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PubMedDate not supplied

Differential dissection of the rat E16 ventral mesencephalon and survival and reinnervation of the 6-OHDA-lesioned striatum by a subset of aldehyde dehydrogenase-positive TH neurons.

The retinoic acid-generating enzyme, aldehyde dehydrogenase (AHD), is expressed in a subpopulation of dopaminergic neurons found in the substantia nigra. Using AHD and tyrosine hydroxylase (TH) as immunohistochemical markers, we determined whether differential dissection of the embryonic (E16) ventral mesencephalon (VM) into its lateral and medial portions contributed equally to the number of TH cells surviving transplantation, if grafted AHD/TH neurons reinnervate the host striatum according to their normal projection patterns, and examined the functional recovery caused by the implanted cells as assessed by amphetamine-induced rotation in a 6-OHDA-lesioned model of Parkinson's disease. The embryonic tissue was transplanted as solid pieces injected via a 20-gauge lumbar puncture needle into the center of the deafferented striatum. Groups received either one complete ventral mesencephalic piece (VM), two medial pieces of ventral mesencephalic tissue (MVM), or two lateral pieces of ventral mesencephalic tissue (LVM). Both VM and MVM groups showed a significant decrease in amphetamine-induced rotation over time and, there was no difference in the degree of reduction observed between the two groups. Histological evaluation of the transplants revealed a much larger total number of surviving TH cells in grafts from the VM and MVM groups compared to the LVM group. Surviving AHD/TH neurons were found in all groups. Whereas TH staining of the transplanted striatum displayed a halo of graft-derived fibers all around the transplant and integration of these fibers into the host neuropil, AHD staining showed a preferential reinnervation of the dorsolateral striatum corresponding to the normal projection pattern of AHD/TH neurons. In summary, selective dissection of the embryonic ventral mesencephalon is possible, functional recovery as assessed by amphetamine-induced rotation in animals transplanted with MVM is similar to that seen in animals grafted with VM, and AHD/TH neurons have a selective reinnervation pattern in the PD transplantation paradigm. These findings may have implications for the grafting of fetal mesencephalic tissue in PD patients.

Age Factors↗
PubMed2002

Immunohistochemical localization of nerve growth factor, glial fibrillary acidic protein and ciliary neurotrophic factor in mesencephalon, rhombencephalon, and spinal cord of developing mongolian gerbil.

The distribution of the nerve growth factor (NGF), the glial fibrillary acidic protein (GFAP) and the ciliary neurotrohic factor (CNTF) was performed in coronal sections of the mesencephalon, rhombencephalon and spinal cord in the developing Mongolian gerbils. Generally, NGF specifically recognizes neurons with the NGF receptor, whereas GFAP does the glia, and CNTF does the motor neurons. The receptor expression was examined separately in gerbils between embryonic days 15 (E15) and postnatal weeks 3 (PNW 3). The NGF-IR was first observed in the spinal cord at E21, which might be related to the maturation. The GFAP reactivity was peaked at the postnatal days 2 (PND2), while the highest CNTF-reaction was expressed at PNW 2. The GFAP stains were observed in the aqueduct and the spinal cord, which appeared to project laterally at E19. The CNTF was observed only after the birth and found in both the neurons and neuroglia of the substantia nigra, mesencephalon, cerebellum and the spinal cord from PND1 to PNW3. These results suggest that NGF, GFAP and CNTF are important for the development of the neurons and the neuroglia in the central nervous system at the late prenatal and postnatal stages.

Animals↗
PubMed2003

Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector.

PURPOSE: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. METHODS: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. RESULTS: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). CONCLUSIONS: AAV may be a useful vector for the transduction of oncogenes to produce cell lines.

1-Methyl-3-isobutylxanthine↗
PubMed1992

A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin.

A dopaminergic neuroblastoma was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell line N18TG2. The resulting interspecies hybrid, named MES23.5, has retained a stable phenotype and karyotype for a continuous culture period of 1 year. The hybrid exhibits several properties that suggest that the parent primary neurons originated in the substantia nigra. The cell line contains tyrosine hydroxylase, which is identifiable both by biochemical and immunological methods and synthesizes dopamine, but no other catecholamine. Additionally, the cell line expresses apparent voltage-gated CA2+ channels as measured by high-affinity omega-conotoxin binding. The MES23.5 omega-conotoxin receptors are of similar affinity class to those found in adult rat mesencephalon. No dihydropyridine receptors, as measured by PN200-100 ligand binding, are present. None of these properties are found in the N18TG2 parent. At least three neuronal features, namely, tyrosine hydroxylase, dopamine synthesis, and omega-conotoxin receptor expression, are quantitatively elevated after sustained treatment with cAMP analogs. The cell line expresses a complex range of neural properties found in the dopaminergic neurons of the substantia nigra, and may therefore be useful elucidating further details of their cell biology.

Animals↗
PubMed1991

Double-labelling data on somatostatin-like and tyrosine-hydroxylase-like immunoreactivities in chick embryo brain stem. I. Pons and mesencephalon.

With the aim of investigating some factors and mechanisms of the chicken brain development, the same thick sections of brain stems from twelve E13-to-E21-aged chick embryos were sequentially tested with a rabbit anti-Somatostatin antiserum, using a PAP-DAB technique, and with anti-tyrosine hydroxylase (-TH) monoclonal antibodies, using an indirect immuno-fluorescence technique. As regards the pons and mesencephalon, the following main results were obtained. At E21 almost the same distribution of the TH-like immunoreactivity (ir) as at E13 was observed. Neuroblasts in a central, relatively wide region of mesencephalic tegmentum and in the central portion of the pons showed TH-like ir. A co-localization of the 2 immunoreactivities was detected only at E18, within some neuroblasts of the mesencephalic and pontine regions with TH-like ir. It is possible that this transitory co-localization plays a role in the development of the pons and mesencephalon of this species.

Animals↗
PubMed1975

Stereotaxic coordinates for the Rhesus monkey thalamus and mesencephalon referencing visual afferents and cytoarchitecture.

When using a stereotaxic instrument for visual field stimulation we found that electrode placements in the thalamus and mesencephalon of prone rhesus monkeys with the aid of avaiable atlases showed considerable errors. As these animals are valuable for primate visual system reseach an atlas was constructed with methods that have not been used before for rhesus. In addition, the specific connections from the visual cortices, superior colliculus and retina to the thalamus and mesencephalon are also shown. Anesthetized monkeys of specific body dimensions had a matrix of pins inserted into the brain before fixation. A matrix was used so that the penetrations seen in the sectioned brain could be cross related as a control for accurate measurements of the stereotaxic planes throughout the brain. The surface of the whole brain frozen blocks were photographed on the microtome just before a cut section was taken. These calibrated pictures formed the "floor plan" of the atlas as they represent more accurately the brain geometry than individual sections which are distorted by cutting, staining and mounting. Cytoarchitectural (Nissl stain) and axonal connectional (Fink-Heimer stain) information was transferred and adjusted onto the block pictures from their corresponding stained sections. Follow up experiments showed that the present coordinates are accurate for these monkeys of restricted body dimensions. In addition, referencing visual axonal projections onto the same cytoarchitectural map in stereotaxic coordinates provides an atlas for localizing areas of the thalamus, on a basis other than cytoarchitecture, which receive combinations of visual inputs for further anatomical and physiological studies of the rhesus monkey visual system. The atlas further demonstrates that projections do not necessarily follow the cytoarchitectural definition of an area, but rather redefine the thalamus on the basis of specific axonal connections.

Animals↗
PubMed1994

Overlapping and distinct actions of the neurotrophins BDNF, NT-3, and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon.

The neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) were compared for their effects in promoting the survival and/or regulation of expression of phenotypic markers of dopaminergic and GABAergic neurons in cultures derived from embryonic rat ventral mesencephalon. Dopaminergic neuron number and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunocytochemistry, and measurement of high-affinity dopamine uptake activity and dopamine content, respectively. High-affinity GABA uptake, glutamic acid decarboxylase (GAD) activity, and endogenous GABA content were used to detect GABAergic neurons. Seven days of treatment with either BDNF or NT-3 resulted in dose-dependent increases in the number of TH-positive neurons, with maximal responses of 3-fold and 2.3-fold, respectively. Dopamine uptake activity and dopamine content were similarly increased. The effects of BDNF and NT-3 on dopamine uptake activity showed no additivity. NT-4/5 treatment elicited the greatest increase (7-fold) in the number of TH-positive neurons, as well as a 2.6-fold increase in dopamine content. In marked contrast to BDNF or NT-3, NT-4/5 had no effect on dopamine uptake capacity. BDNF, NT-3, or NT-4/5 also produced dose-dependent elevations of 2-3-fold in GABA uptake activity. These effects were not additive. GAD activity was increased by BDNF (1.8-fold) and NT-3 (threefold) treatment, but not by NT-4/5, whereas GABA content was increased to a similar extent by all three neurotrophins. NGF had no effect on any of the parameters measured in this study. Northern analyses indicated that the mRNAs encoding TrkB and TrkC, the functional high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively, are expressed in the substantia nigra of adult rat brain, as well as in cultures of developing ventral mesencephalon. Taken together, our results indicate that BDNF and NT-3 have broadly similar effects in promoting the survival and differentiated phenotype of both dopaminergic and GABAergic neurons of the developing substantia nigra. Although BDNF and NT-4/5 are thought to act through the same high-affinity receptor, TrkB, it is evident that these two neurotrophins have distinct as well as overlapping actions toward mesencephalic dopaminergic or GABAergic neurons.

Animals↗
PubMed1995

Morphological study of microglia in human mesencephalon during the development and aging.

To assess the cytogenesis and the structure of the microglial cells, we studied mesencephalons in 47 human fetuses at 7th-40th week of gestational age, and in 18 adult brains from 20 to 70 years. The microglial cells were identified and characterized by morphological criteria using immunohistochemical and histochemical techniques. As early as in the 8th week of gestational age RCA-1 positive cells were detected, mainly in form of amoeboid microglial cells. These microglial cells were observed around the germinal matrix, and at or near the wall of blood vessels. RCA-1 positive cells which were detected within leptomeninges were large but without processes. At the 16th-40th week of gestational age we observed in mesencephalon, amoeboid microglial cells, and also RCA-1 positive and ferritin reactive ramified microglial cells.

Adult↗
PubMed1997

Ontogenesis of embryonic porcine ventral mesencephalon in the perspective of its potential use as a xenograft in Parkinson's disease.

Human fetal neural dopaminergic tissue can be transplanted and can ameliorate neurological deficiencies in patients with Parkinson's disease (PD). Donor tissue from other species has been used experimentally for several years in animal experiments and is now being considered an attractive alternative, particularly from a donor species that breeds in large litters, e.g., the pig. We have studied the early ontogenetic development of the mesencephalic dopaminergic system in the pig, utilising an anti-tyrosine hydroxylase (TH) immunocytochemical technique, and demonstrated the earliest appearance of its cell bodies at embryonic day 20 (E20). We compared the porcine data with those of human fetal development, as revealed by the same technique. Embryonic dopaminergic cell groups resembling the A8, A9, and A10 of the rat are present in the pig and differentiate into the homologous cell groups of human, although interesting quantitative differences are apparent. In the pig, prolonged presence of immature characteristics of TH-immunoreactive (TH-i.r.) cell bodies was observed, notwithstanding the early outgrowth of TH-i.r. axons into the ganglionic eminence. In the human, on the other hand, cell divisions and maturation of dendrites have progressed to a further degree than in the pig, before such distinct outgrowth of axons takes place. In pig embryos of 28 days, cells in the ventral mesencephalon had differentiated into TH containing neurons, which indicates their potential to synthesize dopamine. In spite of their differentiation, these cells still showed immature morphological features (rounded cell bodies with undifferentiated, short processes). Dopamine synthesis by these cells was demonstrated in previous studies by the high performance liquid chromatographic technique (HogenEsch et al. [1993] Can. J. Neurol. Sci. 20(suppl. 4):P.S. 235). In a separate paper, we have described that these porcine 28-day dopaminergic cells retain their potential for development and outgrowth in culture (van Roon et al. [1995] Res. Neurol. Neurosci. 7:199-205). We conclude that the ventral mesencephalon in pig embryos of 28 days is a potential source of dopaminergic neurons to be used as a xenograft in PD.

Animals↗
PubMed1978

Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat.

Amygdalotegmental projections were studied in 26 cats after injections of horseradish peroxidase (HRP) in the diencephalon, midbrain and lower brain stem and in 6 cats after injection of 3H-leucine in the amygdala. Following HRP injections in the posterior hypothalamus, periaqueductal gray (PAG) and tegmentum many retrogradely labeled neurons were present in the central nucleus (CE) of the amygdala, primarily ipsilaterally. Injections of HRP in the posterior hypothalamus and mesencephalon also resulted in the labeling of neurons in the basal nucleus, pars magnocellularis. Following 3H-leucine injections in CE and adjacent structures autoradiographically labeled fibers were present in the stria terminalis and ventral amygdalofugal pathways. In the mesencephalon heavily labeled fiber bundles were located lateral to the red nucleus. Labeled fibers and terminals were distributed to the mesencephalic reticular formation, substantia nigra, ventral tegmental area and PAG. In the pontine and medullary tegmentum the bulk of passing fibers was located laterally in the reticular formation. Many labeled fibers and terminals were distributed to the parabrachial nuclei, locus coeruleus, nucleus subcoeruleus and lateral tegmental fields. Many terminals were also present in the solitary nucleus and dorsal motor nucleus of the vagus nerve. The location of the cells of origin and the distribution of the terminals of the amygdalotegmental projection suggest that this pathway plays an important role in the integration of somatic and autonomic responses associated with affective defense.

Amygdala↗
PubMed1995

Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon.

Embryonic substantia nigra cells when transplanted into the striatum can reverse many of the defects of Parkinson's disease. The efficacy of such grafts is compromised by the poor survival of grafted dopaminergic neurones; typically, 3-10% survive transplantation. We used three tissue culture models to identify stages in the procedure for the preparation and insertion of grafts which might be responsible for this cell death and to identify environments in which survival is optimised. (1) The ventral mesencephalon was dissected from the donor brain, then placed immediately into culture contained in a collagen gel. (2) The dissected tissue fragments were enzymatically dissociated, then the cells placed into monolayer culture. (3) Enzymatically dissociated tissue was packed into 0.5-mm-diameter porous tubes, to simulate the compaction of cells into a graft deposit in the host brain. Dissociation of the tissue by itself caused the death of approximately 30% of dopaminergic neurones, as judged by the difference in cell counts between the intact embryonic day 14 (E14) mesencephalon, and cells dissociated then packed into tubes. Of the dissociated neurones approximately 60% died during the first 24 h and 87% during the first 3 days in monolayer culture, while only 7% of dopaminergic neurones in three-dimensional cultures and 11% of neurones in explant cultures died over the first 3 days. Embryonic dopaminergic neurones are clearly very vulnerable to adverse conditions during the first days after their removal from the donor brain. The excellent survival of neurones in three-dimensional and explant cultures indicates that close association with other cells, which may provide greatly improved access to trophic factors, can enable the cells to survive this period of vulnerability. In contrast to its effects in monolayer cultures, bFGF had no effect on dopaminergic neuronal survival in either explant or three-dimensional cultures.

Animals↗
PubMed1990

Facilitation of sexual receptivity in hamsters by simultaneous progesterone implants into the VMH and ventral mesencephalon.

Intracranial implantation experiments have shown that the ventromedial hypothalamus (VMH) is the most sensitive site for the facilitation of female sexual behavior by progesterone in estrogen-primed rats. However, similar implantation techniques have been much less successful in hamsters. Several lines of evidence indicate that both hypothalamic and midbrain structures are important for hamster lordosis. Therefore we compared the effect of progesterone (P) implants administered simultaneously to VMH and ventral midbrain on opposite sides of the brain to the effects of bilateral implants to each of these sites separately. Ovariectomized female hamsters were stereotaxically implanted with 24-gauge thin-wall guide tubes according to one of five patterns. Bilaterally symmetrical cannulae were aimed at VMH or ventral mesencephalon (vMES) or asymmetrical implants were aimed at one of the following pairs of sites, on opposite sides of the brain: VMH-vMES, VMH-preoptic area (VMH-POA), or anterior hypothalamus-anterior mesencephalon (AH-aMES). After recovery from surgery, females were primed with 10 micrograms estradiol benzoate and given pellets of P or cholesterol through a 30-gauge injector in the targeted sites. Latency, frequency, and duration of lordosis were recorded in 10-min tests with sexually active male hamsters. Sexual receptivity was significantly facilitated by simultaneous contralateral P implants into the VMH-vMES. P implants in any other combination of sites did not significantly facilitate lordosis compared to cholesterol control implants, nor did bilateral administration of this dose of P in either VMH or vMES have a reliable effect. The results support the hypothesis that P action is required in both VMH and vMES to reliably stimulate receptivity in hamsters.

Animals↗
PubMed2001

The expression of mRNAs for the proteasome complex is developmentally regulated in the rat mesencephalon.

The proteasome is a large protease complex that recognizes, unfolds and degrades ubiquitinated proteins. Evidence is now accumulating that the ubiquitin-proteasome system may play an important role in neuronal apoptosis. However, little is known about the involvement of the proteasome in neuronal death in vivo, and there has been no prior analysis of the developmental expression of proteasome subunits in brain during periods of natural and inducible apoptotic death. We therefore studied the mRNA expression levels, using Northern analysis, of a subunit from each of the three key components of the proteasome in the rat mesencephalon from E21 through development and in adulthood. We measured mRNA expression for RC6 (a subunit of 20S), p112 (a subunit of 19S) and PA28-alpha (a subunit of 11S). The expression of PA28-alpha in rat mesencephalon was highest at the earliest times studied, and then decreased at PND 21, 28 and adult, in comparison to E21 (P<0.05) and PND 2, 4 and 7 (P<0.01). The expression of RC6 was lower in adult in comparison to PND 2, 4 and 21 (P<0.05) and PND 14 (P<0.01). There were no significant differences in the mRNA levels of p112 at various times studied. In situ hybridization at PND 7 indicated that all the subunits studied are particularly abundant in the SNpc. Thus, PA28-alpha and RC6 are developmentally regulated, and they may therefore play a role in developmental cell death or differentiation in neurons of the SN.

Animals↗
PubMed1995

Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

Aged↗
PubMed1992

Light and Electron Microscopic Localization of the Neutral Metalloendopeptidase EC 3.4.24.16 in the Mesencephalon of the Rat.

The topographic and cellular distribution of the neurotensin-hydrolysing neutral metalloendopeptidase 24.16 (EC 3.4.24.16) was examined by light and electron microscopic immunohistochemistry in adult rat mesencephalon. Light microscopic immunoradioautography revealed a ubiquitous distribution of the enzyme throughout the midbrain with a relative enrichment of grey matter areas including the substantia nigra, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, rostral and caudal linear raphe nuclei, central grey and superficial grey of the superior colliculus. Peroxidase - antiperoxidase immunocytochemistry revealed two distinct cellular patterns of immunostaining: (1) weakly labelled neuronal perikarya more or less uniformly distributed throughout the grey matter, and (2) intensely immunoreactive glial cells heterogeneously distributed across the mesencephalon. Areas exhibiting dense concentrations of endopeptidase 24.16-containing glial cells corresponded to those displaying enhanced immunoreactivity in immunoradioautographs, suggesting that a major proportion of brain endopeptidase 24.16 is associated with glia. Electron microscopic examination of the substantia nigra and ventral tegmental area confirmed the association of the enzyme with a subpopulation of neurons and allowed identification of labelled glial cells as protoplasmic astrocytes. In neurons, endopeptidase 24.16 immunoreactivity was distributed heterogeneously within the cytoplasm of perikarya, dendrites and axons. Reaction product was also characteristically associated with restricted zones of the plasma membrane and underlying neuroplasm. In astrocytes, endopeptidase 24.16 immunostaining was densely and uniformly distributed throughout the cytoplasm of cell bodies and processes. Many of these processes were in direct contact with endopeptidase 24.16-immunopositive neuronal elements. The present results demonstrate that within the midbrain, endopeptidase 24.16 is both intracytoplasmic and membrane-associated in neurons and predominantly intracytoplasmic in glia. The presence of a large number of immunostained elements within areas of the midbrain known to display high levels of neurotensin and/or neurotensin receptors, together with the demonstrated catabolic activity of the enzyme on neurotensin in vitro, is consistent with a role of endopeptidase 24.16 in the functional inactivation of endogenous neurotensin in this region of the brain.

Journal Article↗
PubMed1984

VIP-like and GFAP-like immunoreactivities in the chicken brain stem. II. The pons and the tegmentum of the mesencephalon.

An immunocytochemical analysis was performed on the chicken pons and mesencephalon (except the optic tectum) according to the PAP-DAB procedure, to study the distribution here of the neurons reacting to anti-VIP antibodies and the gliocytes reacting to anti-GFAP antibodies. Positive and negative controls were carried out in both the immunoreactions. The VIP-immunoreactive neurons showed a distribution essentially corresponding to that observed in other species by various Authors. They appeared scattered mainly in 3 sites: (a) the subventricular grey between pons and mesencephalon; (b) the periaqueductal grey, up to the diencephalon; (c) the rostro-ventral portion of the mesencephalic tegmentum, up to the diencephalon. Furthermore, some perivascular VIP-immunoreactive neuronal processes were seen. No differences have so far been detected as regards the GFAP-like immunoreactivity distribution, in comparison with the data reported by the authors in the chicken medulla and by others in in the brain stem of some other species.

Animals↗
PubMed1998

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: a double labeling immunohistochemical study.

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP.

Animals↗
PubMed1999

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins.

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.

Animals↗