament protein shows many labeled cells and fibers. No similar region is identified in atlases of the cat, mouse, rat or monkey brain, nor does immunoreactivity to any of the markers that delineate it in the human reveal a comparable region in those species. The territory that PMD occupies is included in PH in other species. Since anatomical and physiological data in animals suggest that PH may have multiple subregions, we suggest that the PMD in human may be a further differentiation of PH and may have functions related to the vestibular control of eye movements.

Differential analysis of membrane proteins in mouse fore- and hindbrain using a label-free approach.

The ability to quantitatively compare protein levels across different regions of the brain to identify disease mechanisms remains a fundamental research challenge. It requires both a robust method to efficiently isolate proteins from small amounts of tissue and a differential technique that provides a sensitive and comprehensive analysis of these proteins. Here, we describe a proteomic approach for the quantitative mapping of membrane proteins between mouse fore- and hindbrain regions. The approach focuses primarily on a recently developed method for the fractionation of membranes and on-membrane protein digestion, but incorporates off-line SCX-fractionation of the peptide mixture and nano-LC-MS/MS analysis using an LTQ-FT-ICR instrument as part of the analytical method. Comparison of mass spectral peak intensities between samples, mapping of peaks to peptides and protein sequences, and statistical analysis were performed using in-house differential analysis software (DAS). In total, 1213 proteins were identified and 967 were quantified; 81% of the identified proteins were known membrane proteins and 38% of the protein sequences were predicted to contain transmembrane helices. Although this paper focuses primarily on characterizing the efficiency of this purification method from a typical sample set, for many of the quantified proteins such as glutamate receptors, GABA receptors, calcium channel subunits, and ATPases, the observed ratios of protein abundance were in good agreement with the known mRNA expression levels and/or intensities of immunostaining in rostral and caudal regions of murine brain. This suggests that the approach would be well-suited for incorporation in more rigorous, larger scale quantitative analysis designed to achieve biological significance.

Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain.

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.

Hindbrain chemical mediators of reflex-induced inhibition of gastric tone produced by esophageal distension and intravenous nicotine.

The purpose of this study was to activate a vagovagal reflex by using esophageal distension and nicotine and test whether hindbrain nitric oxide and norepinephrine are involved in this reflex function. We used double-labeling immunocytochemical methods to determine whether esophageal distension (and nicotine) activates c-Fos expression in nitrergic and noradrenergic neurons in the nucleus tractus solitarii (NTS). We also studied c-Fos expression in the dorsal motor nucleus of the vagus (DMV) neurons projecting to the periphery. Esophageal distension caused 19.7 +/- 2.3% of the noradrenergic NTS neurons located 0.60 mm rostral to the calamus scriptorius (CS) to be activated but had little effect on c-Fos in DMV neurons. Intravenous administration of nicotine caused 19.7 +/- 4.2% of the noradrenergic NTS neurons 0.90 mm rostral to CS to be activated and, as reported previously, had no effect on c-Fos expression in DMV neurons. To determine whether norepinephrine and nitric oxide were central mediators of esophageal distension-induced decrease in intragastric pressure (balloon recording), N(G)-nitro-L-arginine methyl ester microinjected into the NTS (n = 5), but not into the DMV, blocked the vagovagal reflex. Conversely, alpha2-adrenergic blockers microinjected into the DMV (n = 7), but not into the NTS, blocked the vagovagal reflex. These data, in combination with our earlier pharmacological microinjection data with nicotine, indicate that both esophageal distension and nicotine produce nitric oxide in the NTS, which then activates noradrenergic neurons that terminate on and inhibit DMV neurons.

A hematopoietic growth factor, thrombopoietin, has a proapoptotic role in the brain.

Central nervous and hematopoietic systems share developmental features. We report that thrombopoietin (TPO), a stimulator of platelet formation, acts in the brain as a counterpart of erythropoietin (EPO), a hematopoietic growth factor with neuroprotective properties. TPO is most prominent in postnatal brain, whereas EPO is abundant in embryonic brain and decreases postnatally. Upon hypoxia, EPO and its receptor are rapidly reexpressed, whereas neuronal TPO and its receptor are down-regulated. Unexpectedly, TPO is strongly proapoptotic in the brain, causing death of newly generated neurons through the Ras-extracellular signal-regulated kinase 1/2 pathway. This effect is not only inhibited by EPO but also by neurotrophins. We suggest that the proapoptotic function of TPO helps to select for neurons that have acquired target-derived neurotrophic support.

Immunohistochemical distribution of neuropeptide Y in the mesencephalon and rhombencephalon of carp, Cyprinus carpio L. (Cyprinidae: Teleostei).

The localization of neuropeptide Y (NPY)-immunoreactive elements was investigated in the mesencephalon and rhombencephalon of carp, Cyprinus carpio, by using antisera raised against porcine NPY and the immunoperoxidase technique. Concurrently, to identify the distribution of NPY-immunoreactivity, we developed an atlas of the studied areas based on Nissl-stained sections. The NPY-immunoreactive (NPY-ir) elements were located in many zones of the mesencephalon and rhombencephalon. In the mesencephalon, positive fibers were the most abundant elements while neurons were scarce. The rhombencephalon rostral part was characterized by a low to moderate fiber density, distributed in the ventro-medial and ventro-lateral region. Differently the caudal part of the rhombencephalon exhibited several NPY-ir elements. In particular, a high density of immunoreactivity was located in the gustatory area at the level of the nucleus (n.) originis nervi glossopharyngei, in the n. nervi vagi, and in the vagal lobe. The latter can be considered a valid neuroanatomical model for the study of gustatory signal processing in vertebrates. Our results regarding the primary gustatory centers give neuroanatomical support to the view that NPY may act as a neurotransmitter and/or a neuromodulator in a wide neural network for feeding behavior control.