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The benzodiazepine receptor tracer [123I]iomazenil (Ro 16-0154, IMZ) can be prepared in close to theoretical specific activity by the reaction of its tributyltin precursor (IMZ-SnBu3) with [123I]NaI in the presence of Iodogen. However, the labeling reaction is associated with variably high amounts of a volatile 123I byproduct. The purpose of these experiments was to characterize the volatile byproduct and to examine the effect on the course of the reaction of the following variables: solvent (MeOH, EtOH, HOAc, H2O), pH (2-7), oxidizing agent (chloramine-T, Iodogen, AcO2H), reaction temperature (22-128 degrees C) and structure of ArSnR3. The order of reactivity of oxidizing agents, at 22 degrees C for 30 min, was chloramine-T > Iodogen >> AcO2H. Raising the pH above 5.8 reduced the labeling, whereas raising the temperature increased the yield up to a maximum at 120 degrees (90%); at higher temperatures, decomposition occurred. The best conditions were 50 micrograms precursor, 50 microL 0.02 M chloramine-T; in aqueous HOAc (pH 3.3) at 120 degrees for 30 min. Variable amounts of volatile byproduct were observed for chloramine-T at different temperatures (2-21%) and for the three oxidizing agents at room temperature (3-22%). By contrast, the Bu3Sn derivatives of IBF, epidepride, beta-CIT and the Me3Sn derivative of beta-CIT gave high labeling yields with peracetic acid at room temperature, and < or = 2% volatile radioactivity. The volatile byproduct from the [123I]IMZ preparations was identified as I-[123I]iodobutane by its trapping characteristics and by its retention time in two different HPLC systems. Volatile activity was not generated in the absence of Bu3Sn precursor and was not due to the presence of impurities in the tributylstannyl precursor.

[123I][(1R)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane] ([123I]beta-CIT) labels dopamine transporters and is, therefore, a marker of neurons that degenerate in Parkinson disease. Single photon emission computed tomography imaging with [123I]beta-CIT showed that radioactivity in striatal regions in healthy subjects increased during a 2-day imaging study, whereas that in Parkinsonian patients peaked earlier at reduced levels relative to healthy subjects. Kinetic analyses of radioactivity in plasma and brain suggest that this decrease was due to an approximately 65% loss of target sites in patients compared with healthy subjects; greater losses occurred in putamen than in caudate. All patients showed lateralized differences in striatal uptake, with greater losses in the striatum contralateral to the side of the body with initial symptoms. These preliminary results suggest that [123I]beta-CIT is a marker for the loss of striatal dopamine terminals in patients with Parkinson disease. Single photon emission computed tomographic imaging with [123I]beta-CIT may be useful for early diagnosis of the disorder, for monitoring the progression of the disease, and for distinguishing the idiopathic disorder from other Parkinsonian syndromes with more widespread pathology.

We have developed and validated in a phantom a method of computer-assisted coregistration using multislice SPECT and MR images. Reusable fiducial markers were fabricated from nylon-based plastic and consist of two parts: a base that remains fixed to the skin with adhesive between scans and a removable, spherical cavity insert that can be filled with contrast agents appropriate for multiple imaging modalities. Markers external and internal to a three-dimensional brain phantom provided a means of quantifying the method's accuracy. A computer algorithm was used to derive transformation matrices for image sets by minimizing the root mean squared deviations obtained for multiple permutations (n = 10) of increasing numbers (range 3-11) of external SPECT/MRI point pairs. As defined by the average +/- SEM mean residual deviations of noncoregistered internal fiducials, the minimal accuracy was 2.4 +/- 0.3 mm (no marker > 3.6 mm) for three coregistration points and did not improve beyond seven to eight fiducials (2.2 +/- 0.1 mm; no marker > 3.2 mm). The method's true accuracy is likely to be better than estimates of minimal accuracy, however, since such measures reflect surmountable random errors in fiducial location. With use of identical MRI (or SPECT) data sets, measures of intraoperator (0.5 +/- 0.1 and 0.5 +/- 0.1 mm, respectively) and interoperator (0.5 +/- 0.3 and 0.8 +/- 0.1 mm) reliability were also obtained, establishing the method as highly reproducible and objective. Preliminary results in a human subject suggest its feasibility for clinical studies.

Objective: The purpose of this study was to compare the memory function of patients with posttraumatic stress disorder (PTSD) to that of matched comparison subjects., Method: Vietnam veterans with combat-related PTSD (N = 26) were compared to physically healthy comparison subjects (N = 15) matched for age, race, sex, years of education, handedness, socioeconomic status, and alcohol abuse. Memory and intelligence were assessed with a battery of neuropsychological tests, including the Russell revision of the Wechsler Memory Scale, the Selective Reminding Test, and subtests of the Wechsler Adult Intelligence Scale-Revised (WAIS-R)., Results: The PTSD patients scored significantly lower than the comparison subjects on the Wechsler Memory Scale logical memory measures for immediate recall (mean = 11.6, SD = 3.3 versus mean = 20.9, SD = 6.6) and delayed recall (mean = 8.0, SD = 3.3 versus mean = 17.8, SD = 6.4). The PTSD patients also scored significantly lower on the total recall, long-term storage, long-term retrieval, and delayed recall measures for the verbal component of the Selective Reminding Test and on the recall, long-term storage, long-term retrieval, and continuous long-term retrieval measures for the visual component of the Selective Reminding Test. There was no significant difference between the PTSD patients and comparison subjects in prorated full-scale IQ as measured by the WAIS-R., Conclusions: Patients with PTSD may have deficits in short-term memory. Counseling and rehabilitation that address these deficits may be of value for PTSD patients.

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.

Single photon emission computed tomography (SPECT) imaging can provide useful measurements of brain receptors and endogenous neurotransmitters and may have significant experimental and clinical applications. This presentation reviews the use of SPECT for neuroreceptor imaging. Studies of receptors for benzodiazepines, dopamine D2 agents, and dopamine reuptake sites will be used to exemplify the capabilities of SPECT. Tracers labeled with the radioisotope 125I have high affinity, high brain uptake, and high ratios of specific to nonspecific binding. Imaging studies of human and nonhuman primate brain will be presented, and the potential clinical applicability of these agents will be discussed.

SPECT imaging of benzodiazepine (BZ) receptor using the antagonist [123I]iomazenil in nonhuman primates was correlated with ex vivo autoradiography and in vitro receptor autoradiography of postmortem tissue sections taken at the time of maximal brain uptake of the radiotracer. Cryostat sections prepared from brain tissue blocks at the orientation of the SPECT imaging plane showed high activity in gray matter regions, with gray to white matter ratios of greater than 30:1. After at least one week decay of 123I, these same tissue sections were used to localize and quantify the distribution of BZ receptors with the standard technique of in vitro receptor autoradiography using [125I]iomazenil. The ex vivo autoradiographic distribution of activity was highly correlated (r = 0.89) with the distribution of BZ receptors, although a few brain regions showed reproducible discrepancies between ex vivo and in vitro results. The ex vivo autoradiograms provided quantitative data from a realistic "biological phantom," which may be used to assess the accuracy of image reconstruction and to investigate differences between the distribution of an intravenously (i.v.) administered tracer and that of its target in brain tissue.

Relative to dopaminergic innervation of cortex, dopamine D1 and D2 receptors may be located on presynaptic terminals and/or postsynaptically on cortical neurons. To assess the relative distribution of these sites, quantitative in vitro receptor autoradiography was performed following injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle (MFB; which lesions presynaptic DA terminals) and ibotenic acid into the prefrontal and anterior cingulate cortices (which lesions neurons whose cell bodies are intrinsic to cortex). Receptor autoradiography was performed ten days after injection of neurotoxins with [3H]SCH 23390 (a D1 probe) and [125I]epidepride (a D2 probe). Both DA receptor subtypes were found in all layers of anterior cingulate and prefrontal cortices but were concentrated in deeper layers V and VI. Ibotenic acid lesion of cortex reduced D1 and D2 receptors by 55-80%, although the concentrations of DA and its major metabolite dihydroxyphenylacetic acid (DOPAC) were unchanged. Lesion of MFB produced no significant change in D1 and D2 receptors, but was associated with a 49-52% decrease in DA and DOPAC levels relative to the contralateral side. These results suggest that the majority of D1 and D2 receptors in prefrontal and anterior cingulate cortices are located postsynaptically on neurons intrinsic to the cortex.

We used the reversibly binding D2 dopamine receptor radioligand [123I]IBZM (iodobenzamide) to test whether the endogenous neurotransmitter dopamine competes in vivo for radiotracer binding measured with single photon emission computed tomography (SPECT). In a series of nonhuman primate experiments (n = 27), the effects of temperature, amphetamine, haloperidol, and reserpine on brain uptake of [123I]IBZM were measured. Specific brain uptake of [123I]IBZM reached a peak by 100 min postinjection of radioligand and demonstrated a gradual, apparent "steady-state" washout over the next 2 hr. Brain uptake was temperature dependent, with rates of washout of specifically bound radioligand greater under normothermic conditions (26%/hr: core body temperature 35-37 degrees C) than under conditions of controlled hypothermia (11%/hr; 32-34 degrees C). Given the greater retention of radioactivity, low-temperature conditions were used in all other experiments. Administration of haloperidol (0.02 mg/kg IV) during the period of apparent steady state resulted in a dramatic increase in washout (60%/hr; p less than 0.0001), consistent with its potent D2 receptor antagonist properties. d-Amphetamine (1.0 mg/kg IV), which has negligible affinity for the D2 receptor but mediates the release of endogenous stores of dopamine, also enhanced washout (34%/hr; p less than 0.0005). Reserpine pretreatment at doses (1.0 mg/kg) sufficient to cause greater than 90% depletion of striatal dopamine levels blocked this amphetamine-enhanced washout (10%/hr; p less than 0.05). Reserpine did not block the increased washout induced by the direct-acting D2 receptor antagonist haloperidol. These results are consistent with the hypothesis that endogenous dopamine may effectively compete for radioligand binding in vivo in neuroreceptor imaging studies using PET and SPECT.

Iodine-123-labeled Ro 16-0154 is a high affinity, reversibly binding radiotracer for the benzodiazepine (BZ) receptor. Brain uptake of this radioligand was relatively stable and showed high ratios of specific-to-nonspecific uptake, with greater than 90% displaced by intravenous administration of BZ receptor agents. Repeated injections of increasing doses of each of five BZ drugs (Ro 16-0154, Ro 15-1788, clonazepam, alprazolam, and diazepam) yielded stepwise displacement curves, which were analyzed to measure the in vivo potencies of these agents. The relatively long half-life of 123I and the stable biologic uptake of the radiotracer allowed such potency estimations in just one experiment following a single injection of radioligand. The in vivo potencies of these five agents were highly correlated with their affinities for the BZ receptor determined with in vitro homogenate binding. A single crystal probe provided potency measurements virtually identical to simultaneously performed SPECT imaging studies. In conclusion, stepwise displacement by agents administered following the injection of the radioligand 123I-Ro 16-0154 provided a reliable means of measuring the in vivo potencies of BZ receptor agents. This in vivo determination may predict the clinical potency of BZ drugs than in vitro homogenate estimations, because the in vivo measure provides the summed effects of receptor affinity, plasma protein binding, penetration of the blood-brain barrier, and metabolism of the displacing agent.

The mechanisms which contribute to the actions of atypical antipsychotic drugs, such as clozapine and the putative atypical agents remoxipride and raclopride, are reviewed. Examination of available preclinical and clinical data leads to two hypotheses concerning the mode of action of atypical antipsychotic drugs. The first hypothesis is that antagonism of the dopamine D2 receptor is both necessary and sufficient for the atypical profile, but that interaction with subtypes of the D2 receptor differentiates typical from atypical antipsychotic drugs. The second hypothesis has been previously advanced, and suggests that a relatively high ratio of serotonin 5-HT2:dopamine D2 receptor antagonism may subserve the atypical profile. It seems likely that the atypical antipsychotic drug profile may be achieved in more than one way.

Autoradiographic images can be analyzed with computer-assisted microdensitometry relative to radioactive reference standards to provide quantitative measurements of regional radioactivity concentrations. [123I]Iodine containing sections of brain paste have been calibrated relative to plastic-embedded tritium(3H) and 125Iodine standards. For exposure times of 8, 12 and 24 h, plastic-embedded standards covered a range concentrations of [123I]iodine from 1000 to 100,000 dpm/mg wet weight. Iodine-123 radioactivity was linear with section thickness from 5 to 30 microns. These studies confirmed the feasibility of using commercially available longer-lived reference standards to provide [123I]iodine equivalent values in absolute units of dpm (or microCi) per mg tissue.

Cholecystokinin binding sites were labeled with [3H]cholecystokinin-8, [125I]cholecystokinin-33, and [125I]cholecystokinin-8 in major structures of macaque basal ganglia by in vitro receptor autoradiography. Analysis of autoradiograms revealed areas of heavy cholecystokinin binding in the neostriatum and substantia nigra that were set off, often quite sharply, from the adjacent globus pallidus and subthalamic nucleus where labeling was, by contrast, very light. Heavy label characterized the ventromedial and posterior parts of the caudate nucleus and adjacent putamen, binding was of moderate intensity in central areas of these regions, while, the dorsolateral margin of the head of the caudate and precommissural putamen, the dorsolateral one-third of the body of the caudate, and all but the most medial and ventral portions of the posterior putamen lateral to the pallidum were sparsely labeled. The pattern of cholecystokinin binding within the neostriatum was mottled; patches of reduced label stood out from the background of more prominent binding. However, those patches were only imperfectly correlated with the striosomal organization of both the caudate nucleus and putamen as revealed by acetylcholinesterase staining. Cholecystokinin binding in the substantia nigra was also intricately patterned. Moderately dense, vertically orientated bands of label were found in the dorsal one-third to half of the pars reticulata, providing a marked contrast to the near background levels in the ventral pars reticulata and overlying pars compacta. The present study shows that heavy cholecystokinin binding is confined to particular areas within the primate basal ganglia; the pattern of label within the substantia nigra and neostriatum can be linked to intrinsic and afferent connections of these structures. The confinement of binding sites to the dorsal pars reticulata suggests an association with dendrites of pars compacta neurons which invade this region; this interpretation is consistent with recent evidence of depletion of nigral cholecystokinin binding sites in macaques following chemical lesion of dopaminergic cells of the par compacta. In the neostriatum the distribution of binding shows overlap with its topographically organized corticostriatal innervation; portions of heavily labeled striatum coincide with regions innervated by association cortex of the frontal and temporal lobes, whereas regions of diminished binding correspond to areas innervated mainly by sensory and motor cortex. These latter findings suggest that cholecystokinin may have a particularly strong influence on cognitive aspects of striatal function.

The binding of an iodinated benzodiazepine (BZ) radioligand has been characterized, particularly in regard to its potential use as a neuroreceptor brain imaging agent with SPECT (Single Photon Emission Computed Tomography). Ro16-0154 is an iodine-containing BZ antagonist and a close analog of Ro15-1788. In tissue homogenates prepared from human and monkey brain, the binding of 125I-labeled Ro16-0154 was saturable, of high affinity (Kd = 0.5 nM at 37 degrees C), and had high ratios of specific to non-specific binding (approximately 40:1). Physiological concentrations of NaCl (150 mM) enhanced specific binding approximately 15% compared to buffer without this salt. Kinetic studies of association and dissociation demonstrated a temperature dependent decrease in affinity with increasing temperature. Drug displacement studies confirmed that 125I-Ro16-0154 binds to the "central" type BZ receptor: binding is virtually identical to that of 3H-Ro15-1788 except that 125I-Ro16-0154 shows an almost 10 fold higher affinity at 37 degrees C. These in vitro results suggest that 123I-labeled Ro16-0154 shows promise as a selective, high affinity SPECT probe of the brain's BZ receptor.

Cholecystokinin (CCK) receptor binding sites have been localized by autoradiography in the guinea pig and rat central nervous system. [125I]CCK-triacontatriapeptide labeled the sites in brain slices with an observed association constant equal to 0.041 min-1 and a dissociation constant equal to 0.008 min-1. CCK-triacontatriapeptide (CCK-33) and the C-terminal octapeptide of CCK-33 (CCK-8) potently inhibited [125I]CCK-33 binding with Ki's of 2 nM, whereas desulfated CCK-8 (CCK8-ds) and the C-terminal tetrapeptide of CCK-33 (CCK-4) were much weaker. Receptors were concentrated in the olfactory bulb, in the superficial laminae of the primary olfactory cortex, in the deep laminae of the cerebral cortex, and in the pretectal area. Substantial numbers of sites were also found in the basal ganglia, in the amygdala, and in the hippocampal formation. [125I]CCK-33 binding sites appear to be located on fibers of the optic tract and probably on olfactory tract fibers as well. These results are discussed in terms of physiological functions associated with CCK, presynaptic receptors, and axonal flow of CCK receptors.

To examine the role of guanine nucleotide binding (G) proteins in receptor-mediated inhibition of serotonin (5-HT) neurons, we intracerebrally injected pertussis toxin (0.5-1.0 microgram) into rat midbrain in a region immediately rostral to the dorsal raphe nucleus. The baseline firing rate of extracellularly recorded 5-HT neurons was not significantly affected by pertussis toxin treatment. However, in comparison to saline-injected controls, pertussis toxin-injected animals showed markedly blunted sensitivity to agonists that act at 5-HT autoreceptors (isapirone, 5-HT and LSD) and to baclofen, a GABAB agonist. This pertussis toxin-induced blunting of sensitivity was demonstrated in vivo (with intravenous and iontophoretic application of drugs) and in vitro in the dorsal raphe brain slice preparation. The sensitivity of iontophoretically applied GABA itself was not significantly decreased with pertussis toxin treatment, consistent with evidence that GABA administered in this manner acts on dorsal raphe cells mainly through GABAA receptors. Our data provide strong evidence for the role of a pertussis toxin substrate(s) (presumably a G protein(s] in mediating the inhibition induced by the autoreceptor and GABAB receptor on 5-HT neurons in rat dorsal raphe nucleus.

An apparent involvement of dopamine in the regulation of cognitive functions and the recognition of a widespread dopaminergic innervation of the cortex have focused attention on the identity of cortical dopamine receptors. However, only the presence and distribution of dopamine D1 receptors in the cortex have been well documented. Comparable information on cortical D2 sites is lacking. We report here the results of binding studies in the cortex and neostriatum of rat and monkey using the D2 selective antagonist [3H]raclopride. In both structures [3H]raclopride bound in a sodium-dependent and saturable manner to a single population of sites with pharmacological profiles of dopamine D2 receptors. D2 sites were present in all regions of the cortex, although their density was much lower than in the neostriatum. The density of these sites in both monkey and, to a lesser extent, rat cortex displayed a rostral-caudal gradient with highest concentrations in the prefrontal and lowest concentrations in the occipital cortex, corresponding to dopamine levels in these areas. Thus, the present study establishes the presence and widespread distribution of dopamine D2 receptors in the cortex.

3H-Imipramine (3H-IMI) binding to platelet membranes was measured in 19 patients with agoraphobia with panic attacks, 9 patients with major depression, and 22 healthy subjects. In comparison to healthy subjects, the maximal number of binding sites (Bmax) was significantly decreased in depressed patients but not in panic disorder patients, and the apparent affinity of binding was slightly decreased in depressed patients but not in panic disorder patients. The Bmax and Kd of 3H-IMI platelet binding did not differ between panic disorder patients with and without a history of a major depressive episode. Thus, 3H-IMI platelet binding is clearly different in patients with panic disorder compared to those with an active depression. Because 3H-IMI binding is associated with the serotonin reuptake site in platelet and brain membranes, these findings give further support to abnormalities in serotonergic function in patients with major depression.

Cholecystokinin (CCK) binding sites were localized in the hippocampus, amygdala, and medial temporal cortices of macaque monkeys by using techniques of in vitro receptor autoradiography. Binding sites were labeled with 3H-CCK-8 and 125I-CCK-33, and nonspecific binding was assessed in the presence of 1 microM CCK-8. Comparison of autoradiograms with Nissl-stained sections allowed precise correlation of autoradiographic grain distribution with cytoarchitecture. CCK binding in the amygdala varied among nuclear subdivisions. It was dense in the lateral, basomedial, endopiriform, and cortical nuclei, in the parvicellular portion of the accessory basal nucleus, the periamygdaloid cortex, the cortical transition area, and in the amygdalohippocampal area. Labeling was sparse in the central, medial, and basolateral nuclei as well as in the magnocellular accessory basal nucleus. In the hippocampal formation, a single dense band of CCK binding was observed over the granule cell layer and adjacent few millimeters of the molecular layer of the dentate gyrus, while in the polymorph and remaining portions of this layer binding was of very low density. Prominent label over the pyramidal layer in the presubiculum clearly distinguished this region from the adjacent subiculum in which binding just exceeded background levels. Moderate to light label was observed in the hilus and stratum pyramidale of CA3, CA2, and CA1, while other hippocampal layers showed minimal specific binding. Variation in CCK binding in the medial temporal cortex showed close correspondence to cytoarchitectonic subdivisions. In entorhinal cortex, for example, binding was concentrated in layers III-VI while label in area 35 was prominent in all laminae except layer IV. Area TH of von Bonin and Bailey ('47) was distinguished from other regions by evenly distributed binding across all layers, while in area TF a bilaminar pattern of label in layers II and IV was observed. The highly specific patterns of CCK binding in amygdala and transitional cortices of the medial temporal lobe can be related to terminal fields of neo- and allocortical afferents to these regions, while label in the hippocampal formation coincides with the terminals of intrinsic neurons which ramify among the somata of cells that are targets of neocortical afferents. Thus, in all structures of the medial temporal lobe the disposition of peptidergic binding sites suggests that CCKergic systems may be important in the modulation of cortical afferents.

Radioligand binding studies of neurotransmitter receptors have provided discrimination at the molecular level, permitting the differentiation of multiple receptor subtypes for several biogenic amines. Using this paradigm we have labeled two distinct receptors each for cholecystokinin (CCK) and for adenosine. Adenosine receptors were labeled in brain with [3H]N6-cyclohexyladenosine (3H-CHA) and [3H]1,3-diethyl-8-phenylxanthine (3H-DP). The adenosine receptor labeled by 3H-CHA appears to be an A1 site, associated with reduction of adenylate cyclase activity, while 3H-DP sites resemble A2 receptors linked to adenylate cyclase enhancement. Cholecystokinin-33 labeled by the Bolton-Hunter procedure with 125I(125I-BH-CCK) labels different receptors in brain and pancreas. The pancreatic receptor does not react with CCK derivatives of fewer than eight amino acids, while the brain receptor does recognize pentagastrin, the carboxyl-terminal five amino acids of CCK. The "brain type" CCK receptor may normally interact with CCK-4, the carboxyl-terminal tetrapeptide of CCK, recently identified as a unique neuropeptide highly concentrated in the brain. CCK-8, the other major molecular form of CCK, may be the endogenous ligand for the "pancreatic type" receptor.

Bradykinin-like immunoreactive structures were localized in rat brain by the indirect immunofluorescence method. Specificity of staining was demonstrated by: (i) the absence of fluorescence when preimmune serum was used, (ii) the disappearance of fluorescence when sera were preadsorbed with bradykinin, and (iii) the presence of identical staining with two different antisera. Immunoreactive neuronal cells are observed only in the hypothalamus, with especially dense clusters overlying the periventricular and dorsomedial nuclei. Fibers and varicose processes are observed in the periaqueductal gray matter, hypothalamus, perirhinal and cingulate cortices, the ventral portion of caudate-putamen, and the lateral septal area.

Cholecystokinin (CCK) is a putative peptide neurotransmitter present in high concentration in the cerebral cortex. By using techniques of in vitro receptor autoradiography, CCK binding sites in primate cortex were labeled with 125I-Bolton-Hunter-labeled CCK-33 (the 33-amino-acid C-terminal peptide) and 3H-CCK-8 (the C-terminal octapeptide). Biochemical studies performed on homogenized and slide-mounted tissue sections showed that the two ligands labeled a high-affinity, apparently single, saturable site. Autoradiography revealed that binding sites labeled by both ligands were anatomically indistinguishable and were distributed in two basic patterns. A faint and diffuse label characterized portions of medial prefrontal cortex, premotor and motor cortices, the superior parietal lobule, and the temporal pole. In other cortical areas the pattern of binding was layer-specific; i.e., binding sites were concentrated within particular cortical layers and were superimposed upon the background of diffuse label. Layer-specific label was found in the prefrontal cortex, anterior and posterior cingulate gyrus, somatosensory cortex, inferior parietal lobule, retrosplenial cortex, insula, temporal lobe cortices, and in the primary visual and adjacent visual association cortices. The areal and laminar localization of layer-specific CCK binding sites consistently coincided with the cortical projections of thalamic nuclei. In prefrontal cortex, CCK binding sites were present in layers III and IV, precisely paralleling the terminal fields of thalamocortical projections from the mediodorsal and medial pulvinar nucleus of the thalamus. In somatosensory cortex, the pattern of CCK binding in layer IV coincided with thalamic inputs arising from the ventrobasal complex, while in the posterior cingulate gyrus, insular cortex, and retrosplenial cortex, layer IV and lower III binding mirrored the laminar distribution of cortical afferents of the medial pulvinar. CCK binding in layers IVa, IVc alpha, IVc beta, and VI of primary visual cortex corresponded to the terminal field disposition of lateral geniculate neurons, whereas in adjacent visual association cortex, binding in layers III, IV, and VI faithfully followed the cortical distribution of projections from the inferior and lateral divisions of the pulvinar nucleus of the thalamus. We interpret the diffusely labeled binding sites in primate cortex as being associated with the intrinsic system of CCK-containing interneurons that are distributed throughout all layers and areas of the cortex. The stratified binding sites, however, appear to be associated with specific extrinsic peptidergic projections.(ABSTRACT TRUNCATED AT 400 WORDS)

125I-Labeled (Bolton-Hunter) cholecystokinin triacontatriapeptide (CCK-33) binds saturably and reversibly to distinct receptors in brain and pancreatic membranes. The peptide specificity of pancreatic CCK binding is the same as that for pancreatic amylase release. In brain, gastrin and pentagastrin display nanomolar affinity for binding sites, whereas in pancreas these two peptides are virtually inactive. Though these differences indicate that brain and pancreas possess distinct CCK receptors, the two tissues show some similarities. Both pancreas and brain receptors show greater sensitivity to sulfated than to desulfated COOH-terminal octapeptide of CCK and display dissociation constants of 0.3-9.5 nM. The pancreas possesses about 300 times more binding sites than does brain. CCK binding in both brain and pancreas is enhanced by divalent cations and reduced by monovalent cations. Receptor binding in both tissues is regulated in a selective fashion by guanyl nucleotides.

Dansyl derivatives of the opiate antagonist naloxazone and the alpha-adrenergic blocking drug 2-[2',6'-dimethoxy)phenoxyethylamino]methyl-benzodioxan (WB-4101) were synthesized as potential in vivo fluorescent labels for opiate and alpha-adrenergic receptors, respectively. When assayed in vitro dansyl analogs display Ki-values for [3H]naloxone and [3H]WB-4101 binding of 8 nM and 80 nM, respectively. The same patterns of histofluorescence are observed in rat brain slices after the intravenous or intracerebroventricular injection of both dansyl drugs. The same patterns are also observed in dansyl-propranolol and 9-aminoacridine-propranolol (9-AAPN) treated animals as well as in untreated control rats. Accordingly, the observed fluorescence does not reflect the labeling of any receptor but is consistent with the distribution of lipofuscin, an endogenous fluorescent compound.

The source of enkephalin in neuronal terminals of the rat globus pallidus was evaluated with electrolytic and knife cut lesions. Straight knife cuts medial to the globus pallidus or a curved knife cut just below the corpus callosum do not reduce immunofluorescence staining in the globus pallidus, indicating that cerebral cortex is not a major source of pallidal enkephalin. Electrolytic lesions of dorsal and ventral portions of the globus pallidus, and even lesions which sever medial from lateral globus, fail to reduce pallidal staining, ruling out long enkephalin neurons traversing the globus. Large electrolytic lesions of the head of the caudate do decrease dorsal pallidal fluorescence, though multiple small caudate lesions are without observable effect. Large pallidal lesions elicit a build-up of enkephalin fluorescence in the caudate. These results suggest that the caudate is the sole source of pallidal enkephalin and that the innervation diffusely converges on the smaller globus pallidus.

A radioreceptor assay for tricyclic antidepressants described here is based on the ability of these drugs to compete with [3H]-3-quinuclidinyl benzilate (3H-QNB) for binding to muscarinic cholinergic receptors in rat brain membranes. The assay is sensitive, in that in can detect, for example, 2 ng/ml nortriptyline in plasma. Seven plasma samples from depressed patients treated with nortriptyline were assayed with the radioreceptor and gas liquid chromatographic methods, and the results from these two methods were almost identical. This assay should be used cautiously, if at all, in patients treated with other drugs that have potent anticholinergic effects.

Rat substantia nigra was injected with 1 microgram of purified pertussis toxin, a substance which inactivates the inhibitory guanine nucleotide regulatory unit of adenylate cyclase. In comparison to saline-injected animals, the pertussis toxin-treated animals showed almost no inhibition of dopaminergic neurons in response to dopamine applied iontophoretically or the dopamine agonist (-)-apomorphine given intravenously. These results provide evidence that a guanine nucleotide regulatory protein is involved as a transducer in mediating the physiological actions of dopamine autoreceptors.

In vitro intracellular recording techniques in the rat brain slice preparation demonstrate that both serotonin (5-HT) and baclofen (a GABAB-receptor agonist) inhibit 5-HT neurons in the dorsal raphe nucleus by inducing a hyperpolarization of membrane potential and a decrease in apparent input resistance (Rin). Similar to previous results with 5-HT, baclofen-mediated inhibition of 5-HT neurons also shows an apparent reversal potential (Erev) of approximately -90 mV, consistent with mediation by K channels. In slices from rats that had previously received a local injection of pertussis toxin (0.5 microgram) immediately rostral to the dorsal raphe nucleus, there was a virtually complete blockade of inhibition induced by both the serotonin autoreceptor and the GABAB-receptor. Intracellular injection of the stable GTP analog (guanosine-5'-O-(3-thiotriphosphate); GTP gamma S) mimicked the actions of both 5-HT and baclofen. The inhibitory actions of GTP gamma S were not additive with those of either 5-HT or baclofen, suggesting they share some common effector system. The stable cAMP analog (8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP] had no effect on membrane potential or apparent input resistance and did not block the inhibitory actions mediated by 5-HT or baclofen. The local injection of pertussis toxin (0.5 microgram) caused a far greater blockade of 5-HT and baclofen-mediated inhibition than the intracerebroventricular (i.c.v.) injection of pertussis toxin (1.0 micrograms). In parallel sets of animals with i.c.v. and local injections, we measured the pertussis toxin-mediated ADP-ribosylation of G proteins in membranes prepared from dorsal raphe nucleus. These biochemical studies showed that sensitivities to 5-HT and baclofen correlated with the concentration of remaining non-ADP-ribosylated G proteins following in vivo pertussis toxin injection. In summary, these results provide evidence for the role of a G protein(s) in the mediation of the cAMP-independent increase in potassium conductance in 5-HT neurons of dorsal raphe nucleus induced by both 5-HT1A- and GABAB-receptors.

Cholecystokinin (CCK), a neuropeptide which fulfills almost all criteria for neurotransmitter status, has been co-localized with dopamine in midbrain mesolimbic and mesocortical neurons that have been implicated in the pathogenesis of schizophrenia. Preclinical research suggests that CCK may in part act to enhance central dopaminergic activity. In an attempt to evaluate the role of CCK relative to the dopamine hyperactivity hypothesis of schizophrenia, in the present investigation the putative CCK receptor antagonist, proglumide, was administered to four schizophrenic patients in a double-blind, placebo-controlled study. All patients were receiving concurrent neuroleptic medication, but were still significantly symptomatic. Proglumide was without effect on the patients' psychosis ratings. Potential reasons for this negative finding are discussed.

Substance K (SK) is a newly discovered neuropeptide which is a member of the tachykinin family that includes substance P (SP). As it is present in high concentrations in substantia nigra, we have investigated the electrophysiological activity of SK microiontophoresed onto single neuronal units in this region. SK microiontophoresed onto single units in the substantia nigra caused excitation of approximately 50% of both dopaminergic and non-dopaminergic neurons. This relatively high proportion of responsivity correlates with the reported high density of SK receptors in the substantia nigra.

The putative peptide neurotransmitter cholecystokinin (CCK) is co-localized with substance P (SP) in a dense cluster of neurons located in the rat Edinger-Westphal (EW) nucleus. In an attempt to record electrophysiologically from these CCK-containing neurons, we have identified a group of nociceptive neurons located within the confines of the EW nucleus. The firing pattern of these nociceptive neurons is erratic, sometimes with a bursting pattern and at other times with fairly regular rates which vary generally from 1 to 7 Hz. These neurons respond to noxious stimuli, such as toe pinch, with an increase in rate and sometimes enter an apparent depolarization blockade (preceded by an increase in the duration and a decrease in the amplitude of the action potential). Systemically administered morphine suppresses both the spontaneous firing rate and the toe pinch-induced increase in firing rate. Naloxone is able to reverse the effects of morphine. Although we have identified in the EW area a moderate density of terminals containing enkephalin-like immunoreactivity, morphine locally applied via microiontophoresis is largely without effect on the firing of these neurons. We hypothesize that the opiate-induced suppression of these nociceptive neurons is not mediated directly on the EW cells, but rather indirectly through afferent systems.

[3H]Bradykinin binds to membranes from a variety of mammalian tissues in a saturable fashion with a dissociation constant of about 5 nM. Highest levels of binding are detected in guinea pig ileum, colon, and duodenum and in the oestrus rat uterus. The relative potencies of bradykinin derivatives in competing for these binding sites in guinea pig ileum membranes correlate with their contractile potencies in the ileum better than with contractile effects in the uterus. Thus, receptor recognition sites may differ in ileum and uterus. Monovalent and divalent cations at physiological concentrations reduce [3H]bradykinin binding. Of the monovalent cations, medium lowers binding 50% at about 80 mM. Among the divalent cations, calcium lowers binding about 50% at 5 mM, suggesting a link to the calcium conductance channel.

The function of the alpha-2-receptor and intracellular effector systems was examined in 39 panic disorder patients and 30 healthy subjects using the platelet as a model system. Alpha-2-receptor density, as reflected by 3H-yohimbine binding, was not different between the two groups. Platelet alpha-2-receptor affinity was decreased (higher Kd) in the panic disorder patients. Other significant abnormalities in the patients included a decreased basal adenylate cyclase activity, reduced EC50 for the epinephrine inhibition of adenylate cyclase activity, and decreased stimulation of adenylate cyclase activity by prostaglandin E1 and sodium fluoride. These results are consistent with a dysfunction in some panic disorder patients at the level of the stimulatory GTP binding regulatory protein, Gs, or the adenylate cyclase catalytic unit. The relationship of these findings to previous studies of noradrenergic function in panic disorder patients is discussed.

Cholecystokinin octapeptide-like (CCK-OP-like) immunoreactivity was localized in the rat brain by using the indirect immunofluorescence method. Specificity in immunohistochemical studies was demonstrated by the virtual elimination of staining with either preimmune sera or sera preadsorbed with CCK-OP and by the achievement of similar fluorescent patterns with two different primary anti-CCK-OP sera. CCK-OP-like fluorescence was localized in neuronal cell bodies, fibers, and varicose terminals. The most dense collections of CCK-OP cells occurred in the periaqueductal gray and in the dorsomedial hypothalamus. Substantial numbers of cells and fibers also were present in the medial/dorsal and perirhinal cortex; more limited groups of cells were found in the pyramidal layer of the hippocampus and in the dorsal raphe.