Tyrosine Hydroxylase-immunoreactive Neurons Research Articles

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

Transplantation of fetal dopaminergic (DA) neurons offers an experimental therapy for Parkinson's disease (PD). The low availability and the poor survival and integration of transplanted cells in the host brain are major obstacles in this approach. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with growth- and survival-promoting capabilities for developing DA neurons. In the present study, we examined whether pretreatment of ventral mesencephalic (VM) free-floating roller tube (FFRT) cultures with GDNF would improve graft survival and function. For that purpose organotypic cultures of E14 rat VM were grown for 2, 4 or 8 days in the absence (control) or presence of GDNF [10 ng/ml] and transplanted into the striatum of 6-hydroxydopamine-lesioned rats. While all groups of rats showed a significant reduction in d-amphetamine-induced rotations at 6 weeks posttransplantation a significantly improved graft function was observed only in the days in vitro (DIV) 4 GDNF pretreated group compared to the control group. In addition, no statistical significant differences between groups were found in the number of surviving tyrosine hydroxylase-immunoreactive (TH-ir) neurons assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts. In sum, our findings demonstrate that GDNF pretreatment was not disadvantageous for transplants of embryonic rat VM with the FFRT culture technique but only marginally improved graft survival and function.

Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the rock hyrax, Procavia capensis

The nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems within the brain of the rock hyrax ( Procavia capensis) were identified following immunohistochemistry for acetylcholinesterase, tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems by comparing those of the rock hyrax to published studies of other mammals. The rock hyrax belongs to the order Hyracoidea and forms part of the Afroplacentalia mammalian cohort. For the most part, the nuclear organization of these three systems closely resembled that described for many other mammalian species. The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals. The nuclear organization of the putative catecholaminergic system was very similar to that seen in rodents except for the lack of a C3 nucleus and the compact division of the locus coeruleus (A6c). In addition, the diffuse locus coeruleus (A6d) appeared to contain very few tyrosine hydroxylase immunoreactive (TH+) neurons. The cholinergic system showed many features in common with that seen in both rodents and primates; however, there were three differences of note: (1) cholinergic neurons were observed in the anterior nuclei of the dorsal thalamus; (2) cholinergic parvocellular nerve cells, probably representing interneurons, forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were observed at the midbrain/pons interface; and (3) a large number of cholinergic nerve cells in the periventricular grey of the medulla oblongata were observed. Thus, while there are many similarities to other mammalian species, the nuclear organization of these systems in the rock hyrax shows specific differences to what has been observed previously in other mammals. These differences are discussed in both a functional and phylogenetic perspective.

Phenotype of neurons in the nucleus of the solitary tract that express CCK-induced activation of the ERK signaling pathway

The satiating potency of CCK has been well characterized, including its mediation by capsaicin-sensitive vagal primary afferents. We have previously shown that peripherally administered CCK activates the MAPK-signaling cascade in a population of nucleus of the solitary tract (NTS) neurons and that preventing ERK1/2 phosphorylation partly attenuates CCK's satiating potency. The aim of this study was to identify the neurochemical phenotypes of the NTS neurons that exhibit CCK-induced activation of ERK1/2. Using confocal microscopy, we demonstrate that intraperitoneal CCK administration increases the number of neurons that express phosphorylated ERK1/2 (pERK1/2) in the medial and commissural subnuclei of the NTS and that CCK-induced expression of ERK1/2 is increased in tyrosine hydroxylase-immunoreactive neurons. Using Western blot analysis, we show that the robust increase in tyrosine hydroxylase phosphorylation obtained with intraperitoneal CCK is significantly attenuated in rats pretreated with the ERK-pathway blocker U0126 injected into the 4th ventricle. In addition, CCK injections increased pERK1/2 expression in POMC neurons in the NTS. In contrast, only the rare GAD67, neuronal nitric oxide synthase, and leptin-responsive neuron exhibited CCK-induced pERK immunoreactivity. We conclude that activation of POMC-immunoreactive neurons and tyrosine hydroxylase activity via the ERK-signaling pathway in the NTS likely contributes to CCK's satiating effects.

Morphological Analyses of Sex Differences and Age-Related Changes in C3H Mouse Midbrain

While it is commonly hypothesized that sexual differentiation in the mammalian brain is initiated mainly by gonadal sex steroids, recent evidence has suggested that dopaminergic (DA) neurons within the rodent midbrain have sex differences independent of gonadal secretions. More recently, it has been reported that Sry (the sex-determining region of the Y chromosome) is directly involved in this difference. The possibility of sexual dimorphism in the mouse midbrain needs to be elucidated. In the present study, the midbrain of C3H mice, which is little understood, was investigated histologically and immuno-histoplanimetrically to reveal sexual and developmental differences. The female ventral tegmental area appeared to contain higher immunoreactivity to tyrosine hydroxylase (TH) than that of males at 11 weeks of age, whereas general histological differences between the sexes were not clearly found. The TH-immunoreactive (TH-ir) neurons within the A8, A9, and A10 mesencephalic areas were examined separately. There was the sex difference in the time period when TH-ir cell numbers significantly increased, indicating that the growth rate of midbrain DA nuclei also differs and that the midbrain DA system may trace different processes of sexual maturation between the sexes. These differences between female and male may reflect the direct regulation by Sry or the multiple effects of both Sry and sex steroids. Further experiments are needed to determine which factor forms this difference in the growth pattern in the numbers of TH-ir neurons.

Immunolocalization of alpha-synuclein in the rat spinal cord by two novel monoclonal antibodies

This study provides the first immunohistochemical evidence of the presence and distribution patterns in the rat spinal cord of α-synuclein (α-Syn), a soluble acidic protein, widely expressed in the CNS and closely associated to the pathogenesis of neurodegenerative conditions such as Parkinson's and Alzheimer's diseases. We used two novel homemade monoclonal antibodies (2E3 and 3D5) recognizing two different epitopes of α-Syn. Both antibodies localized α-Syn within the nerve terminals, whereas 3D5 alone also localized it within the neuronal nuclei. α-Syn-immunoreactive nervous elements were widely recognized throughout rat spinal cord and in almost all the gray matter laminae. However, they appeared particularly concentrated within laminae I, II, VII and X and more scattered in the others. Double immunofluorescent labeling showed that α-Syn colocalized with synaptophysin in the presynaptic nerve terminals, with neuropeptide Y (NPY) in lamina I, II, IX and X, and had close relationships with tyrosine hydroxylase (TH) immunoreactive neurons in laminae VII and X. Interestingly, the α-Syn-immunoreactive nerve elements, in lamina X, contained little of calbindin-28KD and calretinin-31KD. Our findings could help in understanding the genesis of some early clinical symptoms of Parkinson's disease (PD), such as pain and dysautonomic disorders, and indicate the spinal cord as their probable starting point, according to the ascending theory of PD, proposed by Braak.

Species-specific distributions of tyrosine hydroxylase–immunoreactive neurons in the prefrontal cortex of anthropoid primates

In this study, we assessed the distribution of cortical neurons immunoreactive for tyrosine hydroxylase (TH) in prefrontal cortical regions of humans and nonhuman primate species. Immunohistochemical methods were used to visualize TH-immunoreactive (TH-ir) neurons in areas 9 (dorsolateral prefrontal cortex) and 32 (anterior paracingulate cortex). The study sample included humans, great apes (chimpanzee, bonobo, gorilla, orangutan), one lesser ape (siamang), and Old World monkeys (golden guenon, patas monkey, olive baboon, moor macaque, black and white colobus, and François' langur). The percentage of neurons within the cortex expressing TH was quantified using computer-assisted stereology. TH-ir neurons were present in layers V and VI and the subjacent white matter in each of the Old World monkey species, the siamang, and in humans. TH-ir cells were also occasionally observed in layer III of human, siamang, baboon, colobus, and François' langur cortex. Cortical cells expressing TH were notably absent in each of the great ape species. Quantitative analyses did not reveal a phylogenetic trend for percentage of TH-ir neurons in these cortical areas among species. Interestingly, humans and monkey species exhibited a bilaminar pattern of TH-ir axon distributions within prefrontal regions, with layers I–II and layers V–VI having the densest contingent of axons. In contrast, the great apes had a different pattern of laminar innervation, with a remarkably denser distribution of TH-ir axons within layer III. It is possible that the catecholaminergic afferent input to layer III in chimpanzees and other great apes covaries with loss of TH-ir cells within the cortical mantle.

Persistent MDMA‐induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter-immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH-immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose-dependent long-lasting decrease in TH- and DA transporter-immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA-containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac-1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post-treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.

Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial activation

Parkinson's disease is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra. Accumulating evidence has suggested that inflammation in the brain participates in the pathogenesis of Parkinson's disease. Luteolin, a polyphenolic compound found in foods of plant origin, belongs to the flavone subclass of flavonoids, and has been shown to possess antimutagenic, antitumorigenic, antioxidant and antiinflammatory properties. In this study, we found that luteolin concentration-dependently attenuated the lipopolysaccharide (LPS)-induced decrease in [ 3H]dopamine uptake and loss of tyrosine hydroxylase-immunoreactive neurons in primary mesencephalic neuron–glia cultures. Moreover, luteolin also significantly inhibited LPS-induced activation of microglia and excessive production of tumor necrosis factor-α, nitric oxide and superoxide in mesencephalic neuron–glia cultures and microglia-enriched cultures. Our results demonstrate that luteolin may protect dopaminergic neurons from LPS-induced injury and its efficiency in inhibiting microglia activation may underlie the mechanism.

Preventive effects of genistein on motor dysfunction following 6-hydroxydopamine injection in ovariectomized rats

Estrogen and phytoestrogens such as the isoflavones have received considerable attention in Parkinson's disease (PD) research. Because they have been reported to possess neuroprotective effects on dopaminergic neurons in the substantia nigra (SN), isoflavones appear particularly promising for post-menopausal women at risk for PD. However, most previous studies were limited to morphological investigation, and the preventive effects of isoflavones on motor function have not been evaluated. The aim of the present study was to elucidate the prevention by an isoflavone against motor dysfunction after injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle of ovariectomized rats, which mimics post-menopausal status in women. Pretreatment with genistein, an isoflavone, significantly preserved motor function in rats injected with low-dose 6-OHDA, as evaluated by the stepping and cylinder tests. An estrogen receptor antagonist, ICI182780, reversed the effects of genistein, indicating that this effect of genistein is mediated through estrogen receptors. The functional effects of genistein were accompanied by preservation of tyrosine hydroxylase-immunoreactive neurons in the SN after injection of low-dose 6-OHDA. These findings suggest that genistein may be useful for the prevention of PD in post-menopausal women.

Manganese Induces Dopaminergic Neurodegeneration via Microglial Activation in a Rat Model of Manganism

Manganese is an essential trace element required for normal development and bodily functions. However, exposure of the brain to excessive amounts of manganese results in neurotoxicity. Although previous studies examining manganese neurotoxicity have focused on neuronal injury, especially direct injury to dopaminergic neurons, the effects of manganese-induced neurotoxicity on glial cells have not been reported. The current study was designed to examine the effect of manganese on microglial activation, and the underlying mechanism of manganese-induced dopaminergic neuronal injury in vivo. We established an animal model of manganism by intrastriatal injection of MnCl(2).4H(2)O into male Sprague-Dawley rats. One day after administration of manganese, a few microglial cells in the substantia nigra (SN) were activated, although the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the SN was unaffected. Seven days after administration of manganese, a marked reduction in the number of TH-immunoreactive neurons was observed in the SN, and the majority of microglial cells were activated. We found that manganese upregulated inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha) gene expression, as well as iNOS, TNF-alpha, and interleukin-1beta (IL-1beta) protein levels in the SN. Furthermore, treatment with minocycline, an inhibitor of microglial activation, attenuated microglial activation and mitigated IL-1beta, TNF-alpha, and iNOS production as well as dopaminergic neurotoxicity induced by manganese. These results suggested that dopaminergic neurons could be damaged by manganese neurotoxicity, and that the activated microglial cells and their associated activation products played an important role in this neurodegenerative process.

Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson’s disease

Parkinson's disease is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. We investigated whether cell therapy with human mesenchymal stem cells (hMSCs) had a protective effect on progressive dopaminergic neuronal loss in vitro and in vivo. In primary mesencephalic cultures, hMSCs treatment significantly decreased MG-132-induced dopaminergic neuronal loss with a significant reduction of caspase-3 activity. In rats received systemic injection of MG-132, hMSCs treatment in MG-132-treated rats dramatically reduced the decline in the number of tyrosine hydroxylase (TH)-immunoreactive cells, showing an approximately 50% increase in the survival of TH-immunoreactive cells in the substantia nigra compared with the MG-132-treated group. Additionally, hMSC treatment significantly decreased OX-6 immunoreactivity and caspase-3 activity. Histological analysis showed that the number of NuMA-positive cells was 1.7% of total injected hMSCs and 35.7% of these cells were double-stained with NuMA and TH. Adhesive-removal test showed that hMSCs administration in MG-132-treated rats had a tendency to decrease in the mean removal time. This study demonstrates that hMSCs treatment had a protective effect on progressive loss of dopaminergic neurons induced by MG-132 in vitro and in vivo. Complex mechanisms mediated by trophic effects of hMSCs and differentiation of hMSCs into functional TH-immunoreactive neurons may work in the neuroprotective process.

Effects of Sildenafil on Nigrostriatal Dopamine Neurons in a Murine Model of Parkinson's Disease

The objective of this study was to determine if the phosphodiesterase 5 (PDE-5) inhibitor, sildenafil, could be used as a neuroprotective agent in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of Parkinson's disease. The underlying hypothesis of these studies is that blockade of PDE-5 catabolism of cGMP will attenuate the loss of nigrostriatal dopamine (NSDA) neurons following chronic neurotoxin exposure. Chronic MPTP-treated mice were administered sildenafil using three different regimens. Animals were: 1) treated with sildenafil and then exposed to chronic MPTP; 2) treated concurrently with sildenafil and MPTP; and 3) first exposed to MPTP and subsequently treated with sildenafil. End points of neurotoxicity included dopamine (DA) and tyrosine hydroxylase (TH) concentrations in NSDA axon terminals in the striatum, and stereological cell counts of TH immunoreactive neurons in the substantia nigra. Results reveal that sildenafil did not prevent neurotoxicity produced by chronic MPTP exposure regardless of the treatment paradigms employed. On the other hand, sildenafil did not produce any deleterious effect on NSDA neuron function nor did it potentiate the neurotoxic effects of MPTP. These results suggest that sildenafil would not accelerate DA cell loss when used as a treatment for erectile dysfunction in men diagnosed with Parkinson's disease.

The dopaminergic system in the brain of the native Thai chicken, Gallusdomesticus: Localization and differential expression across the reproductive cycle

Dopamine (DA) has a pivotal role in avian prolactin (PRL) secretion, acting centrally through D 1 DA receptors to stimulate PRL secretion by operating through vasoactive intestinal peptide (VIP). DA also inhibits PRL secretion by activating D 2 DA receptors at the pituitary level. This study was designed to investigate the distribution of DA neurons in the native Thai chicken, utilizing tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. The differential expression of hypothalamic TH immunoreactive (TH-ir) neurons was also compared across the reproductive cycle. The results revealed that TH-ir neurons and fibers were found throughout the brain of the laying hen and were predominantly located within the diencephalon and mesencephalon. The observed distribution pattern of TH immunoreactivity was consistent with that reported previously in several avian species. However, changes in the number of TH-ir neurons in the nucleus intramedialis (nI) were observed across the reproductive cycle and correlated directly with variations in PRL levels. The population of TH-ir neurons in the nI increased significantly during the egg incubation period, where circulating PRL levels were the greatest. This study indicates, for the first time, that an association exists between DA neurons and the regulation of the reproductive system in the native Thai chicken. There is a paucity of information about the reproductive neuroendocrine regulation of tropical non-seasonally breeding avian species and it is suggested that the differential expression of DA neurons in the nI might play a role in the control of VIP secretion and subsequent PRL release in such birds.

脂多糖预处理改善脂多糖诱导的中脑脑片神经元损伤

To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA). In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05). Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Gender‐related changes in increase of dopaminergic neurons in the olfactory bulb of Parkinson's disease patients

Gender differences in dopaminergic related neurodegenerative diseases have hardly been studied until now. It is generally accepted that more men than women suffer from Parkinson's disease. One of the most prevalent symptoms in Parkinson's patients, hyposmia, does not show gender differences, while normally the sense of smell is better developed in females. Whether the change in dopamine in the olfactory bulb contributes equally to hyposmia in male and female Parkinson's patients is the subject of the present study. In a stereological study the total number of tyrosine hydroxylase immunoreactive neurons in the olfactory bulbs of male and female Parkinson's patients and age-matched controls has been estimated. The present stereological study shows that the number of tyrosine hydroxylase positive cells in control females is significantly lower than those in control males. The number of dopaminergic cells in the olfactory bulbs of both male and female Parkinson's patients equals that of healthy males of the same age group. We therefore conclude that the hyposmia in Parkinson's disease patients cannot simply be ascribed to dopamine in the olfactory bulb.

Combined use of BDNF, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells

Freshly isolated fetal midbrain neural precursor cells (NPCs) that maintain the potential to differentiate into dopamine (DA) neurons represent a valuable source for cell therapy in Parkinson's disease. However, it is poorly understood why midbrain NPCs lose their dopaminergic differentiation potential after long-term culture. Here we report that human fetal midbrain NPCs can be extensively proliferated with fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) and efficiently differentiated into tyrosine hydroxylase-immunoreactive (TH-ir) neurons. We tested differentiation conditions including the use of low oxygen, ascorbic acid, and prolonged in vitro differentiation time which resulted in a 10-fold increase in the number of MAP2-positive neurons (up to 40–50% of total cells as compared to controls). Under these conditions TH-ir cells constituted 4.3±0.5% of the neuronal population and displayed immature morphologies. Notably, the use of brain-derived neurotrophic factor (BDNF) further increased the proportion of TH-ir neurons (up to 15% of total neurons). In contrast to previous reports, our findings demonstrate that long-term expanded fetal NPCs can generate TH-expressing cells under the appropriate culture conditions and without genetic manipulations.

Tyrosine hydroxylase immunoreactivity in the locus coeruleus is elevated in violent suicidal depressive patients

Our postmortem study aimed to determine the impact of suicide on the number of noradrenergic neurons of the locus coeruleus (LC) in suicidal depressive patients. Noradrenergic neurons were shown by immunostaining tyrosine hydroxylase in the LC of 22 non-elderly patients with mood disorders compared to 21 age- and sex-matched normal controls. Eleven patients were suicide victims and the other eleven died of natural causes. Seven violent suicide victims revealed an increased number of tyrosine hydroxylase immunoreactive (TH-ir) neurons compared with non-violent suicide victims and controls. No difference was found between the number of TH-ir neurons in all suicidal patients and controls and between non-suicidal patients and controls. The differences of TH-immunoreactivity could neither be attributed to medication nor to the polarity of depressive disorder (unipolar/bipolar). The numbers of TH-ir neurons in suicidal patients correlated negatively with the mean doses of antidepressants. The study suggested a presynaptic noradrenergic dysregulation in the LC related to the level of self-aggression. Traditional antidepressants may, therefore, regulate noradrenergic activity of the LC in suicide patients, however, without demonstrating the suicide-preventing effect.

Neurochemical characterization of tyrosine hydroxylase-immunoreactive interneurons in the developing rat cerebral cortex

Understanding the development of cortical interneuron phenotypic diversity is critical because interneuron dysfunction has been implicated in several neurodevelopmental disorders. Here, tyrosine hydroxylase (TH)-immunoreactive neurons in the developing and adult rat cortex were characterized in light of findings regarding interneuron neurochemistry and development. Cortical TH-immunoreactive neurons were first observed 2 weeks postnatally and peaked in number 3 weeks after birth. At subsequent ages, the number of these cell profiles was gradually reduced, and they were seen less frequently in adults. No DNA fragmentation or active caspase 3 was observed in cortical TH cells at any age examined, eliminating cell death as an explanation for the decrease in cell number. Although cortical TH cells reportedly fail to produce subsequent catecholaminergic enzymes, we found that the majority of these cells at all ages contained phosphorylated TH, suggesting that the enzyme may be active and producing L-DOPA as an end-product. Morphological criteria and colocalization of some TH cells with glutamic acid decarboxylase suggest that these cells are interneurons. Previously, parvalbumin, somatostatin, and calretinin were demonstrated in non-overlapping subsets of interneurons. Cortical TH neurons colocalized with calretinin but not with parvalbumin or somatostatin. These findings suggest that the transitory increase in TH cell number is not due to cell death but possibly due to alterations in the amount of detectable TH present in these cells, and that at least some cortical TH-producing interneurons belong to the calretinin-containing subset of interneurons that originate developmentally in the caudal ganglionic eminence.

Estradiol-Induced Synaptic Remodeling of Tyrosine Hydroxylase Immunopositive Neurons in the Rat Arcuate Nucleus

Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.

Dopaminergic mesencephalic systems and behavioral performance in very old rats

Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson's disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4–6 months), old (22–24 months) and senile (30–32 months) Sprague–Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.