Localization and regulation of mRNAs in the nervous tissue as revealed by in situ hybridization

Cony. Eiochem.Physiol.Vol. 98C, No. 1, pp. 41-50, 1991 Printedin Great Britain

LOCALIZATION NERVOUS

0306-4492/91 $3.00 + 0.00 0 I99 1 PergamonPressplc

AND REGULATION OF mRNAs IN THE TISSUE AS REVEALED BY IN SITU HYBRIDIZATION

MITSUHIRO KAWATA,

KAZUNARI YURI

and

YUTAKA SANO

Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan (Received 9 February 1990) Abstract-l. In situ hybridization histochemistry permits the study of specific mRNAs of neuropeptides, enzymes involved in the synthesis of neurotransmitters, receptors and proteins associated with glial cells in nervous tissue. 2. The central and peripheral nervous systems are composed of heterogeneous elements and specific regulatory mechanisms occur in specific cells. 3. This review will focus on the localization and regulation of different mRNAs in the nervous system from Drosophila to human, as revealed by in sifu hybridization histochemistry.

INTRODUCTION

transmission, neuromodulation, and neurohormone in the nervous system. Before reviewing each mRNA of peptides or proteins, we will briefly outline the properties of in situ hybridization histochemistry from methodological points of view.

Recent progress in molecular biology has made it possible to elucidate gene structures of peptides and proteins which function to keep internal milieu of the body. Nervous and endocrine systems play a key role to maintain the homeostasis in response to various external stimuli and changing conditions of environments. This distinction of systems is arbitrary, and these two systems share many common features; one of the major characteristics is the presence of bioactive substances, e.g. amines or proteins, both in neurons and endocrine cells. The nervous system contains a heterogeneous population of highly specialized cells which contribute to its structural organization and functional complexity. A wide variety of proteins is required for these diverse specialized functions, as well as for functions common to all cells in the body. These proteins are all derived from specific genes which encode the sequences of animo acids through gene expression. Four steps are involved in the molecular basis of gene expression, such as transcription, processing of RNA, translation, and post-translational processing. The primary regulation of gene expression is at the transcriptional level reflecting the synthesis of mRNAs, although controls also operate at the other three steps. Measurements of the contents of particular RNA have been made to use Northern blotting or solution hybridization methods. The localization and relative quantitation of particular messenger RNA (mRNA) must be taken into account in an anatomical context because of the heterogeneous population of cells in the nervous system. For that reason, in situ hybridization is a powerful histochemical method to detect specific mRNA at the cellular level with microscopic resolution. This article reviews the localization and regulatory mechanisms of mRNAs of neuropeptides and proteins relevant to the cellular mechanism of neuro-

IN

SITU

HYBRIDIZATION HISTOCHEMISTRY

In situ hybridization histochemistry uses nucleotide probes to detect specific nucleotide sequences on slide-mounted or even free-floating sections by incubating with labeled probes. The resulting DNA:RNA or RNA:RNA hybrids are visualized by identifying the site of the probe hybridized in conjunction with autoradiography or histochemistry. As a probe there are three nucleotides available at present; complementary DNA (cDNA), complementary RNA (cRNA), and synthetic oligonucleotide. Each type of probe has advantages and disadvantages, but in these days synthetic oligonucleotide probe labeled with radioisotope is frequently used. This technique has allowed us to discriminate mRNAs from other members of its gene family in which a large part of nucleotide sequence is similar, and quantitate by counting grain density or number in a particular tissue or cell because the number of silver grains developed by autoradiography reflect the copy number of mRNAs. In combination with immunohistochemistry, and physiological and pharmacological manipulations, one can detect which cells or tissues express the genes of interest or how they respond to changing internal and external milieu. Particularly, the simultaneous demonstrations by in situ hybridization and immunohistochemistry permit the discrimination between the site of peptide/protein biosynthesis and storage or uptake (Coghlan et al., 1985; Jones et al., 1986; Kawata et al., 1988b; Lewis et al., 1985; McAllister et al., 1983; Mengod et al., 1988; Saffen et al., 1988; Sprang and Brown, 1987; Young, 1986).

41

MITSUHIRO KAWATA

42 LHRH

Labeled cells containing LHRH mRNA are distributed in the medial preoptic area, diagonal band of Broca, medial septum.and anteroventral ~~ventricular nucleus. This distribution is basically consistent with that of LHRH immunoreactivity. Since perikarya of neurons containing LHRH mRNA are small in size and message level is low, 32P has been shown to be an appropriate isotope for labeling the probe. Estrogen treatment to ovariectomized rats for seven days causes a si~ifi~ant increase of LHRH message by showing a metric proportional to LHRH mRNA content to multiply the num,ber of LHRH mRNAcontaining neurons by the average number of grains per cell. It has been considered that long-term treatment with estrogen is sufficient to replace the LHRH used for generating LHRH surge, consistent with a fa~ilitatory action on the LHRH system (Kelly et al., 1989; Pfaff, 1986; Rothfeld et al., 1989; Shivers et al., 1986a). SOMATOSTATIN

Somatostatin mRNA-containing cells are seen in the anterior periventricular nucleus, medial parvocellular subdivision of the paraventricular nucleus, suprachiasmatic nucleus, anterior and lateral hypothalamic areas, amygdala (central nucleus), hippocampus, striatum, and cerebral cortex (II and V layers). Hybridization densities over individual neurons in the periventricular nucleus are two to three times greater than hyb~~zation densities over individual neurons in the striatum and cerebral cortex. Ontogenetic studies show that neurons of strong signals are first seen only in the amygdala and hypothalamus at the embryonic day (E) 20, while by the postnatal day (P) 1 a moderate number of hybridized neurons appear in the cerebral cortex. After the many appearances of neurons containing somatostatin mRNA in the cerebral cortex (layer II/III and V/VI) at PlO-P12, they are gradually decreased. Similar observations are made in the cerebellum. In the dorsal root ganglia, about 10% of small neurons contain somatostatin mRNA. In Snell dwarf mice which have severely diminished anterior pituitary function with low or no production of growth hormone, prolactin, or thyroid-stimulating hormone, an approximately 40% decrease of hybridization intensity is observed in the hypothalamus, whereas an increase of about 45% is seen in the cerebral cortex (Arentzen et al., 1985; Card et al., 1988; Henken et al., 1988; Hoefler et al., 1986; Inagaki et al., 1989; Naus et al., 1988; Nishimori et al., 1988; O’Hara et al., 1988; Ohsako et al., 1986; Uhl and Sasek, 1986; Weiss and Chesselet, 1989). CRF

Many of the labeled cells containing CRF mRNA are found in the paraventricular nucleus (medial parvocellular and magnocellular subdivisions) and the supraoptic nucleus, and the literature summarizes that CRF is co-localized with oxytocin in about one-third of magnocellular neurons and with vasopressin in the parvocellular neurons. In addition to

et al

these hypothalamic nuclei, labeled cells are also present in the spinal nucleus of the trigeminal nerve, reticular formation, nucleus of tractus solitarius and raphe nuclei. Following removal of glucocorticoid negative feedback produced by adrenal~tomy, which elicits a profound increase in pituitary ACTH production and secretion, CRF mRNA is markedly increased by 90% in the medial parvocellular subdivision of the paraventricular nucleus, while magnocellular neurons in the supraoptic nucleus do not respond to this ma~pulation. Repeated electroconvulsive shock treatment for seven days induced an approximately 40% increase of CRF mRNA in the medial parvocellular subdivision of the paraventricular nucleus. In the supraoptic nucleus there is a progressive increase in CRF mRNA with 2% NaCl solution loading which is significant within 48 hr, whereas the parvocellular and mag~ellular subdivisions of the paravent~cular nervous show no significant change in CRF mRNA in response to this manipulation. Only four hours after intraperitoneal injection of hypertonic saline, there is a very marked increase of CRF mRNA in the paraventricular nucleus, but no significant change in the supraoptic nucleus (Cummings et ai., 1989; Herman et al., 1989; Lightman and Young, I987b; Swanson and Simmons, 1989; Young et al., 1986b). TRH

Labeled cells containing TRH mRNA are widely distributed in the central nervous system. The largest number of hybridized cells is observed in the hypothalamus, which includes the preoptic suprachiasmatic nucleus, medial and lateral preoptic areas, anterior hypothalamus, medial parvocellular subdivision of the paraventricular nucleus, dorsomedial nucleus, and the posterior portion of the arcuate nucleus. Besides these areas, hybridized cells are also observed in the amygdala (medial and cortical nuclei), caudate-putamen, diagonal band of Broca, bed nucleus of the stria terminalis, raphe magnus, pallidus, and obscurus as well as accessory groups on the periolivary and peripyramidal regions, and the dorsai motor nucleus of the vagus. These regions were previously recognized as containing TRH immunoreacti~ty in their neuronal perikarya by immunohistochemistry, but several areas show specific hybridization signals of TRH mRNA, which include the olfactory bulb, anterior commissural nucleus, reticular nucleus of the thalamus, midbrain periaqueductal gray and the dorsal motor nucleus of the vagus. On the contrary, neither cells in the cerebral cortex nor hippocampus show evidence of specific hybridization of TRH mRNA, although these areas are the site where many TRH-immunoreactive neurons are present. Chemical thyroidectomy produced by the administration of 6-(~-propyl~-2-thiouracil reduces plasma thyroxine and significantly increases hyb~dization signals of TRH mRNA in the paraventricular nucleus. Ontogenetical studies show that the first nucleus containing TRH mRNA is found in the lateral hypothalamus at El4 and up to P21 the number and signal intensity of TRH mRNA-containing neurons increases, and thereafter decreases to show the adult pattern

Localization and regulation of mRNAs in the nervous (Burgunder and Taylor, 1989; Koller et al., 1987; Segerson et al., 1987a; Segerson et al., 1987b). VASOPRESSIN

Labeled neurons containing vasopressin mRNA are present in the ventral portion of the supraoptic nucleus, dorsolateral magnocellular portion of the paraventricular nucleus, and dorsomedial portion of the suprachiasmatic nucleus. Drinking 2% NaCl solution results in a progressive increase of vasporessin mRNA hybridized in the magnocellular neurons of the supraoptic and paraventricular nuclei. Levels of vasopressin mRNA changes to hyperosmotic stimuli in the supraoptic nucleus are stronger than that in the paraventricular nucleus. Adrenalectomy induces a significant increase of vasporessin mRNA in the medial parvocellular division of the paraventricular nucleus, not in the magnocellular division of this nucleus and the supraoptic nucleus. At the cellular level, increased silver grains are detected in CRF-immunoreactive neurons in the paraventricular nucleus, and treatment with dexamethasone prevents the increase of vasopressin mRNA in these neurons produced by adrenalectomy, indicating that neuronal vasopressin genome is modulated by endocrine environment even at the adult stage. Gene sequence analysis of diabetes insipidus (Brattleboro) rat shows a single-base pair deletion in the second exon region of the preprovaropressin gene and several attempts have been made to investigate the vasopressin message level of this animal by using in situ hybridization. So far, there are still controversial findings about the levels of vasopressin mRNA in neurons of the supraoptic and paraventricular nuclei of the Brattleboro rat, due to the use of different probes hybridizing different portions of its mRNA. Vasopressin mRNA in the suprachiasmatic nucleus does not change in response to manipulations such as hypertonic solution loading, adrenalectomy and dexamethasone treatment, but displays a typical diurnal circadian rhythm; higher values in the morning and lower values at the onset

Fig. 1. Oxytocin

mRNA

in the paraventricular

tissue as revealed

by in situ hybridization

43

of the dark phase. Ontogenetically, vasopressin mRNA is first detected at El6 in the supraoptic nucleus, at El8 in the paraventricular nucleus, and E21 in the sunrachiasmatic nucleus. Develoumental studies on the iegulation of vasopressin mRNA in the supraoptic nucleus also show that its message level at E21 of fetal rat is increased by osmotic stimulation of the mother rat, while its level at El9 is not significantly increased by osmotic stimulation of the mother rat, indicating that the mechanisms for osmotic regulation of the vasopressin mRNA level during fetal life develops between El9 and E21. Ultrastructural localization of vasopressin mRNA in the magnocellular neurons of the supraoptic and paraventricular nuclei is visualized, and their signals are generally associated with the rough endoplasmic reticulum of their cell bodies (Arai et al., 1988; Bloch et al., 1986; Card et al., 1988; Davis et al., 1986; Fuller et al., 1985; Guitteny and Bloch, 1989; Guitteny et al., 1988a; Guitteny et al., 1988b; Guitteny et al., 1989; Herman et al., 1989; Kawata et al., 1988~; Laurent et al., 1989; Lewis et al., 1986a; Lightman and Young, 1987b; 1988; McCabe et al., 1986ae; 1988; McEwan and Pfaff, 1985; Nojiri et al., 1985; Reppert and Uhl, 1988; Sherman et al., 1986; Uhl and Reppert, 1986; Uhl et al., 1985; Wolfson et al., 1985; Young et al., 1986a). OXYTOCIN

Labeled cells containing oxytocin mRNA are found in the dorsal portion of the supraoptic nucleus and peripheral portion of the paraventricular nucleus, which are comparable to the findings obtained by immunohistochemistry. Levels of oxytocin mRNA progressively increase not only to hypertonic stimulation, but also during late pregnancy and lactation. Opioids inhibit the release of oxytocin from the axonal terminals, and an opioid antagonist, naloxone, induces the reverse effect. In contrast to the stimulatory effect of naloxone on the secretion, naloxone does not affect the levels of oxytocin mRNA in the supraoptic nucleus. Estrogen and progesterone treatment induce a significant increase

nucleus.

Dark

field microphotograph.

x 120.

MITSUHIROKAWATA et al.

Fig. 2.

Oxytocin mRNA in the supraoptic nucleus. Bright field microphotograph. x 500.

of oxytocin mRNA in neurons of the paraventricular nucleus, but not in the supraoptic nucleus. Since levels of oxytocin mRNA in the supraoptic and paraventricular nuclei do not change after adrenalectomy, a specific effect of sex steroid on oxytocin gene expression in specific areas is considered. In the Brattleboro rat, hybridization signals of oxytocin mRNA increase in the magnocellular regions. Ontogenetically, oxytocin mRNA is first detected at El7 in the supraoptic nucleus and at El8 in the paraventricular nucleus (Jirikowski et al., 1989; Kawata et al., 1988a,c; Laurent et al., 1989; Lightman and Young, 1987b; 1988; McCabe et al., 1986a-e; Sumner, 1989; Young et al., 1986a). POMC Proopiomelanocortin (POMC) mRNA is localized in approximately 3% of cell (corticotrophs) in the anterior lobe and more than 90% of cells in the intermediate lobe. Haloperidol, a dopamine receptor antagonist, induces significant increase in hybridization signals of POMC mRNA in the anterior and intermediate lobes, indicating the dopaminergic inhibitory effect on POMC gene expression. Adrenalectomy specifically results in both increased numbers and intensities of hybridization-positive corticotrophs in the anterior lobe. In combination with in situ hybridization and retrograde transport methods, it is demonstrated that cells containing POMC mRNA in the hypothalamic arcuate nucleus project to the preoptic area (Bloch et al., 1985, 1986; Fremeau et al., 1989; Gee and Roberts, 1983; Hudson et al., 1981; Kelsey et al., 1986; Larsson, 1989; Larsson et al., 1988; Lewis et al., 1986b; Roberts et al., 1982; Shivers et al., 1986b; Wilcox et al., 1986).

neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (central nucleus), hippocampus, anterior and lateral hypothalamic areas, paraventricular nucleus, dorsomedial and ventromedial nuclei, arcuate nucleus, mamillary complex, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, tegmental nucleus of Gudden, parabrachial nucleus, raphe nuclei, nucleus of the solitary tract, nucleus of the trigeminal tract, cochlear nuclei, vestibular nuclei, cuneate nucleus, gracile nucleus, superior olive, nucleus of the trapezoid body, cerebellar nuclei and cortex, and most laminae of the spinal cord. These regions contain many more neurons than have been appreciated previously on the basis of immunohistochemistry. Recent studies identify efferent nuerons in the striatum that are segregated into “patch” and “matrix” components arranged in a mosaic pattern. In the striatum 65% of patch neurons and 58% of matrix neurons contain PPE mRNA and these neurons project to the globus pallidus, not to the substantia nigra. Treatments of haloperidol or 6-hydroxydopamine, dopamine synthesis blocker, induce a significant increase of PPE mRNA in the striatum, whereas 5,7-dihydroxytryptamine, serotonin synthesis blocker, does not affect the levels of PPE mRNA in the striatum, indicating specific inhibitory action of the dopaminergic neuron on PPE gene expression (Bloch, 1985, 1986; Gerfen and Young, 1988; Harlan et al., 1987; Lightman and Young 1987a, 1988; Morris et al., 1988a,b; Nishimori, 1988; 1989; Roman0 et al., 1987; Young et al., 1986~). PREPRODYNORPHIN

PREPROENKEPHALIN Labeled cells containing preproenkephalin(PPE) mRNA are widely distributed in the brain, which ventral tenia tecta, includes piriform cortex,

Labeled cells containing preprodynorphin mRNA are found in the hypothalamic supraoptic and paraventricular nuclei, striatum, nucleus accumbens, dentate gyrus, and nucleus caudalis of the trigeminal

Localization and regulation of mRNAs in the nervous tissue as revealed by in siru hybridization nuclear complex. Neurons containing preprodynorphin mRNA also contain vasopressin mRNA in the supraoptic and paraventricular nuclei. Drinking 2% NaCl solution and acute morphine treatment result in a marked increase of preprodynorphin mRNA in magnocellular neurons of the supraoptic and paraventricular nuclei. In the striatum 52% of patch neurons and 45% of matrix neurons contain preprodynorphin mRNA and these neurons project to the substantia nigra, not to the globus pallidus. Chemical lesioning by 6-hydroxydopamine injection into the mesencephalon does not affect the levels of preprodynorphin mRNA in the striatum, while the levels of preprodynorphin mRNA in the striatum are significantly reduced by the destruction of dorsal raphe nuclei of 5,7-dihydroxytryptamine microinjection, indicating the specific enhancement of preprodynorphin gene expression by the activity in the raphestriatal serotonin pathway (Gerfen and Young, 1988; Lightman and Young, 1987b, 1988; Morris et al., 1986, 1988a,b; Nishimori et al., 1988; Young et al., 1986~). PREPROTACHYKININ Substance P and substance K (neurokinin A) are encoded by the same preprotachykinin(PPT)-A gene, which gives rise to three PPTs through alternative splicing, whereas another PPT-B gene encodes neurokinin B. Cells containing PPT-A and PPT-B mRNAs are present in the neocortex, hippocampus, olfactory bulb and associated areas, caudateputamen, hypothalamus, medial habenula, superior colliculus, central gray, and dorsal horn of the spinal cord. However, each mRNA containing cells shows a quite different distributional pattern in these areas, except for the caudate-putamen where 59% of PPT-B mRNA containing cells have PPT-A mRNA. Other areas contain only one tachykinin cell type, e.g. the nucleus of the lateral olfactory nucleus contains only PPT-B mRNA while the raphe nuclei have only PPT-A mRNA. In the striatum, 61% of patch neurons and 54% of matrix neurons contain PPT-A mRNA and they project to the substantia nigra, not to the globus pallidus. Chemical lesioning by injection 6-hydroxydopamine induces an increase of numbers and signals of PPT-B mRNA in the striatum, whereas the levels of PPT-A mRNA are depressed (Burgunder and Young, 1989; Chesselet et al., 1987; Get-fen and Young, 1988; Harlan et al., 1989; Henken et al., 1988; Noguchi et al., 1989; Warden and Young, 1988; Young et al., 1986~). CHOLECYSTOKININ Cholecystokinin (CCK) mRNA is localized in neurons of the neocortex, olfactory bulb, claustrum, amygdala, dentate gyrus and hippocampus, several subnuclei of the thalamus (ventral group) and hypothalamic supraoptic and paraventricular nuclei. The most abundant and most heavily labeled cells are found in the endopiriform/piriform cortex, tenia tecta, and the ventral tegmental areas. A small number of cells containing CCK mRNA is also scattered in the pons and medulla oblongata (Burgunder and CBPC 9*,*-o

45

Young, 1988; Ingram et al., 1989; Siegel and Young, 1985). CALCITONINGENE-RELATED PEPTIDE There are species-different distributions of cells containing calcitonin gene-related peptide (CGRP) mRNA, but in both guinea-pig and rat, CGRP mRNA is localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. In the rat CGRP mRNA is found in neurons of the ambiguus motor nucleus, parabrachial and peripendicular nuclei, whereas in guinea-pig CGRP mRNA-containing neurons are distributed in the abducens, trigeminal, trochlear and oculomotor nuclei (Guitteny et al., 1988b; Rethelyi et al., 1989). NEUROPEI’TIDEY Neuropeptide Y (NPY) mRNA-containing cells are present in the nucleus accumbens, caudate-putamen, substantia innominata, amygdala, dentate gyrus, and neocortex in the human brain. In the rodent brain, hypothalamic arcuate nucleus also contains NPY mRNA. In the human cortex, hybridization signals of NPY mRNA are found over the soma of small neurons in laminae IV and VI (Brene et al., 1989; Chan-Palay et al., 1988; Gehlert et al., 1987; Terenghi et al., 1987). VASOACTIVEINTESTINALPEPTIDE AND HISTIDINE

PEPTIDE

ISOLEUCINE

Recent studies identify that the nucleotide sequence of the precursor of vasoactive intestinal peptide (VIP) within the central nervous system contains another biologically-active peptide, peptide histidine isoleucine (PHI), in addition to containing VIP itself. VIP/PHI mRNA is localized in neurons of the ventrolateral portion of the suprachiasmatic nucleus, and the message levels show typical diurnal cycle; VIP/PHI mRNA occur in high concentrations shortly after the onset of darkness then 5 h after the onset of the light phase. VIP mRNA is also found in neurons of the dorsal root ganglia (Card et al., 1988; Noguchi et al., 1989; Stopa et al., 1988). PROLACTIN Hybridization signals of prolactin mRNA occur over weakly acidophilic cells in the adenohypophysis, while other acidophils with dark cytoplasm do not contain hybridization signals (Pochet et al., 1981; Shivers et al., 1986b). PROGLUCAGON/GLUCAGON-LIKE PEPTIDE The proglucagon gene encodes glycentin, including glucagon itself, and a glucagon-like peptide. Cells in the nucleus of the tractus solitarius contain proglucagon mRNA, indicating that there is de nooo synthesis of glucagon and glucagon-like peptide in neurons of the medulla oblongata (Han et al., 1986).

MITSUHIRO KAWATAef nl.

46 ANGIOTENSINOGEN

Significantly labeled cells containing angiotensinogen mRNA are present in the area surrounding the anterior recess of the third ventricle, preoptic area, diagonal band of Broca, bed nucleus of the stria terminalis, ventral pallidurn, hypothalamic supraoptic and paraventricular nuclei, arcuate nucleus, amygdala (medial nucleus), superficial laminae of the superior colliculus, central gray, substantia nigra, parabrachial nuclei, pontine nuclei, pedunculopontine nuclei, inferior olive, nucleus of the tractus solitarius, vestibular nucleus, facial motor nucleus, and cerebellar nuclei and cortex. Area postrema and subfornical organ do not contain significant hybridization signals of angiotensinogen mRNA (Lynch et al., 1987). TYROSINE HYDROXYLASE

Cells containing mRNA of tyrosine hydroxylase(TH), the rate-limiting enzyme in the synthesis of catecholamine, are distributed in the anteroventral periventricular nucleus, cellular layer of the hippocampus, substantia nigra pars compacta, A8, AlO, neocortex, and sympathetic ganglia. The anteroventral periventricular nucleus contains over three times as many TH mRNA-containing cells in female rats compared to males. In combination with in situ hybridization and retrograde transport methods, it is identified that neurons containing TH mRNA in the substantia nigra project to the striatum. Ontogenetic studies show that TH mRNA is first detected in sympathetic ganglia at El 1.5, the age corresponding to the initial immunohistochemical expression of TH protein. Reserpine treatment increases the labeling intensity of TH mRNA in sympathetic ganglia, while 6-hydroxydopamine microinjection induces the suppression of TH gene expression in mesencephalon (Chesselet et al., 1987; Jonakait et al., 1989; Schalling et.al., 1986; Simerly, 1989; Young et al., 1986~). GLUTAMIC ACID DECARBOXYLASE

Hybridization signals of mRNA of glutamic acid decarboxylase(GAD), the synthetic enzyme of gamma-aminobutyric acid (GABA) are found in Purkinje, Golgi II, stellate, and basket neurons of cerebellum, neurons in the substantia nigra zona reticularis, and medium-sized neurons in the striatum (Chesselet et al., 1987; Sarthy and Fu, 1989; Wuenschell et al., 1986). CHOLINE ACETYLTRANSFERASE

There are no successful studies of the distribution of mRNA of choline acetyltransferase(CAT), the key enzyme of synthesis of acetylcholine, in vertebrate brain. In Drosophila hybridization signals of CAT mRNA are present in cell-rich cortical regions of the cerebrum (Barber et al., 1989). NERVE GROWTH FACTOR RECEPTOR

Nerve growth factor receptor (NGF) mRNA is localized in neurons of the media1 septum, diagonal

band of Broca, nucleus basalis of Meynert, and superior cervical ganglia which correlate nicely with the distribution of NGF receptor immunoreactivity. In addition, NGF receptor immunoreactivities are found in the meningial, ependymal, and hypothalamic tissue, while no NGF receptor mRNA-containing cells are present in these areas (Gibbs et al., 1989). AMYLOID-BETA-PROTEIN

Amyloid-beta-protein constitutes major components of neurofibrillary tangles and neuritic markers of neuropathological plaques, two Alzheimer disease. Amyloid-beta-protein mRNA is found in the neocortex from Alzheimer disease as well as control, indicating that an overproduction of its mRNA is unlikely to be responsible for amyloid-betaprotein deposition in Alzheimer diseased brain (Lewis et al., 1988; Spillantini et al., 1989). PROTEINS ASSOCIATED WITH GLIAL CELLS

Glial fibrillary acidic protein(GFAP) is a marker of astrocyte and its mRNA is present in the subpial area, white matter of the cerebrum and cerebellum, gray matter of the spinal cord, thalamus, pontine reticular formation and white matter of brain stem. S-100 protein is another marker of astrocytes and ependymal cells, and ontogenetical studies demonstrate a caudal-rostra1 gradient in expression of S-100 mRNA during brain development as well as pronounced differences reflecting the differentiation of subpopulations of astrocytes. Astrocytes metabolize glutamate to glutamine by glutamine synthetase, and its mRNA is first detected at El4 and thereafter its message increases. Myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs stay high until P20 of the mouse spinal cord, but myelin-associated glycoprotein (MAG) and MBP exon 2 mRNAs decrease sharply between P8 to 20. 2’,3’-cyclic nucleotide phosphohydrolase (CNP) mRNA maintains high expression until P20 and then decreases sharply. All these mRNAs are localized in oligodendrocytes, and mRNA of PLP, MAG, and CNP are clustered on cell bodies while mRNA of MBP is scattered throughout the cell bodies and processes. PO glycoprotein is expressed in Schwann cells and its mRNA is only found until P8. No signals of PO mRNA are detected at P30 or adult Schwann cells, indicating PO mRNA is expressed only early in postnatal development (Griffin et al., 1983; Jordan et al., 1989; Kitamura et al., 1987; Lamperth et al., 1989; Landry et al., 1989; Mearow et al., 1989). REFERENCES Arai H., Emson P. C., Agrawal S., Christodoulou C. and Gait M. J. (1988) In situ hybridization histochemistry: localization of vasopressin mRNA in rat brain using a biotinylated oligonucleotide probe. Molec Bruin Res. 4, 6349. Arentzen R., Baldino F., Jr., Davis L. G., Higgins G. A., Lin Y., Manning R. W. and Wolfson B. (1985) In sifu hybridization of putative somatostatin mRNA within hypothalamus of the rat using synthetic oligonucleotide probes. J. Cell. Biochem. 27, 415422.

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Cham-Palay V., Yasargil G., Hamid Q., Polak J. M. and Palay S. L. (1988) Simultaneous demonstrations of neuropeptide Y gene expression and peptide storage in single neurons of the human brain. Proc. natn Acad. Sci. U.S.A. 85, 3213-3215.

Chesselet M.-F., Weiss L., Wuenschell C., Tobin A. J. and Affolter H.-U. (1987) Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain. J. camp. Neurol. 262, 125-140. Coghlan J. P., Aldred P., Haralambidis J., Niall H. D., Penschow J. D. and Tregear G. W. (1985) Hybridization histochemistry. Analyt. Biochem. 149, 1-28. Cummings S. L., Young S. W. III., Bishop G. A., de Souza E. B. and King J. S. (1989) Distribution of corticotropinreleasing factor in the cerebellum and precerebellar nuclei of the opossum: a study utilizing immunohistochemistry, in sifu hybridization histochemistry and receptor autoradiography. J. camp. Neurol. 280; 501-521. _ Davis L. G.. Arentzen R.. Reid J. M.. Mannine R. W.. Wolfson B., Lawrence K. L. and Baldino F. &. (1986) Glucocorticoid sensitivity of vasopressin mRNA levels in the paraventricular nucleus of the rat. Proc. nutn Acud. Sci. U.S.A. 83, 1145-1149.

Fremeau R. T. Jr., Autelitano D. J., Blum M., Wilcox J. and Roberts J. L. (1989) Intervening sequence-specific in situ hybridization: detection of the pro-opiomelanocortin gene primary transcript in individual neurons. Molec. Bruin Res. 6, 197-201.

Fuller P. J., Clements J. A. and Funder, J. W. (1985) Localization of arginine vasopressin-neurophysin II messenger ribonucleic acid in the hypothalamus of control and Brattleboro rats by hybridization histochemistry with

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