Chiara Cirelli

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

SUMMARY  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.

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.

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.

The locus coeruleus and immediate early genes in spontaneous and forced wakefulness

In this study, we mapped the expression of two immediate-early genes to examine the functional activation of the locus coeruleus and other regions of the rat brain after periods of spontaneous wakefulness or sleep and after sleep deprivation. c-fos and NGFI-A are two immediate-early genes that are rapidly induced by physiological stimuli and can be used as molecular markers of neural activation. We used immunocytochemical detection of Fos and NGFI-A proteins associated with double labeling for tyrosine hydroxylase to identify activated noradrenergic cells. We found that the expression of Fos and NGFI-A was markedly increased in the locus coeruleus and other brain areas both after spontaneous wakefulness and after short periods (3-24 h) of sleep deprivation. Several Fos-positive cells and most NGFI-A positive cells found in the locus coeruleus after periods of spontaneous wakefulness were shown to be noradrenergic. This study demonstrates that wakefulness per se, whether spontaneous or induced by total sleep deprivation, results in the functional activation of identified noradrenergic locus coeruleus cells.

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.

Suppression of desynchronized sleep through microinjection of the α2-adrenergic agonist clonidine in the dorsal pontine tegmentum of the cat

The relationships between sleep-waking states and the activity of the noradrenergic system are controversial. In particular, according to an influential model of desynchronized sleep (DS) generation, the arrest of firing of noradrenergic neurons in the locus coeruleus should enhance DS, due to the release from inhibition of executive neurons located in the nearby pontine tegmentum. Since locus coeruleus neurons are strongly inhibited by α2-adrenergic agonists like clonidine, this agent would be expected to increase DS. Yet clonidine powerfully decreases DS when injected systemically in several species. In this study, clonidine was microinjected locally into the dorsal pontine tegmentum of the cat, a region which comprises anatomically the whole locus coeruleus complex and which plays a key role in the generation of DS. In accord with the results of systemic experiments, bilateral injections of clonidine almost suppressed DS and unilateral injections consistently reduced it. The effects were dose dependent and site specific. It is suggested that clonidine may suppress DS by acting additionally on non-noradrenergic cell groups located in the dorsal pontine tegmentum.

Modulation of desynchronized sleep through microinjection of β-adrenergic agonists and antagonists in the dorsal pontine tegmentum of the cat

Brain noradrenergic systems have often been implicated in the regulation of desynchronized sleep (DS). In particular, the reciprocal interaction model of DS generation postulates that noradrenergic neurons in the locus coeruleus inhibit DS-executive cells located in the pontine reticular formation. Accordingly, since noradrenergic inhibition is generally mediated by β-receptors, one should expect β-agonists to decrease and β-antagonists to increase DS. However, systemic injection experiments yielded just the opposite results. Assuming that local microinjection techniques were better suited to testing the model, β-agonists and antagonists were directly infused into the dorsal pontine tegmentum (DPT), a region crucially implicated in the generation of DS. Cats were implanted with standard electrodes for polygraphic recordings and with guide tubes for chemical microinjections. It was observed that, when injected into the DPT, the β-agonist isoproterenol almost suppressed DS, while the β-antagonist propranolol consistently enhanced it, the latter largely due to an increased number of DS episodes. These effects were dose-dependent and strictly site-specific, since injections in immediately neighbouring structures were ineffective. These results: (a) confirm that cell groups located in the DPT play a key role in the generation of DS, and (b) indicate that they undergo a strong noradrenergic modulation, being inhibited by β-receptor stimulation and disinhibited by β-receptor blockade as predicted by the reciprocal interaction model.

Effects of local pontine injection of noradrenergic agents on desynchronized sleep of the cat.

Brain noradrenergic (NA) systems have often been implicated in the regulation of desynchronized sleep (DS). The present experiments investigate the effects on DS of the microinjection, into the cat dorsal pontine tegmentum (DPT), of the alpha 2-agonist clonidine (CLON), the beta-agonist isoproterenol and the beta-antagonist propranolol. The DPT comprises most NA neurons belonging to the locus coeruleus (LC) complex, as well as other cell groups thought to be crucially involved in DS generation. Cats were implanted with standard electrodes (electroencephalogram, electrooculogram and electromyogram, PGO waves, hippocampal activity) and with guide tubes aimed at the DPT. Unilateral or bilateral injections (0.25 microliter) were performed by way of thin cannulae inserted through the guide tubes. Polygraphic activity was then recorded in daily sessions lasting 4 h and scored according to standard criteria. Bilateral injections of CLON into the DPT greatly reduced DS, while unilateral injections were much less effective. Since CLON is known to powerfully inhibit NA LC neurons, its effect was thus opposite to that expected on the basis of the reciprocal interaction model of DS generation, which postulates that NA neurons in the LC inhibit DS-executive cells located in the pontine reticular formation. Bilateral injections of the beta-agonist isoproterenol also reduced DS, while the beta-antagonist propranolol consistently enhanced it, the latter largely due to an increased number of DS episodes. These effects were dose-dependent and strictly site-specific, since injections in immediately neighboring structures were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)

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 , 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.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.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 .