Organization of dopamine D1 and D2 receptors in human striatum: receptor autoradiographic studies in Huntington's disease and schizophrenia

The technique of quantitative autoradiography was used to examine the effects of Huntington's disease (HD) and schizophrenia on the organization of striatal dopamine (DA) D1 and D2 receptors. Whereas the striatum of HD cases showed a reduction in the density of D1 ([3H]SCH 23390) and D2 ([3H]spiroperidol) receptors, the patterning of D2 receptor loss did not match that of the D1 receptor loss. The HD loss of D1 D1 receptors (65%) is far greater than the loss of D2 receptors (28%). Whereas there was a dorsal-ventral gradient of effect on both receptor subtypes, the effects of HD on D2 receptors in the ventral putamen (PUT) and nucleus accumben septi (NAS) were minimal. Similarly, muscarinic M1 and M2 receptors demonstrate different patterns of alteration in HD. The M2 subtype, labeled with [3H]N-methylscopolamine (in the presence of excess pirenzepine to occlude M1 sites), was depleted far more than the M1 receptor subtype, labeled with [3H]pirenzepine. Although the effects of HD on [3H]mazindol labeling of DA terminals were more heterogeneous, there appeared to be a relative preservation of this afferent input to the striatum of the HD cases. In the schizophrenic cases, our autoradiographic studies confirm previous reports of an elevation of D2 receptor density in the striata of many schizophrenics. This increase was evident even though two of the three cases were known to have not been treated with neuroleptics, and the third case may also have been drug naive. However, the increase was far greater in the NAS (164%) and ventral PUT (173%) than more dorsally in the striatum (68%). The density of D1 receptors and DA terminals labeled with [3H]mazindol in the striatum of schizophrenics was not significantly different from that of control cases. Thus in both HD and schizophrenia, the ratio of D2/D1 receptors is altered in favor of the D2 population, particularly in the NAS.     

Differential response of striatal dopamine and muscarinic cholinergic receptor subtypes to the loss of dopamine. II. Effects of 6-hydroxydopamine or colchicine microinjections into the VTA or reserpine treatment.

In the previous paper it was demonstrated that striatal dopamine (DA) D1 and D2 receptor subtypes and muscarinic M1 and M2 receptor subtypes show differing responses to lesions of the mesostriatal DA system. To examine this differential regulation further rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) or colchicine into the ventral tegmental area (VTA), or treated chronically with reserpine or saline. Two weeks later the animals were tested for their behavioral response to a subthreshold dose of apomorphine and 24 h later their brains were removed and processed for quantitative autoradiography or for analysis of DA levels by high-performance liquid chromatography. The 6-OHDA-lesioned animals showed a supersensitive rotational response to apomorphine. The loss of DA, loss of DA uptake sites, regulation of DA D1 and D2 receptors and regulation of the muscarinic cholinergic system was similar to the previous paper. Injection of colchicine in the VTA resulted in incomplete loss of striatal DA (50%), [3H]mazindol binding (50%), and no behavioral supersensitivity to apomorphine. There was a small loss of presynaptically located D2 receptors (13%). Similar to the 6-OHDA lesions there was a loss of D1 (12%) and M1 receptors. Reserpine treatment produced an 86% decrease in DA levels, an enhanced stereotyped responsiveness to apomorphine, and an increase of both D2 (28%) and D1 receptors (26%). There was a loss of muscarinic M1 but not M2 receptors. Thus removal of DA terminals or blockade of transport of proteins in the mesostriatal axons can lead to a reduction in D1 receptor density in the striatum. In contrast, loss of DA without removal of DA terminals leads to a significant up-regulation of the D1 receptor. D2 receptors show increases following removal of DA or of DA terminals. Alteration in the muscarinic cholinergic system following damage to the mesostriatal DA system is a complex response not mimicked by either reserpine or colchicine treatment.     

Decreased striatal D1 binding density following mesotelencephalic 6-hydroxydopamine injections: an autoradiographic analysis

Autoradiographic experiments performed on rats with unilateral mesotelencephalic 6-hydroxydopamine (6-OHDA) injections revealed reduced binding of [3H]SCH23390 to D1 receptors in the striatum ipsilateral to the neurotoxin as well as increased binding of [3H]spiroperidol to D2 receptors in that hemisphere. These opposite influences of injury on the dopamine receptor subtypes occurred in rats sacrificed at 2 weeks or 11 months postoperatively, but neither change was evident at 4 days postoperatively. Equilibrium saturation analysis performed on rats sacrificed at 8 weeks postoperatively indicated that D1 and D2 receptor changes reflected altered Bmax values without KD modifications. Topographic analysis of the D1 decline by quantitative autoradiography revealed that the D1 decrease was greater in dorsal striatum than ventrally. Those striatal regions that showed greater declines in D1 density correspondingly had the greater losses of [3H]mazindol binding after the denervation, suggesting that the decline of D1 binding is a postsynaptic consequence of the reduced mesostriatal dopaminergic innervation. The findings indicate opposite influences of injury on D2 and D1 receptor levels and raise important questions concerning the mechanism by which 6-OHDA injection affects the D1 sites.     

Autoradiography of dopamine receptors and dopamine uptake sites in the spontaneously hypertensive rat

We examined the status of dopamine (DA) D1 and D2 receptors by using [3H]SCH 23390 and [3H]spiperone binding, respectively, and DA uptake sites by using [3H]mazindol binding in spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats. SHR showed significantly higher [3H]SCH 23390 and [3H]spiperone binding in the caudate-putamen (CPu), the nucleus accumbens (NAc) and the olfactory tubercle (OT) in comparison to the SD rats. There were no significant differences in [3H]mazindol-labeled DA uptake sites between the two strains. Unilateral 6-hydroxydopamine (6-OHDA) injection into the striatum resulted in more than 90% depletion of DA uptake sites in the CPu in both strains. 6-OHDA-induced DA depletion was associated with significant increases in striatal [3H]spiperone binding which were of similar magnitude in the SD rats (+64.1%) and SHR (+51.3%). There were only small decreases (-5.4%) in D1 receptor binding in the dorsolateral aspect of the CPu in the SHR, whereas there were no changes in striatal D1 receptors in the SD rats. These results indicate that, although the SHR have higher concentrations of both D1 and D2 receptors in the basal ganglia, these receptors are regulated in a fashion similar to DA receptors in SD rats after 6-OHDA-induced striatal DA depletion.     

Retrograde degeneration of nigrostriatal neurons induced by intrastriatal 6-hydroxydopamine injection in rats.

Quantitative receptor autoradiography was used to assess the effects of unilateral intrastriatal injections of 6-hydroxydopamine (6-OHDA) on the distribution of D1 and D2 dopamine (DA) receptors and of DA uptake sites in the mesostriatal pathway. [3H]Mazindol-labeled DA uptake sites were reduced both in the striatum (-97%) and in the substantia nigra pars compacta (SNpc) (-88%) on the injected side. There were also significant decreases of dopamine uptake sites in the nucleus accumbens (NAc) (-73%) and in the ventral tegmental area (VTA) (-70%). Changes in [3H]mazindol binding were also found within the contralateral VTA (-30%) and SNpc (-13%) but not in the contralateral-striatum. [3H]SCH23390-labeled D1 receptors were significantly reduced in the dorsomedial (-18%) and ventromedial (-14%) aspects of the striatum ipsilateral to the side of the lesions. In contrast, the concentration of [3H]spiperone-labeled D2 receptors was not altered. There were also significant decreases in D1 (-18%) and of D2 (-27%) receptors in the SNpc and of D1 (-10%) in the SN pars reticulata (SNpr). These results suggest that oxyradical-induced damage in striatal DA terminals could lead to retrograde changes in the SNpc. In addition, the data indicate that unilateral striatal damage can result in bilateral changes in the SNpc, thus confirming the interdependence of the two nigrostriatal pathways in rats.     

Striatal dopamine innervation and receptor density: regional effects of the weaver mutation

Mice homozygous for the autosomal recesive gene weaver (wv) exhibit a regionally specific depletion of forebrain dopamine (DA). DA is reduced approximately 70% in the dorsal striatum of homozygotes (wv/wv) relative to heterozygous (+/wv) controls while DA content in ventral striatum is relatively unchanged. The goal of the present study was to determine the regional effects of the weaver mutation on striatal DA receptors and DA uptake sites using quantitative autoradiography. Catecholamine histofluorescence was used to examine midbrain DA-containing cell bodies. Compared to behaviorally normal (+/-) littermates, the binding of [³H]spiroperidol to D₂ sites was significantly increased in the dorsal but not ventral striatum of wv/wv mice. Binding of the D₁ ligand, [³H]SCH23390, was significantly decreased throughout the striatum of wv/wv mice. The binding of [³H]mazindol to DA uptake sites was dramatically reduced in all wv/wv striatal regions except the ventrolateral portion. Compared to +/-littermates, wv/wv mice had far fewer fluorescent cell bodies in the substantia nigra and a less pronounced reduction of ventral tegmental area fluorescent somata. These findings support the hypothesis that heterogeneities exist in the genetic control of the mesotelencephalic DA system. The results underscore the usefulness of the weaver mouse in the study of mesostriatal sub-systems, receptor regulation, and potentially as a model of human neuropathologies that affect distinct populations of cells in the mesotelecephalic system.     

Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathways in the rat

The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from rats which had received unilateral 6-hydroxydopamine (6-OHDA) injections destroying ascending nigrostriatal neurones. The binding of both ligands to striatal sections was first shown to be saturable, reversible and of high affinity and specificity [( 3H]SCH 23390: Bmax 2.16 pmol/mg protein, Kd 1.4 nM; [3H]sulpiride; Bmax 0.67 pmol/mg protein, Kd 10.7 nM). After unilateral stereotaxic 6-OHDA injections, rats rotated contralaterally when challenged with apomorphine (0.5 mg/kg), or specific D1 or D2 agonists, SKF 38393 (1.0-5.0 mg/kg) and LY 171555 (0.05-0.5 mg/kg), respectively. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A loss of approximately 90-95% of uptake sites was reproducibly accompanied by an enhanced density of binding ipsilaterally for the D2 ligand, [3H]sulpiride, in all areas of the striatum, but most markedly in the lateral areas. An increase in the D2 binding site density was also seen in the ipsilateral nucleus accumbens and the olfactory tubercle. In contrast, in the same animals, the striatal D1 receptors were far less affected by dopaminergic denervation, with no consistent changes seen in the binding of [3H]SCH 23390. These results suggest that dopamine D2 receptors are more susceptible than D1 receptors to changes after dopaminergic denervation, which is expressed as an increase in the density of binding sites revealed here with [3H]sulpiride.     

The relationship between loss of dopamine nerve terminals, striatal [H]spiroperidol binding and rotational behavior in unilaterally 6-hydroxydopamine-lesioned rats

The relationship between the destruction of dopamine-containing nerve terminals, specific binding of [³H]spiroperidol and contralateral rotation in response tol-DOPA, was studied in rats with unilateral lesions of the nigrostriatal dopamine (DA) system, induced by intracerebral injections of the neurotoxin 6-hydroxydopamine (6-OHDA). Animals with significant rotational behavior in response tol-DOPA had both a greater amount of specific binding of [³H]spiroperidol in the lesioned striatum compared to the non-lesioned striatum, and at least 90% destruction of DA terminals in the lesioned striatum (less than 10% of control uptake). The non-rotators tol-DOPA had considerably less destruction of DA terminals and no significant increase in specific binding on the lesioned side. The data from this study suggest that beforel-DOPA is effective as an inducer of contralateral rotational behavior, there must be both unilateral damage to the DA terminals greater than 90%, and increased specific binding.     

Neonatal 6-OHDA lesions differentially affect striatal D1 and D2 receptors.

Lesions to the dopamine (DA) system in early postnatal development have different behavioral consequences compared to lesions made in adulthood. Intrastriatal injections of the neurotoxin 6-hydroxydopamine (6-OHDA) on the day of birth (PO) or postnatal day 1 (P1) produce a selective supersensitivity to D1 receptor agonists and a subsensitivity to D1 antagonists (Neal and Joyce, 1991a). In this paper, we describe the long-term effects of early DA loss on DA receptor regulation. Pups received bilateral intrastriatal injections of the neurotoxin 6-OHDA (4 micrograms per striatum) on PO or P1. Adult rats were killed at 90 days of age and the brains were processed for quantitative autoradiography (QAR) or tyrosine hydroxylase (TH) immunocytochemistry. Cohorts were tested for the behavioral responses to the selective D1 receptor agonist SKF38393 (10 mg/kg). Neonatally lesioned rats exhibited increases in abnormal perioral movements in response to D1 receptor stimulation. There was a heterogenous and patchy loss (40-50%) of [3H]mazindol binding to high-affinity DA uptake sites (a marker of DA terminal density) and a similar loss of TH-like immunoreactivity within the striata of the neonatally lesioned rats. There was also a reduction in the number of mu-opioid receptor patches (labelled with [3H]naloxone), a marker for the striatal patch compartment, and a similar patchy loss of D1 binding sites (labeled with [3H]SCH23390). The binding of [3H]spiroperidol to D2 sites was not altered. This is in contrast to the changes observed following adult 6-OHDA lesions, wherein there is a significant increase in the number of D2 binding sites (Joyce, 1991a,b). The results are discussed with respect to the behavioral consequences of neonatal lesions and the differences between neonatal and adult lesions.     

The effects of serotonergic and dopaminergic lesions on sodium-sensitive [3H]mazindol binding in rat hypothalamus and corpus striatum

The effects of intracerebroventricular administration of 6-hydroxydopamine (6-OHDA) and 5,7-dihydroxytryptamine (5,7-DHT) on sodium-sensitive [3H]mazindol binding were investigated in the rat hypothalamus and corpus striatum. In the hypothalamus, specific [3H]mazindol binding was inhibited by low concentrations of sodium and stimulated by high-sodium concentrations, whereas in the corpus striatum, only a sodium-dependent stimulation of [3H]mazindol binding was observed. Lesions with 6-OHDA significantly reduced sodium-dependent [3H]mazindol binding in the corpus striatum, but had no effect on the binding of [3H]mazindol in the absence of sodium. Lesions of serotonergic neurons with 5,7-DHT, however, had no effect on [3H]mazindol binding in the striatum, but resulted in a significant increase in the number of [3H]mazindol binding sites in the hypothalamus. These data suggest that [3H]mazindol may bind to two anatomically distinct binding sites, one that is stimulated and the other inhibited by sodium. The sodium-stimulated binding sites appear to be located on dopaminergic terminals in the striatum, and in the hypothalamus, the sodium-inhibited sites appear to be regulated by serotonergic neuronal activity.     

Plasticity of neostriatal dopamine receptors after nigrostriatal injury: Relationship to recovery of sensorimotor functions and behavioral supersensitivity

Rats given unilateral injections of 6-OHDA along the course of the mesotelencephalic dopaminergic projection show impairments in contralateral sensorimotor functions from which they often recover. Such rats also display an enhanced sensitivity to DA receptor stimulants, e.g. apomorphine, as revealed by contralateral turning, and an increased binding of neuroleptic compounds (e.g. [3H]spiroperidol) to the denervated striatum. This research examines the relationship of these receptor changes to both behavioral supersensitivity and recovery of sensorimotor functions by quantifying the time course of each phenomenon after injury. The supersensitivity to apomorphine and the behavioral recovery developed with a similar time course after injury, being evident within 1.5-3 days and reaching nearly maximal levels by 2 weeks postoperatively. A significant increase in in vivo [3H]spiroperidol binding to the denervated striatum occurred by 4 days postoperatively, and the magnitude of this change increased linearly during the first postoperative month. In contrast, the in vitro binding of this ligand to membranes of the denervated striatum was not increased until 3 weeks after the lesion. The results suggest that a proliferation of DA receptors may contribute to the pharmacological supersensitivity and the recovery of function, and that these early receptor changes may be revealed with greater sensitivity using in vivo binding techniques.     

Long-term behavioral and biochemical effects of 6-hydroxydopamine injections in rat caudate-putamen

Unilateral injections of 6-hydroxydopamine into the rat striatum result in amphetamine-induced circling behavior. This rotational behavior was associated with an almost complete disappearance of desmethylimipramine-insensitive [3H]mazindol binding sites--which represent dopamine uptake sites-in the ipsilateral caudate-putamen (CPu), the substantia nigra pars compacta (SNpc), and in the ventral tegmental area (VTA). There were significant increases in [3H]spiperone-labeled dopamine (DA) D2 receptors in specific subdivisions of the ipsilateral CPu, with the dorsolateral (DL) and ventrolateral (VL) regions showing significant increases in DA D2 receptors. There were nonsignificant increases in the dorsomedial (DM) aspects of the ipsilateral CPu whereas there were no changes in the ventromedial (VM) aspects of that structure. In contrast, there were no significant changes in [3H]SCH 23390-labeled DA D1 receptors in any of the subdivisions of the CPu ipsilateral to the 6-OHDA-induced lesions. These results provide evidence that intrastriatal injections of 6-OHDA result in biochemical changes in rat brain which are almost identical to those observed after 6-OHDA-induced lesions of the substantia nigra. These long-term biochemical effects caused by intrastriatal 6-OHDA injections provide further support for the idea that the nigral DA cell loss observed in the brains of parkinsonian patients could be secondary to retrograde changes due to oxyradicals generated during the metabolism of catecholamines within the caudate-putamen.     

Non-additivity of D2 receptor proliferation induced by dopamine denervation and chronic selective antagonist administration: evidence from quantitative autoradiography indicates a single mechanism of action

Experiments were conducted to investigate whether chronic dopamine (DA) D2 receptor blockade and DA denervation exert additive effects on striatal D2 receptor density. We employed for the first time chronic treatment with a pure D2 antagonist, metoclopramide, and measured regional striatal DA receptor binding with quantitative receptor autoradiography. Rats with extensive unilateral DA denervation induced by intracerebral 6-hydroxydopamine (6-OHDA) were injected daily for 21 days with either metoclopramide (30 mg/kg i.p.) or saline. Following a 72-h drug wash-out period, rats were sacrificed and brain sections through the caudate-putamen and nucleus accumbens were incubated with [3H]spiroperidol or [3H]SCH 23390 to assay D2 and D1 receptors, respectively. Autoradiographic analysis revealed that chronic metoclopramide treatment increased the density of D2 sites in the intact hemisphere for all regions examined without further augmenting the already increased density of D2 receptors seen in the 6-OHDA-treated hemisphere. In addition, chronic metoclopramide and 6-OHDA treatment by themselves exhibited remarkably parallel anterior-posterior gradients in their effects on D2 receptor density. D1 receptor density was not affected by metoclopramide treatment but was slightly reduced in the DA-denervated hemisphere. [3H]Mazindol labelling of high-affinity DA uptake sites indicated that the extent of DA denervation was greater than 98% in both saline- and metoclopramide-treated rats. These findings are consistent with the view that chronic D2 receptor blockade and DA denervation act via a single, common mechanism to increase D2 receptor density. Work from other laboratories, in which additive effects of denervation and chronic neuroleptic treatment have been purported, may have resulted from incomplete denervation. Experimental discrepancies may also be due to differing means by which the mesotelencephalic dopaminergic neurons are injured.     

Transport of [H]mazindol binding sites in mesostriatal dopamine axons

6-Hydroxydopamine injections along mesostriatal dopaminergic axons can be used to interrupt axonal transport from cell bodies in the substantia nigra pars compacta to terminal fields in the striatum. Such lesions produce accumulations of high-affinity dopamine uptake sites (as measured by [³H]mazindol binding) and acetylcholinesterase proximal to the injection, suggesting that at least a portion of the [³H]mazindol binding and acetylcholinesterase activity seen in the striatum is located presynaptically on the mesostriatal dopaminergic fibers.     

Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

Quantitative autoradiography using [3H]-SCH 23390, [3H]-sulpiride and [3H]-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both [3H]-SCH 23390 and of [3H]-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). [3H]-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral [3H]-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect [3H]-SCH 23390 or [3H]-forskolin binding, but largely removed [3H]-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce [3H]-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.     

Contrasting tissue factors predict heterogeneous striatal dopamine neurotoxicity after MPTP or methamphetamine treatment

Treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or (+)-methamphetamine (METH) results in regionally heterogeneous patterns of dopaminergic depletion. The magnitude of the MPTP-induced dopamine (DA) depletion corresponds directly to the density of [3H]mazindol binding to DA transport sites, but not the DA concentration, in intact mouse striatal regions. In contrast, the extent of METH-induced DA depletion corresponds to the intact dopamine concentration, not the [3H]mazindol binding, in the same striatal regions. The findings provide a rationale for testing different hypotheses regarding the neurobiological substrates of mesostriatal injury in idiopathic Parkinson's disease (PD).     

Autoradiographic analysis of muscarinic receptors and choline-uptake mechanisms within foetal basal forebrain transplants

The pharmacological characteristics of both muscarinic receptors and high-affinity choline uptake sites were examined within intracerebral implants of foetal basal forebrain cell suspensions. Approximately 12 weeks after implantation, the transplants were identified by acetylcholinesterase histochemistry. Muscarinic receptors were labelled by [3H]quinuclidinyl benzylate (QNB) autoradiography. The M1 and M2 receptor components of QNB binding were differentiated by pirenzepine competition. The distribution of the high-affinity choline uptake site was examined using [3H]hemicholinium-3 (HC3) autoradiography. Unilateral lesion of the nucleus basalis reduced [3H]QNB (8-25%) and [3H]HC3 (19-43%) binding throughout host frontoparietal cortex ipsilateral to the lesion but did not significantly alter these cholinergic markers within cingulate cortex, subcortical white matter, striatum or septum. Saturation analysis of the implanted tissue revealed the presence of a single population of [3H]QNB and [3H]HC3 binding sites with affinities similar to those of the host tissue (KD = 0.43 nM and 20.4 nM respectively). However, the receptor profile which developed appeared to be intrinsic to the implant; it was unaffected by the site of implantation and was dissimilar to that which ultimately developed in the donor tissue when left in situ.     

Striatal dopamine denervation decreases the GDP binding affinity in rat striatal membranes

The role of G-proteins in D2 receptor supersensitivity was studied in striatal membranes from rats with unilateral 6-hydroxydopamine (6-OHDA) induced lesions of the nigral dopamine (DA) system. Thirteen months after the lesion the number of [3H]raclopride binding sites was increased in the DA denervated striatum, but no changes in ligand binding affinities and in proportion of high-affinity agonist binding sites could be detected. The affinity of [35S]GTPgammaS binding was unaltered after the striatal DA denervation, whereas the binding affinity of GDP was decreased in the DA denervated as compared to the intact striatum. It is proposed that the decrease in GDP binding affinity to D2 DA receptor-coupled G proteins is an important factor in the D2 receptor supersensitivity following degeneration of the striatal DA terminals.     

Postnatal development of striatal dopamine function. II. Effects of neonatal 6-hydroxydopamine treatments on D1 and D2 receptors, adenylate cyclase activity and presynaptic dopamine function

To evaluate the influence of patch and matrix ingrowth of DA terminals upon striatal DA (dopamine) receptor function, we performed bilateral intrastriatal (i.s.) or single intracisternal (i.c.) injections of 6-hydroxydopamine (6-OHDA) into rat pups at various postnatal ages and determined D1 and D2 receptor binding, adenylate cyclase activities and markers for presynaptic DA terminal density and turnover as the animals matured. All injection schedules yielded: (a) variable and partial loss of DA, (b) increased DA turnover, (c) small (15-40%) increases in D1 receptor number but no change in affinity for antagonist ([3H]SCH 23390), (d) 2-3-fold increases in affinity of D1 receptors for agonist (SKF 38393) with preserved regulation of agonist affinity by guanine nucleotide, (e) no significant changes in DA-, guanine-nucleotide-, manganese- and forskolin-stimulated AC (adenylate cyclase) activity. D2 receptor binding was evaluated between 1 and 7 weeks of age in animals with i.s. treatment and 7 and 10 weeks of age in animals with i.c. treatment and was reduced by 40-50% with both treatment regimens. [3H]mazindol binding, a marker for presynaptic terminal DA transport sites, was reduced 30-40% by multiple i.s. or i.c. treatment regimens. In animals treated with one i.s. injection, [3H]mazindol binding was reduced 70% at 1 week of age, equal to control by 2 weeks and 14-46% greater than control between 3 and 7 weeks. We conclude that striatal D1 receptor sites maintain their density and second messenger function independently of postsynaptic DA terminal ingrowth, whereas the development of D2 receptor sites is sensitive to disruptions of DA terminal ingrowth.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.