Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [³H]quinuclidinyl benzilate (QNB), M₁ receptors were directly labeled with [³H]pirenzepine (PZ) and non-M₁ receptors were labeled by the competition of 100 nM PZ with [³H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [³H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [³H]QNB. Subtype analysis showed that there was a reduction of both M₁ (−26%) and non-M₁ (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (B_max) and not affinity (K_d). There was no significant change in [³H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.     

Pirenzepine-insensitive muscarinic autoreceptors regulate acetylcholine release in human neocortex

The release of [³H]acetylcholine ([³H]ACh) and its modulation mediated by autoreceptors were investigated in synaptosomes prepared from fresh human cerebral cortex prelabelled with [³H]choline ([³H]Ch) and depolarized in superfusion with 15 mM KCl. The K⁺-evoked release of tritium was almost totally accounted for by unmetabolized [³H]ACh and was largely calcium-dependent. Exogenous ACh decreased the depolarization-evoked release of [³H]ACh in a concentration-dependent manner (EC₅₀ = 1.5 μM). The inhibitory effect of ACh on [³H]ACh release was counteracted by the non-selective muscarinic antagonist atropine. In contrast, the selective M₁ receptor antagonist pirenzepine was ineffective. It is concluded that muscarinic autoreceptors regulating the release of ACh are present on cholinergic nerve terminals of human cerebral cortex and appear to belong to a pirenzepine-insensitive subtype.     

Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients

We assessed muscarinic M₁, M₂ and M₄ receptor subtypes in the hippocampus of Alzheimer’s and control brains by receptor autoradiography using ligands such as [¹²⁵I]muscarinic toxin-1 ([¹²⁵I]MT-1, M₁ selective), [³H]AFDX-384 (M₂ partially selective) and [¹²⁵I]muscarinic toxin 4 ([¹²⁵I]M₄ toxin-1, M₄ selective). Our results revealed a significant decrease in muscarinic M₄ receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimer’s patients compared to controls. No changes in the density of M₁ or M₂ receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M₄ receptor could be the subtype which is selectively compromised in Alzeheimer’s disease (AD).     

Sensitization of stress-induced feeding in rats repeatedly exposed to brief restraint: the role of corticosterone

The effects of 5-500 μM concentrations of neutral ammonium salts on the binding of ligands to components of the GABA_A receptor complex were investigated. [³H]Flunitrazepam binding to the benzodiazepine receptor was enhanced by ammonium (10–500 μM), but not sodium tartrate with EC₅₀ = 98 μM and E_max = 31%. Further increasing ammonium tartrate concentrations (500–2500 μM) decreased [³H]flunitrazepam binding to control levels. The ammonium tartrate-induced increase in [³H]flunitrazepam binding was manifested as a 50% decrease in K_d. Furthermore, GABA increased the potency of ammonium tartrate in enhancing [³H]flunitrazepam binding by 63%. [³H]Ro 15-1788 and [³H]Ro 15-4513 binding to the benzodiazepine receptor was not significantly enhanced by ammonium tartrate (E_max ≈ 13%). Ammonium tartrate also increased, then decreased the binding of 500 nM [³H]muscimol to the GABA_A receptor (EC₅₀ = 52 μM, E_max = 30%) in a concentration-dependent manner, but had no effect on [³H]SR 95-531 binding (E_max < 16%). The ammonium tartrate-induced alterations in [³H]muscimol binding were demonstrated in saturation assays as the loss of the high affinity binding site and a 27% increase in the _Bmax of the low affinity binding site. These results indicate that ammonia biphasically enhances, then returns ligand binding to both the GABA and benzodiazepine receptor components of the GABA_A receptor complex to control levels in a barbiturate-like fashion. This suggests that ammonia may enhance GABAergic neurotransmission at concentrations commonly encountered in hepatic failure, an event preceding the suppression of inhibitory neuronal function observed at higher ( > 1 mM) ammonia concentrations. This increase in GABAergic neurotransmission is consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia.     

The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets

The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [³H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na⁺,K⁺-ATPase activity was determined in a brain cell membrane preparation. [³H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5–50 nM [³H]MK-801 in the presence of 100 μM glutamate and glycine; and (2) binding assays at 10 nM [³H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 μM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na⁺,K⁺-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (B_max), but the apparent affinity was significantly increased, as indicated by a decrease in the K_d (dissociation constant) from the control value of 8.1 ± 1.6 nM to 5.5 ± 2.1 nM (P < 0.05). Augmentation of [³H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals. These data suggest that acute hypoglycemia may enhance the excitotoxic effects of glutamate in the newborn.     

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [³H]ACHC and [³H]GABA into cultured neurons and astrocytes was studied. [³H]ACHC uptake was less efficient than that of GABA in both cell types and K_m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V_max values were 0.321 and 0.405 nmol·min⁻¹·mg⁻¹ cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K_i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K_i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.     

Regulation of peripheral-type benzodiazepine receptors in cultured astrocytes by monoamine and amino acid neurotransmitters

Exposure of primary cultured astrocytes for 3 days to 1 μM of either dopamine, serotonin or norepinephrine resulted in upregulation (25–34% increase in B_max) of the peripheral-type benzodiazepine receptors (PBRs) labeled with [³H]Ro5-4864. A similar treatment with γ-aminobutyric acid [GABA] caused a 2-fold increase in the affinity (K_d) of [³H]Ro5-4864. The monoamines tested and GABA had no effect on the binding parameters of [³H]PK 11195, another selective PBR ligand. The present study indicates that Ro5-4864 binding sites are susceptible to regulation by specific neurotransmitters and provides further evidence for the distinction between Ro5-4864 and PK 11195 binding sites of the PBRs in cultured astrocytes.     

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

1. The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity.

2. [³H]PZ has been used extensively to identify and characterize the putative M₁ (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia.

3. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists.

4. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M₁ and M₂ subtypes.

5. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.     

Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum.

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats

In order to clarify the roles of hippocampal M₁ and M₂ muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M₁ muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 μg/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 μg/side) on working memory was attenuated by concurrent injection of 10 μg/side AF102B, the selective M₁ muscarinic receptor agonist. Intrahippocampal injection of the M₂ muscarinic receptor antagonist methoctramine at doses up to 1.0 μg/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 μg/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 μg/side AF102B. Concurrent injection of 0.32 μg/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 μg/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 μg/side, respectively. These results suggest that processes mediated by M₁ muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M₂ muscarinic receptors ameliorates working memory deficits produced by M₁ muscarinic blockade, possibly by increasing acetylcholine release.     

Histamine H1-receptors mediate phosphoinositide hydrolysis in astrocyte-enriched primary cultures

Astrocyte-enriched primary cultures of newborn rat brain hemispheres, prelabeled with [³H]inositol, accumulated [³H]inositol phosphate but not [³H]inositol bis-and tris-phosphate, after exposure to histamine for 60 min in the presence of 10 mM LiCl. The response to histamine was not a function of contaminating meningeal fibroblasts since no accumulation of [³H]inositol phosphate was elicited by histamine in meningeal cultures. The stimulation of phosphoinositide hydrolysis by histamine in astrocytes was dose-dependent (EC₅₀ = 1.7 μM, maximal effect = 345% over basal levels) and was mimicked by several H₁-receptor agonists. The use of selectiver receptor antagonists confirmed that the histamine response was the result of activation of H₁-receptors. The histamine-induced [³H]inositol phosphate accumulation was completely abolished by omission of Ca²⁺ from the incubation medium. Astrocyte membranes specifically bound the radiolabeled H₁-antagonist, [³H]mepyramine with an affinity (K_d = 5.9 nM) and a density of binding sites (B_max = 113 fmol/mg protein) similar to rat brain. These results demonstrate the presence of functional histamine H₁-receptors in rat brain astrocytes and suggest a role for histamine as a neuromodulator of astrocyte function.     

Brain nicotinic cholinoceptor binding in spontaneous hypertension

To study the role of central cholinergic mechanisms in hypertension, we have determined nicotinic and muscarinic agonist binding sites in the brain regions of stroke-prone spontaneously hypertensive rats (SHRSP), using [³H]nicotine and [³H]cismethyldioxolane (CD). There was a significant decrease in specific [³H]nicotine binding in the cerebral cortex, thalamus, midbrain, cerebellum and medulla oblongata of SHRSP at 16–24 weeks of age compared to that of age-matched Wistar Kyoto rats (WKY). Scatchard analysis revealed 35% decrease in theB_max value for [³H]nicotine binding in the SHRSP medulla oblongata without a change in theK_d value, suggesting a change in the receptor density. Similar reduction of nicotinic cholinoceptor binding sites was also observed in the discrete brain regions of young (5-week-old) SHRSP. In contrast, there was no alteration in specific [³H]CD binding in the SHRSP brain regions, except the hypothalamus which showed a significant increase. The SHRSP medulla oblongata showed no change in the ChAT activity. Thus, the present study suggests an important role for medullary nicotinic cholinoceptors in the pathogenesis of spontaneous hypertension.     

Brain slices in radioligand binding assays: Quantification of opiate, benzodiazepines and beta-adrenergic ([H] CGP-12177) receptors

1. We have characterized and quantified specific binding of [³H]-flunitrazepam (FNZ: (benzodiazepine), [³H]-naloxone (NAL: (opiate) and [³h]cgp-12177(CGP: (beta-adrenergic) to thick slices (230–400 μm) of mouse and rat brain.

2. The binding sites are stereospecific, saturable and of high affinity. In all cases, the binding of the ligands is readily reversible and demonstrates the appropriate drug specificity.

3. In mouse brain [³H]-NAL binding is elevated by chronic treatment with naloxone (via capsules).

4. We have been unsuccessful in quantifying beta adrenoreceptors with the archetypal ligand [³H]-dihydroalprenolol (DHA). However, the use of [³H]-CGP 12177 enabled us to detect high-affinity beta adrenoreceptors in brain slices.

5. [³H]-CGP also permits the demonstration of rapid and reversible agonist-induced down-regulation (internalization) of beta binding sites.

6. We have been successful in quantifying beta adrenergic sites in single pineal glands of rat and hamster.     

[H]Nipecotic acid binding to GABA uptake sites in human brain

The binding of [³H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with γ-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [³H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to high affinity site. The dissaciation constants (K_d) measured in kinetic and equilibrium experiments were in the same range (0.5–0.6 μM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [³H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of 6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Quantitative autoradiography of the dopamine uptake complex in rat brain using [H]GBR 12935: binding characteristics

The dopamine uptake complex was examined in the rat central nervous system using [³H]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [³H]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [³H]GBR 12935 can be used to specifically label the dopamine uptake complex. [³H]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25 μM) was used as a blank. The specific binding and autoradiographic appearance of [³H]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75 μM) to displace binding to a previously desrribed piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 ± 0.7nM), maximal number of binding sites (6.0 ± 1.3pmol/mg protein), and Hill coefficient (1.1 ± 0.1) of [³H]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75 μM). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 ± 2.3pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 ± 0.3nM) or Hill coefficient (1.1 ± 0.1). A series of drugs that bind to the dopamine uptake complex displaced [³H]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 > mazindol > nomifensine > benztropine > desipramine > amphetamine > dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35, 428 (a cocaine congener) were unique in their competition for [³H]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D₁ receptor. [³H]GBR 12935 can be used as an effective ligand to label the dopamine uptake complex for quantitative autoradiographic studies. It offers a number of advantages over previous autoradiographic assays for this complex including high specificity (> 95% specific binding in rat striatum), high sensitivity (detection of mazindol displaceable sites in the cerebral cortex), low background (comparable to film background), and low cost. This assay also supports the existence of two binding sites for cocaine on the dopamine uptake complex. The exact nature and differences between these two cocaine sites remains to be determined.     

Alterations in neurotransmitter receptors and glucose use after unilateral orbital enucleation

Serotonin (5-hydroxytryptamine; 5-HT), acetylcholine and γ-aminobutyric acid (GABA) are neurotransmitters in the rat visual system. Using quantitative autoradiography, the effect of unilateral orbitral enucleationon [^3H]5-HT, [³H]ketanserin, [³H]quinuclidinyl benilate (QNB) and [³H]muscimol binding to 5-HT₁, 5-HT₂, muscarinic and GABA_A receptors has been examined within anatomical components of the visual pathway at 4 time points up to 20 days after the lesion. The functional deficit was assessed in the same animals using quantitative [¹⁴C]2-deoxyglucose autoradiography. At 1 day after unilateral orbital enucleation, there were no significant alterations in ligand binding although local cerebral glucose use was reduced in primary visual structures in the visually deprived hemisphere. At 5 days post-enucleation, however, [³H]5-HT binding was significantly reduced in both the visually deprived superior colliculus (by 17%) and dorsal lateral geniculate body (DLG) (by 33%). There were similar alterations in the binding of this ligand in these primary retinal projections areas at 10 and 20 days after orbital enucleation, but there were no changes in secondary areas (e.g. visual cortex) at any time point. [³H]Muscimol binding was significantly reduced in the visually deprived DLG (30%) and visual cortex (21%) only at 20 days post-lesion, whilst [³H]ketanserin and [³H]QNB were not altered in any region in the visually deprived hemisphere at any time point post-enucleation. At 10 and 20 days post-enucleation, the degree of [³H]5-HT, and [³H]muscimol binding deficits in visually deprived structures correlated significantly with the level of reduced metabolic activity in these areas (r = 0.700andr = 0.543respectively). The specificity and regional and temporal heterogeneity of neurotransmitter receptor binding alterations provides evidence of selective adjustments within visual system component6s in response to orbital enucleation.     

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M₁- and M₂-receptor subtypes through semi-quantitative in vitro autoradiography. [³Quinuclidinyl benzilate] (QNB) and [³H]pirenzepine (PZ) were used for labeling total mAChR and M₁-receptors, respectively. In the cerebral cortex of adult rats, [³H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M₁-receptors were diffusely observed in all the layers. This means that M₂-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M₁- and M₂-receptor first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M₁-type in the adult rat brain, ontogenic patterns of M₁-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M₂-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M₁-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M₁- and M₂-receptors show distinct developmental behaviors in the rat brain.     

Endogenous ligands for the quisqualate receptor: presence in rat brain cortex synaptic vesicles

The presence in highly purified rat brain cortex synaptic vesicles of endogenous ligands for rat brain quisqualate receptors was investigated. The vesicles were extracted, and their contents fractionated by high voltage electrophoresis. Endogenous ligands were detected by a radioreceptor assay in which such ligands competed with 50 nM -[³H]glutamate for binding to quisqualate receptors present in rat brain postsynaptic densities (PSDs). Binding of -[³H]glutamate to (NMDA) receptors, also present in PSDs, was blocked by 100 μM NMDA. We found that the endogenous ligands present in brain cortex synaptic vesicles for quisqualate receptors, were glutamate and aspartate, in a molar ratio of about two to one. The quisqualate receptor had an affinity 130-fold higher for glutamate (K_d 0.3 μM) than for aspartate, and the latter amino acid also showed a marked negative cooperativity for binding (Hill number 0.29, against 0.67 for glutamate). These findings suggest that glutamate is the natural transmitter that activates quisqualate receptors at some central excitatory synapses, and also that aspartate may be a classical transmitter, the receptor for which still remains to be shown.