Triorthocresyl phosphate-induced neuronal losses in lumbar spinal cord of hens--an immunohistochemistry and ultrastructure study.

To investigate the neuronal losses of hens' spinal cords in the model of organophosphate-induced delayed neuropathy (OPIDN) and to analyze the impact of apoptosis on the pathogenesis of OPIDN. Adult hens were challenged with triorthocresyl phosphate (TOCP) at a single dose (750 mg/kg). Neuronal losses in the 3rd lumbar spinal cord (L3) were assessed by light-microscopy and electron-microscopy methods at different days post exposure, respectively. The typical OPIDN signs were seen in the TOCP-exposed hens at about 9th day. The number of large nerve cells declined gradually. And these cells were verified as neurons by immunostained with neuronal marker NeuN. The expression of FasL reached proximal at about 9th day, decreased from 14th day. Neurons in TOCP exposed groups displayed degenerative morphologies in electronic microscopy. Some neurons showed apoptotic-like ultrastructure profiles at 5th day. The nuclear membrane was complete with chromatin condensed to the margins of nuclear membrane like a crescent-shaped body. Mitochondria morphologic changes appeared early (5 d) following exposure to TOCP, and developed in a time-dependent fashion. Apoptosis might be involved in the development of OPIDN, and play a role in the pathogenesis of OPIDN.

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.

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.

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.

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).

Time-dependent astroglial changes after gamma knife radiosurgery in the rat forebrain.

OBJECTIVE: Using an experimental rat model and a clinically relevant treatment dose, we performed gamma knife radiosurgery to define the hyperacute radiation effects in normal rat forebrain, the time dependence of the astrocytic reaction, and the participation of astrocytes in the healing process after single-dose gamma radiation injuries. METHODS: Seventy-one rats underwent radiosurgical treatment (4-mm collimator) of the caudate-putamen nucleus (single-fraction maximal dose of 100 Gy) and were killed at times ranging from 3 hours to 90 days. Serial cryostat brain sections were processed with the immunohistochemical avidin-biotin complex technique, using anti-glial fibrillary acidic protein as the primary antibody (to identify astrocytes). RESULTS: Vascular changes, including endothelial hyperplasia and vessel wall thickening, were identified as the earliest postradiation manifestations and continued throughout the observation period. Astrocytes reacted to the radiation injury with hyperplasia and hypertrophy. At earlier time points (3-24 h), proliferation was the predominant reaction. The expression of glial fibrillary acidic protein in the proliferating and hypertrophic astrocytes formed an initial peak in the adjacent corpus callosum 3 days after radiosurgery and peaked within the target site between 14 and 30 days. Astrocytic proliferation and hypertrophy were also observed in distant cortices (frontal, parietal, insular, and piriform cortices) and in the hippocampus. No necrosis was observed less than 30 days after irradiation. By Day 90, necrotic lesions with a mean diameter of 4 mm were identified, with glial scar at their peripheries. Astrocytic morphological features varied according to the distance from the necrosis. The irradiated side contained more glial fibrillary acidic protein-containing cells than did the nonirradiated contralateral side. CONCLUSION: During the early phase after radiation, vasculopathy was the first morphological change and may serve as the initiating factor for subsequent changes. Reactive astrocytes appeared not only at the target site but also in the surrounding regions; the severity of injury was determined by the distance from the target.

Fos protein expression in the medulla oblongata and changes in size of spinal lateral horn neurons after 4-wk simulated weightlessness in rats.

Responses of the neurons in medulla oblongata and C8-T1 spinal cord lateral horn of rats induced by simulated weightlessness were investigated using anti-Fos protein and anti-tyrosine hydroxylase (TH) double staining immunohistochemical methods, and Nissl-staining technique respectively. After four weeks of tail-suspension, many Fos-like positive neurons were localized in the medullary visceral zone (MVZ), predominantly in the nucleus of tractus solitarii and ventrolateral medulla, and some of them showed TH-like immunoreactivity. Sizes of the cell bodies of the lateral horn neurons in C8-T1 segment were significantly increased in 4-wk tail-suspended rats (P<0.05) as compared with that in controls. The results suggest that the neurons in MVZ and the spinal lateral horn may be involved in the adaptation of central cardiovascular regulation during weightlessness.

Expression and changes of Fos protein in the rat forebrain after gamma knife irradiation targeted to the caudate putamen.

OBJECTIVE: Forebrain expression and changes of Fos protein immunoreactivity were investigated in rats subjected to gamma knife irradiation (dose, 100 Gy), focusing on the right caudate nucleus. METHODS: Serial cryostat forebrain sections were processed with Fos protein antiserum and then by avidin-biotin-peroxidase complex immunohistochemistry after gamma knife irradiation. RESULTS: Some neurons, glial cells, and endothelial cells were positive for Fos-like immunoreactivity (LI). Fos-LI was mainly distributed in the target area of irradiation, as well as in white matter surrounding lateral ventricles, cortex, hippocampus, and certain nuclei reportedly implicated in the regulation of stress responses. From 0.5 hour to 30 days after irradiation, expression of Fos-LI displayed two peaks. The first peak arose from 3 hours to 24 hours, with cell nuclei being positive for Fos-LI. The second peak was noticeable at the 14th day postirradiation. Subsequently, three types of Fos-LI-positive cells were identified during the period between 14 and 30 days: in the first type, only the nucleus was positive; in the second type, the cytoplasm was stained; and in the third type, the cells were positive in both the nucleus and the cytoplasm. CONCLUSION: During the early stages after gamma knife irradiation, a specific stress response (significant Fos expression with two peaks) is observed not only in the target region but also in the surrounding forebrain regions. The characteristics and significance of two types of Fos-LI-positive cells are discussed in the context of this investigation.

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.

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.

[Advancement on neurotransmitter transporters].

The neurotransmitter transporter (NTT), a kind of glycoprotein situating on the presynaptic membrane, glial membrane, or vesicle membrane, has become the focusing point of neuroscience research in recent years. They could combine selectively with transmitters released into synaptic cleft and carry them back into cells to aid the termination of synaptic transmission. In this way, NTT took an important part in modulation of information between neurons. Study on NTT has not been reported domestically. This article reviewed recent studies on molecular structure, classification, study methods, distribution, function, modulating factors and gene modulation, focus of study and also unresolved problems of NTT.

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.

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.

Medullary catecholaminergic neurons projecting to lateral hypothalamic area and expressing Fos after chemical stimulation of the stomach in the rat.

Catecholaminergic neurons in the medulla projecting to lateral hypothalamic area (LH) and expressing Fos were investigated in the rat by a triple labelling method, in which horseradish peroxidase (HRP) was injected into the LH, and visceral noxious stimulation was induced by formalin injection into the stomach, sections of the medulla were stained histochemically for HRP and immunohistochemically for Fos and tyrosine hydroxylase (TH). Some neurons labeled with HRP and showing both Fos- and TH-like immunoreactivities were mainly found in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) at the middle and caudal levels of medulla, only a few of them were located in the reticular formation between NTS and VLM. These results indicated that some medullary catecholaminergic neurons projected to the LH and some of them might be involved in the transmissing processes of stress responses to the visceral nociceptive information produced by chemical stimulation.

Fos expression in catecholaminergic medullary neurons induced by chemical stimulation of stomach projecting to the paraventricular nucleus of the hypothalamus in rats.

AIM: To determine whether medullary catecholaminergic neurons expressing Fos induced by chemical stimulation of the stomach project to the paraventricular nucleus of hypothalamus (PVH) in rats. METHODS: Horseradish peroxidase (HRP) was introduced stereotaxically into the PVH of rats. Histochemical analysis of coronal sections through the medulla were analyzed using triple-label immunohistochemistry to identify cells that were retrogradely labeled with HRP, Fos (ABC method), and tyrosin hydroxylase (TH) (PAP method). RESULTS: Seven kinds of labeled neurons were found in the nucleus tractus solitarii (NTS), the ventrolateral medulla (VLM) and the reticular formation (RF) of the medulla: Fos-like immunoreactive (FosLI) neurons, TH-like immunoreactive (TH-LI) neurons and HRP retrogradely single-labeled neurons, FosLI/HRP, FosLI/TH-LI and HRP/TH-LI double-labeled neurons, and FosLI/HRP/TH-LI triple-labeled neurons. CONCLUSION: Ascending projections from the NTS, VLM and RF to the PVH might be involved in the transmitting process of visceral noxious stimulation.

GABAergic synapses upon neurons expressing substance P receptors in the nucleus of the solitary tract: an immunocytochemical electron microscope study in the rat.

Morphological substrates for interactions between gamma-aminobutyric acid (GABA) and substance P upon neurons expressing substance P receptor (SPR) in the nucleus of the solitary tract (NST) were investigated by immunocytochemical electron microscopy. In the NST of the rat, many GABA-like immunoreactive axon terminals were in symmetric synaptic contacts with dendritic profiles; they were observed on nearly a half of the SPR-like immunoreactive dendritic profiles in the medial part of the caudal half of the NST.

Catecholaminergic neurons in the nucleus tractus solitarii which send their axons to the midbrain periaqueductal gray express Fos protein after noxious stimulation of the stomach: a triple labeling study in the rat.

In rats which were injected with horseradish peroxidase (HRP) into the midbrain periaqueductal gray (PAG) and then administered with formaldehyde into the stomach, Fos-like immunoreactivity was found in tyrosine hydroxylase-like immunoreactive neurons in the nucleus tractus solitarii (NTS) which were retrogradely labeled with HRP. The results indicate that catecholaminergic NTS neurons may mediate nociceptive visceral information to the PAG.