Localization and measurement of neurotransmitter receptors in rat and human brain by quantitative autoradiography

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Compumrrzed Medical Imaging and Graphics. Vol. 13. No. I, pp. 37-45, 1989 Printed in the U.S.A. All rights reserved.

LOCALIZATION AND MEASUREMENT OF NEUROTRANSMITTER RECEPTORS IN RAT AND HUMAN BRAIN BY QUANTITATIVE AUTORADIOGRAPHY G. N. Ko, B. J. Wilcox, F. M. Petracca, M. A. Miller, M. M. Murburg,

D. G. Baskin and D. M. Dorsa Geriatric Research, Education and Clinical Center, and Psychiatry Service, V.A. Medical Center, Seattle, WA 98 108, and Departments of Psychiatry and Behavioral Sciences, Medicine and Pharmacology, and Biological Structure, University of Washington School of Medicine, Seattle, WA 98 195, U.S.A. (Received I3 November 1987) Abstract-Receptors for neurotransmitters can be visualized and characterized using in vitro tissue slice binding techniques and quantitative autoradiography. In this article, the general methods used in studies of this type are outlined and specific application to the study of catecholamine and neuropeptide receptors in rat and human brain tissue are described. Receptor autoradiography is used to examine regulation of dopamine receptor density in response to denervation and replacement of dopamine using brain transplants. Morphological and pharmacological aspects of vasopressin receptor ontogeny in the rat brain are examined. Finally, autoradiographic data on catecholamine receptor localization and characterization in the human hypothalamus, locus coeruleus, and frontal cortex are presented and discussed with reference to their applications in the study of neuropsychiatric disorders such as schizophrenia and senile dementia of the Alzheimer’s type. Key Words: Autoradiography, Catecholamine, Alpha adrenergic, Vasopressin, Hypothalamus, Frontal cortex, Locus coeruleus, Alzheimer’s disease, Schizophrenia, Receptors

INTRODUCTION

mitter and the degree of activation or inhibition of firing rate of the receptive neuron or group of neurons. Each neuron in the brain does not express all of the various neuroreceptors, and as a consequence, these recognition sites are regionally distributed in the brain sometimes in very discrete structures only a few cell layers thick. With the advent of “test-tube” assays of receptors in tissue homogenate preparations using radioactively labeled neurotransmitters (ligands) it became clear that one could measure these molecules relatively easily. It soon became apparent, however, that this method was only of limited use when information on the anatomical location of receptors in the brain or on the pharmacologic properties of receptors in discrete neural structures was desired. For these reasons, quantitative autoradiography of receptors in brain tissue slices was explored and developed by various investigators including Young and Kuhar [2] and Herkenham and Pert [3]. Neurotransmitter receptors can be visualized using rather simple methods. Generally cryostat (frozen) sections, 10 to 20 pm thick are placed on glass microscope slides and are incubated in a solution containing a radioactively labeled ligand. These sections are then washed to remove unbound ligand and rapidly dried under a stream of cool dry air to prevent

During the past few years, a large number of studies investigating neurochemical aspects of the central nervous system (CNS) have used computer-assisted quantitative autoradiographic methodologies. The use of these techniques to measure cerebral metabolic activity has been discussed in another article in this volume [I]. In this article, we discuss the use of these techniques to study the location and pharmacologic properties of neurotransmitter receptors in the brain.

Quantitativein vitro autoradiography of neuroreceptors Neurotransmitters modulate the activity of other neurons by binding to specific recognition sites on the surface of these cells called “receptors.” These proteinaceous membrane components bind neurotransmitters or their analogs with high affinity and in a specific and completely reversible fashion. The number of these binding sites per neuron is limited, and there is a direct (but sometimes complex) relationship between the number of receptors occupied by trans-

Address for correspondence: Grant N. Ko, M.D., GRECC182B, VA Medical Center, 1660 South Columbian Way, Seattle, WA 98 108. 37

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the ligand from dissociating from the receptor. Because ligands may associate with many nonreceptor elements in the section. adjacent sections are incubated in a solution containing ligand plus a thousandfold excess unlabeled ligand to compete out any of the labeled transmitter bound to the high-affinity receptor sites. The difference in binding between the sections incubated in ligand alone (i.e., total binding) and those incubated with excess unlabeled compound (nonspecific binding) is called specific binding. These slices can then be placed in contact with a photographic emulsion either directly or on a photographic film, and the resulting images can be used to localize receptors. When appropriate kinetic analysis are applied, one can quantitatively measure the number of binding sites present as well as their affinity for the ligand being used to label them (see reference 4 for recent review of these methods). QUANTITATION OF AUTORADIOGRAMS: USE OF COMPUTERS When information beyond simple localization of receptors is required, computer-assisted densitometry can be performed on the images resulting from binding studies if steps are taken to provide a means of standardization. That is, there must be provisions made to relate the optical density of films exposed to the sections to known amounts of radioactivity which will produce those optical densities. This is currently accomplished in one of two ways. Some investigators will incorporate known amounts of radioactivity into brain tissue paste standards and coexpose the brain section of interest with these standards. More recently, plastic standards containing radioactivity have become commercially available. Using these standards, a standard curve can be established that relates radioactivity to optical density of film exposed to that source. All that is required of a computerbased image analysis system is to accurately measure optical density. The basic requirements of such a system have been previously described [5] and several such systems are now available. Usually this includes a video camera and a digital imaging board with facilities built in to handle data that are recorded as gray level per pixel. By relating gray level to optical density that can be read from optical density step wedges obtained from photographic suppliers, the computer can read unknown quantities of radioactivity from the standard curve. There are many methodological issues and problems involved with this sort of calibration procedure, including white matter quenching [6].

.4PPLICATIONS OF COMPUTER-ASSISTED QUANTITATIVE AUTORADIOGRAPHY TO STI.!DIES IN RAT BRAIN We would like to discuss some of the ways we have used this methodology to study receptor localization and pharmacologic properties in the rat bram.

Regulation oj receptor densit) Unilateral lesions of the rat substantia mgra eliminate the ipsilateral dopaminergic innervation to the striatum and result in supersensitivity of dopaminergic receptors [7]. It is believed that this supersensitivity is brought about by an increase in receptor density rather than by an increase in receptor affinity. When animals with these lesions are given apomorphine, a dopamine receptor agonist, they rotate away from the lesioned side, presumably because apomorphine stimulates the supersensitive striatum more than the intact side [8]. Transplanting dopamine neurons from sources such as fetal substantia nigra or adult adrenal medulla to the denervated striatum decreases this rotation effect and increases the concentrations of dopamine in parts of the striatum adjacent to the grafted tissue. In the striatum near the grafted tissue, dopamine receptor density is normalized. compared with the nonlesioned striatum. in animals in which lesion induced turning behavior is normalized by transplanted tissue. On the other hand. dopamine receptor density in the striatum near the grafted tissue is increased in animals whose turning behavior is not returned to normal, thus indicating that there is an association between dopamine receptor density and lesion-induced turning behavior [9] (Fig. 1). Because the changes following lesion of these dopamine producing cells in the brain is similar to the degenerative changes that occur in Parkinson’s disease, these studies provide an experimental model in which to study the neurochemical changes that occur and to explore the potential clinical utility of tissue transplants into the brain to treat this disorder.

Developmental changes of receptors The rat brain is also an interesting model in which to examine the roles of neurotransmitter receptors in ontogeny. The rat pup is born with a relatively immature brain. Various brain structures undergo dramatic structural and neurochemical changes during the first 10 to 20 days of life. For example, the distribution of neurons in the brain that synthesizes the neuropeptide neurotransmitter vasopressin changes radically during this period [ IO].

Neurotransmitterreceptors in rat and human brain 0 G. N. KO et al.

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Fig. I. Spiroperidol binding in the striatum of substantia nigra-lesioned rats with fetal brain grafts that reduced rotational behavior, compared with controls. Differences in film density (gray levels) are shown between the dorso-medial quadrant of the lesioned (right) and normal (left) striatum in rats with reductions in rotations of 20% or more (N = 6, 15 sections) and in animals without significant reductions (N = 4, nine sections). The ordinate represents in absolute difference in mean gray level per pixel (2 standard error) over 5 12 X 5 12 pixels. The difference between groups is statistically significant at p < .O1 (two-tailed t test).

We have used the autoradiographic method to monitor the ontogeny of receptors for the neuropeptide vasopressin during this period of neural development. There were several questions we felt could be addressed appropriately using this technique. For example, in a brain structure such as the septum where innervation by vasopressin neurons is absent at one point and present at another [lo], are receptors for this neurotransmitter present throughout the period, or do they only appear as innervation arrives at the structure? To answer this question, we performed invitro binding studies on rat pup brains obtained from the day of birth through postnatal day 12 [ 111. We found three different patterns of receptor ontogeny during this period. In some brain regions, such as the central nucleus of the amygdala, autoradiographic evidence of vasopressin binding sites could be visualized at birth and was maintained into adulthood. In the septum, binding sites were present in the dorsal aspects of the structure early in development but appeared later in the lateral and ventral aspects of the structure at a time just prior to the appearance of vasopressin containing fibers in those regions (i.e., postnatal day 8-12) (Fig. 2). Interestingly, in other brain regions such as the cingulate cortex and dorsal hippocampus, tritiated vasopressin binding sites we observed very early in development (i.e., postnatal day 7-9) but disappeared by adulthood. These morphological studies suggested that neurotransmitter

receptors are expressed by neurons in various regions of the brain at specific times during the developmental process, and that the timing of expression and repression may (as in the septum) or may not (as in the cingulate gyrus) be related to innervation of these binding sites by neurotransmitter itself. It appears that receptors may subserve quite different roles from those in the adult brain. These studies also allowed us to examine yet another aspect of receptor ontogeny. In addition to the changes in receptor distribution in the brain that we found using the DUMAS (Drexel’s Unix-based Microcomputer Image Analysis System), we studied the pharmacologic characteristics of these binding sites [ 121. In this type of study, several tissue slices are incubated either with tritiated vasopressin, or tritiated vasopressin plus increasing concentrations of competitors that define the specificity of the binding site for this neurotransmitter. Displacement of the labeled compound by competitor can be measured as a determination of optical density of film overlying the binding sites. Plots such as those shown in Figs. 3 and 4 can be constructed. We were able to show that the transitory vasopressin binding sites present in the cingulate gyrus of the developing brain show a different specificity (Fig. 3) than those in the adult brain (Fig. 4). Thus, not only are anatomical locations but pharmacological characteristics of these receptors varied in development.

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Fig. 2. Anatomic distribution of binding sites for the neuropeptide vasopressin in the septal region(s) of the developing (postnatal day 8, A) and adult (90 day old, B) male Long-Evans rat. In each case, 10 pm frozen tissue sections were thaw-mounted to glass slides and incubated with 3Harginine ‘-vasopressin, washed, dried, and placed in contact with LKB Ultrofilm for approximately one month. The resulting images are shown. Arrows indicate regions of highest binding. These studies indicate that these “receptors” are present in the dorsal aspects of the septum at birth and proliferate to include the lateral portions of the structure as the animal matures, perhaps in response to the appearance of nerve fibers that contain and secrete vasopressin into the lateral septum.

THE USE OF QUANTITATIVE AUTORADIOGRAPHY IN POSTMORTEM HUMAN BRAIN TISSUE Receptor autoradiography is a powerful tool that allows, for the first time, a quantitative assessment of pharmacologic characteristics of tissue in precise, anatomically localized areas. Thin serial sections can be alternately stained with standard histologic techniques or radioactive ligands known to bind to receptors, extending knowledge of synaptic and neurochemical events in the human brain. Because the

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technique ofreceptor autoradiography can he apphed to thin serial sections, binding in very discrete brain nuclei can be studied and estimations of receptor characteristics within a single postmortem human brain specimen can be obtained. Thus. charactenstrcs such as the maximal number of receptors (&,,,I and the dissociation constant (k;) can be obtained for experimental and control animals or pathological postmortem human specimens and normal control postmortem specimens. Group comparisons of these pharmacologic characteristics can then be performed to verify or refute hypotheses involving receptor up and down regulation as causes or contributors to neuropathologic and neurobehavioral states. Receptor autoradiography can be used tar two purposes: ( 1) it can be used to map where receptors are in the central nervous system: and (2) it can be used to actually quantify the number of receptors m very small tissue areas such as the locus ceuruleus of the brain stem or the paraventricular nucleus in the hypothalamus. This section describes three ways m which we have used receptor autoradiography as applied to human postmortem brain tissue: CI 1 to visualize alpha-2 adrenergic receptors in human frontal cortex, (2) to provide morphological evidence of the existence of alpha- 1 receptors in the hypothalamus in the paraventricular nucleus and the supraoptic nucleus of the hypothalamus, and (3) for quantification of the number of receptors in locus coeruleus of patients with schizophrenia compared with a comparable group of normal control patient brain tissue obtained at autopsy. Human frontal cortex alpha-2 adrenergic recepton In nonhuman primate experiments, it has been demonstrated that the alpha-2 adrenergic agonist drug clonidine can lead to improvement in the cognitive function (e.g., memory) of aged monkeys [ 131. This improvement in cognition seems to be localized to the dorsolateral prefontal cortex by experiments designed to ablate a variety of regions prior to clonidine treatment of these nonhuman primates. These data have prompted us to ask whether alpha-2 receptors are also present in human frontal cortex. One way to examine this question is to perform alpha-2 adrenergic receptor autoradiography on the dorsolatera1 prefrontal cortex of postmortem control specimens. Figure 5 illustrates the binding of the alpha-2 adrenergic ligand, tritiated paraaminoclonidine (PAC), to the frontal cortex of a normal control specimen. This binding is displaceable with an excess of the alpha receptor competitor, phentolamine, demonstrating that the binding of PAC is specific for receptors rather than nonreceptor tissue sites. Sections

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Fig. 3. Localization and characterization of vasopressin binding sites in the central nucleus of the amygdala (Ca) of the adult (90 day old) Long-Evans rat. Panel A shows LKB Ultrofilm image of a coronal section of rat brain containing the central amygdala (arrow). Panel B shows the results of competition studies designed to measure the specificity of binding of vasopressin. In these studies, serial sections are incubated in a solution containing 3H-arginine *-vasopressin alone or labeled a vasopressin plus a competitor peptide. The sections are then washed, dried, and placed in contact with film for one to two months. The resulting images are analyzed using the DUMAS image analysis system by relating optical density of the film overlying the central amygdala to a radioactive standards that were coexposed with the sections. The data are plotted as the percent of total specific binding of 3H-vasopressin measured in the presence of the concentrations of each competitor shown on the x axis. The competitors were unlabeled arginine ‘-vasopressin itself (AVP), oxytocin (OXY) a structurally similar peptide, and luteinizing hormone releasing hormone (LHRH) a structurally different peptide of similar size to AVP. The data depict the clear ability of the adult brain receptors to discriminate between vasopressin, oxytocin and LHRH.

were incubated in the presence of 2.5 nMo1 PAC. Identical adjacent tissue sections can be incubated in a range of PAC concentrations to allow for estimations of the B,,, as well as the & in frontal cortex. Thus, it appears that alpha-2 receptors are present in human frontal cortex. If, as in the monkey, these receptors are involved in cognition, it is possible that their dysfunction underlies human diseases with

prominent cognitive impairment such as Alzheimer’s disease. This hypothesis can be tested by measuring alpha receptor density and affinity in Alzheimer’s patients’ frontal cortex and comparing the values obtained to those of a control population. Important issues to consider in this experiment are a careful matching of age, postmortem delay, gender, and medication status at the time of death.

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Fig. 4. Localization and characterization of vasopressin binding sites in the cingulate gyrus (cg) of the developing (postnatal day 8) Long-Evans rat brain. Panel A depicts a film image of a coronal section of rat pup brain containing the cingulate binding sites. When specificity of these binding sites was characterized using quantitative autoradiographic methods identical to those described in the legend to Fig. 2, it was found that although developmental binding sites still discriminated well between vasopressin (AVP) and luteinizing hormone releasing hormone (LHRH), they do not distinguish between AVP and oxytocin well. Thus they appear to be less “specific” than adult receptors.

Autoradiographic localization of alpha-l adrenergic receptors in human hypothalamus There is ample animal information available on neurotransmitter regulation of hypothalamic hor-

mone secretion, and it is generally assumed that similar anatomy underlies human neuroendocrine function. We have recently begun to test this notion by mapping neurotransmitter receptors in human hypothalamus. Neurons that produce and secrete the neurotransmitter norepinephrine richly innervate neuroendocrine cells of two nuclei of the rat by hypothalamus: the supraoptic (SON) paraventricular nuclei

(PVN) [ 141. In addition, application of agonists of alpha-l adrenergic receptors increases the firing rate and secretory activity of neuroendocrine cells in these nuclei, particularly those that synthesize and secrete vasopressin [ 151. These receptors appear to exist on the vasopressin neurons themselves. We recently used tritiated prazosin, an alpha- 1 specific antagonist, to map alpha- 1 adrenergic receptors in human hypothalamic tissue. Figure 6 shows an LKB film autora-

diogram of a 20-p section of hypothalamic tissue obtained at autopsy 8 h postmortem from an individual dying of disease without neurologic complications. Prazosin binding sites were found to be almost exclu-

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Fig. 5. Binding of the alpha-2 adrenergic receptor against ‘H-paraaminoclonidine (PAC) to human frontal cortex (B). (A) is an adjacent tissue section incubated with an excess of phentolamine, a potent antagonist of alpha-2 receptors, which is used to define nonspecific binding.

sively located in the supraoptic and paraventricular nuclei. This suggests that the receptors which have been documented to exist in the rat are also present in the human brain.

BRAIN ALPHA-2 ADRENERGIC IN SCHIZOPHRENIA

Fig. 6. Localization of alpha-l adrenergic receptors in human hypothalamus. The image shown is that obtained on LKB Ultrofilm placed in contact with a 20-p thick tissue section that had been incubated with 3H-prazosine (an alpha- 1 antagonist). Alpha receptors are discretely localized in this tissue section to the supraoptic (SON) and paraventricular (PVN) nuclei. 3V = the third cerebral ventricle.

RECEPTORS

Increased norepinephrine has been postulated to be a pathophysiologically significant finding in paranoid schizophrenia [ 161. One possible explanation for the finding of increased norepinephrine in the cerebrospinal fluid as well as limbic brain areas of deceased schizophrenia patients is that inhibitory alpha-2 receptors on norepinephrine producing cells in the locus ceuruleus are diminished in numbers. In support of this possibility, clinical studies have been performed in schizophrenic patients looking at plasma norepinephrine metabolites and the platelets of these patients revealing a decreased number as well as decreased responsiveness of the inhibitory alpha-2 adrenergic receptor [ 17, 181. With in-vitro quantitative autoradiography, a direct examination of locus coeruleus binding of alpha-2 adrenergic ligands is possible. In a small pilot study [ 191, we demonstrated that schizophrenia patients had a nonsignificant

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Fig. 7. Binding of paraaminoclonidine (PAC) to the locus coeruleus of 18 postmortem human brain specimens by groups with mean f SEM. Horizontal lines represent means and standard error. PAC binding to LC, expressed as femtomoles/mg protein, in normal controls was 887 f 3 15 (n = 6) in suicide victims was 647 + 270 (n = 7), and in schizophrenic patients was 190 + 19 (n = 5). Schizophrenic brains had a lower mean alpha-2 receptor number than both control groups, but this did not reach statistical significance (p = .08, ANOVA).

toward lower alpha-2 adrenergic receptors at the locus coeruleus than comparable normal controls or suicide victims (Fig. 7). The definite answer to the hypothesis of subsensitive locus coeruleus alpha-2 adrenergic receptors awaits a confirmatory study with a larger number of schizophrenic subjects. The approach of in-vitro quantative autoradiography as applied to postmortem human specimens, however, appears to be a promising tool by which to further explore clinical and biochemical abnormalities in neuropsychiatric disorders. trend

in rodents were examined. Dopamine receptor auroradiography before and after transplantation of viable fetal substantia nigra tissue into host animals demon strated an association between turning behavior and changes in dopamine receptor density. In-vitro autoradiography was also used to examine the ontogen\. of vasopressin receptors in the rat brain, From the day of birth through postnatal date 12, different patterns of receptor ontogeny were observed during this period. Although some vasopressin binding sites appeared to be distributed comparably from birth snto adulthood, in areas such as the septum, binding sites appeared gradually early in development coincident with the appearance of vasopressin-containing tibers in this region of the brain. Still. in other areas such as the cingulate cortex and the dorsal hippocampus. ~asopressin binding sites were present early in development but disappeared by adulthood. Cingulate vase. pressin binding sites showed different specificit! in the developing brain as compared with the adult brain To extend the knowledge ofthe chemicai organrzation of the brain to neuropsychiatric conditions. \vt* then examined human postmortem tissue. In the normal hypothalamus, we found a high density of alpha- 1 adrenergic receptors in the paraventricular nucleus and the supraoptic nucleus. Our interpretation of this finding is that alpha-l adrenergic receptors are probably involved in the regulation of the neurosecretory cells of these nuclei. Since noradrenergic transmission is known to be disturbed in schizophrenia. we examined alpha-,! adrenergic receptors in the locus coeruleus and found a trend toward decreased receptor density in the locus coeruleus of schizophrenic patients compared with normal controls and suicide victims. These findings are discussed briefly in the context of other studies that have examined norepinephrine dysfunction in schizophrenia. Preliminary data were presented on localization of alpha-2 adrenergic receptors in the prefrontal cortex of the human brain analogous to those that have been described in nonhuman primates and may subserve a role in memory and learning. .4ckno~~IPd~~,z~nr.c--The authors wish to thank John Brelmnger. Paul Jones. Jackson Smood. and Debbie Felt for their skillful technical assistance in performance of the studies outlined herein. This work was supported by the Veterans .4dministration. and by NIH Grant NS2031 I-05 (D.M.D.)

SUMMARY The use of in-vitro quantitative

autoradiography in exploring the chemical organization of the brain has been discussed in this article. First, receptor den-

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2. W. Young and M. Kuhar, A new method for receptor autoradiography: ‘H-opioid receptors in rat brain, Brain Res. 179, 255-270 (1979). 3. M. Herkenham and C. Pert, Light microscopic localization of brain opiate receptors: A general autoradiographic method which preserves tissue quality, J. Neurosci. 2, 1129-l 149 (1982). 4. D. M. Dorsa and D. G. Baskin, Identification of neuropeptide receptors, In Neuromethods, Vol. 6: Peptides. Eds. A. A. Boulton, G. B. Baker, and Q. J. Pittman. The Humana Press, Clifton, NJ, pp. 203-244 (1987). 5. D. G. Baskin and D. M. Dorsa, Quantitative autoradiography and in vitro radioligand bindings, Expl. Biol. Med. 11, 204-234 (Karger, Basel) ( 1986). 6. M. Herkenham, Autoradiographic methods for receptor localization. In Computerized Image Processing for Functional Neuroanatomical Mapping. Ed. D. L. McEachron. Society for Neuroscience, Washington, DC, 50-66 (1986). 7. L. Creese, D. R. Burt and S. H. Snyder, Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science 197, 596 (1977). 8. J. F. Marshall and U. Ungerstedt, Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: Quantification using the rotational model, Eur. _I Pharmacol. 41, 36 1 (1977). 9. W. J. Freed, G. N. Ko, D. L. Niehoff, M. J. Kuhar, et a/.. Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra, Science 222,937-939

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1 I. F. M. Petracca, D. G. Baskin, J. Diaz and D. M. Dorm, Ontogenie changes in vasopressin binding site distribution in rat brain: an autoradiographic study, Dev. Brain Res. 28, 63-68 (1986).

12. F. M. Petracca, D. G. Baskin, J. Diaz and D. M. Dorsa, Ontogenie changes in vasopressin binding site characteristics in rat brain: a quantitative autoradiographic study, Sot. Neurosci.

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undertook a fellowship in the Neuropsychiatry Branch of the National Institute of Mental Health. He is currently an Assistant Professor of Psychiatry and Behavioral Sciences at the University of Washington School of Medicine and a research investigator at the Seattle Veterans Administration Medical Center. About the Author-BARBARA J. WILCOX received her B.S. Degree from the University of Wisconsin in 1975 and her Ph.D. in anatomy from the University of Minnesota in 1984. From 1984 to 1987, Dr Wilcox was a Postdoctoral Fellow in the Department of Medicine at the University of Washington. At present, she is a Research Associate in the Department of Neurology at Case Western Reserve University. About the Author-FRANCES M. PETRACCAobtained her Ph.D. in physiological psychology from the University of Washington in 1985. She then accepted a position as postdoctoral fellow at the Mental Retardation Center of the UCLA School of Medicine. About the Author-MARGARET ANN MILLER received her B.A. degree from Mount Holyoke College in 1972, an M.S. degree from Idaho State University in 1976, and a Ph.D. in physiology and psychology in 1985 from the University of Washington. She is currently working as a postdoctoral research associate under Dr Daniel Dorsa. She studies the physiology and pharmacology of the vasopressin system using receptor quantification methods and in situ hybridization techniques. MICHELE MURBURG received her B.A. About the Author-M. from the University of Wisconsin in 1974 and her M.D. from the Albert Einstein College of Medicine in 1978. She completed her postdoctoral training in psychiatry at Yale University, where she was Chief Resident on the Clinical Neuroscience Research Unit at the Connecticut Mental Health Center from 1981 to 1982. She then joined the faculty at the University of Washington, where she is currently an Assistant Professor in the Department of Psychiatry and Behavioral Sciences. In 1987, she received a Career Development Award from the Veterans Administration. As a Research Associate, her current research at the Seattle Veterans Administration Medical Center focuses on human neurotransmitter-neuropeptide relationships in normal and pathological states.

Abst. 12(2), 827 (1986).

13. A. F. Arnsten and P. S. Goldman-Rakic, o(~Adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates, Science 230, 1273 (1985). 14. J. R. Slade, Central catecholamine pathways to vasopressin neurons. In Vusopressin, Ed. R. W. Schrier. Raven Press, New York, pp. 343-35 1 (1985). 15. J. B. Wakerley. R. Noble and G. Clarke, In vitro studies of the control of phasic discharge in neurosecretory cells of the supraoptic nucleus, Prog. Brain Res. 60, 53-59 (1983). 16. 0. Hornykiewicz, Brain catecholamines in schizophrenia: A good case for noradrenaline, Nature 299,484 (1982). 17. D. E. Sternberg, D. S. Chamey, G. R. Heninger, et al., Impaired presynaptic regulation of norepinephrine in schizophrenia. Effects of clonidine in schizophrenic patients and normal controls, Arch. Gen. Psvchiatrv 39.285 (1982). 18. H. E. Rice, C. B. Smith, K.-R. Silk and J.‘Rosen, Platelet alpha*-adrenergic receptors in schizophrenic patients before and after phenothiazine treatment, Psychiatry Res. 12, 69 (1984). 19. G. N. Ko, J. R. Unnerstall, M. J. Kuhar, R. J. Wyatt and J. E.

Kleinman. Alphaz-adrenergic agonist binding in schizophrenic brains, Psychopharmacol. Bull. 22, 101 l-1016 (1986). About the Author-GRANT N. Ko, M.D. obtained his medical degree from the University of Washington School of Medicine and did a residency in psychiatry at Yale University School of Medicine, including a year of medicine and neurology training. He then

About the Author-DENIs G. BASKIN received a Ph.D. degree in zoology from the University of California at Berkeley in 1969 and did postdoctoral work in anatomy at Albert Einstein College of Medicine and at the University of Minnesota. He is presently Research Associate Professor of Medicine and Biological Structure at the University of Washington School of Medicine in Seattle. His laboratory and research program is centered at the Seattle Veterans Administration Medical Center and is supported by grants from the NIH and the Veterans Administration. His research activities focus on the anatomical and biological functions of receptors for insulin and insulin-like growth factors in the central nervous system. Doctor Baskin is a member of the Society for Neuroscience, Endocrine Society, American Association of Anatomists, and the Histochemical Society. He is presently an officer of the Histochemical Society and a member of the Editorial Board of the Journal of Histochemistry and Cytochemistry. About the Author-DANIEL M. DORSA obtained his Ph.D. degree in endocrinology and physiology at the University of California at Davis and then undertook a year of postdoctoral study at the Rudolf Magnus Institute for Pharmacology in Utrecht, The Netherlands. He then did a year of postdoctoral work in the Department of Physiology at Stanford University School of Medicine. He took a faculty position at the University of Washington School of Medicine, where he is currently Research Associate Professor of medicine and Pharmacology and Associate Director for Research of the Geriatric Research, Education and Clinical Center at the Seattle Veterans Administration Medical Center.