Neurokinin A and substance P in striato-nigral neurons in rat brain

The effect of an ibotenic acid lesion in rostral striatum on tissue levels of neurokinin A and substance P in striatum and substantia nigra was studied in rat brain. A total of 32 micrograms (10 mg/ml) ibotenic acid was injected in four positions to lesion striatal cell bodies rostral to bregma. The neurokinin A level was reduced to a third of the control value in striatum and to less than half of the control level in substantia nigra. Neurokinin A, in addition to substance P, is shown to be possibly present in striato-nigral neurons, which provides further evidence for the existence of a striato-nigral tachykinin pathway.     

Subchronic haloperidol increases CB(1) receptor binding and G protein coupling in discrete regions of the basal ganglia

The present study was designed to test whether chronic neuroleptic treatment, which is known to alter both expression and density of dopamine D(2) receptors in striatal regions, has effects upon function and binding level of the cannabinoid CB(1) receptor in the basal ganglia by using receptor autoradiography. As predicted, subchronic haloperidol treatment resulted in increased binding of (3)H-raclopride and quinpirole-induced guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) in the striatum when compared to that measured in control animals. This increased D(2) receptor binding and function after 3 days washout was normalized after a 2-week washout period. Effect of haloperidol treatment was studied for CB(1) receptor binding and CP55,940-stimulated [(35)S]GTPgammaS in the striatum, globus pallidus, and substantia nigra. (3)[H]CP55,940 binding levels were found in rank order from highest to lowest in substantia nigra > globus pallidus > striatum. Furthermore, subchronic haloperidol treatment resulted in elevated binding levels of (3)[H]CP55,940 in the striatum and the substantia nigra and CB(1) receptor-stimulated [(35)S]GTPgammaS bindings in the substantia nigra after 3 days washout. These increased binding levels were normalized at 1-4 weeks after termination of haloperidol treatment. Haloperidol treatment had no significant effect on CB(1) receptor or [(35)S]GTPgammaS binding levels in globus pallidus. The results help to elucidate the underlying biochemical mechanism of CB(1) receptor supersensitivity after haloperidol treatment.     

In vivo detection of immunoreactive neurokinin A release within rat substantia nigra and its dependency on a dopaminergic input

In the striatum, the tachykinin peptide neurokinin A (NKA) is thought to coexist with substance P in the γ-aminobutyric acid-containing spiny neurones which project to the substantia nigra. We have used in vivo antibody-coated microprobes to directly monitor the release of NKA-like immunoreactivity (NKA-LI) within substantia nigra during various pharmacological manipulations. The data clearly illustrates a basal or resting extracellular presence of NKA-LI restricted to substantia nigra reticulata which was found to be largely dependent on a dopaminergic input. Acute administration of haloperidol (0.1–0.2 mg/kg i.p.) considerably reduced this basal NKA-LI whereas depot administration (14 mg/kg i.m. released over 2 weeks) produced a less substantial reduction. Lesion of nigro-striatal dopamine neurones with the neurotoxic agent 6-hydroxydopamine produced significant reductions in the nigral NKA-LI detected. However, d-amphetamine administration (4 mg/kg i.p.) did not alter the pattern of NKA-LI release for up to 4 h posttreatment. These results indicate that changes in peptide mRNA levels do not necessarily reflect changes in peptide release and suggest that NKA may be the more physiologically relevant tachykinin within the substantia nigra of the rat.     

Autoradiographic localization of a novel peptide binding site in rat brain using the substance P analog, eledoisin

Using slide mounted sections of rat brain sausage, we have characterized the binding of [125I]Bolton Hunter conjugated eledoisin and [125I]Bolton Hunter conjugated substance P. Structure activity studies suggest that the two radiolabeled peptides bind to different binding sites. Autoradiographic studies support this notion. Whereas [125I]BH-SP sparsely labels the interpeduncular nucleus and does not label the substantia nigra at all, [125I]BH-ED densely labels the former and sparsely labels the latter structure. Further, the cortical labeling patterns obtained with the two peptides are strikingly different. These data support the hypothesis that there exist two classes of tachykinin binding sites in rat nervous tissue.     

Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex

The autoradiographic distribution of the 3 neurokinin (NK) receptor sub-types, NK-1, NK-2 and NK-3, was compared in rat cerebral cortex during post-natal development using [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands. Throughout brain development, NK-1 receptor sites are present in low densities with some enrichment seen in lamina III while NK-3 binding sites are concentrated in layers IV and V. However, it appears that NK-2 receptors are mostly expressed in lamina VI and only during the first two postnatal weeks. These results demonstrate further the existence and differential ontogeny of 3 classes of NK receptors in rat brain cortex.     

Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra

The injection of kainic acid into the substantia nigra causes, 3 weeks after treatment, a 40% decrease in the total number of binding sites for [³H]diazepam with an increase in the dissociation constant. This decrease was restored to approximately normal by the subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The injection of kainic acid into the striatum of intact animals failed to modify the number of [³H]diazepam binding sites but increased the K_D. The results indicate that benzodiazepine binding sites in the substantia nigra are partly located on kainic acid sensitive elements (probably interneurons) and, partly, on kainic acid resistant ones. These binding sites become supersensitive after degeneration of striato-nigral pathways. The possible role of GABAergic denervation of the substantia nigra in the development of benzodiazepine binding sites supersensitivity is discussed.     

Binding sites for pituitary adenylate cyclase activating polypeptide (PACAP): comparison with vasoactive intestinal polypeptide (VIP) binding site localization in rat brain sections.

Pituitary adenylate cyclase activating polypeptide (PACAP) is structurally similar to vasoactive intestinal polypeptide (VIP). We investigated the characteristics and topographical distribution of [125I]PACAP binding sites compared with those of [125I]VIP binding sites in the rat brain. Radiolabeled PACAP and VIP showed highly specific binding to sections at the level of the dorsal hippocampus. The specific binding of [125I]PACAP was 10 times higher than that of [125I]VIP in hippocampal sections. [125I]PACAP binding was scarcely displaced by unlabeled VIP, while [125I]VIP binding was effectively displaced by unlabeled PACAP. Therefore, PACAP binding sites may reflect both PACAP specific binding sites and VIP/PACAP binding sites. However, the amount of VIP/PACAP binding sites was negligibly low. Autoradiography revealed that [125I]PACAP binding sites were dense in the piriform cortex, diagonal band, accumbens nucleus, anterior part of the striatum, hippocampal formation, habenular nucleus, lateral hypothalamic area, superior colliculus and dorsal raphe nucleus. Moderate to high labeling was observed in the medial septal nucleus, olfactory tubercle, caudal part of the striatum, most parts of the thalamus, supraoptic and periventricular hypothalamic nuclei, central gray, substantia nigra pars compacta, locus coeruleus, pontine reticular nucleus and cerebellum. Distribution pattern was remarkably different from that of [125I]VIP binding sites in the hippocampal formation, lateral hypothalamic area, substantia nigra pars compacta, pontine reticular nucleus and cerebellum. The present results suggest that PACAP may have a physiological role in the regulation of the central nervous system.     

Huntington's disease: changes in tachykinin content in postmortem brains

Substance P, neurokinin A, neuropeptide K, and neurokinin B were measured in both control (neurologically normal) and Huntington's disease brains obtained post mortem. All four peptides were significantly reduced in the substantia nigra of Huntington's disease patients compared with the control group. No differences were observed in frontal or temporal cortex except that neuropeptide K was significantly reduced in the frontal cortex of Huntington's disease cases. Correlation of the cell loss observed in the striatum and the tachykinin depletions detected in the substantia nigra in the Huntington's disease brains showed that the degree of cell loss agreed well with the extent of tachykinin depletion. Results of double-staining immunocytochemistry were consistent with the coexistence of substance P and neurokinin A in the substantia nigra of control brains and showed a marked depletion of immunoreactivity to both in Huntington's disease brains.     

Localization of substance P binding sites in submucous plexus of guinea pig ileum, using whole-mount autoradiography

Whole mounts of guinea pig ileum submucosa were incubated with radiolabeled tachykinins, and binding sites were visualized using autoradiography. Very dense specific binding for [125I]-Bolton-Hunter substance P (BHSP) was observed over ganglia of the submucous plexus, with weaker binding over internodal strands. Dense specific binding was also seen over occasional strands of circular muscle, with weak binding over clumps of mucosa. Although very weak binding was seen over some large blood vessels, no binding was associated with smaller blood vessels. Localization of binding was absent in whole-mounts coincubated with 1 microM substance P, used to define nonspecific binding. Localization of BHSP-specific binding was also abolished in whole-mounts coincubated with 1 nM substance P, but not with 1 nM neurokinin B, suggesting that binding was probably to an NK-1 tachykinin receptor. In whole-mounts incubated in [125I]-iodohistidyl neurokinin A (INKA) or [125I]-Bolton-Hunter neurokinin B (BHNKB), no specific binding over ganglia was observed. These binding sites for BHSP are probably identical with the neuronal substance P receptors mediating mucosal ion transport.     

A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain

The relative potencies of tachykinin peptide analogs competing for binding of [125I]Bolton Hunter-conjugated substance P ([125I]BH-SP) or [125I]Bolton Hunter-conjugated eledoisin ([125I]BH-ED) in slide-mounted rat brain sections are very different, indicating the presence of two distinct tachykinin binding sites. The structure-activity profiles resemble those described in peripheral bioassay studies in which two tachykinin receptors have been postulated. Autoradiography of the two iodinated ligands bound with selective and one-site in vitro incubation conditions shows two discrete and distinctly different distribution patterns in brain. Binding sites for [125I]BH-ED are densely distributed in the accessory olfactory bulb, intermediate layers of the cerebral neocortex, portions of the hippocampal CA fields, hypothalamic supraoptic and paraventricular nuclei, central portions of the interpeduncular nucleus, sphenoid nucleus, medial subdivision of the solitary tract complex, and the substantia gelatinosa of the spinal cord. Binding sites for [125I]BH-SP are present in many of these same structures, but the densities and distribution patterns are different. In addition, [125I]BH-SP binds in numerous structures not labeled by [125I]BH-ED. Neither pattern matches the locations of terminations of endogenous tachykinin pathways marked by immunohistochemistry. The results suggest that it would be inappropriate to name brain tachykinin receptors according to the endogenous ligand which binds with highest affinity.     

Dramatic increase in nigral t-[35S]butylbicyclophosphorothionate binding sites elicited by the degeneration of the striato-nigral GABAergic pathway: reversal by diazepam

In rats, the degeneration of the striato-nigral GABAergic pathway caused by the intrastriatal injection of kainic acid induced a marked decrease (65%) of GABA content and glutamic acid decarboxylase (GAD) activity and a dramatic increase (225%) in the binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a membrane preparation from the substantia nigra homolateral to the injected striatum. The increase in [35S]TBPS binding in the denervated substantia nigra was exclusively due to an increased density of binding sites (Bmax) with no change in the dissociation constant (kd). The enhancement in [35S]TBPS binding was almost completely reversed by the intraperitoneal administration of diazepam (3 mg/kg) to kainic acid-lesioned rats. Moreover, diazepam produced a significant decrease (30%) in the density of [35S]TBPS binding sites also in the sham-operated side. In contrast the 'in vitro' addition of the GABAA receptor antagonist bicuculline (1 microM) to the membrane preparation from the denervated substantia nigra further increased [35S]TBPS binding. These findings suggest the view that the increase of nigral [35S]TBPS binding is directly related to the inhibition in the function of nigral GABAergic synapses following the loss of the striato-nigral GABAergic pathway. Our results indicate that [35S]TBPS binding to brain structure is a potential tool to reveal alteration in the function of GABAA receptor complex elicited by physiological, pharmacological and pathological conditions.     

Brain dopamine transporter: gender differences and effect of chronic haloperidol

Gender differences and the effect of chronic haloperidol on the rat brain dopamine transporter is reported. The density of striatal dopamine transporter sites labelled with [³H]GBR 12935, and of substantia nigra dopamine transporter mRNA measured by in situ hybridization were higher in female compared to male rats whereas striatal D2 specific binding labelled with [³H]spiperone was not significantly higher. Daily haloperidol treatment (1 mg/kg, i.p.) for 21 days increased striatal [³H]spiperone specific binding but left unchanged striatal [³H]GBR 12935 binding density and affinity as well as substantia nigra dopamine transporter mRNA levels. A reduce clearance rate of dopamine in the striatum after acute and chronic haloperidol was previously reported; the present results indicate that this may occur without changes in the sites of dopamine transport or in gene expression of this transporter.     

Localization of striatal and nigral tachykinin receptors in the rat

The effects of lesioning mesostriatal dopamine projections or striatal neurons on tachykinin binding in the basal ganglia were assessed in the rat. 6-Hydroxydopamine lesions of the medial forebrain bundle destroyed striatal dopamine terminals as assessed by [³H]mazindol autoradiography, but did not significantly affect the binding of NK-1 ([³H][Sar⁹, Met(O₂)¹¹]substance P) or NK-3 ([³H]senktide) tachykinin ligands in the striatum. 6-Hydroxydopamine lesions significantly reduced NK-3 binding in the substantia nigra pars compacta, but not the ventral tegmental area. In contrast, striatal quinolinic acid lesions reduced both NK-1 and NK-3 binding in the striatum, but failed to affect NK-3 binding in the substantia nigra. These findings suggest that both NK-1 and NK-3 receptors within the striatum are predominantly post-synaptic with respect to dopamine neurons, whereas nigral NK-3 receptors are located on dopaminergic neurons.     

Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.     

Calcitonin gene-related peptide (hCGRP alpha) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences.

The distribution of [125I]hCGRP alpha binding sites was studied in tissue sections from rat brain and, at the level of the nucleus accumbens in the brains of 6 other species. In the rat, very high levels of binding were found in the nucleus accumbens, the amygdaloid complex and mammillary body while high amounts were localized to the superficial layers of the superior colliculus, temporal cortex, cerebellum (molecular layer), frontal cortex and inferior olive. Moderate densities of [125I]hCGRP alpha binding were observed in the medial geniculate nucleus, inferior colliculus and substantia nigra. Regional competition studies in rat brain showed that salmon calcitonin was almost as effective as hCGRP alpha in competing for [125I]hCGRP alpha binding sites in the nucleus accumbens but was mostly inactive in other regions such as the mesolimbic cortex and the striatum. On the basis of their atypical sensitivity to salmon calcitonin, [125I]hCGRP alpha binding sites in the rat nucleus accumbens, which appear between postnatal days 4 and 7, do not seem to correspond to either the CGRP1 or CGRP2 receptor subtypes. Marked species differences were observed in the distribution of [125I]hCGRP alpha binding sites, especially in the nucleus accumbens. In the mouse, low densities of hCGRP alpha sites were observed in striatum and fronto-parietal cortex while low to moderate levels were found in the medial and posterior aspects of the nucleus accumbens. A similar distribution was seen in the guinea pig brain albeit of generally higher density. In the rat, very high amounts of [125I]hCGRP alpha binding were seen in the nucleus accumbens while lower levels were found in the striatum and certain cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of the D-1 dopamine receptor in the substantia nigra of several mammalian species: identification in binding studies using [125I]SCH 23982

The substantia nigra of the rat brain contains a high concentration of binding sites for benzazepine antagonists of the D-1 receptor. Using [125I]SCH 23982, the properties of these binding sites are characterized and shown to resemble the D-1 dopamine receptor occurring in the caudate nucleus. Similar binding sites are identified in the substantia nigra of the cat and guinea pig.     

Neurotensin receptor binding sites in monkey and human brain: autoradiographic distribution and effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment

Neurotensin (NT) receptor binding sites were characterized and localized by using membrane binding assay and in vitro receptor autoradiography in monkey and human brain. Additionally, the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment on NT binding sites were investigated in monkey. [125I]Tyr3-NT ([125I]NT) apparently binds to a single class of high-affinity sites (Kd in nanomolar range) in both species. Ligand selectivity patterns strongly suggest that the structural requirements of both monkey and human brain NT receptors are very similar to those previously reported in other tissues, such as those of the rat brain and rat stomach. In monkey brain, [125I]NT binding sites are discretely distributed with high densities of sites found in the cingulate cortex, amygdala, hippocampus, ventral tegmental area, substantia nigra, and periaqueductal gray matter. A similar pattern is observed in the human brain. However, the laminar distribution of [125]NT binding sites in cortex varies between monkey and human brain. In monkey brain, [125I]NT binding sites are mostly concentrated in deep cortical layers while the laminar distribution of NT sites changes with cortical areas in human brain. The densities of [125I]NT binding sites are markedly decreased in the caudate, putamen, and substantia nigra in MPTP-treated monkeys. These results suggest strong interactions between NT and dopaminergic systems in both monkey and human brain tissues.     

Neurotensin receptors and dopamine transporters: effects of MPTP lesioning and chronic dopaminergic treatments in monkeys.

The effect of denervation with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) of the dopamine (DA) nigrostriatal pathway on neurotensin (NT) receptor and DA transporter (DAT) in basal ganglia of monkeys (Macaca fascicularis) was investigated. The MPTP lesion induced a marked depletion of DA (90% or more vs. control) in the caudate nucleus and putamen. The densities of NT agonist binding sites labeled with [125I]NT and the NT antagonist binding sites labeled with [3H]SR142948A decreased by half in the caudate-putamen of MPTP-monkeys. In addition, the densities of [125I]NT and [3H]SR142948A binding sites markedly decreased (-77 and -63%, respectively) in the substantia nigra of MPTP-monkeys. Levocabastine did not compete with high affinity for [125I]NT binding in the monkey cingulate cortex, suggesting that only one class of NT receptors was labelled in the monkey brain. An extensive decrease of [3H]GBR12935 DAT binding sites (-92% vs. Control) was observed in the striatum of MPTP-monkeys and an important loss of DAT mRNA(-86% vs. Control) was observed in substantia nigra. Treatments for 1 month with either the D1 agonist SKF-82958 (3 mg/kg/day) or the D2 agonist cabergoline (0.25 mg/kg/day) had no effect on the lesion-induced decrease in NT and DAT binding sites or DAT mRNA levels. The decrease of striatal NT binding sites was less than expected from the decrease of DA content in this nucleus, suggesting only partial localization of NT receptors on nigrostriatal DAergic projections. These data also suggest that under severe DA denervation, treatment with D1 or D2 DA agonists does not modulate NT receptors and DAT density.     

The turnover rate of gamma-aminobutyric acid in the nuclei of telencephalon: implications in the pharmacology of antipsychotics and of a minor tranquilizer.

The turnover rate of GABA is measured in substantia nigra, globus pallidus, N. accumbens, and striatum of rats injected with muscimol, a potent GABA agonist, and diazepam. The similarity of action of the two drugs on GABA turnover further supports the theory that diazepam acts as a GABA-mimetic drug. Haloperidol and clozapine affect GABA turnover differently in different nuclei. Haloperidol decreases GABA turnover in caudate but does not affect that in substantia nigra, whereas clozapine increases GABA turnover in both areas. However, both drugs accelerate GABA turnover in globus pallidus and N. accumbens. It is suggested that an increase of GABA turnover and perhaps of GABA release in striatum and substantia nigra may account for the lack of tardive dyskinesia and extrapyramidal side effects of clozapine.     

Positron emission tomography of radioligand binding in porcine striatum in vivo: haloperidol inhibition linked to endogenous ligand release

The ligands N-methylspiperone and haloperidol both bind to D(2)-like dopamine receptors. The competitive nature of the binding over a wide range of haloperidol concentrations and the effect on dopamine release have never been tested in vivo. We determined the competitive interaction between 3-N-[(11)C]methylspiperone ([(11)C]NMSP) and haloperidol binding to striatal dopamine D(2)-like receptors with positron emission tomography (PET) of pig brain. [(11)C]NMSP tomography was performed with haloperidol at five different plasma concentrations maintained constant by programmed infusion. Kinetic parameters of ligand competition for binding in the striatum were determined by deconvolving time-activity curves of the striatum and cerebellum from metabolite-corrected arterial plasma [(11)C]NMSP and haloperidol concentrations. Two types of [(11)C]NMSP-binding sites were evident in the striatum, both saturable by haloperidol administration. The preponderant or primary sites bound [(11)C]NMSP irreversibly, as dopamine D2-like receptors, while the secondary sites bound [(11)C]NMSP reversibly, as do serotonin S2 receptors. Woolf-Hanes plots revealed the predicted approximately linear relationships between the binding indices and the haloperidol plasma concentration. For the irreversible binding sites, this relationship indicated a 50% inhibitory concentration of haloperidol of 2 nM in plasma and a maximum binding capacity of 64 pmol cm(-3) in striatum. For the reversible binding sites, the relationship indicated a 50% inhibitory plasma concentration of haloperidol of 1 nM and a maximum binding capacity of 4.5 pmol cm(-3). Second-order polynomial Eadie-Hofstee-Scatchard plots were consistent with increased competition from an endogenous ligand of the irreversibly binding sites only with increasing doses of haloperidol. At the highest haloperidol dose, this hypothetical endogenous ligand had risen 6-7-fold. We contend that this reveals the release of dopamine by high concentrations of haloperidol.