No abstract

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Possible functional interactions between D1 and D2 dopamine (DA) receptors were examined using extracellular single-cell recording with microiontophoretic application of selective D1 and D2 receptor agonists both postsynaptically, in the rat nucleus accumbens (NAc) and caudate-putamen (CPu), and presynaptically, at impulse-regulating somatodendritic DA autoreceptors in the ventral tegmental area (A10) and substantia nigra pars compacta (A9). In addition, synthesis-modulating nerve terminal DA autoreceptors were studied in both the CPu and NAc using the gamma-butyrolactone (GBL) neurochemical model of isolated nerve terminal autoreceptor function in vivo. In both the NAc and CPu, the inhibition of neurons produced by iontophoresis of the D2 receptor agonists quinpirole or RU-24213 was attenuated by acute DA depletion via the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). However, during iontophoresis of the selective D1 DA receptor agonist SKF 38393, the inhibitory effects of the D2 agonists were again evident, suggesting that the attenuation of D2 agonist-induced inhibition was due to decreased D1 receptor activation. In contrast, the inhibitory effects produced by the non-selective D1/D2 agonist apomorphine or by SKF 38393 were unaffected by AMPT pretreatment. Thus, D1 receptor activation appears necessary for D2 receptor-mediated inhibition of NAc and CPu neurons, whereas D2 receptor activation is not required for the inhibition produced by D1 receptor stimulation. In contrast to postsynaptic D2 receptors, the ability of DA agonists to stimulate D2 DA autoreceptors was not altered by manipulations of D1 receptor occupation. Enhancing D1 receptor stimulation with SKF 38393 or reducing D1 receptor occupation with either the selective D1 receptor antagonist SCH 23390 or AMPT failed to alter the rate-inhibitory effect of i.v. quinpirole on A9 or A10 DA neurons. Similarly, iontophoresis of SKF 38393 failed to alter the inhibitory effects of iontophoretic quinpirole. SKF 38393 also failed to affect the inhibition of GBL-induced increases in DOPA accumulation (tyrosine hydroxylase activity) produced by quinpirole in either the NAc or CPu. Furthermore, reversal of GBL-induced increases in DOPA accumulation by apomorphine or quinpirole was unaffected by pretreatment with SCH 23390. Therefore, D1 receptor occupation appears to be necessary for the expression of the functional effects of postsynaptic D2 receptor stimulation but not presynaptic D2 DA autoreceptor stimulation.

No abstract

Extracellular single unit recording and microiontophoretic techniques were used to determine the sensitivities and interactions of D1 and D2 dopamine (DA) receptors in the caudate putamen (CPu) of rats that were denervated of DA by intraventricular injections of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). Seven to 10 d after the 6-OHDA injection, DA levels in the ipsilateral CPu were reduced to 11.8% of control. Current-response curves revealed that the inhibitory responses of CPu neurons to microiontophoretic administration of both the selective D1 receptor agonist SKF-38393 and the selective D2 receptor agonist quinpirole were significantly increased in 6-OHDA-pretreated rats, suggesting up-regulation of both receptor subtypes. Although our previous studies have established that D1 receptor activation is normally required for (enables) the inhibitory effects of selective D2 agonists in the CPu, this requirement was no longer evident in 6-OHDA- denervated rats. Whereas acute DA depletion [produced by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT)] attenuated the inhibitory effects of quinpirole on CPu neurons, long-term DA denervation (produced by 6-OHDA) enhanced the inhibitory effects of the D2 agonist. The enhanced effects of quinpirole in 6-OHDA-lesioned rats were not due to residual DA stimulating supersensitive D1 receptors (i.e., enabling) since further DA depletion (99.7%), produced by acute administration of AMPT in 6-OHDA-lesioned rats, failed to diminish the inhibitory efficacy of quinpirole. In addition to relieving D2 receptors from the need for D1 receptor-mediated enabling, 6-OHDA lesions also abolished the normal synergistic relationship between the receptor subtypes since low (subinhibitory) currents of SKF-38393 (4 nA) failed to potentiate the inhibitory effects of quinpirole on CPu neurons in lesioned rats. Similar findings (i.e., supersensitivity and loss of synergistic effects) were obtained from rats that had received repeated pretreatment with reserpine (2.5 mg/kg) for 4 d, indicating that these effects of 6-OHDA lesions were due to the depletion of synaptic DA rather than to the structural loss of DA terminals. Therefore, both the quantitative (potentiation) and the qualitative (enabling) synergistic effects between D1 and D2 receptors in the rat CPu were abolished when these receptors were functionally supersensitive. The present study provides electrophysiological support for previous behavioral studies indicating that the requirement of D1 receptor stimulation for D2 receptor-mediated functional effects (enabling) is not maintained in rats chronically depleted of DA by either 6-OHDA lesions or repeated reserpine.