Innervation of histamine neurons in the caudal part of the arcuate nucleus of hypothalamus and their activation in response to food deprivation under scheduled feeding.

It has been well established that histaminergic neurons innervate densely the anterior hypothalamus and regulate several functions through the histamine H1 receptor (H1R). However, the physiological function of the histaminergic neurons in other regions including the posterior hypothalamus has not been fully investigated. Recently, we have found a selective c-Fos expression in the caudal part of the arcuate nucleus of the hypothalamus (cARC) by food deprivation under scheduled feeding in rats. In this study, we histochemically examined the correlation of this c-Fos expression with the activation of histaminergic neurons in this region using an anti-H1R antibody. Strong H1R immunoreactivity was observed in the perikarya of the c-Fos positive cells. Abundant histamine-containing fibers were also found in the cARC and in the area between the cARC and the tuberomammillary nucleus (TM), where the histaminergic neuronal cell bodies are exclusively distributed. Our morphological observations suggest that c-Fos expression in the cARC by food deprivation under scheduled feeding is caused by the activation of histaminergic neurons projected from the TM.

Activation of central 5HT2A receptors reduces the craniofacial nociception of rats.

We assessed the contribution of central 5HT2A receptors to the craniofacial tissue nociception in naïve male rats. First, we tested whether activation of central 5HT2A receptors affected nociceptive neural activities recorded from superficial laminae of the trigeminal subnucleus caudalis (Vc)/upper cervical spinal cord junction (Vc/C2) region. Two types of units, such as deep-nociceptive or skin-wide dynamic range (WDR) units were identified from extracellular recordings. Topical administration of 5HT2A receptor agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) onto the Vc/C2 region significantly reduced deep-nociceptive unit discharges evoked by formalin injection into the masseter muscle. Noxious pinch stimulation to the facial skin-evoked skin-WDR unit discharges was significantly reduced by topical administration of 0.1 mg/rat DOI onto the Vc/C2 region. Second, we tested whether i.c.v. administration of DOI affected Fos-like immunoreactivity (-LI) evoked by formalin injection into the masseter muscle. Fos-LI was significantly induced mainly at the ventrolateral (vl) area of trigeminal subnucleus interpolaris (Vi)/Vc junction (vl-Vi/Vc) region and Vc/C2 region in vehicle-treated rats. Formalin-evoked Fos-LI was significantly reduced in laminae I-II of the Vc/C2, but not vl-Vi/Vc region after i.c.v. administration of DOI. Finally, orofacial nocifensive behavioral activities evoked by formalin injection into the masseter muscle were significantly reduced by intracisternal administration of DOI. These results suggest that 5HT2A receptors in the Vc/C2 region mediate antinociceptive effects in the craniofacial nociception.

Expression of mKirre in the developing sensory pathways: its close apposition to nephrin-expressing cells.

mKirre is a novel member of the immunoglobulin superfamily, which is abundant in the developing and adult brain. In the present study, we showed mKirre gene expression in mouse sensory organs during development using in situ hybridization and immunohistochemistry. At embryonic day (E) 11.5, E15.5, and E17.5, we first detected signals for mKirre mRNA in the developing cochleae, retinae, and olfactory neuroepithelia, respectively. After birth, strong signals were observed in these sensory organs. In addition, at this stage, we found its expression in trigeminal ganglion neurons and neuronal populations forming sensory pathways in the olfactory bulb, midbrain, and pons. Furthermore, double-immunofluorescence staining revealed that nephrin-immunoreactivity was overlapping to mKirre-expressing cells in the developing sensory organs. These results suggest that mKirre may be involved in the establishment of the pathway from sensory organs to the brain not only in a homophilic manner but also with its heterophilic interaction to nephrin.

Complete overlap of interleukin-31 receptor A and oncostatin M receptor beta in the adult dorsal root ganglia with distinct developmental expression patterns.

Interleukin-31 receptor A (IL-31RA) is a newly identified type I cytokine receptor, that is related to gp130, the common receptor of the interleukin (IL) -6 family cytokines. Recent studies have shown that IL-31RA forms a functional receptor complex for IL-31 together with the beta subunit of oncostatin M receptor (OSMRbeta). However, little is known about the target cells of IL-31 because it remains unclear which types of cells express IL-31RA. In our previous reports, we demonstrated that OSMRbeta is expressed in a subset of small-sized nociceptive neurons of adult dorsal root ganglia (DRGs). In the present study, we investigated the IL-31RA expression in the adult and developing DRGs. From a northern blot analysis and in situ hybridization histochemistry, IL-31RA mRNA was found to be expressed in the adult DRGs. According to reverse-transcriptase polymerase chain reaction, IL-31RA mRNA was detected in the DRGs and trigeminal ganglia, while no expression of IL-31RA mRNA was observed in the CNS. Double immunofluorescence staining revealed IL-31RA to be expressed in a subset of small-sized neurons, all of which colocalized with OSMRbeta. In addition, the expression of IL-31 RA was detected in afferent fibers in the spinal cord and the dermis of the skin. We also found that the developmental expression pattern of IL-31RA was different from that of OSMRbeta; IL31RA-positive neurons in DRGs first appeared at postnatal day (PN) 10 and reached the adult level at PN14, whereas OSMRbeta-positive neurons were observed at PN0 for the first time. We previously demonstrated OSMRbeta-expressing neurons to decrease, however, they were not found to disappear in oncostatin M (OSM) -deficient mice. These findings suggest that IL-31 and OSM may thus have redundant functions in the development of OSMRbeta-expressing neurons.

Induction of brain-derived neurotrophic factor by leptin in the ventromedial hypothalamus.

Leptin, an adipocyte-derived hormone, reduces food intake by regulating orexigenic and anorexigenic factors in the hypothalamus. Although brain-derived neurotrophic factor is an important anorexigenic factor in the hypothalamus, little is known about the regulation of brain-derived neurotrophic factor expression by leptin in the hypothalamus. In the present study, we examined the effect of leptin on the expression of brain-derived neurotrophic factor in the hypothalamus. I.V. administration of leptin (10 microg/g) led to the increase in the expression of brain-derived neurotrophic factor mRNA, which was observed in the dorsomedial part of the ventromedial hypothalamic nucleus. The increased expression of brain-derived neurotrophic factor mRNA was detected in phosphorylated signal transducer and activator of transcription 3-positive neurons, suggesting that leptin induced brain-derived neurotrophic factor expression in neurons of the dorsomedial part of the ventromedial hypothalamic nucleus. In addition, the expression of brain-derived neurotrophic factor was increased at the protein level in the ventromedial hypothalamic nucleus of leptin-injected mice. Interestingly, brain-derived neurotrophic factor-positive fibers also increased in the ventromedial hypothalamic nucleus and dorsomedial hypothalamic nucleus of leptin-injected mice, which were in close apposition to tyrosine kinase receptor B-immunoreactive neurons and colocalized with synaptophysin, a marker of presynaptic terminals. These results suggest that leptin induces brain-derived neurotrophic factor expression in the dorsomedial part of the ventromedial hypothalamic nucleus and brain-derived neurotrophic factor may exert as anorexigenic factors possibly through the activation of tyrosine kinase receptor B in the ventromedial hypothalamic nucleus and dorsomedial hypothalamic nucleus.

Contribution of peripheral 5-HT2A or 5-HT3 receptors to Fos expression in the trigeminal spinal nucleus produced by acute injury to the masseter muscle during persistent temporomandibular joint inflammation in rats.

We investigated the contribution of peripheral 5-HT2A or 5-HT3 receptors to Fos expression in the trigeminal spinal nucleus (VSP) following acute masseter muscle injury in male rats with or without temporomandibular joint (TMJ) inflammation persisting for 7 days. TMJ inflammation was evoked by an injection of complete Freund's adjuvant (CFA). Two hours after formalin injection into the masseter muscle produced Fos-like immunoreactivity (Fos-LI) in several regions of the VSP and upper cervical spinal cord (C2), such as ventrolateral (vl) area of the trigeminal subnucleus caudalis (Vc)/subnucleus interpolaris (Vi) transition (vl-Vi/Vc), paratrigeminal nucleus (dPa5), middle portion of the Vc (mid-Vc) and Vc/C2 transition (Vc/C2) regions in both groups. Significant increases in the number of Fos-LI were observed in these areas in CFA group compared with non-CFA group. TMJ inflammation alone did not induce a significant level of Fos-LI in the VSP. In order to assess the effect of antagonizing 5-HT2A or 5-HT3 receptors on formalin-induced Fos-LI, rats were pre-treated with local (masseter muscle) administration of ketanserin or tropisetron (0.01, 0.1 mg/rat) 20 min prior to formalin injection. In CFA group, these antagonists given locally reduced the Fos-LI response in the laminae I-II at the mid-Vc and Vc/C2 regions. These antagonists reduced the Fos-LI response in the dPa5, but not in the vl-Vi/Vc region. The Fos-LI response was not affected by i.v. administration of ketanserin (0.01, 0.1 mg/rat) or tropisetron (0.01 mg/rat). In non-CFA group, these antagonists given locally did not reduce the Fos-LI response. These results suggest that peripheral 5-HT2A and 5-HT3 receptors contribute to nociceptive processing in the masseter muscle in TMJ inflammatory conditions.

TRPV2, a capsaicin receptor homologue, is expressed predominantly in the neurotrophin-3-dependent subpopulation of primary sensory neurons.

TRPV2, a member of transient receptor potential ion channels, responds to high-threshold noxious heat, but neither to capsaicin nor to proton. Although TRPV2 is expressed in medium- to large-sized dorsal root ganglion (DRG) neurons with myelinated fibers in adult rodents, little is known about the neurotrophin dependence of TRPV2-positive neurons in the developing and adult DRGs of mice. In the present study, using immunohistochemistry, we found that TRPV2 was first expressed in DRG neurons at embryonic day (E) 11.5, when neither TRPV1 nor TRPM8 was detected yet. Double-immunofluorescence staining revealed that tyrosine kinase receptor C (TrkC) was expressed in most of TRPV2-positive DRG neurons at E11.5 and E13.5. In addition, the percentage of TRPV2-positive neurons in the total DRG neurons at E13.5 reached the same as that of adulthood. In adult DRGs, TrkC and Ret were expressed in 68% and 25% of TRPV2-positive neurons, respectively. These results suggest that TRPV2 is expressed predominantly in the NT-3-dependent subpopulation of DRG neurons throughout development and in adult mice.

Up-regulated phosphorylation of signal transducer and activator of transcription 3 and cyclic AMP-responsive element binding protein by peripheral inflammation in primary afferent neurons possibly through oncostatin M receptor.

Oncostatin M (OSM), a member of interleukin-6 family cytokines, contributes to the development of nociceptive sensory neurons. However, little is known about the role of OSM in dorsal root ganglia (DRGs) of adult mice after peripheral inflammation. In the present study, we showed that OSM mRNA was highly expressed in the inflamed skin during acute inflammation induced by complete Freund's adjuvant (CFA), while the expression of oncostatin M receptor (OSMR) did not change in the ipsilateral DRG. Although peripheral inflammation induced significant increases in the number of neurons with phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated p38 mitogen-activated protein kinase (p-p38) in ipsilateral DRGs, OSMR-positive neurons exhibited neither p-ERK nor p-p38. In addition, we found significant increases in the number of neurons with phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and phosphorylated cAMP-responsive element binding protein (p-CREB) in the ipsilateral DRGs. Interestingly, OSMR-positive neurons with p-STAT3 and p-CREB were significantly increased after peripheral inflammation. Thus, our results suggest that acute inflammation induce the phosphorylations of several signal molecules, including ERK, p38, cAMP-responsive element binding protein, and STAT3. Among them, the up-regulation of p-STAT3 and p-CREB may be induced possibly through OSMR.

The role of peripheral 5HT2A and 5HT1A receptors on the orofacial formalin test in rats with persistent temporomandibular joint inflammation.

The role of peripheral serotonin (5HT) 2A and 5HT1A receptors on the orofacial nocifensive behavioral activities evoked by the injection of formalin into the masseter muscle was evaluated in the rats with persistent temporomandibular joint (TMJ) inflammation evoked by Complete Freund's Adjuvant (CFA). The orofacial nocifensive behavioral activities evoked by the injection of formalin into masseter muscle were significantly enhanced at 1 day (CFA day 1 group) or 7 days (CFA day 7 group) during TMJ inflammation. Pretreatment with local administration of 5HT2A receptor antagonist, ketanserin (0.01, 0.1 mg/rat) into the masseter muscle or systemic administration of ketanserin via i.p. injection (1 mg/kg) reduced the orofacial nocifensive behavioral activities of the late phase evoked by formalin injection into masseter muscle on the side of TMJ inflammation (CFA day 7 group). However, local (0.001-0.1 mg/rat) or systemic (1 mg/kg) administration of 5HT1A receptor antagonist, propranolol, into masseter muscle did not produce the antinociceptive effect in CFA day 7 group. Moreover, local administration of ketanserin (0.1 mg) or propranolol (0.1 mg) into masseter muscle did not inhibit nocifensive orofacial behavior in rats without TMJ inflammation. These data suggest that persistent TMJ inflammation causes the elevation of the orofacial nocifensive behavior, and peripheral 5HT2A receptors play an important role in mediating the deep craniofacial tissue nociception in rats with TMJ inflammation.

Central serotonin 3 receptors play an important role in the modulation of nociceptive neural activity of trigeminal subnucleus caudalis and nocifensive orofacial behavior in rats with persistent temporomandibular joint inflammation.

The role of central serotonin 3 receptors on neural activities recorded from superficial laminae of trigeminal subnucleus caudalis/upper cervical spinal cord junction region was investigated using rats with (Complete Freund's Adjuvant day 7 group) or without (non-Complete Freund's Adjuvant group) persistent temporomandibular joint inflammation evoked by Complete Freund's Adjuvant for 7 days. We identified two types of units, Deep-wide dynamic range units and Skin-wide dynamic range units from extracellular recordings. Deep-wide dynamic range units have mechanoreceptive fields in the deep craniofacial tissues including masseter muscle but do not have cutaneous mechanoreceptive fields. Deep-wide dynamic range unit discharges evoked by the formalin injection into masseter muscle were significantly enhanced in the late phase in Complete Freund's Adjuvant day 7 group. Discharges of Skin-wide dynamic range units evoked by the noxious pinch stimulation to facial skin in Complete Freund's Adjuvant day 7 group were significantly enhanced compared with those in non-Complete Freund's Adjuvant group. Topical administration of central serotonin 3 receptor antagonist, tropisetron, onto trigeminal subnucleus caudalis/upper cervical spinal cord junction region significantly reduced both formalin-evoked Deep-wide dynamic range unit and pinch-evoked Skin-wide dynamic range unit discharges in non-Complete Freund's Adjuvant and Complete Freund's Adjuvant day 7 groups significantly. The inhibitory effects of tropisetron on pinch-evoked Skin-wide dynamic range unit discharges were prolonged in Complete Freund's Adjuvant day 7 group compared with those in non-Complete Freund's Adjuvant group. The role of central serotonin 3 receptors in trigeminal subnucleus caudalis/upper cervical spinal cord junction region was also tested by orofacial formalin test in Complete Freund's Adjuvant day 7 group. Intracisternal administration of tropisetron decreased the orofacial nocifensive behavior in the late phase evoked by the injection of formalin into the masseter muscle. These results suggest that central serotonin 3 receptors in trigeminal subnucleus caudalis/upper cervical spinal cord junction region are involved in mediating pronociceptive effects in both superficial and deep craniofacial tissues nociception during persistent temporomandibular joint inflammation.

Expression of mKirre, a mammalian homolog of Drosophila kirre, in the developing and adult mouse brain.

mKirre, a mammalian homolog of the Drosophila kirre, is expressed in bone marrow stromal cells and the brain. Although mKirre has been shown to support the hematopoietic stem cells, little is known about the function of mKirre in the brain. In the present study, to gain insights into the function of mKirre, we investigated the expression pattern of mKirre gene in the developing and adult mouse brain using in situ hybridization. In the adult brain, mKirre mRNA was highly expressed in the olfactory bulb, the piriform cortex, the cochlear nucleus, and the cerebellum. At embryonic day (E) 11.5, we could observe mKirre mRNA in the differentiating zones of various regions, such as the caudate-putamen, the geniculate body, the thalamus, the amygdala, and the brainstem. Its gene expression in these regions at E11.5 also persisted to the adult, in which its expression levels were much less prominent. After birth, we could first observe high expression of mKirre mRNA in the glomerular and mitral layers of the olfactory bulb, the cortical plate of the neocortex, the cochlear nucleus, and the molecular and granule cell layers of the cerebellum. In the hippocampus, its gene expression was first observed in the dentate gyrus at postnatal day 7. The spatiotemporal expression pattern of mKirre mRNA suggests important roles of mKirre in later developmental processes, especially the synapse formation.

Spontaneous synaptogenesis in ex vivo sympathetic ganglion and the blockade by serum treatment.

Central denervation for more than 1 month has been shown to cause an increase in the number of adrenergic synapses in sympathetic ganglia in vivo. Here, we report several lines of evidence that adrenergic synapses may be generated de novo in ex vivo superior cervical ganglion (SCG) of adult rats only several hours after the isolation. Structures immunoreactive for synaptophysin, a marker of presynaptic elements, were drastically decreased 6 days after the preganglionic denervation. A significant increase in number of synaptophysin positive boutons was observed over 3-8 hours in the denervated SCGs maintained ex vivo at 36 degrees C in oxygenated physiologic saline, and this increase was blocked by adding normal serum in the saline. Electron microscopic analysis confirmed that the number of adrenergic synapses specifically labeled with 5-hydroxydopamine was increased by several-fold under the same condition. Intracellular labeling of SCG neurons revealed an increase in the incidence (from 8 to 50%) of neurons having dendritic plexus after the in vitro incubation. No evidence of axonal sprouting within the ganglion was observed. Intracellular recordings from single neurons of denervated SCGs revealed that maximum amplitudes of inhibitory postsynaptic potentials, which were completely blocked by yohimbine, an alpha2-adrenoceptor antagonist, in response to focal stimulation were increased over the several hours. These results suggest that dendrites of SCG neurons rapidly develop and exhibit local efferent characteristics that underlie the inhibitory synaptic transmission once they are subjected to serum deprivation.

Organization of excitatory and inhibitory local networks in the caudal nucleus of tractus solitarius of rats revealed in in vitro slice preparation.

Morphological and physiological properties of neurons in the caudal nucleus of tractus solitarius (NTS) of rats were studied in vitro by whole-cell recording and intracellular staining with biocytin. Synaptic responses following the solitary tract stimulation were also investigated to elucidate anatomical substrates of the underlying local circuits. Biocytin-filled NTS cells were divided into three groups according to the pattern of their axonal arborization: (1) local circuit neurons whose axon collaterals were extensively distributed within the NTS with the main axons leaving the NTS; (2) presumed interneurons whose axon collaterals seemed to be restricted within the NTS; and (3) projection neurons whose axons had few, if any, collaterals. Both local circuit neurons and presumed interneurons had small cell bodies (< 150 microns2 in somal area) and exhibited tonic regular spiking at depolarized membrane potentials. Polysynaptic excitatory background activity was increased and lasted for 300-1000 msec in these neurons following solitary tract stimulation. The projection neurons had medium to large cell bodies (> 150 microns2 in somal area). Inhibitory postsynaptic responses produced by an increased CI-conductance were recorded in these projection neurons. These findings suggest that excitatory local networks are organized by an assembly of the local circuit neurons in the caudal NTS, and that the interneurons are arranged to connect the excitatory local network with medium to large projection neurons via inhibitory synapses. Visceral afferent information is probably processed in the highly organized excitatory and inhibitory local networks within the caudal NTS and conveyed to other brain regions.

Principal neurons as local circuit neurons in the rat superior cervical ganglion: the synaptology of the neuronal processes revealed by intracellular injection of biocytin.

To analyze the local circuitry of the sympathetic ganglion, the synaptic relations of the neuronal processes of the principal neurons in the rat superior cervical ganglion were investigated by correlated light and electron microscopy combined with intracellular injection of biocytin. Intracellular iontophoresis of biocytin followed by avidin-biotinylated horseradish peroxidase cytochemistry allowed complete visualization of the neuronal processes of the principal neurons. The stained principal neurons have a single process (axon), which leaves the ganglion, and several intraganglionic processes (dendrites), some of which show specific terminal arborizations. Some terminals of the dendritic collaterals formed pericellular plexuses or intercellular glomerular plexuses. Electron microscopically, the dendrites and their collaterals contain numerous small vesicles. Synaptic membrane specializations were observed between the stained dendritic collaterals and unlabeled neurites. These may be both preganglionic axon terminals and processes of principal neurons. The likely direction of neurotransmission often could not be determined because of the bidirectional synaptic structures. Our findings show that the dendritic collaterals of principal neurons appear to make both post- and presynaptic contacts with both the principal neurons and the preganglionic axons. It is suggested that the principal neurons might participate in local circuits involving not only preganglionic axons but also neighboring principal neurons.

Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb.

The distribution of neuropeptidelike immunoreactivities in the adult guinea pig olfactory bulb was studied immunohistochemically with antisera raised against neurotensin (NT), substance P (SP), methionine-enkephalin-Arg6-Gly7-Leu8 (ENK), somatostatin (SOM), neuropeptide Y (NPY), and cholecystokinin-8 (CCK). In the main olfactory bulb, NT-like immunoreactive (NT-IR) neurons were found among periglomerular cells. In addition, a few periglomerular cells showed ENK-like immunoreactivity. Granule cells displaying SP- or ENK-like immunoreactivities and short axon cells with SOM- or NPY-like immunoreactivities were observed in the deeper half of the granule cell layer. SOM-IR short axon cells were also seen in the external plexiform layer. Dense NT- or NPY-IR fibers were distributed in superficial lamina of the granule cell layer, and sparse SP- or CCK-IR fibers were found in the glomerular layer. In the accessory olfactory bulb, some mitral, periglomerular, and granule cells showed NT-like immunoreactivity. SP- or ENK-IR granule cells were also observed. These results are discussed in relation to laminar organization of the olfactory bulb. The most characteristic features of peptide distribution in guinea pigs, as compared with that of rats in previous studies, were the relative abundance of NT-IR structures and the lack of SP- and CCK-IR juxtaglomerular and tufted cells.

Neuropeptide- and neurotransmitter-related immunoreactivities in the developing rat olfactory bulb.

The development of neuropeptide and neurotransmitter-related immunoreactivities in the rat olfactory bulb were investigated immunohistochemically by using antisera raised against substance P (SP), cholecystokinin-8 (CCK), neurotensin (NT), leucine-enkephalin or methionine-enkephalin-Arg6-Gly7-Leu8 (ENK), somatostatin (SOM), neuropeptide Y (NPY) and tyrosine hydroxylase (TH). Results obtained for the adult olfactory bulb confirmed previous observations, except for SP-like immunoreactive (SP-IR) granule cells in the main olfactory bulb (MOB) and NT-IR neurons around the modified glomerular complex (MGC) (Teicher et al., Brain Res. 194:530-535, 1980). SP-, CCK- and NT-IR neurons were observed in the MOB of the rat fetus. SP-IR neurons also appeared in the accessory olfactory bulb (AOB). Among them, NT-IR neurons in the MOB and SP-IR neurons in the AOB were observed on embryonic day 16. SP- and CCK-IR neurons in the MOB appeared on embryonic day 18. Most of these neurons were presumed to be projecting neurons. SOM-, NPY-, ENK- and TH-IR neurons appeared in the newborn rats. The number and intensity of immunostaining of these neurons continued to increase with age, producing the adult pattern, except for NT-IR neurons in the MGC and SP-IR neurons in the mitral cell layer of the AOB, which were more numerous and intensely stained in young animals.

Terminal field of cholecystokinin-8-like immunoreactive projection neurons of the rat main olfactory bulb.

The terminal field of cholecystokinin-8 (CCK)-like immunoreactive (CCK-IR) tufted cells in the rat main olfactory bulb was examined by means of immunohistochemistry combined with either an anterograde tracer or a degeneration method. CCK immunostaining was carried out in animals in which Phaseolus vulgaris agglutinin (PHA) had been injected into the main olfactory bulb. Pairs of adjacent sections were processed for CCK and PHA immunostaining, respectively. Dense CCK-IR terminallike staining was noted in layer Ia of the anterior olfactory nucleus and lateral part of the olfactory tubercle; weaker staining was also observed in the transitional area between the anterior olfactory nucleus and the piriform cortex, in the medial part of the olfactory tubercle, and in the cortical amygdaloid nucleus. The CCK-IR staining was limited to the area containing PHA-labeled terminals and was diminished in these sites after unilateral olfactory bulbectomy. Immuno-electron microscopic analysis showed that CCK-IR profiles in such regions made asymmetric synaptic contacts, mainly with dendritic spines. These results suggest that CCK-IR tufted cells project mainly to the anterior olfactory nucleus and lateral part of the olfactory tubercle, and act mainly via axospinous synapses.

Transient expression of adenosine deaminase in facial and hypoglossal motoneurons of the rat during development.

Immunohistochemical and retrograde tracing techniques were employed to demonstrate a changing pattern of adenosine deaminase (ADA) immunoreactivity in cranial motoneurons during their ontogenesis in the rat. Immunostaining for ADA was observed only in motoneurons of hypoglossal and facial motor nuclei and only at certain stages during development. Moreover, ADA immunoreactivity was restricted to subpopulations of motoneurons within each nucleus. In the hypoglossal nucleus ADA-immunostained neurons were seen only in the dorsal subnucleus, where they appeared at about 15 days of gestation, reached maximal staining intensity early after birth, and disappeared by the 25th postnatal day. In the facial motor nucleus, immunoreactive neurons were detected only in the intermediate subnucleus, where ADA immunostaining was first detected at 18 days of gestation and was maximal during the first few postnatal days, and in the lateral subnucleus, where immunostaining appeared perinatally. In both facial motor subnuclei, ADA immunoreactivity was no longer detectable by the 15th postnatal day. Retrograde tracing with WGA-HRP or fluorescent dye injected into various muscles of the face or tongue in young animals indicated that ADA-immunoreactive motoneurons in the hypoglossal and facial motor nuclei innervate retractor muscles of the tongue and perioral or nasal muscles, respectively. In view of the critical role of these muscles in suckling and sniffing behavior, it is suggested that metabolic pathways associated with ADA may be involved in the early maturation of the motoneurons projecting to these muscles. Alternatively, the transient presence of ADA in these neurons may reflect a developmental period during which purine nucleosides and/or nucleotides may serve as neuromodulators at their peripheral terminations.

Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscopic study.

By using both light and electron microscopic immunocytochemical methods, Met5-Enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive structures were detected in the pelvic ganglion of male rats. Denervation studies were carried out to determine the origin of these immunoreactive fibers and the projection of immunoreactive neurons within the pelvic ganglion. MEAGL-like immunoreactivity was found in numerous axon boutons, some small, intensely fluorescent (SIF) cells, and a few principal ganglion neurons. Most of the immunoreactive nerve fibers formed pericellular plexuses surrounding the ganglion cells. In addition, there were a few scattered varicose fibers. These fiber plexuses could be classified into two types: type I (approximately 90% of fibers), which consisted of 80-120 small boutons that synapsed on either the dendrites (80% of cases) or somata (20% of cases) of principal neurons; and type II (approximately 10% of fibers), which consisted of 20-40 larger boutons that formed axodendritic synapses exclusively. After transection of the hypogastric and pelvic nerves, virtually all of the pericellular fiber plexuses disappeared, whereas the scattered varicose fibers remained. According to their ultrastructure, these remaining fibers were considered to arise from SIF cells. Following the injection of Fast Blue into the bladder wall, some of the MEAGL-like immunoreactive principal neurons were retrogradely labeled. The results of this study indicate that there are two origins for the MEAGL-like immunoreactive fibers detected in the pelvic ganglion: most arise from preganglionic neurons in the spinal cord, and a small proportion may originate from intraganglionic MEAGL-like immunoreactive SIF cells or principal neurons. Some MEAGL-like immunoreactive principal neurons may project to the urinary bladder.

Fine structure of synapses and retinal innervation of substance P and adenosin deaminase containing neurons in the superior colliculus of the rat.

The fine structure of substance P (SP) and adenosine deaminase (ADA) immunoreactive structures in synaptic contacts localized to the superficial layers of the superior colliculus of the rat was investigated by means of immunoelectron microscopy. We also examined the possibility of retinal innervation of SP- and ADA- containing neurons by immunohistochemistry after degeneration of retinal terminals caused by enucleation. SP-like immunoreactive presynaptic terminals of the stratum griseum superficiale (SGS) formed both asymmetric and symmetric synaptic contacts. Presynaptic dendritelike structures were also observed. SP immunoreactive postsynaptic elements made contacts with terminals showing diverse features. ADA-like immunoreactive structures were seen only as postsynaptic elements to different kinds of nonimmunoreactive terminals and were mostly localized in the ventral third of the SGS and the dorsalmost stratum opticum (SO). After enucleation, degenerating retinal terminals were found to form synaptic contacts with SP and ADA immunoreactive structures. The highest number of such degenerating terminals on ADA immunoreactive structures was observed 2 days after retinal denervation, very few being seen after 5 days. These degenerating terminals were restricted to the ventral SGS and dorsal SO. SP immunoreactive structures postsynaptic to degenerating retinal terminals were most numerous 5 days after enucleation and mainly localized in the dorsal SGS. Occasionally, SP immunoreactive dendritelike processes forming synapses with degenerating retinal terminals were simultaneously presynaptic to other nonimmunoreactive profiles, defining, therefore, serial synapses. The present results suggest that SP-I and ADA-I collicular neurons may be part of distinct channels carrying visual information to the lateral posterior and lateral geniculate nuclei of the thalamus, respectively.