Evidence for the localization of 5HT1A binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain

Precise anatomical distribution of 5HT1 binding sites has been investigated in the nuclei raphe dorsalis, raphe centralis and locus caeruleus of the rat brain. An original pattern of distribution was observed in the raphe nuclei, closely correlated to the already known distribution of 5HT containing elements. This pattern, more pronounced when 5HT1A sites were labelled, completely disappeared after lesioning by 5, 7DHT indicating the presence of this subtype of 5HT1 binding sites on 5HT containing neurons. It is postulated that these 5HT1A sites correspond in these raphe nuclei to 5HT autoreceptors.     

Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B

The nature of the receptor mediating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT1 or 5HT2 receptors. We have explored the possibility that such receptors in the rat fundus may better correlate with 5HT1A or 5HT1B receptor subtypes as defined by radiolabeled ligand binding studies with brain cortical membranes. Meta chlorophenylpiperazine (CPP) and meta trifluoromethylphenylpiperazine (TFMPP), selective ligands for the 5HT1B receptor and LY165163, a selective ligand for the 5HT1A receptor, have been evaluated for their agonist and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY165163 showed no agonist activity in this smooth muscle in concentrations up to 10(-4)M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors in the rat fundus was similar for all three phenylpiperazines in spite of the reported selectivity of MCPP and TFMPP for 5HT1B and of LY165163 for 5HT1A receptors. Furthermore, the affinity of these agents for serotonin receptors in the rat stomach fundus did not agree with their reported affinity for either 5HT1A or 5HT1B binding sites in rat cortical membranes. Thus, the similarity in affinities of these phenylpiperazine derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HT1A and 5HT1B binding sites argues against the possibility that the serotonin receptor in the rat fundus is of the 5HT1A or 5HT1B subtype of serotonin receptor.     

5-Hydroxytryptamine binding to synaptic membranes from rat brain.

The ³H-5HT binding capacity of rat brain synaptic membranes prepared by density gradient centrifugation has been investigated using a rapid ultrafiltration technique. A saturable, high affinity (K_D = 1.10^?9 M), 5HT displaceable binding has been found. It is thermosensitive, temperature dependent and pH dependent. 5HT and related tryptamines are the most effective displacers of bound ³H-5HT, whereas compounds which are not structurally related to 5HT (chlorpromazine, imipramine, cyproheptadine and cinanserine) and other neuro-transmitters (noradrenalin, dopamine) are ineffective. The distribution of 5HT-specific binding sites in the brain is related to serotonergic input. We conclude that these 5HT binding sites might possibly represent 5HT receptor sites.     

Synthesis and pharmacological study of radioiodinated serotonin derivative specific of 5-HT1B and 5-HT1D binding sites of the central nervous system

We describe here the synthesis of a new serotonin conjugate, S-CM-GTNH2, and its radioiodinated derivative. Quantitative autoradiographic studies on rat and guinea pig brain sections incubated with 2 nM [3H]5-HT showed a preferential affinity of S-CM-GTNH2 for 5-HT1B and 5-HT1D sites. Autoradiograms from brain sections incubated with 0.02 nM S-CM-G[125I]TNH2 showed a heterogeneous anatomical distribution of the labelling with high densities in regions rich in 5-HT1B or 5-HT1D binding sites, and with no labelling of those rich in 5-HT1A or 5-HT1C sites. The pharmacological profiles of the binding sites corresponded to those of 5-HT1B and 5-HT1D receptor subtypes. The radioligand S-CM-G[125I]TNH2 is a good probe for the study of these sites and will be used for their subcellular localization in electron microscopy.     

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using [I]FK33824

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites [25,35], consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, [³H]oxymorphone, [³H]D-ala²-MePhe⁴, Gly-ol⁵-enkephalin (DAGO), and [¹²⁵I]D-ala²-Me-Phe⁴-met(o)-ol]enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu binding sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand [¹²⁵I]FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.     

Different effects of serotonin antagonists on 3H-mianserin and 3H-ketanserin recognition sites

In minces prepared from the frontal cortex of rats treated with ketanserin (10 mg/kg i.p.) or mianserin (5 mg/kg i.p.) twice daily for 21 days, the Vmax of the adenylate cyclase stimulated by NE (100 microM) is attenuated, suggesting that ketanserin and mianserin share with a number of antidepressants the ability to attenuate the adenylate cyclase stimulation by NE. Ketanserin, given with the above mentioned dose schedule for 7 consecutive days, reduced the Bmax of 5HT2 recognition sites but failed to change either the Bmax or the apparent Kd of H-mianserin binding. A significant decrease in the Bmax of 5HT2 binding sites is elicited also by a single injection of mianserin (1). This drug also down-regulates its own binding when given twice daily for 3 weeks. From this and other information (2,3), it is concluded that ketanserin and mianserin bind to distinct recognition sites. The possibility that 5HT2 and mianserin recognition sites are functionally related and that serotonergic synapses are modulated by multiple chemical signals might be considered.     

Diazepam increases 5-hydroxyindole concentrations in rat brain and spinal cord

Diazepam elevates serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) concentrations in rat brain and spinal cord. The maximal effect occurs 1–2 hrs after drug injection and is dose related between 5–20 mg/kg (intraperitoneal). The action of diazepam on brain 5HT and 5HIAA concentrations is modified by previous food consumption: the ingestion of a diet that raises brain 5HT and 5HIAA one hour before drug injection enhances the diazepam-induced increase in brain indoles; consumption of a diet that lowers brain 5HT and 5HIAA partially blocks the elevation in brain indoles that follows diazepam injection.     

Characterization and distribution of bombesin binding sites in the goldfish hypothalamic feeding center and pituitary

Bombesin (BBS)/gastrin-releasing peptide (GRP) binding sites were characterized and their distribution examined in the goldfish brain and pituitary by radioligand binding and autoradiography. Binding of ¹²⁵I-[Tyr⁴]-BBS-14 to tissue sections was found to be saturable, reversible, time-dependent and displaceable by BBS/GRP-like peptides. Analysis of saturable equilibrium binding revealed a one-site model fit with a K_d of 0.665 ± 0.267 nM. This binding site displayed high affinity for members of the BBS subfamily of peptides, including GRP10 (K_i; 0.292 ± 0.038 nM) and GRP27 (K_i; 2.034 ± 1.597 nM), but showed no affinity for the BBS_8–14 fragment. While an approximate 100-fold lower binding affinity was displayed by the binding site for neuromedin B (K_i; 61.5 ± 28.2 nM), litorin was highly effective in displacing radiolabeled BBS binding (K_i; 1.469 ± 0.427 nM). The localization of saturable and high affinity BBS/GRP binding sites in specific areas of the goldfish brain and pituitary generally revealed a similar anatomical distribution to BBS/GRP-like immunoreactive material reported previously by our laboratory. Quantitative densitometric analysis of radiolabeled BBS binding to brain nuclei and the pituitary revealed a moderate concentration of BBS/GRP binding sites in the hypothalamic feeding area, including the nucleus diffusus lobi inferioris, nucleus recessus lateralis, nucleus lateral tuberis, and nucleus anterior tuberis. Other brain nuclei known to influence the brain feeding center which contained a high density of BBS/GRP binding sites included nuclei of the dorsal and ventro-medial telencephalon, the preoptic hypothalamus, and the optic tectum. High densities of BBS/GRP binding sites were also localized in the dorsal cerebellum, and nucleus habenularis. In the pituitary, BBS/GRP binding sites were present in high concentration in the neurointermediate lobe, with a relatively lower density localized in the pars distalis. The present study further supports a role for BBS/GRP-like peptides in the regulation of feeding behavior and anterior pituitary hormone secretion in teleosts.     

INJECTION OF 6-HYDROXYDOPAMINE AND HYDROGEN PEROXIDE INTO THE SUBSTANTIA NIGRA AND LATERAL VENTRICLE OF THE CAT: SPECIFIC AND NONSPECIFIC EFFECTS ON STRIATAL BIOGENIC AMINES1

Attempting to clarify the mechanism by which intracerabral injection of 6-hydroxydopamine (60HDA) reduces catecholamines in the caudate nucleus (CN), we have tested two hypotheses: (1) 60HDA specifically attacks catecholaminergic neurons; (2) 60HDA liberates hydrogen peroxide (H₂O₂) which destroys neurons indiscriminately. To this end, we have injected high or low doses of 60HDA or equimolar amounts of H₂O₂ stereotaxically into the substantia nigra (SN) or the lateral ventricle of cats and have placed electrocoagulative lesions in the SN. We determined the CN levels of dopamine (DA), norepinephrine (NE) and serotonin (5HT) 7-10 days later. Nigral injections of high doses (8 μ mol) of either agent or low doses (80 nmol) of 60HDA decreased both DA and NE and induced similar histologic damage in the SN with neuronal drop-out at the periphery of the lesions. Injection of 80 nmol of H₂O₂ into the SN did not decrease CN amine levels and did not produce histologic damage in the SN. Electrocoagulation of the SN decreased CN DA and NE, but the histologic lesions failed to show any peripheral neuronal drop-out. Ventricular injections of high doses (16 μmol) of 60HDA or H₂O₂ reduced not only DA and NE but also 5HT levels in the ipsilateral CN. Low intraventricular doses (0-16 μmol) of 60HDA decreased only DA and NE without affecting 5HT levels in the CN whereas 0.16 μmol of H₂O₂ had no effect on any of the CN amines. The catecholamine-depleting effects of low doses (80 nmol) of 60HDA were significantly potentiated by inhibiting brain monoamine oxidase by 90 percent or more at the time and site of injection of 60HDA. These results suggest that the extracellular liberation of H₂O₂ from 60HDA could explain some possibly nonspecific effects of high doses of 60HDA; at lower doses, however, 60HDA may act via selective uptake into catecholaminergic neurons with subsequent intracellular release of H₂O₂.     

Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Differential regulation of multiple neuroreceptors in a somatic cell hybrid by inhibitors of glycoprotein processing

Specific binding of [3H][D-Ala2,D-Leu5]enkephalin, [3H]ethylketocyclazocine, 5-[3H]hydroxytryptamine, and [3H]spiperone was examined in neuroblastoma-brain hybrid cell line NCB-20 following exposure to inhibitors of N-linked protein glycosylation (tunicamycin, TM) and oligosaccharide processing (swainsonine, SW). TM treatment reduced ligand binding at delta- and sigma-opiate receptors and neuroleptic binding sites (20 to 50% of control), with no discernible effect on the binding properties of 5HT1-serotonin receptors. In contrast, exposure to SW resulted in a three-fold increase in binding capacity of sigma-receptors, while decreasing receptor affinity for ligand. SW treatment did not alter ligand interactions with either sigma-receptors or neuroleptic binding sites, but did reduce specific binding of serotonin to 5HT1-receptors. The effects of TM and SW on distinct receptor subpopulations were further demonstrated by attenuation of opiate and serotonin-mediated regulation of intracellular cyclic AMP.     

Neuronal localization of the neutral endopeptidase ''enkephalinase'' in rat brain revealed by lesions and autoradiography.

The cellular localization of rat brain enkephalinase was studied after induction of selective unilateral lesions using in vitro quantitative autoradiography of the specific binding of the enzyme inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly). Twenty-one days following injection of kainic acid in the caudate-putamen (CP) [3H]HACBO-Gly binding was locally decreased by 52% with a concomitant reduction of 67 and 78% in the ipsilateral substantia nigra (SN) and globus pallidus (GP), respectively. Inhibition of axonal transport in the CP by unilateral stereotaxic injection of colchicine induced a large (30-60%) and progressive decrease in enkephalinase labelling within the ipsilateral GP and SN. Taken together these results strongly suggest that in the CP a large fraction of enkephalinase is localized on intrinsic striatal neurones, and that the enzyme present both in the GP and the SN is partly localized on nerve terminals originating from neurones in the CP. No change in [3H]HACBO-Gly binding was observed in the CP following injection of 6-hydroxydopamine in the nigrostriatal bundle, contrasting with the 30% depletion in opioid receptors. This would indicate that enkephalinase is present in only very low amounts, if at all, on striatal dopaminergic nerve terminals.     

Protein synthesis at synapse versus cell body: Enhanced but transient expression of long‐term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein‐synthesis dependent long‐term facilitation (LTF) produced by 5‐HT that decays rapidly. Changes in expression of a SN‐specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5‐HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5‐HT was blocked by anisomycin or was reversed 48 h after 5‐HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long‐term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 275–286, 2003     

8-OH-DPAT abolishes the pulmonary C-fiber-mediated apneic response to fentanyl largely via acting on 5HT1A receptors in the nucleus tractus solitarius

Intravenous bolus injection of morphine causes a vagal-mediated brief apnea (～3 s), while continuous injection, via action upon central μ-opioid receptor (MOR), arrests ventilation (>20 s) that is eliminated by stimulating central 5-hydroxytryptamine 1A receptors (5HT(1A)Rs). Bronchopulmonary C-fibers (PCFs) are essential for triggering a brief apnea, and their afferents terminate at the caudomedial region of the nucleus tractus solitarius (mNTS) that densely expresses 5HT(1A)Rs. Thus we asked whether the vagal-mediated apneic response to MOR agonists was PCF dependent, and if so, whether this apnea was abolished by systemic administration of 8-hydroxy-2-(di-n-propylamino)tetral (8-OH-DPAT) largely through action upon mNTS 5HT(1A)Rs. Right atrial bolus injection of fentanyl (5.0 μg/kg, a MOR agonist) was performed in the anesthetized and spontaneously breathing rats before and after: 1) selective blockade of PCFs' conduction and subsequent bivagotomy; 2) intravenous administration of 5HT(1A)R agonist 8-OH-DPAT; 3) intra-mNTS injection of 8-OH-DPAT; and 4) intra-mNTS injection of 5HT(1A)R antagonist WAY-100635 followed by 8-OH-DPAT (iv). We found the following: First, fentanyl evoked an immediate apnea (2.5 ± 0.4 s, ～6-fold longer than the baseline expiratory duration, T(E)), which was abolished by either blocking PCFs' conduction or bivagotomy. Second, this apnea was prevented by systemic 8-OH-DPAT challenge. Third, intra-mNTS injection of 8-OH-DPAT greatly attenuated the apnea by 64%. Finally, intra-mNTS microinjection of WAY-100635 significantly attenuated (58%) the apneic blockade by 8-OH-DPAT (iv). We conclude that the vagal-mediated apneic response to MOR activation depends on PCFs, which is fully antagonized by systemic 8-OH-DPAT challenge largely via acting on mNTS 5HT(1A)Rs.     

Oligomerization of G-protein-coupled receptors shown by selective co-immunoprecipitation

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.     

Manipulations of synaptic serotonin: discrepancy of effects on serotonin S1 and S2 sites

The effect of repeated treatment (28 day) with D-fenfluramine, a serotonin (5HT) releaser, L-tryptophan, a 5HT precursor, or fluoxetine, a 5HT uptake inhibitor, on 3H-5HT and 3H-spiperone binding in the rat cerebral cortex was investigated. Treatment with fenfluramine and fluoxetine caused a significant decrease in the number of 3H-5HT binding sites (Bmax). Fenfluramine also decreased binding of 3H-spiperone in the cortex, but fluoxetine treatment increased this binding. Treatment with L-tryptophan produced no change in the binding of either 3H-5HT or of 3H-spiperone significantly. The data show that manipulation of synaptic 5HT concentration does not always result in parallel changes in S1 and S2 receptors. This suggests that the 5HT S1 and S2 receptors may be subject to different regulatory mechanisms.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels

To understand the mechanism of allosteric coupling between the ligand-binding domain and the ion channel of the Cys-loop ligand-gated ion channels (LGICs), we fused the soluble acetylcholine-binding protein (AChBP), which lacks an ion channel, to either the cationic serotonin type-3A ion channel (5HT(3A)) or the anionic glycine ion channel. Both linear chimeras expressed in HEK-293 cells display high affinity for the nicotinic agonist epibatidine (K(D) = 0.2-0.5 nM), but are not targeted to the cell surface. Only after substituting a ring of three loops located at the putative membrane side of the AChBP three-dimensional structure by the homologous residues of 5HT(3A), the resulting chimera AChBP(ring)/5HT(3A) (i) still displayed on intact cells an apparent high affinity for epibatidine, yet with a fourfold decrease (K(D) = 2.1 nM), (ii) displayed a high proportion of low affinity sites (11 +/- 7 microM) for the resting state stabilizing competitive antagonist alpha-bungarotoxin and (iii) was successfully targeted to the cell surface, as seen by immunofluorescence labelling. The AChBP(ring)/5HT(3A) chimera forms a pentameric structure, as revealed by sucrose gradient sedimentation. However, no whole-cell patch-clamp currents were detectable. Interestingly, binding assays with membrane fragments prepared from cells expressing AChBP(ring)/5HT(3A) showed a decrease in the apparent affinity for the agonists nicotine and epibatidine (5-fold), concomitant with an increase in the proportion of high-affinity sites (48 +/- 1 nM) for alpha-bungarotoxin. These results indicate that fusion of AChBP to an ion channel forms a pentameric receptor exposed to the cell surface and able to convert between discrete allosteric states, but stabilized in a high affinity state for epibatidine that likely corresponds to a desensitized form of LGICs. These artificial chimeras might offer a useful system to investigate signal transduction in LGICs.     

Heterogeneity of D2 dopamine receptors in different brain regions.

The binding of [3H]spiperone has been examined in membranes derived from different regions of bovine brain. In caudate nucleus, nucleus accumbens, olfactory tubercle and putamen binding is to D2 dopamine and 5HT2 serotonin receptors, whereas in cingulate cortex only serotonin 5HT2 receptor binding can be detected. D2 dopamine receptors were examined in detail in caudate nucleus, olfactory tubercle and putamen using [3H]spiperone binding in the presence of 0.3 microM-mianserin (to block 5HT2 serotonin receptors). No evidence for heterogeneity among D2 dopamine receptors either between brain regions or within a brain region was found from the displacements of [3H]spiperone binding by a range of antagonists, including dibenzazepines and substituted benzamides. Regulation of agonist binding by guanine nucleotides did, however, differ between regions. In caudate nucleus a population of agonist binding sites appeared resistant to guanine nucleotide regulation, whereas this was not the case in olfactory tubercle and putamen.