Number Of TH-ir Neurons Research Articles

Individual Differences in the Expression of Tyrosine Hydroxylase mRNA in Neurosecretory Neurons of the Human Paraventricular and Supraoptic Nuclei: Positive Correlation with Vasopressin mRNA

Previous studies indicated that in the human paraventricular nucleus (PVN) and in the supraoptic nucleus (SON) tyrosine hydroxylase (TH) – the first and rate-limiting enzyme in catecholamine synthesis – is localized mainly in magnocellular neurosecretory neurons. Individual differences were observed among control subjects in number and distribution of TH-immunoreactive (IR) perikarya, indicating that antemortem factors may regulate TH expression. Since a large number of TH-IR perikarya were observed in subjects who suffered from somatic illnesses leading to prolonged osmotic or nonosmotic stimulation of vasopressin (VP) release, we suggested that TH expression is related to the activation of VP neurons. The purpose of our study was to apply (1) in situ hybridization for TH mRNA on human PVN and SON to investigate how the previously reported individual differences in TH protein expression are depicted at the transcriptional level and (2) quantitative TH immunohistochemistry and in situ hybridization for VP mRNA throughout the dorsolateral part of the SON (dl-SON) in order to elucidate whether indeed expression of TH in neurosecretory nuclei depends on activation of VP neurons. Postmortem formalin-fixed, paraffin-embedded hypothalamic sections of 16 control subjects were studied for TH protein and TH and VP mRNAs. For 6 of the above cases, the number of TH-IR neurons and the total VP mRNA levels were estimated throughout the entire dl-SON using an image analysis system. Individual variation was observed in TH mRNA expression which appears to parallel the expression of TH-protein. Using Spearman’s bivariate test, a positive correlation was found between the number of TH-IR- and TH-mRNA-expressing neurons in both PVN and SON (p < 0.01) as well as between the number of TH-IR neurons and the total VP mRNA in the dl-SON (p < 0.05). Our results show (1) that the individual variability in the number of TH-IR neurons within the neurosecretory nuclei might be due to differential expression and/or stability of TH mRNA and (2) that expression of TH-immunoreactivity in human PVN and SON depends on the activation of VP neurons.

Valproate pretreatment protects dopaminergic neurons from LPS-induced neurotoxicity in rat primary midbrain cultures: role of microglia

Parkinson's disease is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Accumulating evidence supports the notion that neuroinflammation is involved in the pathogenesis of this disease. Valproate (VPA) has long been used for the treatment of seizures and bipolar mood disorder. In vivo and in vitro studies have demonstrated that VPA has neuroprotective and neurotrophic actions. In this study, using primary neuron–glia cultures from rat midbrain, we demonstrated that VPA is a potent neuroprotective agent against lipopolysaccharide (LPS)-induced neurotoxicity. Results showed that pretreatment with 0.6 mM VPA for 48 h robustly attenuated LPS-induced degeneration of dopaminergic neurons as determined by [ 3H] dopamine uptake and counting of the number of TH-ir neurons. The neuroprotective effect of VPA was concentration-dependent and was mediated, at least in part, through a decrease in levels of pro-inflammatory factors released from activated microglia. Specifically, LPS-induced increase in the release of TNFa, NO, and intracellular reactive oxygen species was markedly reduced in cultures pretreated with VPA. These anti-inflammatory effects of VPA were time and concentration-dependent correlated with a decrease in the number of microglia. Thus, our results demonstrate that protracted VPA pretreatment protects dopaminergic neurons from LPS-induced neurotoxicity through a reduction in levels of released pro-inflammatory factors, and further suggest that these anti-inflammatory effects may be contributed by VPA-induced reduction of microglia cell number. Taken together, our study reinforces the view that VPA may have utility in treating Parkinson's disease.

220. Prolonged Biologically Active Transgene Expression Driven by HSV Latency Active Promoter 2 (LAP2) in Brain In Vivo

The herpes simplex virus (HSV) latency associated promoter (LAP2) is a transposable element that drives life-long expression of latency associated transcripts (LATs) from latent HSV genomes. We examined the ability of this promoter to drive prolonged expression of a biologically active transgene from latent HSV vector genomes in brain in vivo using the 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of Parkinson disease. Animals injected with an HSV vector with GDNF under the control of LAP2 five and a half months before 6-OHDA injection showed preservation dopaminergic neuron function in the face of 6-OHDA toxicity measured by amphetamine-induced rotation, number of tyrosine hydroxylase (TH) neurons in the substantia nigra (SN), and immunoreactivity for TH and the dopamine transporter (DAT) in striatum determined 6 months after vector inoculation. The amount of GDNF produced in injected SN determined by ELISA remained stable at 1–2 pg/mg over 6 months. Mice inoculated with the LAP2-GDNF HSV vector followed by 3 months of daily low dose MPTP injection were substantially protected against the consequences of that treatment measured by weekly behavioral testing (contralateral pellet retrieval in a staircase apparatus) and by histologic measures including number of TH-IR neurons in SN, and TH-IR and DAT-IR in striatum determined at the conclusion of the experiment 13 weeks after vector inoculation. These studies demonstrate that the HSV LAP2 promoter element provides prolonged expression of relevant amounts of a transgene to produce significant biological effects in brain in vivo over the course of many months.

Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease.

One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions. In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl). However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation.

Overexpression of Bcl-2 Reduces Sex Differences in Neuron Number in the Brain and Spinal Cord

Several sex differences in the nervous system depend on differential cell death during development in males and females. The anti-apoptotic protein, Bcl-2, promotes the survival of many types of neurons during development and in response to injury. To determine whether Bcl-2 might similarly control cell death in sexually dimorphic regions, we compared neuron number in wild-type mice and transgenic mice overexpressing Bcl-2 under the control of a neuron-specific promoter. Three neural areas were examined: the spinal nucleus of the bulbocavernosus (SNB), in which neuron number is greater in males; the retrodorsolateral nucleus (RDLN) of the spinal cord, which exhibits no sex difference in neuron number; and the anteroventral periventricular nucleus (AVPV) of the hypothalamus, in which both overall cell density and the number of tyrosine hydroxylase immunoreactive (TH-ir) neurons are greater in females. Bcl-2 overexpression significantly increased SNB cell number in females, overall cell density of AVPV in males, and RDLN cell number in both sexes. Bcl-2 overexpression did not alter the number of TH-ir neurons in AVPV of males or females. These findings indicate that Bcl-2 can regulate sexually dimorphic cell number in the brain and spinal cord and suggest that Bcl-2 may mediate effects of testosterone on cell survival during neural development. In contrast to the regulation of overall cell density in AVPV, the sex difference in TH cell number apparently is not caused by a Bcl-2-dependent mechanism.

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons.

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.

Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra.

Nuclear receptor-related factor 1 (Nurr1), a member of the nuclear receptor superfamily, is associated with the induction of dopaminergic (DA) phenotypes in developing and mature midbrain neurons. It is well established that dopaminergic nigrostriatal function decreases with age. Whether age-related deficits in DA phenotypic markers are associated with alterations in Nurr1 expression is unknown. The present study found that virtually all of tyrosine hydroxylase-immunoreactive (TH-ir) neurons within the young adult human substantia nigra were Nurr1-immunoreactive (Nurr1-ir) positive. Stereologic counts revealed a significant reduction in the number of Nurr1-ir nigral neurons in middle-aged (23.13%) and aged (46.33%) individuals relative to young subjects. The loss of Nurr1-ir neurons was associated with a similar decline in TH-ir neuron number. In this regard, TH-ir neuronal number was decreased in middle-aged (11.10%) and in aged (45.97%) subjects, and this loss of TH-ir neurons was highly correlated (r = 0.92) with the loss of Nurr1-ir neurons. In contrast, the number of melanin-containing nigral neuron number was generally stable across age groups, indicating that changes in Nurr1 and TH reflect phenotypic age-related changes and not frank neuronal degeneration. In support of this concept, confocal microscopic analyses of Nurr1-ir and TH-ir fluorescence intensity revealed parallel decreases in Nurr1- and TH-immunofluorescence as a function of age. These data demonstrate that age-related decline of DA phenotypic markers is associated with down-regulation of Nurr1 expression in the SN.

Androgen Effects on Tyrosine Hydroxylase Cells in the Northern Leopard Frog, Rana pipiens

The interaction between gonadal steroids and dopamine neurons has been examined extensively in rodent model systems. However, there have been few investigations examining the functional relation between gonadal steroids and dopaminergic systems in nonmammalian taxa, and none in amphibians. We examined the effects of testosterone (T) and dihydrotestosterone (DHT) on changes in tyrosine hydroxylase immunoreactive (TH-ir) neuron number in the fore- and midbrain of male Rana pipiens, the Northern leopard frog, using a whole-mount immunohistochemical procedure. Gonadectomized males had significantly fewer TH-ir neurons in the medial preoptic area (POA), suprachiasmatic nucleus (SCN), and the caudal hypothalamus/posterior tubercular region (HY/TP) compared with T-implanted males. A follow-up study demonstrated that T- and DHT-implanted males had similar numbers of TH-ir neurons in these three regions compared with intact males and that all three of these groups possessed significantly more TH-ir neurons compared with gonadectomized males. These results suggest that circulating sex steroids have a significant impact on the activity of dopaminergic neurons in male R. pipiens. Although the function of these specific dopaminergic neurons is not yet known, the POA, SCN, and TP/DH are known to be integral brain regions underlying the neural control of reproductive behavior in frogs. These results suggest that dopamine may be important in controlling the behavior or neuroendocrine mechanisms of reproduction in these animals and that dopaminergic activity in these areas is regulated by gonadal steroids.

Streptozotocin-induced diabetes and the neurochemistry of vagal afferent neurons

To assess whether diabetes alters the content and/or expression of neuroactive agents and protooncogenes in afferent neurons of the vagus nerve, the nodose ganglia of streptozotocin (STZ)-induced diabetic rats were studied at 8, 16, and 24 weeks after induction of diabetes. Neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), the immediate early gene c-Jun, vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP) content and expression were measured in nodose ganglia of control, diabetic, and diabetic+insulin-treated rats using immunocytochemistry and reverse trancription-polymerase chain reaction (RT-PCR). The numbers of nNOS-immunoreactive (ir) neurons were increased in the nodose ganglion of diabetic compared to control rats at the 8- and 16-week time points. However, no change was noted in the nNOS mRNA content of the diabetic nodose ganglion at either time point. Moreover, no alterations in the numbers of vagal efferent NOS-containing neurons (labeled with NADPH-diaphorase histochemistry) were noted in the dorsal motor nucleus of the vagus (DMV) or the nucleus ambiguus (NA) of control, diabetic, and diabetic+insulin-treated rats at any time point. Neither the numbers of TH-ir neurons nor the content of TH mRNA was altered in the diabetic rats at the 8- and 16-week time points. However, 24 weeks of diabetes resulted in a reduction in the numbers of TH-ir neurons in the diabetic nodose ganglia when compared to control, an effect not seen in diabetic rats receiving insulin. The number of nodose ganglion neurons labeled for the protooncogene, c-Jun, was small yet slightly increased in the diabetic nodose ganglia at the 8-week time point and was reversed with insulin treatment. The increase in c-Jun-ir neurons was not found at 16 or 24 weeks of diabetes. VIP-ir and CGRP-ir were unchanged at any of the time points. These data show that diabetes affects the content of some, but not all, neuroactive agents in the nodose ganglion and may reflect a modest level of diabetes-induced damage and/or alterations in axonal transport in the vagus nerve.

Group I mGluR antagonist AIDA protects nigral DA cells from MPTP-induced injury.

The effects of i.c.v. injection of AIDA, a group I mGluR antagonist, were studied on the nigral DA cells after MPTP-induced injury in the black mouse, using TH immunocytochemistry and unbiased stereology. MPTP reduced the total number of TH-IR neurons by 55.2% and non-TH-IR neurons by 27.5%. A 15 min AIDA pre-treatment (10 nmol) selectively counteracted the loss of TH-IR cells caused by MPTP as evaluated 10 days after the insult without changing the total number of non-neuronal cell nuclei. The results suggest that group I mGluR antagonists may have a neuroprotective role against MPTP-induced degeneration of DA neurons and thus probably also against neurodegenerative processes occurring in Parkinson's disease.

Diminished Survival of Mesencephalic Dopamine Neurons Grafted into Aged Hosts Occurs during the Immediate Postgrafting Interval

The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5–20%, and even poorer in grafts to the aged striatum. Grafted cells die in young adult rats during the first 4 days after implantation. The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals. We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation. At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats. This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting. THir neurons in grafts to the intact striatum possessed a significantly shorter “long axis” than their counterparts on the lesioned side. No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting. In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts.

Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons

Oxidative stress has been implicated in the selective degeneration of dopaminergic (DAergic) neurons in Parkinson’s disease (PD). In this study, we tested the efficacy of EUK-134, a superoxide dismutase (SOD) and catalase mimetic, on the nitration of tyrosine hydroxylase (TH), a marker of oxidative stress, and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP +) and 6-hydroxydopamine (6-OHDA) in primary DAergic neuron cultures. Exposure of cultures to 10 μM MPP + reduced dopamine (DA) uptake and the number of tyrosine hydroxylase immunoreactive (THir) neurons to 56 and 52% of control, while exposure to 30 μM 6-OHDA reduced DA uptake and the number of THir neurons to 58 and 59% of control, respectively. Pretreatment of cultures with 0.5 μM EUK-134 completely protected DAergic neurons against MPP +- and 6-OHDA-induced neurotoxicity. Exposure of primary neuron cultures to either MPP + or 6-OHDA produced nitration of tyrosine residues in TH. Pretreatment of cultures with 0.5 μM EUK-134 completely prevented MPP +- or 6-OHDA-induced nitration of tyrosine residues in TH. Taken together, these results support the idea that reactive oxygen species (ROS) are critically involved in MPP +- and 6-OHDA-induced neurotoxicity and suggest a potential therapeutic role for synthetic catalytic scavengers of ROS, such as EUK-134, in the treatment of PD.

GDNF induces recovery of the nigrostriatal dopaminergic system in the rat brain following intracerebroventricular or intraparenchymal administration.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be a survival and neuroprotective factor for nigrostriatal dopaminergic neurons in vivo. The present study was designed to investigate the possible neuroprotective and restorative role by GDNF for dopaminergic neurons which were exposed to the neurotoxin 6-hydroxydopamine (6-OHDA). We compared neurochemical, morphological and behavioural changes following striatal infusion of GDNF to those following intracerebroventricular (i.c.v.) infusion. Apomorphine-induced rotation showed significant recovery after both types of infusion. Significant recovery of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons and fibers were found in the substantia nigra and striatum following both striatum and i.c.v. infusion except for the number of TH-IR neurons in the i.c.v. infusion group. These results suggest that GDNF induces recovery of the nigrostriatal dopaminergic system, and this indicates a potential usefulness of GDNF for the treatment of Parkinson's disease.

Intrastriatal and Intranigral Grafting of hNT Neurons in the 6-OHDA Rat Model of Parkinson's Disease

The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty-seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT-DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron-specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed.

Effects of prolactin on expression of Fos-related antigens in tyrosine hydroxylase-immunoreactive neurons in subdivisions of the arcuate nucleus

Dual immunohistochemistry was employed to determine the effects of prolactin on expression of Fos and its related antigens (FRA) in tuberoinfundibular dopamine (TIDA) neurons located in the dorsomedial (DM) and ventrolateral (VL) subdivisions of the arcuate nucleus (ARC) in the male rat. Systemic administration of the DA receptor antagonist haloperidol caused a sustained (up to 12 h) increase in plasma prolactin concentrations that was accompanied by a transient increase (at 3 h) in the percentage of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons containing FRA-IR nuclei in the DM-ARC. In contrast, haloperidol caused a prolonged (1.5 to 12 h) decrease in the percentage of TH-IR neurons with FRA-IR nuclei in the VL-ARC. Haloperidol had no effect, however, on the overall number of TH-IR neurons in either of these regions. Co-administration of prolactin antisera (PRL-AB) blocked haloperidol-induced increases in both plasma prolactin concentrations and the percentage of TH-IR neurons expressing FRA in the DM-ARC, but had no effect on haloperidol-induced inhibition of FRA expression in TH-IR neurons in the VL-ARC. Intracerebroventricular (i.c.v.) administration of prolactin also increased the percentage of TH-IR neurons containing FRA-IR nuclei in the DM-ARC, but this effect was of longer duration (up to 6 h) than that of haloperidol in all but the most caudal portion of the DM-ARC. In the VL-ARC, prolactin caused a transient increase (at 1.5 h) in the percentage of TH-IR containing FRA-IR nuclei. These results demonstrate that prolactin regulates immediate early gene expression in TIDA neurons in male rats, and reveal that there are temporal differences in the responsiveness of discrete subpopulations of these neurons to prolactin. Prolactin causes a short-lived increase in FRA expression in TIDA neurons in the VL-ARC which is followed by a more prolonged activation of FRA expression in TIDA neurons in the DM-ARC.

Tyrosine hydroxylase-expressing and/or aromatic L-amino acid decarboxylase-expressing neurons in the mediobasal hypothalamus of perinatal rats: differentiation and sexual dimorphism.

In this quantitative and semiquantitative immunocytochemical study, the authors evaluated the differentiation of neurons expressing tyrosine hydroxylase (TH) and/or aromatic L-amino acid decarboxylase (AADC) in the mediobasal hypothalamus (MBH) of male and female rats on embryonic day 18 (E18), E20, and postnatal day 9 (P9). Four neuronal populations were distinguished according to either enzyme expression or neuron location. The earliest and most prominent first population was represented by TH-immunoreactive (IR)/AADC-immunonegative (IN) neurons that were detected initially at E18 and always were located in the ventrolateral region of the MBH. The second population of TH-IN/AADC-IR neurons was observed first at E20 and, after that time, was distributed dorsomedially. The third minor population of TH-IR/AADC-IR neurons initially was detected at E20 and was located dorsomedially. The fourth population was represented by TH-IR/AADC-IN neurons that were distributed in the dorsomedial region at any studied age. The numbers of TH-IR and AADC-IR neurons increased from their initial detection at E18 and E20 until P9. The area of TH-IR and AADC-IR neurons also increased from E18 to E20 and from E20 to P9, respectively. Both TH-IR and AADC-IR neurons showed sex differences in the neuron number, size, and optic density (OD). The numbers of TH-IR neurons in males exceeded those of females at E20 and at P9, although, at P9, sexual dimorphism was a characteristic only of the ventrolateral population. The area and OD of TH-IR neurons from females exceeded those from males in the entire mediobasal hypothalamus (MBH) at E18 and E20 but only in its dorsomedial region at P9. Sexual dimorphism also was an attribute of AADC-IR neurons at E20 and P9. Their number, size, and OD were significantly higher in females than in males. Thus, the MBH of perinatal rats contained two major populations of TH-IR/AADC-IN or TH-IN-AADC-IR neurons and a minor population of TH-IR/AADC-IR neurons. The differentiating neurons expressing either enzyme showed sexual dimorphism.

Transient Coexistence of Tyrosine Hydroxylase and .GAMMA.-Aminobutyric Acid Immunoreactivities in the Developing Anterior Olfactory Nucleus of the Mouse.

The localization of tyrosine hydroxylase (TH) -immunoreactive (IR) (TH-IR) neurons, which are transiently observed in the anterior olfactory nucleus (AON) of the mouse, was studied. A double-labelling method was used with antibodies against TH, L-3, 4-dihydroxyphenylalanine (DOPA), phenyletha-nolamine-N-methyltransferase (PNMT), γ-aminobutyric acid (GABA), calretinin (CR) and calbindin-D28k (CB). When the total number of TH-IR neurons observed in all regions of the AON obtained from 10- to 14- day-old mice was set at 100%, 30% of TH-IR neurons were observed in the lateral (AOL), 25% in the dorsal (AOD), 20% in the ventral (AOV), 15% in the medial (AOM) and 5% in both the posterior (AOP) and external (AOE) regions. Approximately 40% of the TH-IR neurons in the AOL to the AOV regions also exhibited GABA immunoreactivity. This corresponds to 20% of the total number of TH-IR neurons observed in all regions of the AON, and also corresponds to 12% of the total number of GABA-IR neurons observed in all regions of the AON. However, TH-IR neurons did not exhibit concomitant PNMT, CR, and CB immunoreactivities. These findings suggest that the TH-IR neurons in the AON of the mouse, a proportion of which is also GABA-IR, may function as a source of neuromodulators for the postnatal growth of GABA-IR neurons.

Tyrosine hydroxylase-immunoreactive neurons are decreased in number in the cerebral cortex of Parkinson's disease

Tyrosine hydroxylase (TH)-immunoreactive (IR) neurons and their relationship with Lewy bodies were investigated in Parkinson's disease. Using anti-TH and/or anti-ubiquitin antibodies, we evaluated the cerebral cortices included superior frontal gyrus, precentral gyrus, postcentral gyrus, inferior temporal gyrus, cuneus, cingulate gyrus, short and long gyri of insula, and parahippocampal gyrus from 18 autopsy cases of Parkinson's disease and 16 controls. The appearance of TH-IR neurons in cerebral cortices was suggestive of non-pyramidal interneurons. The mean number of TH-IR neurons and the density of TH-IR fibers in Parkinson's disease were decreased in comparison with the controls. The cognitive impairment in Parkinson's disease has been accounted for by the lesions of the basal nucleus of Meynert, the locus ceruleus, and the cortical Lewy bodies in the cerebral cortex. In addition to these lesions, the global loss of non-pyramidal TH-IR cortical neurons and TH-IR fibers would induce the dysfunction of higher-order control of the neocortex and the limbic system in Parkinson's disease. Double-immunostaining with anti-TH and anti-ubiquitin antibodies did not show the TH-IR neuron with cortical Lewy body in the cerebral cortices of Parkinson's disease. In the cerebral cortices of Parkinson's disease, TH-IR non-pyramidal neurons in which cortical Lewy body is not formed decreased in number.

Isolation and manipulation of rostral mesencephalic tegmental progenitor cells from rat

A technique for isolating mitotic progenitor cells from the embryonic rostral mesencephalon is described. Culture of the progenitor cells in complete media with subsequent staining for neuron specific enolase (NSE) revealed that only 0.6% of the cells were NSE immunoreactive. Coculturing the progenitor cells with established striatal cultures did not result in conversion of any of the cells to the dopamine neuron phenotype (tyrosine hydroxylase immunoreactive (THir) neurons). In contrast, co-culture of progenitor cells with established mesencephalic cultures produced a statistically significant, and in some cases (three of twelve), dramatic increase in the number of THir cells. The THir cells that were present had more pronounced process extension than those observed in mesencephalic mono-cultures. Culturing progenitor cells in transwell baskets that were continuously exposed to media but physically separated from established mesencephalic cultures growing underneath the baskets led to the conversion of only a few progenitor cells to THir neurons in four of twelve transwell studies suggesting that cell-cell contact between progenitor cells and mesencephalic cells is required for the conversion. This co-culture technique also increased the number of THir neurons in the mesencephalic cultures although the increase was not profound enough to explain the increase observed in traditional co-culture. These data suggest that mitotic progenitor cells can be isolated from fetal rat tissue and successfully converted to the dopamine neuron phenotype. Progenitormesencephalic co-culture appears to increase the number of THir cells in both tissue sources mediated in part by soluble factor(s) although cell-cell contact and presumably extracellular matrix proteins play a more substantial role. These progenitor cells may prove useful as a tissue source for transplantation procedures in Parkinson's disease.

Isolation and manipulation of rostral mesencephalic tegmental progenitor cells from rat.

A technique for isolating mitotic progenitor cells from the embryonic rostral mesencephalon is described. Culture of the progenitor cells in complete media with subsequent staining for neuron specific enolase (NSE) revealed that only 0.6% of the cells were NSE immunoreactive. Co-culturing the progenitor cells with established striatal cultures did not result in conversion of any of the cells to the dopamine neuron phenotype (tyrosine hydroxylase immunoreactive (THir) neurons). In contrast, co-culture of progenitor cells with established mesencephalic cultures produced a statistically significant, and in some cases (three of twelve), dramatic increase in the number of THir cells. The THir cells that were present had more pronounced process extension than those observed in mesencephalic mono-cultures. Culturing progenitor cells in transwell baskets that were continuously exposed to media but physically separated from established mesencephalic cultures growing underneath the baskets led to the conversion of only a few progenitor cells to THir neurons in four of twelve transwell studies suggesting that cell-cell contact between progenitor cells and mesencephalic cells is required for the conversion. This co-culture technique also increased the number of THir neurons in the mesencephalic cultures although the increase was not profound enough to explain the increase observed in traditional co-culture. These data suggest that mitotic progenitor cells can be isolated from fetal rat tissue and successfully converted to the dopamine neuron phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)