Colocalization of tyrosine hydroxylase and GAD65 mRNA in mesostriatal neurons

Although dopamine has been considered as the only neurotransmitter in the nigrostriatal pathway, studies carried out in the last two decades have suggested the existence of a nondopaminergic nigrostriatal projection, and more recently, gamma-aminobutyric acid (GABA) has been identified as its neurotransmitter. In this study, we used the combination of immunocytochemistry for tyrosine hydroxylase (TH; a marker of dopaminergic neurons), in situ hybridization (ISH) for two different isoforms of glutamic acid decarboxylase (GAD65 and GAD67, the rate-limiting enzyme in GABA synthesis) and retrograde tracing techniques to investigate the possible existence of nigrostriatal neurons containing both neurotransmitters (dopamine and GABA) in the rat. Our results revealed that approximately 10% of mesostriatal dopaminergic neurons, most of them lying in the medial region of the substantia nigra pars compacta (SNC) and neighbouring A10 region, contain GAD65 mRNA. These findings reveal a third nigrostriatal pathway formed by dopaminergic/GABAergic neurons. Contrasting with the idea that in the basal ganglia, dopamine and GABA are released from different cell populations, the results suggest a more complex dopamine/GABA interaction than previously assumed, probably including cotransmission.     

Effect of pentylenetetrazol on the expression of tyrosine hydroxylase mRNA and norepinephrine and dopamine transporter mRNA

Seizure activity has been shown to have differential effects on the terminal content of the monoamines, norepinephrine (NE) and dopamine (DA). Induction of seizure activity reduces the terminal content of NE, while DA levels remain unchanged or slightly elevated. This study examined the effect of the chemoconvulsant pentylenetetrazol (PTZ) on the mRNA expression of regulatory proteins which maintain the terminal content of NE and DA (i.e., synthesis and re-uptake). The areas examined were the noradrenergic neurons of the locus coeruleus (LC) and dopaminergic neurons of the substantia nigra pars compacta/ventral tegmentum area (SNpc/VTA) in the rat. In the LC, PTZ increased mRNA expression of the immediate early gene, c-fos, and mRNA expression of the synthesizing enzyme, tyrosine hydroxylase (TH), and the re-uptake protein, norepinephrine transporter (NET). This effect on TH and NET was observed only 1 day after the administration of PTZ. In contrast, PTZ did not alter the expression of c-fos mRNA in the SNpc/VTA, but reduced the expression of the dopamine transporter (DAT) mRNA. This effect was observed only 1 day after the administration of PTZ. TH mRNA expression in dopaminergic neurons was elevated initially in a manner similar to that observed in the LC. However, the effect of PTZ on TH mRNA expression in dopaminergic neurons was more prolonged (still elevated 3 days later). These results indicate that the chemoconvulsant PTZ has differential effects on the mRNA expression of regulatory systems (TH and neurotransporter proteins) in noradrenergic and dopaminergic neurons.     

Variations in number of dopamine neurons and tyrosine hydroxylase activity in hypothalamus of two mouse strains

Mice of the BALB/cJ strain have more neurons and greater tyrosine hydroxylase (TH) activity in the midbrain than mice of the CBA/J strain (Baker, H., T. H. Joh, and D. J. Reis (1980) Proc. Natl. Acad. Sci. U.S.A. 77: 4369-4373). To determine whether the strain differences in dopamine (DA) neuron number and regional TH activity are more generalized, regional TH activity was measured and counts of neurons containing the enzyme were made in the hypothalamus of male mice of the BALB/cJ and CBA/J strains. TH activity was measured in dissections of whole hypothalamus (excluding the preoptic area), the preoptic area containing a rostral extension of the A14 group, the mediobasal hypothalamus containing the A12 group, and the mediodorsal hypothalamus containing neurons of the A13 and A14 groups. Serial sections were taken and the number of DA neurons was established by counting at 50- to 60-microns intervals all cells stained for TH through each area. In conjunction with data obtained biochemically, the average amount of TH per neuron was determined. In all areas, TH activity in CBA/J mice was significantly less (p less than 0.001) than in BALB/cJ mice, ranging from 48% in the mediobasal hypothalamus to 71% in the medial and dorsal hypothalamus. The number of TH-containing neurons was also significantly less in the CBA/J strain (p less than 0.001), ranging from 49% in the preoptic area to 74% in the mediobasal hypothalamus (MBH). With the exception of the MBH, enzyme activity per neuron was similar in both strains. In the MBH, strain differences in TH activity were greater than those for neuron number, resulting in less TH activity per neuron in the CBA/J strain. The results suggest that strain differences in the number of DA neurons are widespread and involve DA systems throughout the brain. Therefore, differences in whole brain TH activity cannot be attributed only to differences in specific regions. Our findings further support the view that the number of neurons of a specific chemical class may be under genetic control.     

Quantitative study of tyrosine hydroxylase mRNA in catecholaminergic neurons and adrenals during development and aging

Using a solution hybridization-S1 nuclease protection assay, we quantitatively studied tyrosine hydroxylase mRNA (mRNATH) in catecholaminergic cells of the substantia nigra, hypothalamus, superior cervical ganglion, and adrenal of male rats from early neonatal life to old age. Throughout this time, the lowest level of mRNATH in any tissue was found in the youngest animals, and their development was associated with an increase in the quantity of mRNATH. However, the extent of the increase as well as the pattern of change was dependent on the tissue. The amount of mRNATH in the substantia nigra of 1-day-old pups was 162 +/- 7 attomoles (mean and S.E.M.), increasing to 877 +/- 39 amol at 14 days of age. Then, the amount fell to 480 +/- 25 amol at 6 weeks of age, but changed little between 6 weeks and 23 months of age. In the hypothalamus of 1-day-old pups, the quantity of mRNATH was 24 +/- 3 amol, increasing to 60 +/- 6 amol at 2 weeks and changed little thereafter. mRNATH in the superior cervical ganglion increased gradually until 10 months of age; at which time the amount was 3 times that of neonatal animals. In the adrenal, mRNATH increased continuously throughout the period of observation. The amount of mRNATH in the adrenal of 23-month-old animals was 25 times that in the adrenal of 4-day-old pups. These data suggest that tyrosine hydroxylase gene expression does not diminish in aged rats.     

Up-regulation of D3 dopamine receptor mRNA by neuroleptics

The effects of 14 days neuroleptic treatment on the expression of the D₃ dopamine receptor gene was investigated in rats using a sensitive polymerase chain reaction assay. In olfactory tubercle, D₃ mRNA levels increased following haloperidol (40%), pimozide (56%), and sulpiride (63%) administration, and in nucleus accumbens, levels increased after haloperidol (50%) and sulpiride (50%). D₃ expression in the motor striatum did not change with any antagonist tested. Clozapine did not affect D₃ expression in any brain region. These data suggest that dopamine antagonists can regulate the expression of the D₃ receptor in a brain region selective manner. The findings also suggest that the motor complications of chronic antipsychotic therapy are not due to D₃ receptor up-regulation in the striatum. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease marked by severe loss of dopamine (DA) neurons in the nigrostriatal system, which results in depletion of striatal DA. Transplantation of embryonic ventral mesencephalic (VM) DA neurons into the striatum is a currently explored experimental treatment aimed at replacing lost DA in the nigrostriatal system, but is plagued with poor survival (5-20%) of implanted neurons. Here, we tested the ability of erythropoietin (Epo) to provide neuroprotection for embryonic day 14 (E14) VM DA neurons. Epo was tested in vitro for the ability to augment tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival under normal cell culture conditions. In vitro, Epo did not increase the number of TH-ir neurons when administered at the time of plating the E14 VM cells in culture. We also tested the efficacy of Epo to enhance E14 VM transplants in vivo. Rats unilaterally lesioned with 6-hydroxydopamine received transplants that were incubated in Epo. Treatment with Epo produced significant increases in TH-ir neuron number, soma size, and staining intensity. Animals receiving Epo-treated grafts exhibited significantly accelerated functional improvements and significantly greater overall improvements from rotational asymmetry compared to control grafted rats. These data indicate that the survival of embryonic mesencephalic TH-ir neurons is increased when Epo is administered with grafted cells in a rodent model of PD. As direct neurotrophic effects of Epo were not observed in vitro, the mechanism of Epo neuroprotection remains to be elucidated.     

Induction of tyrosine hydroxylase expression in rat forebrain neurons

Olfactory nerve input is required for the normal expression of tyrosine hydroxylase (TH) by dopaminergic neurons in the glomerular region of the rodent main olfactory bulb. To determine whether the olfactory nerve exerts a similar influence on neurons in other brain regions, we performed unilateral bulbectomies in rat pups on postnatal day 5-7 and examined the brains 2-6 months later, after the regenerated olfactory nerve had penetrated the forebrain. Tissue was stained for TH, dopamine beta-hydroxylase (DBH) and olfactory marker protein immunoreactivity. We observed novel TH-immunoreactivity in neurons located in those areas of the adult forebrain which received olfactory nerve fibers, particularly the rostral extension of the subependymal layer. Many of these neurons resembled the periglomerular cells of the olfactory bulb. No cell staining for DBH was observed in these areas, suggesting the possible dopaminergic phenotype of these neurons. Our data indicate that afferent regulation of neurotransmitter expression by the olfactory nerve is not limited to the cells of the olfactory bulb.     

Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.

We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after beginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral A1 or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur.     

Flunarizine induces a transient loss of tyrosine hydroxylase immunoreactivity in nigrostriatal neurons

Neurotoxic effects of flunarizine (Fz), a selective calcium channel blocker, on the nigrostriatal dopamine system was investigated. Systemic injections of Fz to mice resulted in a transient loss of tyrosine hydroxylase (TH) immunoreactive nigrostriatal neurons without cell loss. TH immunoreactivity in these neurons was greatly reduced as rapidly as one day after drug administration (regardless of dosage used) and thereafter recovered in both dose- and time-dependent manners. Such a novel neurotoxic action of Fz may constitute a morphological substrate for reversible drug-induced parkinsonian signs described in recent clinical case reports.     

Collateral sprouting in central mesolimbic dopamine neurons: Biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons

The olfactory tubercle of adult rats was examined for the development of collateral sprouts from intrinsic dopaminergic axons following unilateral olfactory bulbectomy. In the ipsilateral tubercle tyrosine hydroxylase (TH) activity began to increase by 10–14 days following the lesion, gradually reaching a maximum of 125% of control (P < 0.005) by 21 days where it remained permanently elevated. The rise of TH activity in the tubercle reflected changes of the dopaminergic innervation, since dopamine-β-hydroxylase (DBH) activity was unchanged, and lesions of the dorsal noradrenergic bundle reduced DBH but not TH activity in the tubercle. By immunocytochemical staining the elevation of TH reflected an increased number and altered distribution of TH-containing processes within the olfactory tubercle. By 30 days the uptake of [³H]dopamine into synaptosomes of the olfactory tubercle was also increased to 140% of control (P < 0.05). In the dopaminergic cell bodies of the ipsilateral A10 group (which innervate the tubercle) TH activity was transiently elevated to 121% (P < 0.05) by 4 days, returning to control levels by 10 days. Histologically no change in activity was detected. The results indicate that mesolimbic dopaminergic neurons of A10 which innervate the olfactory tubercle will sprout in response to removal of a major non-dopaminergic input, that the new innervation is sustained, and that during collateral sprouting there is a transient elevation of TH activity in the uninjured cell bodies which precedes the period of axonal growth. The findings suggest that (a) the increase of TH activity in the A10 cell bodies during collateral sprouting may be a reflection of an increase in the amount of enzyme protein required for transport into the enlarging terminal fields, (b) that as in development sprouts are in place before they reach biochemical maturity, (c) the biochemical mechanisms underlying collateral sprouting of uninjured neurons are not necessarily the same as those associated with regenerative sprouting in response to axonal injury, and (d) the development and the acquisition of biochemical maturation of collateral sprouts in the CNS involves complex two-way signalling between terminal field and cell bodies. The development of collateral sprouts of dopaminergic neurons may be the cellular substrate of the development of behavioral hyperactivity and aggression produced by bilateral olfactory bulbectomy in rat.     

Collateral sprouting in central mesolimbic dopamine neurons: biochemical and immunocytochemical evidence of changes in the activity and distrubution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons.

The olfactory tubercle of adult rats was examined for the development of collateral sprouts from intrinsic dopaminergic axons following unilateral olfactory bulbectomy. In the ipsilateral tubercle tyrosine hydroxylase (TH) activity began to increase by 10-14 days following the lesion, gradually reaching a maximum of 125% of control (P less than 0.005) by 21 days where it remained permanently elevated. The rise of TH activity in the tubercle reflected changes of the dopaminergic innervation, since dopamine-beta-hydroxylase (DBH) activity was unchanged, and lesions of the dorsal noradrenergic bundle reduced DBH but not TH activity in the tubercle. By immunocytochemical staining the elevation of TH reflected an increased number and altered distribution of TH-containing processes within the olfactory tubercle. By 30 days the uptake of [3H]dopamine into synaptosomes of the olfactory tubercle was also increased to 140% of control (P less than 0.05). In the dopaminergic cell bodies of the ipsilateral A10 group (which innervate the tubercle) TH activity was transiently elevated to 121% (P less than 0.05) by 4 days, returning to control levels by 10 days. Histologically no change in activity was detected. The results indicate that mesolimbic dopaminergic neurons of A10 which innervate the olfactory tubercle will sprout in response to removal of a major non-dopaminergic input, that the new innervation is sustained, and that during collateral sprouting there is a transient elevation of TH activity in the uninjured cell bodies which precedes the period of axonal growth. The activity in the uninjured cell bodies which precedes the period of axonal growth. The findings suggest that (a) the increase of TH activity in the A10 cell bodies during collateral sprouting may be a reflection of an increase in the amount of enzyme protein required for transport into the enlarging terminal fields, (b) that as in development sprouts are in place before they reach biochemical maturity, (c) the biochemical mechanisms underlying collateral sprouting of uninjured neurons are not necessarily the same as those associated with regenerative sprouting in response to axonal injury, and (d) the development and the acquisition of biochemical maturation of collateral sprouts in the CNS involves complex two-way signaling between terminal field and cell bodies. The development of collateral sprouts of dopaminergic neurons may be the cellular substrate of the development of behavioral hyperactivity and aggression produced by bilateral olfactory bulbectomy in rat.     

Bradykinin elevates tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells

Bradykinin is known to rapidly elevate intracellular calcium leading to secretion of neurotransmitters and short term activation of tyrosine hydroxylase (TH). In this study we examined the effect of bradykinin on mRNA levels of two catecholamine biosynthetic enzymes. Treatment of PC12 cells with 1 μM bradykinin for 3 h markedly elevated both TH and dopamine β-hydroxylase (DBH) mRNA levels.     

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA‐containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA‐expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP‐TH⁺ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole‐cell patch‐clamp recordings revealed at least two distinct neuronal types, termed EGFP‐TH⁺ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine‐ and cyclic AMP‐regulated phosphoprotein of 32 kDa (DARPP‐32), Type A neurons constituted 75% of the EGFP⁺ cells, had dendritic spines (24.6%), contained DARPP‐32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and 3‐nitropropionic acid. The membrane properties and N‐methyl‐d ‐aspartate : non‐N‐methyl‐d ‐aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin‐like, DARPP‐32‐like and dynorphin B‐like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.