Late chronotypes, late mealtimes. Chrononutrition and sleep habits during the COVID-19 lockdown in Italy.

The emerging field of chrononutrition provides useful information on how we manage food intake across the day. The COVID-19 emergency, and the corresponding restrictive measures, produced an unprecedented change in individual daily rhythms, possibly including the distribution of mealtimes. Designed as a cross-sectional study based on an online survey, this study aims to assess the chrononutrition profiles (Chrononutrition Profile Questionnaire, CP-Q) in a sample of 1298 Italian participants, during the first COVID-19 lockdown, and to explore the relationship with chronotype (reduced Morningness-Eveningness Questionnaire, rMEQ), sleep quality (Pittsburgh Sleep Quality Index, PSQI) and socio-demographics. Our findings confirm a change in eating habits for 58% of participants, in terms of mealtimes or content of meals. Being an evening chronotype and experiencing poor sleep imply a higher likelihood of changing eating habits, including a delay in the timing of meals. Also, under these unprecedented circumstances, we report that the timing of breakfast is a valuable proxy capable of estimating the chronotype. From a public health perspective, the adoption of this straightforward and low-cost proxy of chronotype might help in the early detection of vulnerable subgroups in the general population, eventually useful during prolonged stressful conditions, as the one caused by COVID-19 pandemic.

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.

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys.

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.

Effects of unilateral labyrinthectomy on the norepinephrine content in forebrain and cerebellar structures of albino rats.

Albino (Wistar) rats were used to investigate whether unilateral labyrinthectomy (UL) modified the concentration of norepinephrine (NE) as well as of dopamine (DA) and the corresponding metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) in different areas of the cerebral and the cerebellar cortex and the striatum. The results obtained in 38 rats submitted to UL were compared to those of 18 rats submitted to sham-operation. The animals were operated under sodium pentobarbital anesthesia and sacrificed 1.5, 3 and 6 h after surgery. All rats submitted to UL showed phenomena of deficit (1.5-3 h after the lesion) followed by partial vestibular compensation (3-6 h after the lesion). Significant changes in the content of NE were neither found in different areas of the cerebral and the cerebellar cortex, nor in the striatum of rats sacrificed 1.5 h after UL. Three h after the lesion a bilateral increase in the NE content occurred in all the explored areas of the cerebral cortex (i.e., frontal, parieto-temporal and occipital) and the cerebellar cortex (i.e., the vermis and flocculus), as well as in the striatum. This increase, however, was more prominent in the parieto-temporal areas of the neocortex of the intact side, in all the explored areas of the cerebellar cortex of that side, as well as in the striatum of the lesioned side. This asymmetric increase in NE content could not be attributed, at least exclusively, to a generalized activation of the noradrenergic LC nuclei of both sides, due to waking and/or stress which may occur after UL, but did rather depend on asymmetric changes in unit discharge of the vestibular nuclei projecting to the LC of both sides, following UL. In particular, the increased discharge of the vestibular nuclei of the intact side would lead to activation of noradrenergic neurons projecting particularly to the parieto-temporal cortex and the cerebellar cortex of the intact side, as well as to the striatum of the lesioned side. A bilateral increase in NE content was still observed in different areas of the cerebral and cerebellar cortex of rats sacrificed 6 h after UL. This increase, however, was of smaller entity than that observed in the same areas 3 h after UL and quite symmetric. The content of DA and its metabolite DOPAC decreased bilaterally in the striatum of rats sacrificed 1.5 h after UL. This effect was attributed to a reduced synthesis and release of DA, which probably resulted from a reduced facilitatory influence that the deafferented vestibular nuclei exert on the dopaminergic, nigrostriatal system of both sides, although mainly on the intact side. The corresponding values, however, bilaterally recovered to slightly increase with respect to the control values in rats sacrificed 3 and 6 h after UL. In these experiments the content of both DA and DOPAC remained symmetric on both sides after UL, in contrast with the bilateral but asymmetric increase in NE concentration observed in the same structure 3 h the lesion. The present results integrate and extend those of previous experiments showing that: 1) albino rats sacrificed 6 h after UL displayed an increased synthesis of NE, which affected particularly the LC of the intact side as well as the medial vestibular nuclei of both sides (21); and 2) the structures which showed an increased content of NE at given time intervals after UL also displayed an increase in the expression of the immediate early gene c-fos (cf. 16 for ref.). These findings suggest that bilateral but asymmetric activation of the noradrenergic LC neurons following UL may lead to an asymmetric increase in c-fos expression in several target structures, thus contributing to the plastic changes responsible for vestibular compensation. In conclusion, it appears that UL induces in several brain structures of albino rats a short-term increase in synthesis and release of NE. (ABSTRACT TRUNCATED)

Fos-protein expression in noradrenergic locus coeruleus neurons after unilateral labyrinthectomy in the rat.

c-Fos mRNA and the related Fos-protein are rapidly induced by physiological stimuli and can be used as molecular markers of neural activation and plasticity. In a recent study (14), we found that rats submitted to unilateral labyrinthectomy (UL) displayed an asymmetric increase in the expression of both c-fos and Fos-protein not only in several vestibular, precerebellar and cerebellar structures and the caudate-putamen, but also in the locus coeruleus (LC)-complex, whose neurons integrate labyrinthine signals and are apparently involved in the plastic changes which are at the basis of vestibular compensation. In the present study we investigated the putative noradrenergic nature of the Fos-positive LC neurons observed after UL by combining Fos-protein immunocytochemistry with the immunocytochemical detection of tyrosine hydroxylase (TH), a synthetizing enzyme of noradrenaline. The experiments were performed in rats sacrificed 3, 6 and 24 h after surgical lesion of one labyrinth. The results obtained were in agreement with the previous findings, showing that already 3 h after UL an asymmetric increase of the c-fos and/or Fos-protein expression occurred in the vestibular nuclei, the inferior olive, the cerebellar cortex and the caudate-putamen. Most interestingly, the Fos-protein expression markedly increased in the LC-complex of both sides, although mainly ipsilaterally to the intact side. It appeared also that several Fos-positive LC-complex neurons were probably noradrenergic in nature, as they could be double-labeled with the Fos/TH technique. These findings were attenuated 6 h after UL and disappeared after 24 h, when partial compensation of the vestibular syndrome had occurred. Thus, UL results in asymmetric functional activation in the LC region of well identified noradrenergic neurons. This finding is attributed to the fact that asymmetric stimulation of labyrinth receptors gives rise to asymmetric changes in firing rate of LC neurons (45). Since these neurons send noradrenergic afferents to several target structures such as the vestibular nuclei, the inferior olive, the cerebellar cortex and the caudate-putamen, we postulated that the asymmetric labyrinthine activation of the noradrenergic LC system, occurring after UL, could increase the Fos-protein expression in the above mentioned brain structures. This possibility could then represent a key factor in determining the plastic changes, which are at the basis of vestibular compensation.

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.

Role of the spinocerebellum in adaptive gain control of cat's vestibulospinal reflex.

In decerebrate cats, a 3-h period of sustained roll tilt of the head (at 0.15 Hz. +/- 10) leading to selective stimulation of labyrinth receptors, associated with a synchronous roll tilt of the body (at 0.15 Hz., +/- 12.5) leading to 2.5 degrees out-of-phase neck rotation produced an adaptive increase in gain of the vestibulospinal reflex (VSR) elicited by roll tilt of the animal at 0.15 Hz, +/- 10 degrees. This increase reached the maximum at the end of the third h of stimulation and persisted unmodified during the first h after stimulation. Microinjection into zone B of the cerebellar anterior vermis of the GABA-A agonist muscimol (0.25 microliter at 8 micrograms/microliters saline), producing only a slight or negligible depression of the VRS gain in non-adaptive conditions, prevented the occurrence of the adapted increase in gain of the VSR following a 3-h period of sustained head-body rotation. Moreover, intravermal injection of the GABA-A agonist muscimol or the GABA-B agonist baclofen (0.25 microliter at 8 or 2 micrograms/microliters saline, respectively) suppressed the already adapted VSR gain. It is postulated that the adaptive increase in gain of the VSR following a sustained neck-vestibular stimulation depends on plastic changes which affect the Purkinje cells of the cerebellar anterior vermis.

Depression of the vestibulospinal reflex adaptation by intravermal microinjection of GABA-A and GABA-B agonists in the cat.

In decerebrate cats the gain of the vestibulospinal reflex (VSR), elicited by sinusoidal roll tilt of the animal at 0.15 Hz, +/- 10 degrees, was tested every 10-15 min during and after a sustained (3 h) period of roll tilt of the head at the parameters indicated above, associated with synchronous roll tilt of the body at 0.15 Hz, +/- 12.5 degrees; this stimulus led to 2.5 degrees of neck rotation, which was thus out of phase with respect to head rotation. In this condition the gain of the VSR progressively increased during the first h of neck-vestibular stimulation, to reach a plateau level at the end of the third h of stimulation. This adaptive process was followed for at least 1 h after stimulation. Microinjection into the zone B of the cerebellar anterior vermis of the GABA-A agonist muscimol (0.25 microliter at 8 micrograms/microliter saline) producing only a slight or negligible depression of the VSR gain in non-adaptive conditions, prevented the occurrence of the adapted increase in gain of the VSR following a 3-h period of sustained head and neck rotation. In addition, intravermal injection of the GABA-A or the GABA-B agonist muscimol or baclofen, respectively, at the same dose indicated above supressed the adapted increase in gain occurring after a 3-h period of continuous neck-vestibular stimulation. The effective sites were located into the zone B of the cerebellar anterior vermis, from which the direct corticocerebellar projection to the lateral vestibular nucleus originates. In conclusion, the results seem to indicate that the adaptive increase in gain of the VSR which occurs in decerebrate cats depend upon plastic changes which affect the Purkinje cells of the cerebellar anterior vermis. These changes were in fact suppressed by GABAergic inhibition of these neurons. The demonstration that the effects of the GABA agonists occurred suddenly makes unlikely the hypothesis that the cerebellar anterior vermis represents either a relay for adaptive changes occurring before it (for instance in the inferior olive) or else the generator of error signals that elicit plasticity in target structures (as in the vestibular nuclei).

Injections of beta-noradrenergic substances in the cerebellar anterior vermis of cats affect adaptation of the vestibulospinal reflex gain.

In precollicular decerebrate cats, intermittent sinusoidal roll tilt of the whole animal (at 0.15 Hz, +/- 10 degrees) produced a vestibulospinal reflex (VSR), characterized by an increased EMG activity of the forelimb extensor triceps brachii during side-down and a decreased activity during side-up tilt. This reflex was first tested during and after a 3-h period of sustained animal tilt at the same parameters indicated above. An adaptive increase in gain of the VSR progressively developed in some experiments, but not in others. In a second group of experiments, however, sinusoidal roll tilt of the head (0.15 Hz, +/- 10 degrees) was associated with a synchronous roll tilt of the body (at 0.15 Hz, +/- 12.5 degrees). This additional stimulus led to 2.5 degrees of neck rotation, which was thus out of phase with respect to head rotation. In all these experiments, submitted to a 3-h period of sustained neck-vestibular stimulation, the gain of the VSR tested every 10-15 min consistently increased to reach the maximum at the end of the third hour of stimulation. This adaptive process was followed up to 1 h after stimulation. Microinjection into the hemivermal cortex of the cerebellar anterior lobe of the beta-noradrenergic antagonists propranolol or sotalol (0.25-0.50 microliter at 8 micrograms/microliter saline) produced only slight and short-lasting changes in the basic amplitude of the VSR, but always decreased or prevented the occurrence of the adaptive increase in gain of the VSR during sustained out of phase head-neck rotation. The same agents also suppressed the increase in gain of the VSR which occurred in some experiments during sustained roll tilt of the whole animal (at 0.15 Hz, +/- 10 degrees), leading to selective stimulation of labyrinth receptors. On the other hand, the beta-noradrenergic agonist isoproterenol (0.25 microliters at 8 micrograms/microliters saline) brought to the light the adaptive process in other experiments in which no adaptation occurred during sustained animal rotation. These effects occurred when the sites of injection were located within the zone B of the cerebellar anterior vermis, which projects to the lateral vestibular nucleus. In conclusion, the results indicate that the adaptive changes affecting the gain of the VSR in decerebrate cats are facilitated by the noradrenergic afferent system acting on the cerebellar vermis through beta-adrenoceptors.

Depression of the vestibulospinal reflex by intravermal microinjection of GABAA and GABAB agonists in the decerebrate cat.

Experiments performed in decerebrate cats have shown that unilateral microinjection into the cerebellar anterior vermis of a GABAA (muscimol) or a GABAB agonist (baclofen) decreased the gain of the vestibulospinal reflex involving the ipsilateral triceps brachii (iVSR). On the contrary, the phase angle of the reflex was not significantly modified. These effects started 5 to 10 min after the injection and persisted for at least 1 to 2 h before disappearing. Just the opposite changes in gain of the VSR were obtained after local microinjection of the GABAA (bicuculline) or the GABAB antagonist (saclofen). The area on which the GABAergic agents were effective was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.0 to 1.4 mm with respect to the midline. This vermal region corresponded to the zone B of the cerebellar cortex, which receives a labyrinth input and projects to the ipsilateral lateral vestibular nucleus, where it exerts a prominent inhibitory influence. It is suggested that GABA agonists inhibit the Purkinje (P)-cells' activity, thus reducing the labyrinthine-induced modulation of the firing rate of these neurons by mossy and climbing fiber afferents. Since the vermal P-cells discharge outphase with respect to the excitatory VS neurons, we may explain why a reduced output of these P-cells results in a reduced gain of the VSR. These experiments provide evidence that the cerebellar anterior vermis exerts a positive influence on the basic VSR gain.

Noradrenergic agents in the cerebellar vermis affect adaptation of the vestibulospinal reflex gain.

In precollicular decerebrate cats, the vestibulospinal reflex (VSR) was intermittently recorded from the triceps brachii during sinusoidal roll tilt of the whole animal (at 0.15 Hz, +/- 10 degrees), leading to selective stimulation of labyrinth receptors. This reflex, tested during and after a 3-h period of sustained animal tilt at the same parameters indicated above, showed an adaptive increase in gain in some experiments but not in others. In a second group of experiments, however, rotation of the head (at 0.15 Hz, +/- 10 degrees) was associated with a synchronous body rotation (at 0.15 Hz, +/- 12.5 degrees) which led to an additional neck input, due to 2.5 degrees of out-phase body-to-head displacement. In these experiments, the VSR, tested every 10-15 min, consistently showed an adaptive increase in gain during and after a 3-h period of sustained vestibular and neck stimulation. Microinjection into the cerebellar anterior vermis of beta-adrenergic agents (0.25 microliters at 8 micrograms/microliters saline) produced slight and short-lasting changes in the basic amplitude of the VSR, due to the neuromodulatory influence of these agents on the Purkinje cells activity. In addition, the beta-adrenergic agonist isoproterenol brought to the light an adaptive process in those experiments in which no adaptation occurred during a sustained roll tilt of the whole animal. On the other hand, the beta-adrenergic antagonists propranolol or sotalol either suppressed the increase in gain of the VSR which occurred in other experiments during sustained animal rotation, or prevented the occurrence of an adaptive increase in gain during a continuous out-phase head and body rotation. We conclude that the adaptive changes in gain of the VSR are facilitated by the noradrenergic system acting within the cerebellar cortex through beta-adrenoceptors.

Adaptive modification of the cat's vestibulospinal reflex during sustained vestibular and neck stimulation.

In decerebrate cats, rotation about the longitudinal axis of the whole animal at 0.15 Hz, +/- 10 degrees produced an increased electromyogram (EMG) activity of the triceps brachii during side-down tilt and a decreased activity during side-up tilt. This vestibulospinal reflex (VSR) was tested before, during and after a sustained (3-h) period of roll tilt of the head at the parameters indicated above, associated with a synchronous roll tilt of the body at 0.15 Hz, but at the peak amplitude of either 12.5 degrees or 7.5 degrees. This additional stimulus led to 2.5 degrees of neck rotation, which was respectively out of phase (condition A) or in-phase (condition B) with head rotation. In a few instances the peak amplitude of neck rotation was increased to 5 degrees. In the first experimental condition A, the gain of the VSR (tested every 10-15 min) progressively increased, starting from the first hour of out of phase neck-vestibular stimulation to reach, on average, 241% of the control value at the end of the third hour of stimulation. On the other hand, in the second experimental condition B, the mean gain of the VSR first decreased to 82% during the first hour of in-phase neck-vestibular stimulation, but then increased to 165% of the corresponding control during the last hour of recording. In other experiments an adaptive increase in gain of the pure VSR occurred during a sustained (3-h) period of selective roll tilt of the whole animal, but it was less consistent and, on average, smaller in amplitude than that obtained during out of phase neck-vestibular stimulation. The adaptive changes in gain of the VSR described above were not associated with changes in the phase angle of the responses, and were also observed during the post-adaptation period. Further experiments indicated that the gain of the N-VSR, i.e. of the EMG responses to combined neck-vestibular stimulation, displayed a prominent adaptive increase during the sustained out of phase stimulation, but not during the in phase stimulation.

Modulation of vestibulospinal reflexes through microinjection of an alpha 1-adrenergic antagonist in the dorsal pontine tegmentum of decerebrate cats.

1. The possibility that the norepinephrine (NE)-containing locus coeruleus (LC) neurons produce changes in posture as well as in gain of the vestibulospinal (VS) reflexes by acting on the dorsolateral pontine tegmentum (DPT) and the related medullary inhibitory reticulospinal (RS) system through alpha 1-adrenoceptors has been investigated in decerebrate cats. 2. Injection of the alpha 1-adrenergic antagonist prazosin PRZ (0.25 microliter at 0.1-1 microgram/microliter solvent) into the DPT, namely in the dorsal pontine reticular formation (pRF), as well as in the peribrachial nucleus of one side, decreased the postural activity in the ipsilateral limbs while increasing that of the contralateral limbs. In addition, the amplitude of modulation and thus the gain of the multiunit EMG responses of the ipsilateral and to a lesser extent of the contralateral triceps brachii to roll tilt of the animal at 0.15 Hz, +/- 10 degrees, increased. These effects appeared 5-10 min after the injection, reached the highest values in about 40-60 min and persisted for additional 1.5-2 h before disappearing. 3. The effects were site-specific and to some extent dose-dependent. However, neither changes in posture nor in gain of the VS reflexes were obtained after injection in the effective area of an equal volume of solvent. 4. In order to account for these findings it was postulated that the alpha 1-antagonist blocks the tonic inhibitory influence that the NE-containing LC neurons exert on ipsilateral DPT either by exciting through alpha 1-receptors interposed inhibitory interneurons, or by inhibiting presynaptically excitatory afferents to the same pontine tegmental structures. The increased discharge of these neurons and the related medullary inhibitory RS neurons would reduce the postural activity in the ipsilateral limbs. However, since the inhibitory RS neurons show a response pattern to tilt opposite in sign to that elicited by the excitatory VS neurons, we could expect that for a given labyrinth signal, the increased discharge of the RS neurons in the animal at rest would lead to a greater disinhibition of limb extensor motoneurons during ipsilateral tilt. These motoneurons would then respond more efficiently to the same excitatory volleys elicited by given parameters of stimulation, thus leading to an increased gain of the EMG responses of forelimb extensors to labyrinth stimulation. The possibility that the DPT of one side activates inhibitory RS neurons of both sides explains why PRZ increases the gain of the VS reflexes not only ipsilaterally but also contralaterally to the side of the injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicotinic receptors in the cerebellar vermis modulate the gain of the vestibulospinal reflexes in decerebrate cats.

1. The possibility that the cholinergic afferent system terminating in the vermal cortex of the cerebellar anterior lobe acts on the target neurons by utilizing nicotinic receptors has been investigated in decerebrate cats by testing the effects of local microinjection of cholinergic nicotinic agonists and antagonists on posture as well as on the dynamic characteristics of the vestibulospinal (VS) reflexes. 2. Unilateral injection into the vermal cortex of the culmen of nicotine (0.25 microliter at the concentration of 0.05-0.5 microgram/microliter saline) decreased the extensor tonus in the ipsilateral forelimb, while the extensor tonus in the contralateral forelimb increased. The some agent significantly increased the gain of the first harmonic component of the EMG responses of the ipsilateral and more prominently also of the contralateral triceps brachii to animal tilt. However, the phase angle of the responses remained bilaterally unmodified. The effects described above were first observed 5-10 min after the injection, reached the peak after 40-60 min and persisted for at least 2-3 h before disappearing. 3. The effective area was located between the second and the fourth folium of the cerebellar vermis rostral to the fissura prima, at the laterality of 1.4-1.8 mm. This area, which upon cathodal stimulation suppressed the spontaneous EMG activity of the ipsilateral triceps brachii, actually corresponds to the zone B of the cerebellar cortex which exerts a direct inhibitory influence on the lateral vestibular nucleus. Moreover, the effects were dose-dependent. 4. Microinjection of nicotinic antagonists of both the ganglionic type (hexamethonium, 0.25 microliter at 4 micrograms/microliters saline) and the neuromuscular type (d-tubocurarine, 0.25 microliter at 7 micrograms/microliters saline) produced a postural asymmetry opposite in sign to that elicited by nicotine. The same agents also decreased the response gain of the triceps brachii of both sides to animal tilt recorded either under normal conditions or after previous injections of nicotine. 5. The experiments indicate that the cholinergic system is involved in the control of posture as well as in the gain regulation of the VS reflexes. Previous histochemical studies had shown that the cholinergic fibers terminate not only on Purkinje (P)-cells, but also and more prominently as mossy fibers ending on granular cells. This system may thus affect the discharge of P-cells and related inhibitory interneurons not only ipsilaterally but also contralaterally to the side of the injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic receptors in the cerebellar vermis modulate the gain of the vestibulospinal reflexes in decerebrate cats.

1. The Purkinje (P)-cells of the cerebellar vermis, which exert a prominent influence on posture as well as on the gain of vestibulospinal (VS) reflexes, are under the control not only of the classic mossy fibers and climbing fibers which liberate excitatory amino acids as neurotransmitter, but also of cholinergic afferents. The role of these afferents was investigated in precollicular decerebrate cats by using the method of local microinjection of cholinergic agents into appropriate areas of the cerebellar cortex. 2. Unilateral injection into the vermal cortex of the culmen of the non-selective cholinergic agonist carbachol (0.25 microliters at 0.5 micrograms/microliters saline) produced a postural asymmetry, characterized by a slight decrease of the extensor tonus in the ipsilateral forelimb and an increased tonus in the contralateral forelimb. Moreover, the gain of the EMG responses of the ipsilateral and the contralateral triceps brachii to animal tilt increased significantly, while no significant changes in the phase angle of the responses were observed. These effects started 5-10 min after the injection and persisted for at least 2 hours before disappearing. Similar but smaller effects were obtained after injection of eserine, an inhibitor of acetylcholinesterase. Thus, the effects could be produced by increasing the naturally present amount of acetylcholine (ACh). 3. The changes in posture and gain of the VS reflexes described above utilized in part at least muscarinic receptors, since effects similar to those induced by carbachol injection were also obtained after unilateral microinjection into the vermal cortex of the culmen of the muscarinic agonist bethanechol (0.25 microliters at 0.1 micrograms/microliters). On the other hand opposite effects, characterized by an increased postural activity in the ipsilateral forelimb associated with a decreased activity in the contralateral forelimb, as well as by a reduced gain of the EMG responses of the triceps brachii of both sides to animal tilt were observed in other experiments after local microinjection of the muscarinic antagonist scopolamine (0.25 microliter at 4-8 micrograms/microliters saline). Evidence for muscarinic supersensitivity was obtained following repetitive injections of scopolamine into the cerebellar vermis. 4. The area which upon injection of the cholinergic agents modified the postural activity as well as the gain of the VS reflexes was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.4-1.8 mm with respect to the midline.(ABSTRACT TRUNCATED AT 400 WORDS)