TH-ir Neurons Research Articles

Reassessment of caspase inhibition to augment grafted dopamine neuron survival.

One experimental therapy for Parkinson's disease (PD) is the transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted neurons die, many via apoptosis. Activated caspases play a key role in execution of the apoptotic pathway; therefore, exposure to caspase inhibitors may provide an effective intervention strategy for protection against apoptotic cell death. In the present study we examined the efficacy of two different caspase inhibitors, caspase-1 inhibitor Ac-YVAD-CMK and caspase-3 inhibitor Ac-DEVD-CMK, to augment mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival in culture and following implantation into the denervated striatum of rats. We report that treatment with Ac-YVAD-CMK provided partial but nonsignificant protection for TH-ir neurons against serum withdrawal in mesencephalic cultures plated at low density, while neither caspase inhibitor promoted TH-ir neuron survival in higher density cultures, simulating graft density. We demonstrate that plating procedures (full well vs. microislands) and cell density directly affect the degree of insult experienced by TH-ir neurons following serum withdrawal. This varying degree of insult directly impacts whether caspase inhibition will augment TH-ir neuron survival. Our grafting experiments demonstrate that Ac-YVAD-CMK does not augment grafted TH-ir neuron survival when added to mesencephalic cell suspensions prior to grafting or to mesencephalic reaggregates for 3 days in vitro prior to transplantation. These experiments provide further evidence of the failure of these caspase inhibitors to augment TH-ir neuron survival. Furthermore, we suggest that cell culture paradigms used to model grafting paradigms must more closely approximate the cell densities of mesencephalic grafts to effectively screen potential augmentative treatments.

Distribution and ultrastructure of dopaminergic neurons in the dorsal motor nucleus of the vagus projecting to the stomach of the rat

Almost all parasympathetic preganglionic motor neurons contain acetylcholine, whereas quite a few motor neurons in the dorsal motor nucleus of the vagus (DMV) contain dopamine. We determined the distribution and ultrastructure of these dopaminergic neurons with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b (CTb) following its injection into the stomach. A few TH-immunoreactive (TH-ir) neurons were found in the rostral half of the DMV, while a moderate number of these neurons were found in the caudal half. Most of the TH-ir neurons (78.4%) were double-labeled for CTb in the half of the DMV caudal to the area postrema, but only a few TH-ir neurons (5.5%) were double-labeled in the rostral half. About 20% of gastric motor neurons showed TH-immunoreactivity in the caudal half of the DMV, but only 0.3% were TH-ir in the rostral half. In all gastric motor neurons, 8.1% were double-labeled for TH. The ultrastructure of the TH-ir neurons in the caudal DMV was determined with immuno-gold-silver labeling. The TH-ir neurons were small (20.4×12.4 μm), round or oval, and contained numerous mitochondria, many free ribosomes, several Golgi apparatuses, a round nucleus and a few Nissl bodies. The average number of axosomatic terminals per section was 4.0. More than half of them contained round synaptic vesicles and made asymmetric synaptic contacts (Gray's type I). Most of the axodendritic terminals contacting TH-ir dendrites were Gray's type I (90%), but a few contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II).

An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality

Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease. Survival rates of grafted dopamine (DA) neurons are extremely poor (5–20%) and is even poorer in grafts to the aged striatum. Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates. However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions. This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting. Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days. All grafts were placed in the denervated striatum of young adult male Fischer 344 rats. Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts. Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery. Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days. Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts. This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.

Effect of glial cell line-derived neurotrophic factor (GDNF) co-transplantation with fetal ventral mesencephalic cells (VMC) on functional restoration in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson’s disease: neurobehavioral, neurochemical and immunohistochemical studies

Among trophic factors already known, glial cell line-derived neurotrophic factor (GDNF) and other members of its family have potent and specific action on dopaminergic neurons. In the present investigation an attempt has been made to validate the role of GDNF co-transplantation with fetal ventral mesencephalic cells (VMC) on functional viability and restoration using neurobehavioral, neurochemical and immunohistochemical parameters at 6 weeks post-transplantation in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson’s disease (PD). A significant restoration (P<0.01) in d-amphetamine induced rotations, spontaneous and apomorphine induced locomotor activity in rats co-transplanted with VMC and GDNF was observed as compared to VMC alone transplanted rats. Level of dopamine (DA), 3,4-dihydroxy-phenyl acetic acid (DOPAC) and dopamine D2 (DA-D2) receptors in the caudate putamen (CPu) were significantly (P<0.001) restored in co-transplanted group as compared to VMC transplanted or GDNF administered animals. The functional viability of transplanted VMC was confirmed by tyrosine hydroxylase (TH) expression and quantification of TH-positive cells by image analysis revealed a significant restoration in TH-IR fibers density as well as TH-IR neurons counts in co-transplanted animals over VMC transplanted animals. Results suggest that co-transplantation of VMC and GDNF may be a better approach towards functional restoration in 6-OHDA lesioned rat model of Parkinson’s disease.

Effects of orally administered levodopa on mesencephalic dopaminergic neurons undergoing a degenerative process

Although the issue of in vivo levodopa toxicity appears to be settled by now in the light of recent findings, a crucial aspect was not accounted for the experiments designed to tackle that question. Levodopa could in fact be non-toxic on surviving dopamine neurons, but that could not be the case when the drug is administered at the same time those neurons are undergoing degeneration, which is what happens in the clinical setting. Dopaminergic neurons could in that situation be more vulnerable to levodopa’s potential toxic action. Our aim was to determine if oral administration of levodopa is toxic for mesencephalic dopaminergic neurons that are actively involved in a degenerative process. We induced delayed retrograde degeneration of the nigrostriatal system in rats by injecting 6-hydroxydopamine (6-OHDA) intrastriatally. Treatment was started the day after the injection. Dopaminergic markers were histologically studied at the striatal and nigral levels, to determine degree of damage of the nigrostriatal dopaminergic system in levodopa- and vehicle-treated rats. No significant differences between levodopa or vehicle-treated rats were found in: (i) striatal immunoautoradiographic labeling for tyrosine hydroxylase (TH) and the membrane dopamine transporter (DAT); (ii) cell counts of TH-immunoreactive (TH-ir) neurons remaining in the substantia nigra and ventral tegmental area (VTA); (iii) surface area of remaining TH-immunoreactive neurons in the substantia nigra. The present experiments demonstrate that levodopa does not enhance delayed retrograde degeneration of dopaminergic neurons induced by intrastriatal administration of 6-OHDA.

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.

Angiogenic and neurotrophic effects of vascular endothelial growth factor (VEGF165): studies of grafted and cultured embryonic ventral mesencephalic cells

The present series of experiments investigated the effects of vascular endothelial growth factor (VEGF165) on adult rat striatal cerebrovasculature and embryonic dopamine (DA) neuron allografts in a rat model of Parkinson’s disease (PD). We examined VEGF165′s ability to (1) alter the vascular network of the adult rat striatum, (2) influence the vascular growth of solid embryonic day 14 (E14) ventral mesencephalic (VM) grafts when placed into a VEGF-pretreated host striatum, (3) alter the function and survival of E14 VM grafts when transplanted into an adult DA-deleted striatum, and (4) influence cell survival and neurite growth in cultures of E14 VM cells. We demonstrate here that a single bolus injection of VEGF165 into the adult rat striatum significantly increases the amount of vasculature in the vicinity of the injection site in a delayed and transient manner when compared to saline controls. Transplanting solid E14 VM grafts into the VEGF165-pretreated striatum resulted in a homogeneous distribution of small blood vessels throughout the graft, a pattern that closely resembles mature adult vasculature. In contrast, grafts in the control condition contained a patchy distribution of heavily dilated vessels. Behavioral measurements indicate that VEGF pretreatment of the intrastriatal graft site accelerates recovery of amphetamine-induced rotational asymmetry in unilateral 6-OHDA lesioned rats. Unexpectedly, however, VEGF pretreatments failed to increase survival of tyrosine hydroxylase-immunoreactive (THir) neurons in the grafts. In contrast to this finding in vivo, adding VEGF165 to glial-reduced E14 rat VM cultures produced a fourfold increase in THir cell survival and a doubling in the length of THir neurites. We conclude that with the proper method of delivery, VEGF165 may prove to be one of several strategies necessary to significantly improve the survival and function of fetal VM tissue grafts.

Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex.

Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of alpha-adrenoreceptor antagonists (i.e., alpha1-prazosin or alpha2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to approximately 55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to approximately 27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-l-arginine methyl ester or the beta-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on alpha1- and alpha2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.

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.

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. α2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine β-hydroxylase (DβH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4–L6 dorsal horn. Consistently, greater numbers of both TH- and DβH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by α2-adrenergic receptor agonists.

Localization and functional significance of striatal neurons immunoreactive to aromatic l-amino acid decarboxylase or tyrosine hydroxylase in rat Parkinsonian models

Striatal neurons which are immunoreactive (ir) to aromatic l-amino-acid decarboxylase (AADC) or tyrosine hydrodroxylase (TH) may play a role in the decarboxylation of l-DOPA to dopamine (DA) in advanced stages of Parkinson’s disease (PD). However, the functional significance of these neurons and the mechanisms responsible for their induction remain to be clarified. In this study, rats were subjected to different types of dopaminergic or serotonergic denervation and l-DOPA injection to study the effects on these neurons. AADC-ir neurons were found in both normal and DA-denervated striata, and no significant differences in their number and distribution were induced following different types of denervation or l-DOPA administration. TH-ir neurons were only found in DA-denervated striata. However, TH-ir neurons did not appear in those areas with maximal DA depletion, but rather were observed near spared or partially lesioned DA terminals. The population of AADC-ir neurons may make a significant contribution to the effects of exogenous l-DOPA in advanced stages of PD. In addition, TH-ir neurons may contribute to these effects, since we have detected AADC-ir in TH-ir neurons using confocal laser scanning microscopy. Finally, neither l-DOPA therapy nor serotonergic denervation induces significant changes in the number or distribution of these neurons.

Bradykinin receptor agonist facilitates low-dose Cyclosporine-A protection against 6-hydroxydopamine neurotoxicity

Cyclosporine-A (CsA) is neuroprotective in animal models of Parkinson’s disease (PD), Huntington’s disease and stroke. Because CsA does not easily cross the blood–brain barrier (BBB), high doses (i.e. >10 mg/kg in rats) and chronic administration may be necessary to produce beneficial effects. However, immunosuppressant side effects (including nephrotoxicity and hepatotoxicity) are associated with such CsA dosing regimens. The bradykinin B2 receptor agonist, Cereport ® (labradimil and formerly called RMP-7), transiently increases the permeability of the BBB to facilitate delivery of drugs to the CNS. Here we examined the effects of co-administration of CsA and Cereport in the unilateral 6-OHDA model of PD. Animals were pretreated with vehicle, CsA alone (1 mg/kg, a low dose without either immunosuppressive or neuroprotective effects, or 10 mg/kg, a high dose that produces both immunosuppression and neuroprotection), or CsA (1 mg/kg) in combination with Cereport (9 μg/kg). Behavioral analyses, using elevated body swing and amphetamine-induced rotational tests, revealed that a low dose of CsA was neuroprotective when combined with Cereport, but not when given alone. Tyrosine hydroxylase immunohistochemistry demonstrated that while near complete (>90%) depletions of nigral TH-ir neurons were noted in lesioned animals that received vehicle infusion or low-dose CsA alone, lesioned animals that received low-dose CsA+Cereport exhibited a significant sparing of nigral TH-ir neurons and a marked reduction in the loss of striatal TH-ir fibers. The safer and effective administration of lower doses of CsA combined with enhanced BBB permeability using Cereport, offers a novel way of producing protective effects in the CNS without the toxic liabilities of high-dose CsA.

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.

Catechol- O-methyltransferase inhibition protects against 3,4-dihydroxyphenylalanine (DOPA) toxicity in primary mesencephalic cultures: new insights into levodopa toxicity

Inhibition of catechol- O-methyltransferase (COMT) has protective effects on levodopa ( l-DOPA), but not d-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main l-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3- O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 μM after 24 h, whereas DA was more toxic than l-DOPA with toxicity at concentrations of ≥1 μM. The coenzyme of COMT, S-adenosyl- l-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or l-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect l-DOPA toxicity. l-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 μM). Increased contamination of the cultures with glial cells attenuated l- and d-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against l-DOPA toxicity only. Investigations of l-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular l-DOPA concentrations by Ro 41-0960. Our data confirm that l-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of l-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of l-DOPA.

Cyclooxygenase-2-deficient mice are resistant to 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra

Cyclooxygenases (COX), key enzymes in prostanoid biosynthesis, may represent important therapeutic targets in various neurodegenerative diseases. In the present study, we explored the role of COX in Parkinson's disease (PD) by using 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine (MPTP) as a tool to create a rodent Parkinsonian model. MPTP (20 mg/kg, subcutaneously) was injected daily into COX-1- and COX-2-deficient mice and wild-type (WT) controls for five consecutive days. Immunocytochemical analysis of tissues collected 7 days after the final MPTP treatment showed that MPTP significantly decreased the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNc) of WT (40% decrease) and COX-1 −/− (45% decrease) mutants. However, a much smaller loss of TH-ir neurons in COX-2 −/− mutants (20% decrease) was observed. Furthermore, electrochemical analysis revealed a more than 70% decrease in the levels of dopamine and its metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the striatum of the WT control COX-1 −/− and COX-2 −/− mutant mice. These results indicate that loss of COX-2 activity reduces MPTP-induced damage to the dopaminergic neurons of the SNc, but does not alter the levels of dopamine and its metabolites in the striatum. Interestingly, MPTP caused the same degree of loss of dopaminergic neurons in both COX-2 +/− and COX-2 −/− mice (20% loss). The results of this study indicate an important role of COX-2 in MPTP-induced neuronal degeneration and suggest the possibility that manipulation of the COX-2 could be an important target for therapeutic interventions in PD.

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.

Lentivirally Delivered Glial Cell Line-Derived Neurotrophic Factor Increases the Number of Striatal Dopaminergic Neurons in Primate Models of Nigrostriatal Degeneration

The primate striatum contains tyrosine hydroxylase (TH)-immunoreactive (ir) neurons, the numbers of which are augmented after dopamine depletion. Glial cell line-derived neurotrophic factor (GDNF) strongly modulates the viability and phenotypic expression of dopamine ventral mesencephalic neurons. The effect of GDNF on TH-ir neurons intrinsic to the striatum has yet to be investigated. In the present study, stereological counts of TH-ir striatal neurons in aged and parkinsonian nonhuman primates revealed that GDNF delivered via a lentiviral vector (lenti-) further increased the number of these cells. Aged monkeys treated with lenti-GDNF displayed an eightfold increase in TH-ir neurons relative to lenti-beta-galactosidase-treated monkeys. Unilateral 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine treatment alone in young monkeys resulted in a bilateral eightfold increase in TH-ir striatal cells. This effect was further magnified sevenfold on the side of lenti-GDNF treatment. These cells colocalized with the neuronal marker neuronal-specific nuclear protein. Some of these cells colocalized with GDNF-ir, indicating that an alteration in phenotype may occur by the direct actions of this trophic factor. Thus, GDNF may mediate plasticity in the dopamine-depleted primate brain, which may serve to compensate for cell loss by converting striatal neurons to a dopaminergic phenotype.

Distribution of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) immunoreactivity in the central nervous system of two chondrostean fishes (Acipenser baeri and Huso huso).

To obtain a better understanding of the evolution of the brain catecholaminergic systems of fishes, we have examined the distribution of catecholamine-synthesizing enzymes in two species of sturgeon (Acipenser baeri and Huso huso) using antibodies against tyrosine hydroxylase (TH) and dopamine-beta -hydroxylase (DBH; only analyzed in Acipenser). Both sturgeons showed TH-immunoreactive (THir) neurons widely distributed in most regions of the brain, the highest number of THir cells being located in the forebrain (olfactory bulb, preoptic area, and posterior tuberculum). THir cells were also seen in other forebrain areas (retrobulbar area, dorsal and ventral telencephalic areas, hypothalamus, ventral thalamus, pretectal area) and in the brainstem (locus coeruleus, viscerosensory area, caudal reticular formation, and area postrema). Immunoreactive fibers and varicosities showed a wide distribution, being particularly abundant in the diencephalon and mesencephalon. DBH-immunoreactive (DBHir) cells were observed in the anterior tuberal nucleus, where these cells were TH-negative, and in the locus coeruleus and the caudal rhombencephalon (vagal reticular formation), where the DBHir cells were also THir. DBHir fibers were scarce in the telencephalon and very abundant in the diencephalon, mesencephalon, and rhombencephalon. The comparative analysis of the catecholaminergic systems of chondrosteans and those observed in other groups of fishes and tetrapods indicate a similar organization of many nuclei, as well as characteristics that are probably primitive, such as the presence of a large number of forebrain catecholaminergic groups.

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.