Evidence of gamma-aminobutyric acidergic control over the catecholaminergic projection from the medulla oblongata to the central nucleus of the amygdala.

It is known that the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) project to the central nucleus of the amygdala (Ce), conveying visceral information. Conversely, the Ce sends projections to the NTS and the VLM. To understand better the role of catecholamine and gamma-aminobutyric acid (GABA) in these reciprocal connections, experiments were performed by combining lectin-conjugated horseradish peroxidase (WGA-HRP) anterograde and retrograde transport with preembedding immunocytochemistry to detect tyrosine hydroxylase (TH), and postembedding immunocytochemistry to detect GABA. The light microscopic study suggested that the majority of neurons in the NTS and the VLM projecting to the Ce were TH immunoreactive (TH-IR). Most of them were located at the level of the obex. Under the electron microscope, the GABAergic and non-GABAergic terminals were found to form synaptic contacts with the TH-(IR) or Ce-projecting or TH-IR/Ce-projecting double-labelled neurons of the NTS and VLM. The GABAergic terminals mostly formed symmetrical synaptic contacts with the postsynaptic structure in which perikarya (14-19%), dendrites (79-84%), and spines (2%) were observed. Approximately 94% of the axon terminals in the NTS and 90% of those in the VLM arising from the Ce were GABAergic and appeared not to form synaptic contacts with the TH-IR or Ce-projecting neurons in these regions. The present results demonstrated that the catecholaminergic neurons of the NTS and VLM projecting to the Ce receive an extensive GABAergic innervation and that the amygdala projection to the medulla is mostly GABAergic.

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker.

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammillary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammillary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study.

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.

Evidence for the medullary visceral zone as a neural station of neuroimmunomodulation.

The aim of the present study was to test the possibility that the catecholaminergic projectional pathway from the vagus nerve to the medullary visceral zone (MVZ) thence to the paraventricular nucleus of hypothalamus (PVN) was involved in the cytokine-to-brain communication. A triple labeling method in which WGA-HRP retrograde tracing was combined with anti-Fos and -TH immunohistochemical staining was used. WGA-HRP was stereotaxically injected into unilateral PVN in the rat, after a survival of 48 h, animals received intraperitoneal injection of lipopolysaccharide (LPS). The distribution of the HRP retrogradely labeled neurons, Fos protein positive and catecholaminergic neurons (tyrosine hydroxylase as marker) in the MVZ was observed. Subdiaphragmatic vagotomy (SDV) and sham surgery were also used to observe the different Fos expression in the MVZ after intraperitoneal administration of lipopolysaccharide (LPS) or pyrogen-free saline (NS). Under light microscope, seven types of positively stained neurons could be distinguished within the MVZ, namely neurons single-labeled with Fos, HRP or TH, respectively; neurons double-labeled with Fos/TH, Fos/HRP or HRP/TH separately; and neurons triple-labeled with Fos, HRP and TH staining. Intraperitoneal LPS caused lots of robust Fos expression within the MVZ in the sham surgery groups and this response in the MVZ was markedly inhibited in the vagotomized rats. The results suggested that some catecholaminergic neurons in the MVZ could send projections to the PVN and this pathway might be involved in the relay of peripheral immune information via vagus nerve. MVZ was a neural relay station in the immune-to-brain communication and might play a significant role in the neuroimmunomodulation via vagus-MVZ-PVN pathway.

Expression and changes of HSP70 in the rat forebrain subjected to gamma knife (100Gy) irradiation targeted on the caudate putamen and survived for different times.

Forebrain heat shock protein 70 (HSP70) immunohistochemical reactivity was investigated in rats subjected to gamma knife irradiation focusing on the right caudate putamen nucleus. The forebrain sections of all experimental animals were processed with anti-HSP70 antiserum and then by avidin-biotin peroxidase complex immunohistochemistry after gamma ray irradiation with a dose of 100Gy and they each survived for different times (from 30 min to 30 days). Some neurons, glial cells, and endothelial cells were HSP70-like immunoreactivity (HSP70-LI) positive. HSP70-LI was mainly distributed in the target area of irradiation, as well as in non-target regions, e.g. the cortex, hippocampus, and hypothalamus, etc. The expression and change of HSP70-LI from 3 h to 30 days after irradiation followed the following rules: (1) Within 3 to 24 h, the dilated vessels with HSP70-LI endothelial cells were found at first, and a few lightly stained HSP70-LI neurons and glias were observed in the target and non-target regions; (2) In 3-7 days, darkly stained HSP70-LI neurons and glias were apparently increased and formed an expression peak. From 14 to 30 days, HSP70-LI cells were distinctly decreased and became weakly stained or negative. These results suggested that although the irradiation target of the gamma knife was localized, the response to irradiation occurred extensively.

Fos expression in serotonergic midbrain neurons projecting to the paraventricular nucleus of hypothalamus after noxious stimulation of the stomach: a triple labeling study in the rat.

Horseradish peroxidase (HRP) was stereotaxically injected into the paraventricular nucleus of the hypothalamus (PVH) and visceral noxious stimulation was given by an administration of formalin into the stomach. The brain sections were subjected to HRP histochemistry and immunostained with Fos and serotonin antibodies. The periaqueductal gray (PAG) and dorsal raphe nucleus (DR) contained single-labeled (Fos, 5HT, HRP), double-labeled (Fos/HRP, Fos/5HT, HRP/5HT) and triple-labeled (HRP/Fos/5HT) neurons. Triple-labeled neurons were mainly observed in the DR and in discrete areas of the PAG ipsilateral to side of the HRP injection. These findings suggest that a population of serotonergic midbrain neurons receive nociceptive visceral information and project to the PVH.

Expression and significance of three types of Fos-immunoreactive cells after gamma knife irradiation of the forebrain in the rat.

The expression and significance of three types of Fos-like immunoreactive (Li) cells were investigated after gamma knife irradiation of the forebrain in the rat. Three months after the irradiation, the brain sections were immunostained with an antiserum against Fos protein. It was shown that the Fos-like immunoreactivity (LI) appeared in some of the neurons, glial cells and endothelial cells in the target area, the white matter surrounding the lateral ventricle, the cerebral cortex and the hippocampus. Three characteristic types of Fos-Li cells were identified in these regions. (1). Only the nuclei of the cells were Fos-ir, (2). Only the cytoplasm was immunostained, and (3). Both the nuclei and the cytoplasm showed Fos-LI. It is suggested that type 1 are the normal responsive cells, type 2 are seriously injured cells, so that the Fos translocation mechanism is damaged, and type 3 represents the intermediate form.

The sites of origin of a-melanocyte-stimulating hormone-containing axonal components in the lateral area of the midbrain periaqueductal gray of the rat.

The sites of origin of alpha-melanocyte-stimulating hormone (alpha-MSH)-containing axonal components in the lateral area of the midbrain periaqueductal gray (PAG) were studied in the rat by the retrograde tracing method of horseradish peroxidase, combined with the immunocytochemical technique. The results indicated that alpha-MSH-containing axonal components in the PAG arose not only from the arcuate nucleus of the hypothalamus but also from the ventral zona incerta, the periventricular, perifornical and lateral hypothalamic regions.

Noxious somatic stimulation-induced expression of Fos-like immunoreactivity in catecholaminergic neurons with habenular nucleus projection in the medullary visceral zone of rat.

In order to study the expression of Fos protein in catecholaminergic neurons in the medullary visceral zone (MVZ), which project to the habenular nucleus (HB), a triple-labeling method combining wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunohistochemical staining was used in the rat. WGA-HRP was stereotaxically injected into unilateral HB. Forty-eight hours later, 50 microl of 8% formalin was injected into the foot pad of the right front paw. Two hours after formalin injection, animals were anesthetized and perfused transaortically. Coronal sections (40 micron) were cut from the cervical segment of spinal cord, the medulla oblongata and WGA-HRP injected area with a cryostat. First, sections of the injected area and the medulla oblongata were histochemically processed to demonstrate the presence of retrogradely transported WGA-HRP using the chromogen tetramethylbenzidine (TMB). Then sections of the spinal cord and the medulla oblongata were immunostained with anti-Fos and anti-TH antibodies using the ABC method. Under the light microscope, seven types of variously labeled neurons could be identified in MVZ, namely Fos and TH-immunoreactive (Fos- or TH-IL) neurons, WGA-HRP labeled ones, Fos/HRP, Fos/TH and HRP/TH double-labeled and Fos/HRP/TH triple-labeled cells. The results suggest that some catecholaminergic neurons in MVZ could send projections to HB and this pathway may be involved to relay nociceptive information from spinal cord to brainstem and on to the forebrain.

Projection from the ventrolateral medullary neurons containing tyrosine hydroxylase to the central amygdaloid nucleus in the rat.

By using a double-labeling produce of retrograde horseradish peroxidase (HRP) tracing and the immunocytochemical localization of tyrosine hydroxylase (TH), the present study ascertained that the axonal fibers of catecholaminergic neurons in the caudal ventrolateral medulla projected to the central amygdaloid nucleus in the rat. The majority of double-labeled cells were observed primarily around the level of the obex. 92% of HRP retrogradely labeled cells contained TH-like immunoreactive (TH-IR), but only 31% of TH-IR cells was labeled with HRP.

Tyrosine hydroxylase-, neurotensin-, or cholecystokinin-containing neurons in the nucleus tractus solitarii send projection fibers to the nucleus accumbens in the rat.

A double-labeling method combining the retrograde tracing of horseradish peroxidase (HRP) with the immunocytochemical technique was used in the present study. The results suggest that the neurons containing tyrosine hydroxylase (TH), neurotensin (NT) or cholecystokinin (CCK) in the nucleus tractus solitarii (NTS) of the rat send their axons to the nucleus accumbens bilaterally with ipsilateral dominance. HRP-TH, HRP-NT or HRP-CCK double-labeled cell bodies were mainly located in the medial and the commissural subnucleus of the NTS. HRP-TH double-labeled cells were the largest in number, followed by HRP-NT cells, with HRP-CCK cells present in the lowest numbers.

Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus--an immunocytochemical study.

Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the parabrachial nucleus (PB) of human fetus, fetal age (menstruation age) 11.5 to 40 weeks. The time of initial appearance of SP-LI in the human brainstem PB was between fetal age 11.5 weeks and 16 weeks. While the fetus grew, the density of SP-LI fibers and terminals in the human fetus brainstem PB increased constantly from fetal age 16 weeks to 40 weeks. These findings indicate that substance P (SP) might play an important role in the human parabrachial nucleus development and in its functional establishment during the prenatal period.

Prenatal ontogeny of substance P-like immunoreactivity in the trigeminal spinal caudal subnucleus of the human fetus--an immunocytochemical study.

Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the trigeminal spinal caudal subnucleus (TSCS) of human fetus, fetal age (menstruation age) 11.5-40 weeks. The time of initial appearance of SP-LI in the human brainstem TSCS was between the fetal age 11.5 weeks and 16 weeks. While the fetus grew, the density of SPLI fibers and terminals in the human fetus brainstem TSCS increased constantly from fetal age 16 weeks to 40 weeks. These findings indicated that substance P (SP) might play an important role in the human trigeminal nerve system development and in its functional establishment during the prenatal period.

Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats.

Dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) is a key element of dopamine/D1/DARPP-32/protein phosphatase-1 (PP-1) signaling cascades of mammalian brain. We are interested in the expression patterns of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in DARPP-32-containing neurons, which may constitute morphological basis for interaction between dopamine and ionotropic glutamate receptors in dopaminoceptive cells. Double immunofluorescence was performed to visualize neurons showing coexpression of DARPP-32 with NMDA or AMPA receptor subunits (i.e., NR1, NR2a/b, glutamate receptor subunit 1 [GluR1], GluR2/3, and GluR4) in the forebrains of rats. Distribution of DARPP-32-positive neurons completely or partially overlapped with that of NMDA receptor- or AMPA receptor-immunoreactive ones in the frontal and parietal cortex, hippocampus and neostriatum, and neurons double-labeled with DARPP-32/NR1, DARPP-32/NR2a/b, DARPP-32/GluR1, DARPP-32/GluR2/3, or DARPP-32/GluR4 immunoreactivity were numerously observed. Semiquantification analysis indicated that most of DARPP-32-containing neurons (86-98%) expressed NR1, NR2a/b and GluR2/3, while less of them (14-90%) expressed GluR1 and GluR4. Although high rates (90-98%) of DARPP-32-positive cells expressed NMDA receptors in all regions above, variant percentages of them expressing AMPA receptor subunits were observed among the cortex (54-90%), hippocampus (59-97%) and neostriatum (14-97%). The study presents differential expression patterns of NMDA and AMPA receptors in DARPP-32-postive neurons in these forebrain regions. Taken together with previous reports, the present data suggest that interaction between dopamine and glutamate receptors may occur in the dopaminoceptive neurons with distinct receptor compositions and may be involved in modulating neuronal properties and excitotoxicity in mammalian forebrain.

Serotonergic projections from the midbrain periaqueductal gray and nucleus raphe dorsalis to the nucleus parafascicularis of the thalamus.

By a double-labeling method combining the retrograde tracing of horseradish peroxidase and the immunocytochemical technique, serotonin-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) and nucleus raphe dorsalis (DR) of the rat were observed to send projection fibers to the nucleus parafascicularis of the thalamus bilaterally with an ipsilateral dominance. These serotonin-containing projecting neurons were observed mainly at the middle-caudal levels of the ventrolateral subdivision of the PAG and less at the middle-rostral levels of the DR.

Visceral noxious stimulation induced expression of Fos protein in medullary catecholaminergic neurons projecting to nucleus accumbens in the rat: a study with triple labeling method of HRP tracing combined with Fos and TH immunohistochemistry.

Horseradish peroxidase (HRP) was stereotaxically injected into the nucleus accumbens (Acb), and visceral noxious stimulation given by injecting formalin into the stomach. Sections of the medulla were subjected to HRP reaction combined with immunohistochemical reactions for Fos protein (ABC method) and tyrosine hydroxylase (TH, PAP method). The catecholaminergic neurons of the medulla (including vagal complex, ventrolateral medulla and reticular formation between them) which expressed Fos protein and projected to Acb were studied. The results showed that HRP retrogradely labeled cells were seen in the medulla bilaterally with apparent ipsilateral predominance and TH-LI and Fos-LI single labeled cells were bilaterally distributed; HRP/TH,TH/Fos double labeled neurons were more numerous than HRP/Fos double-labeled neurons. HRP/TH/Fos triple-labeled neurons were small in number and were mainly distributed in the nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM), but only a few labeled cells were located in RF between nTS and VLM. It is concluded that TH-LI neurons in the medulla projected to Acb and some of them expressed Fos protein after noxious stimulation of the stomach.

An indirect projection from the nucleus of the solitary tract to the central nucleus of the amygdala via the parabrachial nucleus in the rat: a light and electron microscopic study.

The morphological basis of how visceral information from the nucleus of the solitary tract (NTS) is relayed from the parabrachial nucleus (PBN) to the central nucleus of the amygdala (Ce) was studied at the light and electron microscopic levels using the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), kainic acid degeneration, and retrograde tracing with horseradish peroxidase (HRP). After injection of PHA-L into the caudal NTS, anterogradely labeled fibers and terminals were predominantly distributed in the external lateral (el) and central lateral (cl) subnuclei of the PBN. After injection of HRP into the Ce, retrogradely labeled neurons in PBN were mainly distributed in the same areas. In double-labeling experiments, there was a clear overlap between neuronal elements labeled with HRP and PHA-L in the el and cl. At the electron microscopic level, the PHA-L-labeled axon terminals from the NTS mainly contained spherical agranular synaptic vesicles and formed asymmetric contacts with the postsynaptic dendrites or dendritic spines in PBN. After the lesioning agent kainic acid was injected into the NTS and HRP deposited in the Ce, it was found the afferent fibers from the NTS made direct synaptic contact with the lateral PBN neurons which in turn projected to Ce. Such evidence adds to our growing knowledge of regulation of visceral function in central nervous system and would be likely helpful for understanding the important roles of the NTS, PBN and Ce in the central control of cardiovascular, respiratory and gastrointestinal functions.

Prenatal ontogeny of substance P-like immunoreactivity in the nucleus tractus solitarii and dorsal motor nucleus of the vagus nerve of the human fetus: an immunocytochemical study.

By using immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the dorsal motor nucleus of the vagus nerve (nX) and the nucleus tractus solitarii (nTS) of the human fetus at fetal age (menstruation age) of 11.5 weeks to 40 weeks. The time of initial appearance of SP-LI in the human brainstem nTS was between the fetal age 11.5 weeks and 16 weeks. At fetal age 16 weeks, the nTS showed moderate density of SP-LI fibers and terminals in subnucleus dorsalis of the nTS and nX. While the fetus grew, the density of SP-LI in the human fetus brainstem nTS and nX increased gradually from fetal age 16 weeks to 40 weeks. According to the Nissl staining, at fetal age 23 weeks, the nTS of human fetus can be subdivided into dorsal, medial, dorsolateral, ventrolateral, ventral and gelatinosus subnuclei. The cytoarchitectonic subdivisions of human fetus nTS is in good agreement with the results obtained by immunocytochemical staining. These findings indicated that substance P (SP) might play an important role in the development of human brainstem nX, nTS, their related cranial nerves, and in their functional establishment during the prenatal period.

Noradrenergic neurons expressing substance P receptor (NK1) in the locus coeruleus complex: a double immunofluorescence study in the rat.

By using a double immunofluorescence method we examined the distribution of noradrenergic neurons expressing substance P receptor (NK1) or neuromedin K receptor (NK3) in the rat brainstem. The distribution of SPR-like immunoreactive (-LI) neurons completely overlapped that of tyrosine hydroxylase (TH)-LI neurons in the locus coeruleus (A6), ventrolateral and lateral reticular formation of pons (A5 and A7). Partially overlapping distribution of SPR- and TH-LI neurons were found in certain regions of the medulla oblongata (A1-A4). Neurons showing both SPR- and TH-like immunoreactivities, however, were only found in the locus coeruleus complex (A5-A7): 100% of these TH-LI neurons displayed SPR-like immunoreactivity. Neurons showing both NKR- and TH-like immunoreactivities were not detected in the aforementioned areas of brainstem. The present study has provided morphological evidence for direct physiological modulation of noradrenergic neurons by tachykinins through SPR in locus coeruleus complex (A5-A7).

Significant up-regulation of nestin protein in the neostriatum of MPTP-treated mice. Are the striatal astrocytes regionally activated after systemic MPTP administration?

We are interested in the possible role of central glial cells in pathogenesis of Parkinson's disease of mammals. Parkinsonism model was induced by systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and the reactive glial cells were examined by immunocytochemical visualization of nestin protein in the brains and spinal cords of C57 mice. Abundant nestin-like immunoreactivity was predominately found in the caudate putamen of MPTP-treated mice and about 481-fold of nestin-like immunoreactive cells increased compared with that of control animals, indicating that significant up-regulation of nestin protein occurred in these regions. Majority of nestin-like immunoreactive cells characterized with astrocytic profiles of multiple, radical and hypotrophic processes, and showed a distribution and dynamic patterns similar to that of glial fibrillary acid protein (GFAP)-immunoreactive cells in the caudate putamen. Double immunofluorescence confirmed that 100% of nestin-like immunoreactive cells exhibited GFAP-immunoreactivity while nestin/GFAP double-labeled cells constituted about 84% of total GFAP-immunoreactive cells in the caudate putamen, indicating these nestin-like immunoreactive cells belong to a reactive population of the astrocytes. On the other hand, no obvious changes of nestin- or GFAP-like immunoreactivities were detected in the globus pallidus, the substantia nigra and the ventral tegmental area after MPTP-treatment. The results have provided morphological evidence for the regional activation of astrocytic glial cells following systemic MPTP administration, suggesting that a large population of reactive striatal astrocytes might play an important role in initial pathogenesis or acute stage of Parkinson's disease in mammals.