SYNAPSE 12~195-205(1992)

Sigma Receptors Are Associated With Cortical Limbic Areas in the Primate Brain Departments

of

DEBORAH C. MASH AND CYRUS P. ZABETIAN Neurology (D.C.M.) and Pharmacology (D.C.M., C.P.Z.), University of Miami School of Medicine, Miami, Florida 33141

KEY WORDS

Limbic system, Sigma ligands, Primate, Rhesus macaque, AutoradiOgraPhY

ABSTRACT Putative sigma receptors are a current target for antipsychotic drug development. Novel antipsychotic agents which possess selective and high affinity for sigma binding sites may serve as an alternative to the principal neuroleptic drugs currently in clinical use which mediate extrapyramidal side effects and dyskinesias through their blockade of dopamine receptors. We have used in vitro autoradiography to localize putative sigma receptors labelled with ( +)-[3Hl-3-(3-hydroxyphenyl)-N-(l-propyl)piperidine [(+ )-[3Hl-3-PPPlin the brain of the rhesus macaque. The binding characteristics of (+)-[3Hl-3-PPPin the primate brain were comparable to those previously described in the rodent. Saturation analysis demonstrated a single class of sites in cerebellar and hippocampal membranes with a Kd value of 28 nM. Sigma receptors labeled with ( +>-f3H]-3PPP in the primate brain displayed the appropriate rank order of potency and stereoselectivity in competition binding assays. Haloperidol displaced (+)-[3H]-3-PPPbinding in the low nanomolar range, and the (+ 1isomer of pentazocine was 50-fold more potent than (-)pentazocine. Computerized densitometric analysis of the autoradiograms demonstrated a striking enrichment of sigma binding sites over the paralimbic belt cortices, including the orbitofrontal, cingulate, insular, parahippocampal, and temporopolar gyri. Peak densities of sigma receptors were seen over the medial and central nuclei of the amygdala and were widely distributed within the hippocampal formation. Sigma binding sites densities were elevated over the suprachiasmatic and supraoptic nuclei of the hypothalamus. Moderate sigma receptor densities were observed over the ventromedial sectors of the caudate and the putamen. Sigma receptors were also elevated over autonomic relay nuclei of the brainstem, including the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. The distribution of sigma receptors in the primate brain suggests that the paralimbic belt cortices, amygdala, hippocampus, hypothalamus, and autonomic relay nuclei of the brainstem may be interrelated by a topographic chemical linkage. The autoradiographic visualization of sigma receptor distributions in the primate brain provides further support for a role of sigma receptor mechanisms in the functions of the limbic system. o 1992 Wiley-Liss, Inc.

INTRODUCTION The identification and discrimination of sigma and PCP receptors provides potential sites for the action of antipsychotic drugs (Snyder and Largent, 1989). Unique sigma receptor binding sites have been demonstrated in central and peripheral tissues with (+) 13H]NANM (Su,1982; Tam and Cook, 1984; Itzhak et al., 1985), (+H3HI-3-PPP (Gundlach et al., 1986), I3H1DTG (Weber et al., 19861, [3Hl-haloperidol (Weissman et al., 19881, and (+I [3Hl-pentazocine(De Costa et al., 1989; Walker et al., 1990). Many antipsychotic drug classes exhibit high affinity for the sigma binding site (Largent et al., 1988). Recently, subclasses of sigma binding sites have been postulated on the basis of 0 1992 WILEY-LISS, INC.

ligand binding and biochemical studies (Quirion et al., 1992). The demonstration of sigma receptor regulation by chronic neuroleptic treatment (Itzhak and Stein, 1991) and of sigma-sensitive electrophysiological (Kennedy and Henderson, 1990) and second messenger responses (Bowen et al., 1988) provides additional support for functional sigma receptor sites in the central nervous system. Sigma receptor distributions have been visualized with various radioligands in the guinea pig and cat brain. Autoradiographic localization of haloperidol-sen-

Received March 11,1992;accepted in revised form May 29,1992.

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sitive sigma receptors in the guinea pig brain demonstrated high densities of binding sites in the forebrain over neuroendocrine and core limbic brain structures (McLean and Weber, 1988).Lower levels of sigma binding sites were observed over nonlimbic nuclei, with the lowest levels of binding demonstrated over extrapyramidal brain regions. The anatomical pattern for the sigma receptor labeled with [3H]-DTGwas shown to be similar to the distribution of sites labeled by (+)-r3Hl-3PPP (Largent et al., 1986; McLean and Weber, 1988). Recent autoradiographic studies in the guinea pig brain demonstrate further that the distribution pattern for ( +)-[3H]-3-PPPagrees well with the localization of the potent and selective sigma ligand ( +)-[3H1-pentazocine (Walker et al., 1990). Neuroleptic-sensitive sigma binding sites are localized to the substantia nigra pars compacts of the cat (Grabiel et al., 1989). Grabiel et al. (1989) have suggested that the intense [3HI-DTGbinding visualized over the densocellular zone of the substantia nigra implies a special linkage of sigma receptors with the limbic system by way of the dopaminergic projections to the striosomal compartment of the striatum. In keeping with this hypothesis, a functional link between (+ )-3-PPP and dopaminergic neurons has been demonstrated in vivo (Hjorth et al., 19851,further suggesting that sigma-active drugs may modulate dopaminergic neurotransmission. An association between sigma opiates and psychotogenesis has been proposed on the basis of the behavioral spectrum of effects produced by these agents, including hallucinations, delusions, dysphoria, depersonalization, and affective lability (Keats and Telford, 1964; Forrest et al., 1969; Haertzen, 1970). Putative sigma receptor sites bind stereoselectively to dextrorotary (+) isomers of certain psychotomimetic benzomorphans. However, early clinical studies in humans were often performed with racemic mixtures of the synthetic or semisynthetic opiates, indicating the possibility that the psychotomimetic effects of sigma opiates may have been produced, in part, by the action of the levorotatory isomers. This “wrong”stereospecificity complicates the interpretation of these early findings and potentially limits the relevance of the sigma receptor site for psychiatric disorders. However, classical and nonclassical neuroleptics bind with very high affinity to sigma receptor sites, with potencies equivalent to D2 dopaminergic receptors, raising the possibility that certain behavioral effects of these drugs may be related to the targeted actions of the (+) benzomorphans at the sigma receptor site. In the study reported here, we have examined the distribution of sigma receptors in the macaque brain. Special emphasis was placed on an analysis of the distribution of sigma receptor sites labelled with ( +)-[3H]3-PPP within particular sectors of the cortical surface of the primate brain. Autoradiographic visualization of ( +)-[3H]-3-PPPbinding was performed on sagittal and

coronal sections of the brain of the rhesus macaque. A brief report of this work has been published previously (Ciarlegio and Mash, 1990).

METHODS The animals used in this study were 2 juvenile, male monkeys (Macaca mulatta) that weighed approximately 5 kg. The animals were restrained with ketamine and deeply anesthestized with halothane gas and placed in a stereotaxic headholder. Following removal of the calvarium and upper cervical vertebra, cardiopulmonary arrest was induced by a medullary transection. The brain was rapidly removed and frozen in 2-methylbutane at -30°C. Serial coronal and sagittal sections of each hemisphere were cut at 30 p on a Hackermright cryostat and thaw-mounted on gelatincoated slides. Sections were dried under reduced pressure a t 4°C as previously described (Herkenham and Pert, 1982). Receptor binding assays Tissue was homogenized in ice-cold 50 mM Tris buffer (pH 7.8) with a Brinkman Polytron (Westburg, NY).Homogenates were centrifuged at 48,000 X g for 20 minutes, and the pellet was resuspended in 10 mM Tris buffer (pH 7.8) to yield a final concentration of 5 mg tissuelml. All assays were performed in triplicate. Saturation isotherms were obtained by incubating membranes with 1.5-200 nM (+)-[3Hl-3-PPP(sp. act. 101 Ci/mmol). Specific binding was defined as the difference in binding in the absence and presence of 10 WM ( 5 bpentazocine. The incubation was terminated by the addition of 4.5 ml of ice cold buffer followed by rapid filtration over Whatman 934-AH glass fiber filters treated with 0.1% polyethylenimine, and rapidly washing with 3 changes of 4.5 ml ice cold buffer. After air drying, filters were placed in Cytoscint cocktail (ICN Biomedical)and counted in a LKB model 1211 scintillation counter. Competition curves were generated by incubating membranes with 3 nM ( +)-[3Hl-3-PPP, and the indicated concentrations of competitor in 25 mM Tris for 60 minutes at 25°C. Receptor autoradiography Sigma receptors were labeled in slide-mounted tissue sections according to the method of Largent et al. (1986). Briefly, unfixed cryostat sections were labelled with 30 nM (+)-[3Hl-3-PPPin 25 mM Tris buffer (pH 7.8). Under these assay conditions, ( +)-[3Hl-3-PPP occupies 50% of the total number of sigma binding sites. After a 60 minute incubation a t 25”C, the slidemounted sections were rinsed in 3 changes of buffer at 4°C. Nonspecific binding to brain sections was defined as binding occurring in the presence of 100 pM (?)pentazocine. At the end of the incubation period, slides were washed 3 x 3 minutes in buffer at 4°C and dried

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with a stream of cool air. Radioactivity bound to dried sections was estimated by liquid scintillation spectrometry and by exposure to film emulsion for autoradiograPhY. Dried labeled sections and [3H] Micro-scales (Amersham Corp.) were apposed to [3H] Ultrafilm (LKB Instruments, Gaithersburg, MD) for 4 weeks at 4°C. Film autoradiograms were analyzed by using a computerized image analysis system developed at the Center for Cerebrovascular Disease of the Neurology Department at the University of Miami School of Medicine. Autoradiograms were digitized at 100 pm pixel size using an Optronics scanning drum densitometer interfaced to a cluster of MicroVAX-3 computers. Following background subtraction, the optical densities were converted to specific activities obtained by co-placed standards (Unnerstall and Kuhar, 1982). The resulting specific activities are expressed as bound pmoles of radiolabeled ligand per gram dry tissue weight. The specific activities were displayed in 15 pseudocolor codes on a Sony color raster display. Region of interest analysis was based on cytoarchitectonic designations according to Bonin and Bailey (1947) and Mesulam and Mufson (1982).

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isomer in displacing (+ )-[3H]-3-PPPbinding from receptor sites (Fig. 2). The (+)-isomer of pentazocine exhibited a complex binding curve that best fit a 2-site model (nH = 0.58; p < 0.001). The binding curve for the (-)-isomer was monophasic, with a Hill slope (nH)of 0.96. The y value of 30.9 nM (nH= 1.03) derived from the computer-generated IC50value for DTG agrees with the reported range of K, values for DTG binding reported previously in rodent and guinea pig brain membranes (Contreras et al., 1990; Karbon et al., 1991).

Autoradiographic localizations of ( +)-[3Hl-3-PPP binding sites in the primate CNS To visualize neuroanatomical patterns in the primate brain, slide-mounted sections were incubated with 30 nM (+)-[3H]-3-PPP(-50% occupancy) and apposed to tritium sensitive film. Discrete localizations of ( + )-[3Hl-3-PPPbinding sites were observed throughout the primate CNS, confirming earlier results in guinea pig brain (Largent et al., 1986). Densitometric analysis of ( +)-r3H]-3-PPPbinding within 36 sampled brain regions showed that binding site densities ranged between 11.4 and 52.0 pmoVg of original tissue (Table I). In the frontal lobes of the primate cerebral cortex, peak densities of sigma binding sites were visualized over Data analysis the orbitofrontal surface (areas 12 and 13).Intense laData from saturation experiments were transformed belling of sigma sites was demonstrated throughout the by the method of Rosenthal (1967) to estimate KD and subcallosal, anterior, and posterior divisions of the cinB, values. Hill coefficients were determined by least- gulate gyrus.Peak densities were especially noted over squares linear regression analysis. KD and values the dorsal and rostra1 division in area 32 and in the were derived from the iterative, nonlinear, least- adjoining prefrontal areas on the medial and lateral squares regression analysis of the RS-1 program (BBN surfaces of the frontal lobe. The anterior cingulate gySoftware Products Corp., Cambridge, MA) and SCAFIT rus (area 24) had a greater density of (+)-[3Hl-3-PPP and EBDA (McPhearson, 1985). Fifty-percent inhibi- binding than the posterior cingulate gyrus (area 23) tory concentrations (IC5J were derived from binding (Table I). Caudally, intense labeling of sigma receptors was seen also over the retrosplenial cortex. Sigma recurves analyzed with the RS-1 program. ceptors were dense over the temporal pole, insula, and RESULTS perihippocampal gyrus. In contrast, the premotor and Sigma receptors in the primate brain motor regions (areas 6 and 4) had low levels of sigma The saturation binding curve of the sigma receptor receptor site labelling. The lowest densities of cortical for ( +)-[3H]-3-PPPin the monkey cerebellum is shown sigma receptors were seen over the parietal and occipiin Fig. 1. These data yielded a linear Rosenthal plot, tal lobes (Fig. 3, Table I). Sigma receptor densities were elevated throughout indicating that ( +)-13H]-3-PPPinteracts in the primate brain with a single class of receptor sites having an the hippocampal formation, with the highest binding affinity for the radioligand in the nanomolar range seen over the fascia dentata and CAI sector (Fig. 4B (Fig. 1B) In hippocampal membranes, (+)-[3Hl-3-PPP and 4C). All parts of the septa1 complex had moderately of48 * 3 elevated densities of sigma receptors. Within the basal bound with a KD of 28.5 2.0 nM and a B, pmoVg of original tissue (data not shown). The potency ganglia, sigma receptor densities were low. The ventral of drug competitors to inhibit 3 nM (+)-[3Hl-3-PPP and medial parts of the caudate and putamen had binding to putative sigma receptors is shown in Fig. 2. somewhat elevated levels of binding sites as compared Competition studies in cerebellar membranes demon- to the dorsal and lateral sectors. In the nucleus accumstrate that (+ )-t3H1-3-PPPbinding displayed the appro- bens, binding site densities were elevated and were priate rank order of potency and stereoselectivity of the comparable to those of the amygdala. Low to backsigma receptor in the primate brain. Haloperidol was ground levels of sigma binding sites were detected over the most potent drug displacer of ( +)-[3Hl-3-PPPbind- the medial and lateral parts of the globus pallidus (Fig. ing, with a Ki value of 1.3 nM (nH= 0.97). The (+)- 4A). In general, ( + )-[3Hl-3-PPPbinding sites were uniformly low over the thalamus, although elevated densiisomer of pentazocine was more potent that the (-I

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Bound (pmol/g tissue) Fig. 1. Demonstration of high affinity (+)-r3H1-3-PPPbinding to membranes prepared from the monkey cerebellum. A Saturability of (+)-[3H]-3-PPPbinding. Membranes from 10 mg of tissue were incubated with (+)-[3H1-3-PPP(1.5-200 nM) for 60 minutes at 25°C. Nonspecificbinding was determined in the presence of 2,000-fold excess of

(2) pentacozine.

The results shown are from a representative experiment. B: Rosenthal plot of the specific binding of (+)-[3Hl-3-PPP.Saturation analysis revealed that ( +)-L3H1-3-PPPbound to an apparent single class of binding sites with a dissociation constant, KD,of 28.5 & and I a binding sites density, B, of 44 pmoVg original tissue.

ties were seen over the intralaminar complex and the lateral reticular nucleus. In the hypothalamus, very high binding site densities were seen over the supraoptic and suprachiasmatic nuclei (Fig. 4A). In the primate, moderate levels of sigma receptors were visualized over the substantia nigra pars compacts. Within the brainstem, high concentrations of

sigma binding sites were seen over several autonomic relay nuclei, including the nucleus tractus solitarius, nucleus ambiguus, and dorsal motor nucleus of the vagus (Table I). As can be seen in sagittal sections, (+If3H1-3-PPPbinding was moderate to low over the pons and the medulla (Fig. 3A-C). The cerebellum contained moderately elevated receptor densities, with the high-

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Ligand (Log M) Fig. 2. Competition studies of sigma receptors in monkey cerebellar membranes. Membranes were incubated with 3 nM (+)-[3H]-3-PPPand the indicated concentration of unlabeled haloperidol, the isomers of pentazocine,and 1.3 di-o-tolylguanidine.The data points are presented as percent sites occupied and are from a representative experiment. (+)-Pentazocine(@), haloperidol (O),D W and (-1pentazocine (0).

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coloring of experience and in the channeling of drive and affect (Mesulam, 1985). In subcortical regions, high binding site densities occurred over the supraoptic and DISCUSSION suprachiasmatic nuclei of the hypothalamus and over The present study provides biochemical and anatom- autonomic relay nuclei, including the nucleus tractus ical evidence for the existence of putative sigma recep- solitarius (nTS) and dorsal motor nucleus of the vagus. tors in nonhuman primate brain. Sigma receptors have Emotional states are associated with specific patterns been identified previously in the primate cerebellum of autonomic response, and the paralimbic areas are using [3Hl-haloperidol in the presence of spiperone to known to participate in the regulation of autonomic occlude D2 receptor sites (Vu et al., 1990). Saturation tone (Pool and Ransohoff, 1949; Showers and Lauer, binding analysis in primate hippocampal and cerebel- 1961). An association between sigma receptors and core lar membranes indicates that (+ )-[3Hl-3-PPPbinds to a limbic and neuroendocrine structures has been demonsingle class of receptor recognition sites with nanomo- strated previously in the rodent brain using different lar affinity. These data agree with previous studies sigma-selective radioligands. Largent et al. (1986) demwhich demonstrate that L3H1-(+ )-PPP binding does not onstrated high densities of binding sites over core limrecognize multiple subclasses of sigma binding sites bic areas and neuroendocrine nuclei using ( +)-L3H1-3(Largent et al., 1984; Karbon et al., 1991; Quirion et al., PPP and (+)-f3H1-3-SKF10,047 in rats. Similar results 1992). Competition binding assays demonstrate further in guinea pig brain were obtained using i3H1-DTG the appropriate rank order of potency and stereoselec- (McLean and Weber, 1988). In the rodent brain, espetivity for the putative sigma receptor site (Koe et al., cially intense binding site densities were noted within 1989; Karbon et al., 1991). Haloperidol was the most the cerebellum, particularly in the Purkinje cell layer. potent drug identified at sigma receptors assayed in the We observed only moderate levels of sigma binding primate brain membranes. ( +)-[3H]-3-PPP binding sites within the cerebellum, well below those found over sites were blocked with nanomolar potency by the cortical limbic regions. However, somewhat elevated sigma compounds DTG and (+) pentazocine. The (+) densities of sigma receptor sites were seen over the isomer of pentazocine was 50-fold more potent than (-) cerebellar vermis. The results demonstrated in the monkey brain are in general agreement with regional pentazocine. A striking finding is this study was the association of assays of sigma binding sites in human brain (Weisselevated densities of sigma receptors with the paralim- man et al., 1988). Putative sigma receptor sites were bic belt cortices and core limbic structures. The para- labeled in the human brain with [3Hl-haloperidolin the limbic cortical areas play a major role in the affective presence of spiperone to occlude binding to D, receptor

est levels of binding visualized over the cerebellar vermis (Table I).

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Fig. 3. Computer-generated color coding of (+)-13H1-3-PPPbinding sites in sagittal sections of the monkey brain. The sagittal brain sections (A-C) shown are lateral to medial. Pseudocolor codes represent a rainbow scale (red = high density; green = intermediate density; purple = low density). ac, anterior commissure; Acb, accumbens; amg, amygdala; cc, corpus callosum; Cb, cerebellum; Cd, caudate nucleus;

CS, central sulcus; FD, fascia dentata; LF, lateral fissure; OF, orbitofrontal cortex; PS, principal sulcus; SN, substantial nigra; th, thalamus; TP, temporopolar cortex; V1, primary visual cortex; 32, precallosal cingulate cortex; 24, anterior cingulate cortex; 23, posterior cinuglate cortex; 4, primary motor cortex; 6 , premotor cortex; GP, globus pallidus; LS, lunate sulcus.

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Fig. 4. Distribution of (+)-[YH]-3-PPPbinding sites in the hypothalamus and hippocampal formation of the monkey. Same spectral representation as shown in Figure 3. A Coronal section taken at the level of the anterior commissure (ac). Note the marked elevation in ( +)-L3H1-3-

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PPP binding over the superchiasmatic (SCN) and supraoptic nuclei (SO) in the monkey hypothalamus. B, C: Sigma receptors are elevated over the fascia dentata and CA1 sectors of the hippocampal formation. PrS, presubiculum; Put, putamen. Other abbreviations as in Fig. 3.

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TABLE I . Densities and standard errors of sigma binding sites labeled with (t)13HI-PPP Brain region Association and sensorimotor cortices Primary visual cortex Primary somatosensory cortex Auditory association cortex Premotor cortex (area 6 ) Primary motor cortex (area 4) Caudal inferior parietal lobe Primary auditory cortex Dorsolateral prefrontal cortex Cingulate gyrus Cingulate (precallosal, area 32) Cingulate (anterior, area 24) Cingulate (posterior,area 23) Paralimbic areas Paraolfactory cortex Orbitofrontal cortex Temporopolar cortex Entorhinal-prorhinal cortex Insular cortex Core limbic areas Amygdala Central nucleus Medial nucleus Hippocampus Dentate gyrus CA1 sector Claustrum Basal ganglia Caudate Putamen Globus pallidus Nucleus accumbens Comparison areas Substantia nigra Pulvinar Superior colliculus Inferior colliculus Nucleus of the solitary tract Dorsal motor nucleus of the vagus Ambiguus nucleus Inferior olive Cerebellum (hemisphere) Cerebellum (vermis)

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41.9 1.2 43.7 i- 3.5 41.2 i- 1.2 42.4 i- 2.9 43.2 f 1.9 40.3 f 0.9 39.9 t- 1.2 46.2 f 1.0 37.3 i- 1.1 32.7 i- 1.0 22.0 f 0.9 21.1 t- 1.0 11.4 f 0.5 37.3 t- 1.2 28.1 i- 1.2 18.1 t- 1.5 20.5 f 0.6 14.3 f 0.6 40.0 2 1.9 52.0 i- 1.5 41.0 ? 0.5 20.5 f 0.6 27.7 i- 0.8 36.1 i- 0.3

recognition sites. Although core limbic areas demonstrated elevated densities of haloperidol-sensitive sigma receptors, high binding site densities were also seen over the occipital poles and the cerebellum. It shouId be noted that major differences in the pattern of sigma receptor distribution across studies lie outside of structures associated with the limbic system. These anatomical differences may be attributable to a variety of factors, including interspecies variation and differing selectivities of radioligands t o multiple receptor subtypes and/or agonist affinity states. Within the paralimbic areas, sigma receptor densities were highest over the anterior cingulate gyms (areas 32 and 24). It is interesting to point out that the anterior cingulate gyms has extensive connections, mostly reciprocal, with both limbic and neocortical structures. Limbic projections include the orbitofrontal gyrus, insula, amygdala, and intralaminar and midline thalamic nuclei (Pandya and Kuypers, 1969; Vogt et al.,

1979). Neocortical connections include premotor (areas 6 and 8) and the inferior parietal cortex (area 7; Pandya et al., 1981). The functions of the cingulate gyrus in relation to the limbic system are controversial. However, electrical stimulation of area 24 in monkeys elicits a variety of autonomic responses both sympathetic and parasympathetic, including bradycardia, decreased blood pressure, mydriasis, and piloerection (Ward, 1948). Stimulation of area 24 in humans resulted in somewhat different autonomic responses, including mydriasis, rubefaction of the face, tachycardia, and tachypnea (Talairach et al., 1973). Stimulation of the anterior cingulate gyrus in the cat inhibits attack behavior evoked by hypothalamic stimulation and elicits pupillary dilation, whereas stimulation of the posterior cingulate (area 23) does neither (Siege1 and Chabora, 1971). Taken together, these observations suggest that the anterior cingulate gyrus may serve as a higher center of autonomic regulation by way of its projections to the hypothalamus (Domesick, 1969; Kalia and Mesulam, 1980). The precise role of sigma recognition sites in the functions of the cingulate gyrus remain speculative. In addition to alterations in autonomic response, electrical stimulation of the anterior cingulate gyrus in humans resulted in unexpected motor responses (Talairach et al., 1973). This motor behavior consisted of complex, stereotyped movements of the lips, tongue, fingers, and body. These responses are fundamentally different from those elicited by direct stimulation of the primary motor area and suggest the anterior cingulate gyrus may influence motor behavior via its connections to the premotor cortex (Talairach et al., 1973; Pandya et al., 1981). A third function attributed to the anterior cingulate gyrus is the control of affect and emotional reaction to environmental stimuli (Baleydier and Mauguiere, 1980). Ablation of area 24 in monkeys resulted in social indifference to fellow monkeys, cessation of mimetic activity and grooming, and failure to display signs of affection (Ward, 1948; Glees et al., 1950). Early psychosurgical attempts were made to treat a variety of mental disorders in humans with anterior cingulectomy, including depression (Tow and W i t t y , 19531, psychosis (Livingston, 19531, and schizophrenia (Tow and Armstrong, 1954). Postoperative behavioral changes were often rapid in onset and included decreased hostility and fear, diminished social inhibition, and a decline in impulsive actions. Stereotaxic cingulectomy was performed to beneficially modify malfunctions in the limbic bridge through which abnormal behaviors were presumed to be emanating. The targeted actions of sigma antagonists to the cingulate gyrus may account, in part, for their therapeutic usefulness as antipsychotic agents. The insula, another paralimbic region that exhibits high levels of sigma binding sites, has been implicated as playing a role in visceral sensation and autonomic responses (Mesulam and Mufson, 1982b). In rhesus

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monkeys, stimulation of the anterior insula results in a variety of cardiovascular, gastrointestinal, respiratory, and salivatory responses (Hoffman and Rasmussen, 1953; Showers and Lauer, 1961). The nucleus tractus solitarius, also rich in sigma receptors, is the first central synapse for visceral afferent input (Kalia and Mesulam, 1980). The nTS projects via the ventral posteromedial nucleus of the thalamus to the anterior insula, forming a network that may integrate autonomic function (Mesulam and Mufson, 1982). In this regard, it is interesting to point out that much of the parasympathetic outflow exits the brainstem via yet another sigma receptor-rich area, the dorsal motor nucleus of the vagus. The hypothalamus contained strikingly high levels of sigma binding sites within the supraoptic and suprachiasmatic nuclei. Elevated sigma receptor binding over the suprachiasmatic nucleus, “the mind’s clock,” suggests a potential role for sigma-active drugs in the photoneuroendocrine system. The supraoptic nucleus (SON) contains ADH-producing neurosecretory cells, as well as osmoreceptive cells (Thorn, 1970). ADH release from the SON is regulated by input from both osmoreceptors and baroreceptors via the nTS. Self-induced water intoxication has been observed in schizophrenic patients (Dubovsky et al., 1973; Khamnei, 1984; Bremner and Regan, 1991; Muller and Lann, 1991).Water intoxication in these patients results from primary polydipsia and/or the syndrome of inappropriate antidiuretic hormone secretion (SIADH; Kramer and Drake, 1983). Schizophrenia is hypothesized to be associated with absolute or relative cortical hyperdopaminergic activity involving the mesocortical and mesolimbic pathways (Meltzer and Stahl, 1976). Raskind et al. (1975) speculated that the clinical triad observed in their patients having psychotic depression with schizophreniform features, SIADH, and polydipsia may have resulted from dopaminergic hyperactivity within the CNS. There is a close anatomic proximity of the centers regulating thirst (lateral hypothalamus, preoptic area, septum) and ADH release (SON), and these hypothalamic nuclei are interconnected with the limbic system. Dopamine regulates ADH release and extracellular stimulation of thirst, and the destruction of hypothalamic dopaminergic pathways abolished central receptor-induced ADH release in cats (Milton and Paterson, 1973) and produced adipsia in rats (Ungerstedt, 1971). However, a potential role of antipsychotic drug binding to sigma receptors in the supraoptic nucleus should be considered in the etiology of this syndrome. Further studies are clearly needed to clarify what physiological role sigma-active drugs may play in hypothalamic functioning. Despite recent progress in defining the physiological relevance of sigma receptors, a sigma-mediated transduction pathway remains unclear. Sigma-active drugs have been implicated in the modulation of adrenergic,

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cholinergic, and dopaminergic neurotransmission. Kennedy and Henderson (1990) demonstrated that (+)3-PPP depolarized sympathetic neurons of the mouse isolated hypogastric ganglion by inhibition of potassium currents, increasing the evoked release of catecholamines. Several sigma ligands, including (+)-pentazocine, DTG, and haloperidol, potently block stimulation of inositol phosphate production by the full muscarinic receptor agonist, carbachol, in rat brain synaptoneurosomes (Bowen et al., 1988). The rank order of potency for heterologous phosphoinositol inhibition correlates well with binding affinity at sigma receptor sites. To establish that sigma binding sites represent true receptor recognition sites requires the identification of an endogenous neurotransmitter. Polypeptides isolated from guinea pig brain, termed sigmaphins, and a protein found in porcine brain, designated B-endopsycosin, inhibit the binding of sigma ligands, suggesting the possibility of an endogenous ligand in brain (Su et al., 1986; Contreras et al., 1987). In addition, neuropeptide Y and peptide YY have been shown to bind with higher affinity to sigma sites than either haloperidol or pentazocine (Roman et al., 1989). Intrastriatal injection of the neurotoxin 6-hydroxydopamine produced retrograde degeneration of dopaminergic nigral neurons, accompanied by a significant depletion in ( + )-[3Hl-3-PPP binding within the pars compacta of the substantia nigra in guinea pigs (Gundlach et al., 1986). This observation suggests that a t least part of the sigma binding sites within the substantia nigra are located on the cell bodies (or dendrites) of dopaminergic nigrostriatal neurons. Grabiel et al. (1989) demonstrated dense regions of C3Hl-DTG binding within discrete zones of the substantia nigra in cats. The highest binding site densities were visualized over the densocellular zone of the substantia nigra, a chemoarchtectonic zone that contains striosomal projecting dopaminergic neurons. The ‘striosomes’are extensively connected to limbic structures, including the amygdala and insula. In the cat brain, enriched areas of sigma binding sites were observed over midbrain dopamine-containing cell groups which project directly to limbic forebrain regions. In vivo studies have provided evidence that sigma receptors may influence nigrostriatal and mesolimbic dopaminergic projections through a functional interaction between sigma and NMDA receptors or an NMDA-utilizing synapse downstream from neurons modulated by sigma receptors (Iyengar et al., 1990). Taken together these data are in keeping with the anatomical observations in the primate suggesting that sigma agents may modulate, via direct or indirect mechanism(s1, dopaminergic input into cortical limbic areas. A major problem limiting the therapeutic usefulness of neuroleptic medications is the occurrence of extrapyramidal symptoms. The clinical efficacy of conventional antipsychotics is believed to occur via D2 receptor block-

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