Gene expression in autonomic areas of the medulla and the central nucleus of the amygdala in rats during and after space flight.

During space flight astronauts show vestibular-related changes in balance, eye movements, and spontaneous and reflex control of cardiovascular, respiratory and gastrointestinal function, sometimes associated with space motion sickness. These symptoms undergo compensation over time. Here we used changes in the expression of two immediate-early gene (IEG) products to identify cellular and molecular changes occurring in autonomic brainstem regions of adult male albino rats killed at different times during the Neurolab Space Mission (STS-90). Both direct effects of gravitational changes, as well as indirect effects of gravitational changes on responses to light exposure were examined. Regions under the direct control of vestibular afferents such as the area postrema and the caudal part of the nucleus of the tractus solitarius (NTSC) were both directly and indirectly affected by gravity changes. These areas showed no changes in the expression of IEG products during exposure to microgravity with respect to ground controls, but did show a significant increase 24 h after return to 1 G (gravity). Exposure to microgravity significantly inhibited gene responses to light exposure seen after return to 1 G. A similar direct and indirect response pattern was also shown by the central nucleus of the amygdala, a basal forebrain structure anatomically and functionally related to the NTS. The rostral part of the NTS (NTSR) receives different afferent projections than the NTSC. This region did not show any direct gravity-related changes in IEG expression, but showed an indirect effect of gravity on IEG responses to light. A similar pattern was also obtained in the intermediate reticular nucleus and the parvocellular reticular nucleus. Two other medullary reticular structures, the dorsal and the ventral medullary reticular nuclei showed a less well defined pattern of responses that differed from those seen in the NTSC and NTSR. The short- and long-lasting molecular changes in medullary and basal forebrain gene expression described here are thought to play an important role in the integration of autonomic and vestibular signals that ultimately regulate neural adaptations to space flight.

Short-term (Fos) and long-term (FRA) protein expression in rat locus coeruleus neurons during the neurolab mission: contribution of altered gravitational fields, stress, and other factors.

Changes in immediate-early gene (IEG) expression during and after space flight were studied in the rat locus coeruleus (LC) during the NASA Neurolab mission. The LC sends widespread projections throughout the brain and releases the neuromodulator norepinephrine. LC neurons respond to natural vestibular stimulation; their firing rate also increases during waking and decreases or ceases during sleep. LC neurons express IEGs such as c-fos after activation. Adult male albino Fisher 344 rats were killed at four mission time points, and the number of Fos- and Fos-related antigen (FRA)-positive LC cells were counted in flight and ground-based control rats. Half of the subjects at each time point were exposed to light for 60 min prior to killing to standardize their sleep-waking state. FRA-expressing cells were more numerous than Fos-expressing cells in both flight- and ground-based subjects. The difference between FRA- and Fos-expressing cells within individuals was significantly larger 24 h after landing in subjects exposed to both space flight and light pulse than in all other subjects at any mission time point. Fos and FRA responses scaled in proportion to the maximum response observed in any single individual showed similar patterns of variation. Analysis of the scaled and combined responses showed that LC IEG levels responded to both gravity changes and light pulses. Subjects exposed to either single stimulus had equivalent responses, significantly greater than those of control subjects maintained in dim light. The combination of gravity change and light pulse gave significantly higher LC responses than either stimulus alone 24 h after takeoff, and to a lesser extent after 12 days in space; the highest responses were obtained 24 h after landing. By 14 days after landing, animals exposed to space flight and light pulse responded no differently than ground-based subjects. No difference in LC IEG expression was clearly attributable to changes in the sleep-waking state of subjects. Activity of noradrenergic LC neurons has been previously shown to modulate IEG expression in target structures. The increased IEG LC activity (seen most especially 24 h after landing) may reflect large-scale activation of noradrenergic neurons that may serve as a trigger for molecular changes in target structures, and be critical for adaptation to gravity changes.

Gene expression in rat vestibular and reticular structures during and after space flight.

Space flight produces profound changes of neuronal activity in the mammalian vestibular and reticular systems, affecting postural and motor functions. These changes are compensated over time by plastic alterations in the brain. Immediate early genes (IEGs) are useful indicators of both activity changes and neuronal plasticity. We studied the expression of two IEG protein products [Fos and Fos-related antigens (FRAs)] with different cell persistence times (hours and days, respectively) to identify brainstem vestibular and reticular structures involved in adaptation to microgravity and readaptation to 1 G (gravity) during the NASA Neurolab Mission (STS-90). IEG protein expression in flight animals was compared to that of ground controls using Fisher 344 rats killed 1 and 12 days after launch and 1 and 14 days after landing. An increase in the number of Fos-protein-positive cells in vestibular (especially medial and spinal) regions was observed 1 day after launch and 1 day after landing. Fos-positive cell numbers were no different from controls 12 days after launch or 14 days after landing. No G-related changes in IEG expression were observed in the lateral vestibular nucleus. The pattern of FRA protein expression was generally similar to that of Fos, except at 1 day after landing, when FRA-expressing cells were observed throughout the whole spinal vestibular nucleus, but only in the caudal part of the medial vestibular nucleus. Fos expression was found throughout the entire medial vestibular nucleus at this time. While both Fos and FRA expression patterns may reflect the increased G force experienced during take-off and landing, the Fos pattern may additionally reflect recent rebound episodes of rapid eye movement (REM) sleep following forced wakefulness, especially after landing. Pontine activity sources producing rhythmic discharges of vestibulo-oculomotor neurons during REM sleep could substitute for labyrinthine signals after exposure to microgravity, contributing to activity-related plastic changes leading to G readaptation. Reticular structures exhibited a contrasting pattern of changes in the numbers of Fos- and FRA-positive cells suggestive of a major influence from proprioceptive inputs, and plastic re-weighting of inputs after landing. Asymmetric induction of Fos and FRAs observed in some vestibular nuclei 1 day after landing suggests that activity asymmetries between bilateral otolith organs, their primary labyrinthine afferents, and vestibular nuclei may become unmasked during flight.

Immediate early gene expression in the vestibular nuclei and related vegetative areas in rats during space flight.

Changes in neuronal activity resulting in somatic and vegetative deficits occur during different space flight conditions. Immediate early genes (IEGs: c-fos and Fos-related antigen [FRA]) are useful indicators of changes in neuronal activity and plasticity. They are induced within minutes of several extracellular stimulations, while the corresponding proteins persist for hours (Fos) or days (FRAs). Changes in IEG expression are likely to contribute to adaptation to microgravity and readaptation to the terrestrial environment. During the NASA Neurolab Mission (STS-90), changes in IEG expression were studied in adult male albino rats (Fisher 344) sacrificed at flight day (FD) 2 (24 h after launch), FD14 and at similar time points after re-entry (R + 1, 24 h after re-entry, and R + 13). These time points were chosen to maximize the probability of detecting changes in IEG expression related to changes in gravitational fields occurring during the mission, e.g. (i) increase in gravitational force from 1 to 3 g during the launch, before reaching about 0 g at FD2; (ii) adaptation to 0 g at FD14; (iii) increase in gravity from 0 to approximately 1.5-1.8 g before reaching 1 g at R + 1; and (iv) readaptation to 1 g at R + 13. Fos- and FRA-positive cells were identified in the brainstem of flight rats and ground-based controls using immunocytochemistry. With respect to control rats, the number of labeled cells increased in flight animals in the medial and spinal vestibular nuclei (but not in the lateral vestibular nucleus) at FD2, decreased at FD14, greatly increased at R + 1 and returned to baseline levels at R + 13. Similar changes in IEG expression were also observed in the nucleus of the solitary tract, the area postrema and the central nucleus of the amygdala. In particular, in these vegetative areas the number of Fos-positive cells decreased in flight rats with respect to controls at FD14, i.e. after exposure to 0 g, but significantly increased at R + 1, i.e. after return to 1 g. Thus, altered gravitational fields produced molecular changes in vestibular nuclei controlling somatic functions, as well as in related medullary and basal forebrain structures regulating vegetative functions.

Fos-related antigens are involved in the transcriptional responses of locus coeruleus neurons to altered gravitational fields in rats.

Locus coeruleus (LC) neurons, which have widespread projections to the whole brain, respond to natural stimulation of macular receptors. Using immunocytochemistry we investigated whether rats exposed to altered gravitational fields showed changes in Fos and Fos-related antigen (FRA) protein levels in the LC. Fos protein is induced very rapidly and returns to basal levels within hours after stimulation, while FRAs persist for days or weeks after induction. Adult male albino rats (Fisher 344) were sacrificed at different time points during a space flight (NASA Neurolab Mission, STS-90) and the numbers of Fos- and FRA-positive cells in the LC were counted and compared to those in ground-based control rats. No significant changes in Fos protein expression were detected in the LC under different space flight conditions. In contrast, the number of FRA-positive cells increased on average to 167% of that of the controls at FD2, i.e. when gravity increased from 1 to 3 g during the launch before reaching about 0 g. FRA-labeled neurons then decreased to 46% of control values at FD14, i.e. after adaptation to 0 g, but increased again to 317% of control values at R + 1, when the animals were exposed to an increase in gravitational force from 0 to 1.5-1.8 g before reaching 1 g during landing. The number of labeled cells was 193% of the control values at R + 13, i.e. after readaptation to 1 g. Thus gravitational force appears to be very effective in inducing a long-term increase in FRA protein expression in the LC. Because activity in the noradrenergic LC neurons may increase Fos expression in several target structures, we postulate that the long-lasting induction of FRAs in the LC at FD2, and more prominently at R + 1, may contribute to the long-term molecular changes which probably occur in the brain during adaptation to 0 g and readaptation to 1 g.

Decreased apoptosis in proliferative and postmitotic regions of the Caspase 3-deficient embryonic central nervous system.

Caspase 3 (CPP32/Yama/apopain), a mammalian homolog of the Caenorhabditis elegans pro-cell death gene ced-3, is required for normal programmed cell death (PCD) in the nematode. Its prior deletion by homologous recombination in mice resulted in embryonic/early postnatal lethality associated with dramatic central nervous system (CNS) hyperplasia, yet a reported subtle decrease in cell death (Kuida et al. [1996] Nature 384:368-372). By comparison, the magnitude and distribution of dying cells identified using a DNA end-labeling technique, in situ end-labeling plus (ISEL+) (Blaschke et al. [1996] Development 122:1165-1174; Blaschke et al. [1998] J. Comp. Neurol. 396:39-50), supported an alternative explanation where the loss of caspase 3 function produces a more pervasive block in cell death, particularly among neuroblasts. To determine the relationship between loss of caspase 3 and dying cells identified by ISEL+, we analyzed caspase 3 +/+, +/-, and -/- embryos for normal caspase 3 expression and ISEL+ labeling. Both caspase 3 mRNA and active caspase 3 protein are present throughout the +/+ embryonic CNS, and both are absent from -/- embryonic cortices. Quantitation of dying cells identified by ISEL+ reveals a 30% reduction of labeled cells throughout the caspase 3 -/- embryonic cortices relative to +/+ littermates. Associated with this decrease is marked expansion of the total population of actively proliferating neuroblasts identified by 5-bromo-2;-deoxyuridine incorporation that nevertheless appears to maintain histological features of normal neurogenesis rather than dysregulated, neoplastic growth. These data indicate that caspase 3 deficiency results in a pervasive, albeit partial, decrease in embryonic neuroblast apoptosis that can account for the observed phenotypic hyperplasia in -/- embryos, and support the additional operation of caspase 3-independent PCD mechanisms during embryonic CNS development.

Onset of apoptotic DNA fragmentation can precede cell elimination by days in the small intestinal villus.

DNA fragmentation is a hallmark of apoptosis, and has been viewed as a short-lived process (

NGFI-A expression in the rat brain after sleep deprivation.

The effects of total sleep deprivation (SD) on the expression of the immediate-early gene NGFI-A were studied in the rat brain by in situ hybridization. Rats were manually sleep-deprived for 3, 6, 12 and 24 h starting at light onset (08:00 h) and for 12 h starting at dark onset (20:00 h). SD performed during the day induced a marked increase in NGFI-A mRNA levels with respect to sleep controls in many cerebrocortical areas and caudate-putamen, which was most evident after 6 h SD. A decrease was seen in hippocampus and thalamus, particularly after 12 h SD. Rats sleep-deprived for 12 h during the night showed an increase in NGFI-A expression in some cortical areas while rats sleep-deprived for 24 h showed few changes with respect to controls. The pattern of NGFI-A expression after forced wakefulness showed some differences from that observed after spontaneous wakefulness [M. Pompeiano, C. Cirelli and G. Tononi, Immediate early genes in spontaneous wakefulness and sleep: expression of c-fos and NGFI-A mRNA and protein, J. Sleep Res., 3 (1994) 80-96]. These observations are discussed with respect to the functional consequences of wakefulness in specific brain areas.

Neuronal gene expression in the waking state: a role for the locus coeruleus.

Several transcription factors are expressed at higher levels in the waking than in the sleeping brain. In experiments with rats, the locus coeruleus, a noradrenergic nucleus with diffuse projections, was found to regulate such expression. In brain regions depleted of noradrenergic innervation, amounts of c-Fos and nerve growth factor-induced A after waking were as low as after sleep. Phosphorylation of cyclic adenosine monophosphate response element-binding protein was also reduced. In contrast, electroencephalographic activity was unchanged. The reduced activity of locus coeruleus neurons may explain why the induction of certain transcription factors, with potential effects on plasticity and learning, does not occur during sleep.

c-fos Expression in the rat brain after unilateral labyrinthectomy and its relation to the uncompensated and compensated stages.

The expression of the immediate early gene c-fos has been studied in the entire brain of rats 3, 6 and 24 h after surgical unilateral labyrinthectomy. We combined in situ hybridization for c-fos messenger RNA with immunocytochemistry for Fos protein to document very early changes in c-fos expression and to identify with cellular resolution neuronal populations activated by unilateral labyrinthectomy. Three hours after unilateral labyrinthectomy a bilateral increase in both c-fos messenger RNA and protein levels was seen in the superior, medial and spinal vestibular nuclei, nucleus Y, and prepositus hypoglossal nucleus. These changes were asymmetric in the medial vestibular nucleus, being most prominent in the dorsal part of the contralateral nucleus (where second order vestibular neurons are located) and in the ventral part of the ipsilateral nucleus (where commissural neurons acting on the medial vestibular nucleus of the intact side are located). An increase in c-fos messenger RNA expression was seen bilaterally, but with an ipsilateral predominance, in the vermal and paravermal areas of the cerebellar cortex, flocculus and paraflocculus, as well as in the precerebellar lateral and paramedian reticular nuclei. c-fos messenger RNA and protein levels increased in a few regions of the contralateral inferior olive. A predominantly ipsilateral increase in c-fos expression also occurred in the caudate-putamen. A bilateral but not exactly symmetric increase in both c-fos messenger RNA and protein levels was present in several nuclei of the dorsal pontine tegmentum (parabrachial nucleus, locus coeruleus and laterodorsal tegmental nucleus), mesencephalic periaqueductal gray, and several hypothalamic, thalamic and cerebrocortical regions. No change was seen in the cerebellar nuclei, lateral vestibular nucleus and red nucleus. The increased expression of c-fos observed 3 h after unilateral labyrinthectomy, in conjunction with the sudden occurrence of postural and motor deficits, usually declined 6-24 h after the lesion, i.e. during the development of vestibular compensation. In the dorsal part of the medial vestibular nucleus, however, the pattern of c-fos expression observed 3 h after unilateral labyrinthectomy was reversed 6-24 h after the lesion: both c-fos messenger RNA and protein levels increased on the ipsilateral side, but greatly decreased on the contralateral side. In conclusion, asymmetric changes in c-fos expression occurred within 3 h after unilateral labyrinthectomy, but gradually declined or reversed 6 and 24 h after the lesion, thus being temporally related to the appearance and development of vestibular compensation.

Changes in gene expression during the sleep-waking cycle: a new view of activating systems.

Moruzzi pioneered the notion of ascending activating systems that were responsible for the electrophysiological activation characterizing the transition from sleep to waking. This paper proposes to extend the notion of electrophysiological activation to the domain of gene expression. Evidence is reviewed indicating that in the transition between sleep and waking there is, together with a change in neuronal firing patterns, a change in patterns of gene expression in widespread regions of the brain. The hypothesis is presented that changes in the activity of neuromodulatory systems with diffuse projections may subserve the diffuse, tonic and phasic activation of both neuronal responses and of gene expression. Finally, the paper discusses the possibility that such changes in gene expression may be of importance for plastic phenomena and for the functional consequences of sleep.

Effects of sleep deprivation on the postnatal development of visual-deprived cells in the cat's lateral geniculate nucleus.

1. Observations made by Wiesel and Hubel in kittens during the critical period have shown that monocular visual deprivation (MD) produces hypotrophic changes in the deprived layers of the LGN. Since, in addition to retinal inputs, the LGN also receives extraretinal inputs which are particularly active during REM sleep (a phase which is highly represented at birth), we performed experiments to find out whether total sleep deprivation (SD) interferes with the neuronal maturation in the LGN, thus modifying the susceptibility of LGN neurons to MD. 2. Ten groups of twin kittens were submitted to eyelid suture of one side at the age of 12 to 42 days after birth, and maintained into MD for periods of time which produced only slight or negligible changes in the deprived LGN layers. However, one of the twins in each group was allowed to sleep, while the other was submitted to 2 to 6 days of SD obtained by gentle handling during the last period of MD. At the end of the experiments cross-sectional areas of cell bodies were measured in the binocular segment of different layers of the LGN of both sides, at comparable levels. 3. MD, started 25 to 42 days after birth and continued for 11 to 23 days produced a slight but significant reduction of the mean cell area in the visual-deprived magnocellular ventral (C) and/or dorsal (A) layers of the contralateral LGN, but not in the middle (A1) layer of the ipsilateral LGN. This shrinkage, however, was most severe and involved also the layer A1 if kittens were also submitted to 5-6 days of SD during the last period of MD. There was also a tendency towards increased size in the nondeprived geniculate layers, probably due to an increased monocular visual experience resulting from an increased wake time in the light. However, the slight increase in cell size seen in these layers contrasted with the prominent increase in shrinkage of the visual-deprived layers after SD, indicating that this finding might have resulted from removing an influence of sleep. The effects of SD appeared to depend on the age of kittens (critical period) and the duration of MD. 4. In conclusion, shortly after birth, SD enhanced the structural abnormalities produced by monocular eyelid closure in the visual-deprived LGN layers. Since rhythmic discharges of pontine structures impinge on the LGN neurons during REM sleep, it is postulated that they could represent an endogenous source of stimulation leading to periodic read out of the synaptic connections between primary optic fibers and LGN neurons. This extraretinal input may thus collaborate with the retinal input to facilitate neuronal maturation of the LGN. The possibility that specific noradrenergic and cholinergic neurons, normally acting on the visual system during the sleep-waking cycle, intervene in the postnatal development of the LGN neurons has been discussed.

Sleep deprivation and c-fos expression in the rat brain.

This study examined the effects of sleep deprivation on the expression of the immediate early gene c-fos in the brain with both in situ hybridization and immunocytochemistry. Rats were manually sleep-derived for 3 h, 6 h, 12 h, and 24 h starting at light onset (08.00 hours), and for 12 h starting at dark onset (20.00 hours). c-Fos expression was found to be higher in sleep-deprived rats with respect to control animals in several brain areas. The increase was evident both in terms of c-fos mRNA and Fos protein, although with a different time course. Among the areas that showed a consistent induction of c-fos were many cortical regions, the medial preoptic area and the posterior hypothalamic area, some thalamic nuclei, and several nuclei of the dorsal pontine tegmentum. The pattern of c-fos expression after sleep deprivation was very similar to that observed after comparable periods of spontaneous wakefulness (Pompeiano et al. 1994). In general, the increase in c-fos expression was not simply proportional to the amount of previous wakefulness. In many areas, the highest levels of c-fos were seen after 3 h of sleep deprivation. These observations are discussed with respect to the homeostatic regulation of sleep and to the functional consequences of wakefulness in specific brain areas.

Sleep-waking changes after c-fos antisense injections in the medial preoptic area.

The preoptic hypothalamus has been consistently implicated in the homeostatic regulation of sleep and waking. Recently, it was shown that periods of either spontaneous or forced wakefulness result in the induction in this region of the immediate-early gene c-fos. In this study, antisense oligonucleotides complementary to c-fos mRNA were employed to interfere with the expression of Fos protein. Injections in the rat medial preoptic area of c-fos antisense, but not of sense, oligonucleotides blocked the expression of Fos protein detected immunocytochemically. Rats receiving bilateral antisense injections showed a higher percentage of wakefulness the day after the injection than controls receiving sense or sham injections or antisense injections outside the preoptic area. These results suggest that blocking the expression of Fos protein in the preoptic hypothalamus may interfere with the homeostatic regulation of sleep and wakefulness.

c-Fos expression during wakefulness and sleep.

We have recently demonstrated that c-fos expression is strongly induced by both spontaneous and forced wakefulness in many brain regions. c-Fos expression was considerably increased in regions involved in the regulation of arousal states, such as the locus coeruleus (noradrenergic neurons) and the medial preoptic area (non-GABAergic neurons). With c-fos antisense injection in the medial preoptic area, we demonstrated that c-fos expression in this region is causally involved in sleep regulation. c-Fos expression in other areas, such as the cerebral cortex and the hippocampus, may be related to the functional consequences of prolonged wakefulness and to the need of sleep. Further work should explore the mechanisms leading to changes in the expression of c-fos, and possibly of its target genes, during the sleep-wake cycle.

Differential effects of 5,7-dihydroxytryptamine-induced serotoninergic degeneration of 5-HT1A receptors and 5-HT uptake sites in the rat brain.

The time-course of 5,7-dihydroxytryptamine-induced lesions (2, 5 and 14 days after i.c.v. injection of 150 micrograms) and the effects of acute reserpine treatment (10 mg/kg, i.p., one or 5 days before scheduled death), were evaluated by autoradiography of [3H]paroxetine binding sites in the rat brain. Reserpine had no significant effect on [3H]paroxetine binding, indicating that the depletion of serotonin is not sufficient per se to alter the serotonin uptake sites in any region. Two days after the 5,7-dihydroxytryptamine lesion, [3H]paroxetine binding was already decreased in the majority of brain regions. In the caudate putamen these binding sites were significantly decreased only 14 days after the lesion, whereas the ventral tegmental area (or the enclosed median forebrain bundle), the dorsal raphe (mainly the ventral portion) and the median raphe maintained their high density of serotonin uptake sites even after 14 days. Results were similar using [3H]citalopram as ligand for the serotonin uptake sites, in the brains of rats lesioned 5 days before death; an exception was the ventral portion of the dorsal raphe, where there was a significant increase with [3H]paroxetine and a decrease with [3H]citalopram binding. In adjacent sections of the same brains we also measured [3H]8-OH-DPAT binding, confirming that it completely disappears in the dorsal raphe after the lesion. Thus, considering the extent of serotonin cell body degeneration, there appears to be a paradoxical mismatch between the excessive loss of [3H]8-OH-DPAT binding and the resistance of [3H]citalopram or [3H]paroxetine binding in the dorsal raphe, suggesting that the two binding sites may undergo adaptive regulation in surviving neurons.

Immediate-early genes in spontaneous wakefulness and sleep: expression of c-fos and NGFI-A mRNA and protein.

We have recently shown that the expression of two immediate-early genes, c-fos and NGFI-A, is strongly affected by sleep deprivation, In this work, we investigated c-fos and NGFI-A expression after periods of spontaneous wakefulness or sleep. We used in situ hybridization and immunocytochemistry to detect the corresponding mRNA and protein levels, respectively. A first group of rats (S-L) was sacrificed during the light hours at the end of a long period of sleep. A second group (W-L) was sacrificed under similar conditions, except that during the last half hour the animals had been spontaneously awake. A third group (W-D) was sacrificed during the dark hours after a long period of continuous wakefulness. We found that c-fos and NGFI-A expression in several brain areas was increased in W-L and W-D rats with respect to S-L rats. Some of these areas, including the cerebral cortex, basal ganglia, and colliculi, may have been activated by the increased sensory and motor activity associated with waking. The activation of other areas, such as the medial preoptic area of the hypothalamus and some brainstem nuclei, may be more directly related to sleep regulation. These results indicate that many regions showing an increased expression of immediate early genes after wakefulness induced by sleep deprivation are also activated by periods of spontaneous wakefulness.

Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors.

Because of their similarities, serotonin 5-HT2, 5-HT1C, and the recently described 5-HT2F receptors have been classified as members of the 5-HT2 receptor family, and they have been renamed 5-HT2A, 5-HT2C and 5-HT2B, respectively. The regional distribution and cellular localization of mRNA coding for the members of 5-HT2 receptor family were investigated in consecutive tissue sections from the rat brain by in situ hybridization histochemistry. No evidence for the expression of 5-HT2B receptor was found. High levels of 5-HT2A (formerly 5-HT2) receptor mRNA were observed only in few areas, as the frontal cortex, piriform cortex, ventro-caudal part of CA3, medial mammillary nucleus, the pontine nuclei and the motor cranial nerve nuclei in the brainstem, and the ventral horn of the spinal cord. The distribution of 5-HT2A receptor mRNA is generally in good agreement with that of the corresponding binding sites, although discrepancies were sometimes observed. 5-HT2C (formerly 5-HT1C) mRNA was present at very high levels in the choroid plexuses. However, very high levels were also seen in many other brain regions, as the retrosplenial, piriform and entorhinal cortex, anterior olfactory nucleus, lateral septal nucleus, subthalamic nucleus, amygdala, subiculum and ventral part of CA3, lateral habenula, substantia nigra pars compacta, several brainstem nuclei and the whole grey matter of the spinal cord. These results confirm and extend previous observations that 5-HT2C receptor mRNA is present in many brain areas in addition to those autoradiographically shown to have the corresponding binding sites and that 5-HT2C receptor subtype is a principal 5-HT receptor in the brain. From the comparison between their distributions, 5-HT2A and 5-HT2C receptor mRNAs appeared to be expressed in distinct but overlapping sets of brain regions. Both mRNAs coexisted at high levels in the anterior olfactory nucleus, piriform cortex, endopiriform nucleus, claustrum, pyramidal cell layer of the ventral part of CA3, taenia tecta, substantia nigra pars compacta, and several brainstem nuclei. In other regions both mRNAs were present but with different distributions, as the caudate-putamen. These results are also discussed in relation to the physiological meaning of the existence of two so similar receptor subtypes in the brain.