Rat Hindbrain Research Articles

Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.

Recent studies have shown that generation of different kinds of neurones is controlled by combinatorial actions of homeodomain (HD) proteins expressed in the neuronal progenitors. Pax6 is a HD protein that has previously been shown to be involved in the differentiation of the hindbrain somatic (SM) motoneurones and V1 interneurones in the hindbrain and/or spinal cord. To investigate in greater depth the role of Pax6 in generation of the ventral neurones, we first examined the expression patterns of HD protein genes and subtype-specific neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. We found that Islet2 (SM neurone marker) and En1 (V1 interneurone marker) were transiently expressed in a small number of cells, indicating that Pax6 is not directly required for specification of these neurones. We also observed that domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbx1) were shifted and their boundaries became blurred. Thus, Pax6 is required for establishment of the progenitor domains of the ventral neurones. Next, we performed Pax6 overexpression experiments by electroporating rat embryos in whole embryo culture. Pax6 overexpression in the wild type decreased expression of Nkx2.2, but ectopically increased expression of Irx3, Dbx1 and Dbx2. Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescued the development of Islet2-positive and En1-positive neurones. To know reasons for perturbed progenitor domain formation in Pax6 mutant, we examined expression patterns of Shh signalling molecules and states of cell death and cell proliferation. Shh was similarly expressed in the floor plate of the mutant hindbrain, while the expressions of Ptc1, Gli1 and Gli2 were altered only in the progenitor domains for the motoneurones. The position and number of TUNEL-positive cells were unchanged in the Pax6 mutant. Although the proportion of cells that were BrdU-positive slightly increased in the mutant, there was no relationship with specific progenitor domains. Taken together, we conclude that Pax6 regulates specification of the ventral neurone subtypes by establishing the correct progenitor domains.

Neural responses to MSG in rats and monkeys

The issue that has divided opinion over the status of umami as a basic taste quality is its relationship with the taste of sodium salt. That is addressed here, informed by electrophysiological data from the hindbrain of rats and from the forebrain of macaques. In multidimensional spaces, generated from patterns of neural activity evoked in the rat's hindbrain by an array of taste stimuli, MSG does indeed lie near the position of NaCl. Yet when sodium transduction is disrupted by the lingual application of amiloride, the impact on the taste response to MSG is minor, and the pattern of activity it elicits is unaltered. Thus, MSG generates a taste quality that transcends saltiness, one that survives even as saltiness is compromised. This conclusion is in accord with the independent transduction mechanisms ascribed to MSG. In the macaque's taste system, MSG is an effective stimulus with a dynamic range of 0.009 to 0.300 M. Approximately one-third of the taste cells at each synaptic relay respond to MSG at 0.1 M. MSG evoked a neural response profile in primary taste cortex that correlated quite well with that elicited by NaCl, in accord with the pronounced salty component humans report for its taste. At succeeding synaptic relays, however, that relationship deteriorated, becoming increasingly distant and labile. At higher-order gustatory levels, MSG evoked a profile that was no more similar to those of the basic stimuli than they are to each other. This implies that MSG warrants independent status as a basic taste stimulus, serving as the prototype for the umami quality.

Expression patterns of the netrin receptor UNC5H1 among developing motor neurons in the embryonic rat hindbrain.

The axon guidance molecule netrin-1 has been implicated in the midline repulsion of developing cranial motor axons. We have examined expression patterns of the netrin receptors UNC5H1 and DCC in embryonic rat hindbrains, in combination with labelling of developing motor neurons. We found that UNC5H1 expression colocalised with a number of cranial motor neuron subpopulations from embryonic day 11 (E11) to E14, while DCC was expressed by motor neurons at E12.

Local nonpermissive and oriented permissive cues guide vestibular axons to the cerebellum.

Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.

Hindbrain floor plate of the rat: ultrastructural changes occurring during development.

Most of the molecular and experimental studies on the floor plate (FP) have been performed on the FP region extending along the spinal cord. However, little is known about the hindbrain FP. The FP undergoes regional and temporal changes throughout development, but information with respect to the ultrastructural correlate of such changes is missing. The present investigation was focused on the ultrastructural developmental changes occurring in the FP of the rat hindbrain. The FP cells of the hindbrain secrete a material reacting with antibodies against the secretory glycoproteins of the subcommissural organ (AFRU). This antibody was used to perform an ultrastructural immunocytochemical analysis of the rat FP. From E-12 on, there is a progressive increase in the development of the rough endoplasmic reticulum (RER), so that by E-18, it has reached a high degree of hypertrophy. A unique feature of the hindbrain FP cells is the presence of tubular formations and 140-nm vesicles that appear to originate from RER cisternae. The labelling of these two structures with AFRU and Concanavalin A strongly suggests that they are pre-Golgi compartments containing secretory material. Since these structures are present in the basal process and in the apical cell pole of the FP cells, the possibility that they release their content at these sites, is discussed. It is proposed that a secretory mechanism bypassing the Golgi apparatus (constitutive secretion?) operates in the FP cells. The presence of apoptotic cells within the FP of E-20 embryos and newborns suggests that death, and not re-differentiation, is the fate of the FP cells.

Comparative study on the distribution patterns of P2X1–P2X6 receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): Association with catecholamine cell groups

The present study investigated the topographical distribution of P2X1–P2X6 receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double-labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor-like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)-containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X2 subunit), whereas the lowest proportion of double-labeled cells occurred in the C2 region of the NTS for the P2X5 subunit. These findings support a role for extracellular adenosine 5′-triphosphate in fast synaptic neurotransmission within the brainstem. J. Comp. Neurol. 427:485–507, 2000. © 2000 Wiley-Liss, Inc.

Perinatal distribution of galanin and galanin receptor-1 mRNA in the rat hindbrain

In situ hybridisation was used to determine the distribution and levels of mRNA for galanin precursor preprogalanin (ppGAL) and galanin receptor-1 (GAL-R1) in the rat hindbrain before and after birth. Quantification of mRNA levels was performed from E21. Also, immunohistochemistry was used to study GAL-like immunoreactivity (GAL-LI) prenatally. On E16, no expression of ppGAL mRNA could be detected in any areas examined, whereas on E19 low transcript levels were observed. GAL-LI, however, was seen at relatively high levels in nerve fibres already on E16, mainly in the areas receiving primary afferents. Also, GAL-R1 mRNA was expressed at high levels in discrete areas of the hindbrain on E16. On E21 ppGAL mRNA was found in the locus coeruleus (LC), the nucleus of the solitary tract, the dorsal motor nucleus of the vagus (nX), the lateral reticular nucleus (LRn) and superficially along the ventral medullary surface. Expression increased postnatally in all these areas except in nX and LRn. GAL-R1 mRNA, on the other hand, was found to be expressed at high levels on E21 in the LC, where levels then decreased on P1. Expression of GAL-R1 mRNA was also found in other areas of the brainstem, but here no changes were detected around birth. These findings demonstrate that ppGAL and GAL-R1 mRNAs, as well as GAL-LI, are present in the brainstem in the rat fetus and that the changes in expression after birth could be of importance for the newborn in the transition from pre- to postnatal life.

Vagus nerve participates in CCK-induced Fos expression in hindbrain but not myenteric plexus

CCK activates neurons in rat hindbrain and small intestinal myenteric ganglia. Activation of neurons at both sites is mediated through type A CCK receptors. CCK-induced activation of hindbrain neurons is mediated by capsaicin-sensitive vagal fibers. Therefore, it is possible that CCK-induced activation of myenteric neurons also depends upon vagal activation. To test this hypothesis, we examined hindbrain and myenteric neuronal expression of Fos immunoreactivity following CCK injection in rats that had undergone bilateral subdiaphragmatic vagotomy or systemic treatment with capsaicin, a neurotoxin that destroys small unmyelinated primary sensory neurons in the vagus, as well as in other peripheral nerves. We found that CCK (2 or 10 μg/kg) significantly increased Fos expression in both the brains and small intestinal myenteric plexuses of control rats. CCK-induced Fos expression was abolished or attenuated in the brains of vagotomized or capsaicin-treated animals. However, vagotomy or capsaicin treatment did not diminish CCK-induced Fos expression in the small intestinal myenteric plexus. We conclude that CCK-induced activation of intestinal myenteric neurons does not depend on activation of vagal sensory or motor neurons, while activation of neurons in the dorsal hindbrain is mediated, at least in part by CCK-induced activity of small unmyelinated vagal sensory neurons.

Neuroanatomical targets of anxiogenic drugs in the hindbrain as revealed by Fos immunocytochemistry

It is speculated that specific hindbrain transmitter pathways centred on the periaqueductal gray and locus coeruleus are an important integrative neural substrate for the expression of anxiety and the somatic symptoms and cardiovascular changes that accompany severe anxiety states, such as in panic disorder. Here we investigated the effects of various drugs, known to induce panic in humans and to be anxiogenic in animals, on Fos expression in the periaqueductal gray, locus coeruleus and other parts of the rat hindbrain. The drugs tested were the benozodiazepine inverse agonist FG-7142, the α 2-adrenoceptor antagonist yohimbine, the non-selective 5-hydroxytryptamine 2C receptor agonist m-chlorophenyl piperazine, the adenosine antagonist caffeine and the cholecystokinin analogue BOC-CCK 4. A clear-cut finding was that administration of each anxiogenic drug caused a striking region-specific pattern of Fos expression within the hindbrain. In particular, the drugs commonly increased Fos-like immunoreactivity in the periaqueductal gray and locus coeruleus. Increased Fos expression in the periaqueductal gray was specific to the rostral dorsolateral and caudal ventrolateral regions. All the anxiogenic drugs also increased Fos-like immunoreactivity in the lateral parabrachial nucleus and nucleus of the solitary tract and all but one (BOC-CCK 4) increased Fos in the dorsal raphe nucleus. Rats habituated to the test environment and injected with saline vehicle displayed little or no Fos-like immunoreactivity in the hindbrain areas investigated. In summary, each of the anxiogenic drugs tested (FG-7142, yohimbine, m-chlorophenyl piperazine, caffeine and BOC-CCK 4) increased Fos expression in a restricted number of hindbrain regions, including the periaqueductal gray and locus coeruleus. Previous Fos studies have found that these same regions are activated by various fearful environmental stimuli. Therefore, a specific set of hindbrain circuits may be commonly involved in the processing of anxiety-related information evoked by pharmacological and environmental manipulation. The present findings also raise the possibility that measurement of the effect of anxiogenic drugs on Fos expression might be a useful way to model hindbrain pathways activated by anxiety and possibly panic.

Comparative study on the distribution patterns of P2X1-P2X6 receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): Association with catecholamine cell groups

The present study investigated the topographical distribution of P2X(1)-P2X(6) receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double-labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor-like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)-containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X(2) subunit), whereas the lowest proportion of double-labeled cells occurred in the C2 region of the NTS for the P2X(5) subunit. These findings support a role for extracellular adenosine 5'-triphosphate in fast synaptic neurotransmission within the brainstem.

Involvement of adenosine triphosphate-sensitive K+ channels in glucose-sensing in the rat solitary tract nucleus

The presence of adenosine triphosphate-sensitive (ATP-sensitive) K+ channels (KATP channels) in the caudal nucleus tractus solitarii (NTS), and their possible involvement in glucose-sensing, were assessed by extracellular recording of neuronal activity in rat hindbrain slices. In 21 out of 36 recorded cells, firing was increased by sulfonylureas and decreased by K+ channel opener (KCO), indicating the existence of KATP channels in the caudal NTS. In seven out of the nine neurons activated by a 2 mM increase in the glucose level, the effects of sulfonylureas and KCO were consistent with the involvement of KATP channels in the glucose response. Conversely, the mechanism(s) underlying the response of glucose-depressed neurons remains to be clarified. Finally, the presence of KATP channels was also detected in some neurons that were unresponsive to a 2 mM change in the glucose level. Thus, KATP channels were pharmacologically identified in the caudal NTS, where they may be partly involved in glucose sensing.

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Differential regulation of distinct phenotypic features of serotonergic neurons by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are members of the transforming growth factor-beta superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP-2, -6 and -7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5-HT) neuron development, we have generated serum-free cultures of 5-HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP-6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5, 7-dihydroxytryptamine (5,7-DHT) by about two-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short-term treatments. Importantly, BMP-7 selectively induced 5, 7-DHT uptake without affecting TpOH immunoreactivity. BMP-6 and -7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP-6 and -7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial-derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP-mediated effects on serotonergic development, suggesting that the glia-derived factor is probably brain-derived neurotrophic factor (BDNF) or neurotrophin-4. In support of this notion, we detected increased levels of BDNF mRNA in BMP-treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5-HT neuron development.

Neuronal responses to cannabinoid receptor ligands in the solitary tract nucleus

Our previous study showed many neurons in the subpostremal division of the nucleus tractus solitarii to be cannabinoid-sensitive. In order to further investigate this sensitivity, single unit activity was recorded extracellularly in rat hindbrain slices, and the effects of bath application of Δ 9-tetrahydrocannabinol and of two synthetic cannabinoid receptor agonists were analysed and compared to each other. Approximately half the recorded neurons responded to agonists, and most of the neurons exposed to two of the agonists reacted similarly to both. The involvement of cannabinoid CB 1 receptors in neuronal sensitivity to Δ 9-tetrahydrocannabinol is supported by these data and by the effects of N-piperidin-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide, hydrochloride (SR 141716A), a compound which is considered to be a selective antagonist and/or a selective inverse agonist of this receptor type.

Chronic non-peptide neurokinin receptor antagonist treatment alters striatal tachykinin peptide and receptor gene expression in the rat

The neurokinin-1 receptor (NK-1R) and the tachykinin peptide substance P (SP) are found throughout the central nervous system (CNS) and are involved in the regulation of sensory, cardiovascular, and inflammatory function. Selective antagonists for the NK-1R such as CP-122,721 block NK-1R-mediated responses both in vitro and in vivo. This study investigated the effects of long-term daily CP-122,721 treatment on gene expression of SP and the NK-1R in the striatum and hindbrain of the rat. The striatum and hindbrain of rats receiving CP122,721 (5, 30, or 150 mg/kg) once-daily for 30 days were assayed for SP- and NK-1R-encoding mRNAs using solution hybridization–nuclease protection assays. Results show that treatment with CP-122,721 significantly increased SP-encoding mRNA and NK-1R mRNA levels in the striatum, but not in the hindbrain. The ability of CP-122,721 to alter SP and NK-1R gene expression may provide a use for non-peptide neurokinin receptor antagonists in the modulation of systems regulated by NK-1R function.

Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function

It has been shown in cats that gastric motor control by the dorsal vagal complex and nucleus ambiguus is under a tonic GABAergic influence. Since much more work has been performed in rats to define vago-vagal reflexes controlling gastrointestinal function, an understanding of the potential inhibition by candidate neurotransmitters such as GABA (gamma aminobutyric acid) in the rat dorsal vagal complex (DVC) is essential to assess. Multiple-barrelled micropipettes were used to apply to the dorsal vagal complex the GABAA antagonist, bicuculline methiodide (0.1-1 nmol), and a GABAA agonist, muscimol (10 nmol) prior to micro-injection of the GABAA antagonist. Micro-injections of bicuculline (353 pmol and 1 nmol), which were localized primarily in the dorsal motor nucleus of the vagus, produced significant increases in intragastric pressure and pyloric motility. These responses were abolished by vagotomy and by a prior micro-injection of muscimol. To determine whether GABAergic blockade in the dorsal vagal complex results in gastric motor excitation through excitatory amino acid receptors, kynurenic acid (5 nmol), a kainate/NMDA (N-methyl D-aspartic acid) receptor antagonist, was micro-injected prior to bicuculline. This abolished the increase in gastric motor function normally evoked by bicuculline. In the other two important hindbrain nuclei controlling gastric function, the nucleus raphe obscurus and nucleus ambiguus, bicuculline (353 pmol) significantly increased intragastric pressure via vagally mediated pathways. These data demonstrate that all three rat hindbrain nuclei known to influence gastric function via the vagus nerve are under tonic GABAergic control. In addition, in the dorsal vagal complex, relief from GABAergic inhibition results in increases in gastric motor function through kainate/NMDA receptor-mediated excitation.

Principal involvement of cyclooxygenase-1-derived prostaglandins in the c- fos expression of the rat hind brain following visceral stimulation with acetic acid

We investigated the relationship between c- fos expression in the hind brain and peritoneal prostaglandin (PG) synthesis after visceronociceptive stimulation with acetic acid in rats. Time-course studies on the mRNA expression for c- fos in the hind brain and cyclooxygenase (COX) isoforms in the peritoneal cells, as well as on the peritoneal 6-keto-PGF 1 α accumulation, after stimulation indicated that COX-1 but not COX-2 was responsible for the peritoneal synthesis of PGs which were suggested to evoke c- fos expression in the hind brain. Pharmacological experiments using mofezolac, a preferential inhibitor against COX-1, and NS-398, a selective inhibitor against COX-2, confirmed the involvement of COX-1 derived PGs in the induction of c- fos expression in the hind brain following the noxious stimulation.

Amino Acid Levelsin the Brain of Rats with Peripheral Nerve Constriction or Section

BACKGROUND: Both excitatory and inhibitory amino acids modulate central neuronal activity following peripheral nerve tissue injury.OBJECTIVE: To establish whether mononeuropathy induced by chronic constriction injury or by sectioning of the sciatic nerve affects neurotransmitter amino acid levels in the rat brain areas.METHODS: Glutamine, glutamic acid, gamma‐aminobutyric acid (GABA) and aspartic acid in the hindbrain, midbrain and hypothalamus of rats were measured by high performance liquid chromatography 14 days after surgery.RESULTS: After monolateral nerve ligation an increase in glutamine and a decrease in GABA levels in all brain areas were observed, with no side preference. The section of the sciatic nerve did not cause any modification in amino acid levels.CONCLUSIONS: The results allow one to differentiate more clearly classic models of mononeuropathy from a biochemical point of view. Moreover, results indicate that continuous neuronal stimulation induced by nerve constriction is able to modify some amino acid pathways at a supraspinal level.

The floor plate of the hindbrain is a highly specialized gland. Immunocytochemical and ultrastructural characteristics

Although the participation of the floor plate (FP) in the differentiation of neurons in the ventral spinal cord is well established, the function of the FP at the level of the hindbrain is not known. The present investigation was focused on the FP of the rat hindbrain. We used an antibody specifically labeling the rat FP (FP4) and an antiserum raised against the secretory glycoproteins of the subcommissural organ that also reacts with an intracellular material in the FP. Immunocytochemical and ultrastructural evidence was obtained indicating that the rostralmost end of the FP, at the metencephalon, is a highly specialized gland which differentiates early and undergoes regression before birth. The material(s) secreted by these FP cells appear to be transported downward into their basal processes, and upward into ventricular protrusions. It is proposed that the former would participate in the differentiation of the serotonergic neurons, and the latter could be released into the ventricle and reach distant targets. Furthermore, evidence is presented supporting the occurrence of regional and temporal specializations of the FP.

Localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the hindbrain and spinal cord of the rat.

The calcium/calmodulin-dependent protein phosphatase calcineurin was localized at the light microscopic level in the rat hindbrain and spinal cord by using an antibody against the alpha-isoform of the catalytic subunit. Calcineurin was highly concentrated in axons, dendrites, and cell bodies of a subpopulation of alpha-motoneurons in hindbrain motor nuclei and the lateral motor column along the length of the spinal cord. These calcineurin-positive alpha-motoneurons appeared to be randomly distributed and represented approximately 25% of the total alpha-motoneuron pool in the motor trigeminal nucleus and the spinal cord lateral motor column. Within the facial nucleus, calcineurin-containing motoneurons were present in the medial and dorsal subdivision but not in the lateral and intermediate subdivision. In addition to the enrichment in motoneurons, calcineurin was enriched in cells of the superficial laminae of the spinal cord dorsal horn and its extension into the medulla, the caudal spinal trigeminal nucleus. Axonal staining in the white matter of the spinal cord was generally weak, except in the dorsolateral funiculus, where strongly calcineurin-positive axons formed a putative ascending tract that appeared to terminate uncrossed in the caudal lateral reticular nucleus of the medulla. This tract may originate from calcineurin-positive cells in the dorsolateral funiculus. We also compared the distribution of calcineurin with calcium/calmodulin-dependent kinase II in the spinal cord and found that the kinase is more widely expressed. Thus, calcineurin is highly restricted to a few locations in the hindbrain and spinal cord. Selective staining in facial subnuclei that innervate phasically active muscles suggests that calcineurin-positive motoneurons represent a subset of alpha-motoneurons innervating a metabolic subtype of muscle fibers, possibly fast-twitch fibers.