Distribution and expression of CRF receptor 1 and 2 mRNAs in the CRF over-expressing mouse brain

Corticotropin-releasing factor (CRF) acts through CRF 1 and CRF 2 receptors (CRF1, CRF2). To test the hypothesis that CRF controls the expression of these receptors in a brain site- and receptor-type specific manner, we studied CRF1 mRNA and CRF2 mRNA expressions in mice with central CRF over-expression (CRF-OE) and using in situ hybridization. CRF1 and CRF2 mRNAs appear to be differentially distributed across the brain. The brain structures expressing the receptors are the same in wild-type (WT) and in CRF-OE mice. We therefore conclude that chronically elevated CRF does not induce or inhibit expression of these receptors in structures that normally do not or do, respectively, show these receptors. However, from counting cell body profiles positive for CRF1 and CRF2 mRNAs, clear differences appear in receptor expression between CRF-OE and WT mice, in a brain-structure-specific fashion. Whereas some structures do not differ, CRF-OE mice exhibit remarkably lower numbers of CRF1 mRNA-positive profiles in the subthalamic nucleus (-38.6%), globus pallidus (-31.5%), dorsal part of the lateral septum (-23.5%), substantia nigra (-22,8%), primary somatosensory cortex (-18.9%) and principal sensory nucleus V (-18.4%). Furthermore, a higher number of CRF2 mRNA-positive profiles are observed in the dorsal raphe nucleus (+32.2%). These data strongly indicate that central CRF over-expression in the mouse brain is associated with down-regulation of CRF1 mRNA and up-regulation of CRF2 mRNA in a brain-structure-specific way. On the basis of these results and the fact that CRF-OE mice reveal a number of physiological and autonomic symptoms that may be related to chronic stress, we suggest that CRF1 in the basal nuclei may be involved in disturbed information processing and that CRF2 in the dorsal raphe nucleus may play a role in mediating stress-induced release of serotonin by CRF.     

Differential effects of haloperidol,clozapine, and fluperlapine on tuberoinfundibular dopamine neurons and prolactin secretion in the rat

Summary Two atypical neuroleptic agents, clozapine and fluperlapine, produced rapid elevations in plasma PRL concentrations that were similar in magnitude to those produced by haloperidol. However, the PRL response to clozapine or fluperlapine was of much shorter duration than that elicited by haloperidol. Clozapine, but neither fluperlapine nor haloperidol, produced a rapid increase in the activity of tuberoinfundibular dopamine (TIDA) neurons, as evidenced by an enhanced accumulation of dihydroxyphenylalanine (DOPA) in the median eminence after the inhibition of DOPA decarboxylase. The clozapine-induced increase in DOPA accumulation was evident within 30 minutes after its administration and persisted for at least 4 hours. The clozapine-induced increase in the activity of TIDA neurons may account, in part, for the abbreviated PRL response to this neuroleptic. In addition, ability to produce a short-lived increase in PRL secretion in the rat appears to be common to the atypicl neuroleptic drugs.     

Comparative localization of serotonin1A, 1C,and 2 receptor subtype mRNAs in rat brain

Serotonin (5-HT) mediates its effects on neurons in the central nervous system through a number of different receptor types. To gain better insight as to the localization of 5-HT responsive cells, the distribution of cells expressing mRNAs encoding the three 5-HT receptor subtypes 1A,1C, and 2 was examined in rat brain with in situ hybridization using cRNA probes. 5-HT1A receptor mRNA labeling was most pronounced in the olfactory bulb, anterior hippocampal rudiment, septum, hippocampus (dentate gyrus and layers CAI-3), entorhinal cortex, interpeduncular nucleus, and medullary raphe nuclei. 5-HT 1C receptor mRNA labeling was the most abundant and widespread of the three 5-HT receptor subtypes examined. Hybridization signal was denset in the choroid plexus, anterior olfactory nucleus, olfactory tubercle piriform cortex, septum, subiculum, entorhinal cortex, claustrum, accumbens nuclues, striatum, lateral amygdala, paratenial and paracentral thalamic nuclei, subthalamic nucleus, substantia nigra, and reticular cell groups. 5-HT2 receptor mRNA was localized to the olfactory bulb, anteriorhippocampal rudiment, frontal cortex, piriform cortex, entorhinal cortex, claustrum, pontine nuclei, and cranial nerve motor nuclei including the oculomotor, trigeminal motor, facial, dorsal motor nucleus of the vagus, and hypoglossal nuclei. The distributions of mRNAs for the three different 5-HT receptor subtypes overlap with regions that bind various 5-HT receptor-selective ligands and are present in nearly all areas known to receive serotonergic innervation. The results of this study demonstrate that nervous which express these 5-HT receptor subtypes are very widespread in the central nervous system, yet possess unique distributions with in the rat brain. Moreover, previously unreported regions of 5-HT receptor subtype expression were observed, particulary with the 5-HT receptor riboprobe in the brainstem. Finally, several brain areas contain multiple 5-HT receptor subtype mRNAs, which leads to the possibility that individual cells may express more than one 5-HT receptor subtype. © 1995 Willy-Liss, Inc.     

大鼠代谢型谷氨酸受体1亚型基因片段的克隆及其在小脑的表达

目的：克隆大鼠代谢型谷氨酸受体1亚型（mGluR1)基因特异片段，制备cDNA探针。方法：从Wistar大鼠小脑中提取总RNA，以RT－PCR方法得到预期的599bp条带，将这一片段克隆到pGEM-T easy载体上，经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板，通过体外转录的方法 合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验，以检测探针的可靠性。结果：测序证实用RT－PCR的方法获得了mGluR1基因特异片段，成功地构建了pGEM-TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为10ng/μl及30ng/μl。原位杂交实验的结果显示，用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆，用正义探针杂交无阳性信号。结论：本实验克隆了mGluR1基因特异片段，并制备了cRNA探针，并用大鼠小脑进行的原位杂交实验显示，此探针灵敏度高，特异性好。     

Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina

In the retina, the ε2 and ζ1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The ε1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.     

Postnatal development of NR1, NR2A and NR2B immunoreactivity in the visual cortex of the rat

N-Methyl-D-aspartate receptors (NMDARs) are critically involved in some types of synaptic plasticity. The NMDAR subunits NR1, NR2A and NR2B are developmentally regulated, and it has been proposed that developmental changes in their expression may underlie developmental changes in cortical plasticity. Age-dependent change in cortical plasticity is most commonly measured by the monocular deprivation effect, which occurs during a critical period between P22 and P50 in the rat. Although the development of NMDAR subunits has been studied from birth through the fourth postnatal week, there is only meager information from older ages when visual plasticity ends. We hypothesized that there will be significant age-dependent change in expression of NR1, NR2A or NR2B between P22, when the cortex is plastic, and P90, when it is not. We applied specific antibodies recognizing NR1, NR2A and NR2B to the primary visual cortex at P14, P22, P30, P45 and P90. We found age-dependent changes in NR1-IR that were negatively correlated with changes in NR2A-IR; these subunits are not regulated in unison. In contrast, NR2A-IR and NR2B-IR were positively correlated. NR2A-IR and NR2B-IR both passed through a developmental minimum around P45, then recovered to approximately their P22 level. NR1-IR passed through a maximum at P45. There were no significant differences between P22 and P90. These results do not support the simple hypothesis that the loss of plasticity corresponds to a simple transition from juvenile levels of NMDAR subunit proteins to new adult levels. On the other hand, the results do confirm the hypothesis that there are significant changes in processing of NMDAR proteins during the time that plasticity is lost. How these changes of IR relate to synaptic transmission and plasticity needs to be clarified.     

Ontogeny of the striatal neurons expressing the D1 dopamine receptor in humans

We studied D1 dopamine receptor (D1R) gene expression in the human striatum during ontogeny by in situ hybridization, immunohistochemistry, and D1R ligand autoradiography. D1R mRNA, protein, and binding sites ([³H]SCH 23390) were detected in the striatum from week 12 of fetal life. At this time, D1R mRNA was predominant in the striosomal neurons; D1R immunoreactivity (D1R-IR) and D1R binding sites displayed a pattern similar to D1R mRNA. D1R-IR was essentially present in striosomal cell bodies and neuropil, whereas only a few cell bodies were detected in the matrix. From week 20 of fetal life, D1R gene expression developed in the matrix neurons as well, thus leading to an even D1R mRNA expression throughout striosomes and matrix compartments at birth. Comparative analysis of the expression of D1R and dynorphin mRNA show the same developmental patchy pattern up to week 26. Indeed, neurons expressing the D1R gene contain dynorphin mRNA; in contrast, they do not express the preproenkephalin A gene. At birth, the pattern of D1R mRNA expression level was sharply different from that of dynorphin (DYN) gene expression. High DYN mRNA expression was restricted to the striosomes, whereas high D1R mRNA expression was present in the whole striatum. These results demonstrate that, during human ontogeny, functional D1 receptors are expressed as early as week 12 in the striatum, developing initially in the striosomal neurons containing high dynorphin mRNA content. Toward the end of fetal life, there is a dissociation between D1R and DYN expression levels, suggesting that neuroanatomical or neurochemical modifications occur at this period, which may contribute to the regulation of the tone of the striatal D1R and DYN gene with topological specificity. © 1996 Wiley-Liss, Inc.     

Distribution and expression of the subunits of N-methyl-d-aspartate (NMDA) receptors; NR1, NR2A and NR2B in hypoxic newborn piglet brains

The purpose of this study was to identify the distribution and the expression of the NR1, NR2A and NR2B subunits of the NMDA receptor after cerebral hypoxia. Ten piglets were divided into control and hypoxic groups (n=5, each). The control piglets were ventilated with normoxia for 1 h, and the hypoxic piglets were ventilated with hypoxia until p_aO₂ was below 20 mmHg. Tissue samples from the nine different regions of newborn piglet brain were obtained, and the protein amount of the NR1, NR2A, and NR2B subunits measured by immunoblot using the antibody to the NR1, NR2A, and NR2B subunits. The NR1, N2A, and NR2B subunits were distributed very differently; hippocampus and cortical area are more prominent than white matter and cerebellum. But the expression of the NR1, NR2A and NR2B subunits were not significantly different between the control and the hypoxic group, 1 h after hypoxic exposure, indicating no changes in the protein amount of NMDA receptor subunits. These results show a significantly higher amount of the NR1, NR2A and NR2B subunits in the hippocampus and the cerebral cortex of newborn brains, indicating that these structures could be highly vulnerable to excitotoxicity in the newborn brain.     

Differential distribution of the glutamate transporters GLT-1 and GLAST in tanycytes of the third ventricle

The ventral one-third of the ventricular lining in the hypothalamus is formed by specialized ependymal cells called the tanycytes. These cells may serve a neuroendocrine transport function because of their structural specializations, which include apical microvili on the ventricular surface and long basal processes that terminate on blood vessels or on the glia limitans. Here, we describe the expression of mRNA and protein for the glutamate transporters GLT-1 and GLAST in unique tanycyte populations of the third ventricle in rat brain. Using nonisotopic in situ hybridization, we demonstrate GLAST mRNA labeling in tanycytes of the ventral floor and lateral walls in the tuberal and mammillary recess portions of the third ventricle. This GLAST mRNA labeling had a higher intensity than the labeling intensity observed in regular ependymal cells throughout the ventricular system. Furthermore, we have identified strong GLT-1 mRNA labeling in a population of tanycytes situated in the dorsolateral walls of caudal tuberal and mammillary recess portions. Immunocytochemical staining indicates that both GLT-1 and GLAST protein are expressed in the tanycyte populations as well. These data corroborate previous findings that third ventricle tanycytes are functionally heterogeneous. Furthermore, the GLT-1-expressing tanycytes represent a population of tanycytes that, to date, has not been recognized as functionally distinct. The strong GLAST expression by the ventral tanycytes in the hypophysiotropic area suggests a role of tanycyte-mediated glutamate transport in neuroendocrine activity. The functional role of GLT-1 in dorsal wall tanycytes remains to be explored.     

Differential expression of kainate receptors in the basal ganglia of the developing and adult rat brain

Glutamate is the principal excitatory transmitter of the mammalian brain and plays a particularly important role in the physiology of the basal ganglia structures responsible for movement regulation. Using in situ hybridization with oligonucleotide probes, we examined the expression patterns of the five known kainate type glutamate receptor subunit genes, KA1, KA2 and GluR5–7, in the basal ganglia of adult and developing rat brain. In the adult rat, a highly organized and selective pattern of expression of the kainate subunits was observed in the basal ganglia and associated structures as well as in other regions of the brain. KA2 mRNA was abundant in the striatum, nucleus accumbens, subthalamic nucleus and substantia nigra pars compacta, and was present at lower levels in the globus pallidus and substantia nigra pars reticulata. Neither KA1 nor GluR5 expression was observed in the basal ganglia of adult rats, although these messages were present in other regions. GluR6 was highly expressed in the striatum and subthalamic nucleus and to a lesser extent in the substantia nigra pars reticulata, while no hybridization signal was detectable in the large, presumably dopaminergic neurons of the substantia nigra pars compacta. In contrast, GluR7 was strongly expressed in the substantia nigra pars compacta, was present at lower levels in the striatum, globus pallidus and substantia nigra pars reticulata, and was not detectable in the subthalamic nucleus. During postnatal development, expression of the kainate receptor subunits was characteristically highest on postnatal day 1 and declined to adult levels by day 20; however, in the globus pallidus we did observe the transient expression of KA1 and GluR5 between day 1 and day 10. These results demonstrate that the neuronal structures comprising the basal ganglia express a distinct combination of kainate receptor subunit genes, suggesting that the pharmacological properties of the resultant glutamate receptors are likely to be regionally specific. The organization of expression of these genes is established early in life, which is consistent with the important role they may play in establishing the functions of the motor system.     

D1 dopamine receptor-mediated induction of zif268 and c-fos in the dopamine-depleted striatum: Differential regulation and independence from NMDA receptors

Excitatory amino acid afferents from cerebral cortex and dopamine afferents from the substantia nigra synapse on common projection neurons in the striatum. Activation of D1 dopamine receptors increases immediate early gene expression in the striatum and conductance through the N-methyl-d-aspartate (NMDA) receptor. To examine the contribution of NMDA receptor activation to dopamine receptor-mediated responses, we determined the effects of intrastriatal administration of NMDA antagonists on immediate early gene expression in the striatum and rotational behavior induced by stimulation of the D1 receptor in rats with unilateral dopamine depletions. Systemic administration of SKF 38393 increased c-fos and zif268 mRNAs in the striatum and induced contralateral rotation. Intrastriatal infusion of the competitive NMDA receptor antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid caused a dose-dependent attenuation of SKF 38393-induced rotation and partially decreased c-fos mRNA expression. However, D1-mediated increases in zif268 mRNA were not affected, except by the highest concentration of antagonist used (10 mM). Another competitive antagonist, 2-amino-5-phosphonovaleric acid, had similar effects. Like the competitive antagonists, intrastriatal infusion of the non-competitive NMDA antagonist MK-801 partially decreased c-fos, but not zif268, mRNA in the area around the microdialysis probe. However, unlike competitive antagonists, local infusion of 1 mM MK-801 potentiated D1-mediated increases in c-fos and zif268 mRNAs in lateral striatum. These data suggest that 1) some D1 dopamine receptor-mediated effects on striatal function are independent of ongoing NMDA receptor activation, whereas other effects are at least partially mediated by NMDA receptor activity in the striatum, and 2) competitive and non-competitive antagonists of the NMDA receptor differently affect D1-mediated immediate early gene expression in the striatum. © 1996 Wiley-Liss, Inc.     

Modulation of dopamine D1-mediated turning behavior and striatal c-fos expression by the substantia nigra

In order to study the possible contribution of the substantia nigra (SN) in the positive interaction between dopamine D₁ receptor agonists and glutamate antagonists in unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, the effect of the D₁ agonist, SKF 38393, was studied in combination with intranigral infusions of glutamate antagonists of the NMDA (MK 801, CPP) or AMPA (NBQX) type of receptor. Local infusion into the SN of the 6-OHDA lesioned side of MK 801, CPP or NBQX at doses inducing no or minimal behavioral effects significantly increased the turning behavior and the expression of c-fos induced, in the lesioned caudate-putamen (CPu), by a parenteral administration of SKF 38393. The same result was obtained after intra-SN infusion of the GABA agonist, muscimol. High doses of MK 801, CPP or muscimol infused into the SN produced intense contralateral turning per se and induced a sparse c-fos expression in the lesioned CPu which was antagonized by parenteral administration of MK 801. The results indicate that a depression of SN pars reticulata efferent neurons potentiates D₁-mediated responses and suggest that this area may play a role in the positive interaction between glutamate antagonists and D₁ receptor agonists. © 1995 Wiley-Liss, Inc.     

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs, for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly. © 1994 Wiley-Liss, Inc.     

Pentylenetetrazole (PTZ)-induced c-fos expression in the hippocampus of kindled rats is suppressed by concomitant treatment with naloxone

Rats were kindled by repeated injections of pentylenetetrazole (PTZ; 37.5 mg/kg; i.p.) in the presence or absence of the opioid receptor antagonist naloxone. Naloxone (10 mg/kg; i.p.) applied 30 min before each PTZ application had no major effect on the seizure development, although a slight decrease in the seizure expression of fully kindled animals could be observed. In the kindled animals, a pronounced but transient increase in c-fos mRNA level was observed in several brain areas after the injection of PTZ. The magnitude of c-fos induction was related to the seizure stage reached. Detectable c-fos mRNA levels in the cortex were observed in rats showing stage four seizures, whereas the expression of c-fos in the hippocampus required stage five kindled seizures. The induction of c-fos expression in the hippocampus of stage five kindled rats but not in other brain areas was prevented by treatment of naloxone prior to each PTZ application. In contrast, a single injection of naloxone to kindled rats was not sufficient to prevent c-fos mRNA expression in the hippocampus. In addition, a single PTZ application (at the higher dose of 45 mg/kg) to rats that were not kindled also caused c-fos expression in several brain regions, but this was not influenced by naloxone. Assuming that c-fos expression reflects neuronal activity our findings suggest a functional role of endogenous opioid peptides in the development of kindling-induced neuronal excitation in the hippocampus. In addition, the excitation of the hippocampus does not appear to be involved in the seizure activity but may be responsible for the impairment of learning in PTZ-kindled rats which can be prevented by pretreatment with naloxone [A. Becker, G. Grecksch, M. Brosz, Naloxone ameliorates the learning deficit induced by pentylenetetrazole kindling in rats, Eur. J. Neurosci. 6 (1994) 1512–1515].     

Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.     

Effects of haloperidol and clozapine on preprotachykinin-A messenger RNA, tachykinin tissue levels, release and neurokinin-1 receptors in the striato-nigral system.

The effects of haloperidol and clozapine on tachykinin tissue levels, preprotachykinin-A messenger RNA, spontaneous and potassium-evoked tachykinin release, dopamine D2 receptors, and [125I]Bolton-Hunter-substance P binding sites in the striato-nigral system were examined. Chronic administration (10 days) of the dopamine receptor antagonist haloperidol (2 mg/kg i.p.) significantly decreased tissue levels of substance P like-immunoreactivity and neurokinin A like-immunoreactivity in the striatum and the substantia nigra. The corresponding preprotachykinin-A mRNA was decreased in the striatum. Haloperidol did not affect the potassium-evoked tachykinin release in the substantia nigra but significantly increased the spontaneous release. Haloperidol increased the number of D2-receptors but left [125I]Bolton-Hunter-substance P binding sites, representing neurokinin 1 (NK-1) receptors, as determined by competition experiments with selective ligands, unchanged. Clozapine (30 mg/kg, i.m.) did not influence nigral and striatal tachykinin tissue levels, preprotachykinin-A mRNA and potassium-evoked release or spontaneous efflux in the substantia nigra, or D2-receptors and [125I]Bolton-Hunter-substance P binding sites. The present data indicate that neuroleptics influence the striato-nigral tachykinin system in different ways. Tachykinins may, therefore, contribute to the therapeutic and/or untoward effects of certain neuroleptic drugs.