Kazue Semba

Multiple output pathways of the basal forebrain: organization, chemical heterogeneity, and roles in vigilance

Studies over the last decade have shown that the basal forebrain (BF) consists of more than its cholinergic neurons. The BF also contains non-cholinergic neurons, including gamma-aminobutyric acid-ergic neurons which co-distribute and co-project with the cholinergic neurons. Both types of neuron project, in variable proportions, to the cerebral cortex, hippocampus, thalamus, amygdala, and olfactory bulb, whereas descending projections to the posterior hypothalamus and brainstem nuclei are predominantly non-cholinergic. Some of the cholinergic and non-cholinergic projection neurons contain neuropeptides such as galanin, nitric oxide synthase, and possibly glutamate. To understand better the function of the BF, the organization of the multiple ascending and descending projections of BF neurons is reviewed along with their neurochemical heterogeneity, and possible functions of individual pathways are discussed. It is proposed that BF neurons belong to multiple systems with distinct cognitive, motivational, emotional, motor, and regulatory functions, and that through these pathways, the BF plays a role in controlling both cognitive and non-cognitive aspects of vigilance.

Indirect projections from the suprachiasmatic nucleus to major arousal-promoting cell groups in rat: Implications for the circadian control of behavioural state

The circadian clock housed in the suprachiasmatic nucleus (SCN) controls various circadian rhythms including daily sleep-wake cycles. Using dual tract-tracing, we recently showed that the medial preoptic area (MPA), subparaventricular zone (SPVZ) and dorsomedial hypothalamic nucleus (DMH) are well positioned to relay SCN output to two key sleep-promoting nuclei, namely, the ventrolateral and median preoptic nuclei. The present study examined the possibility that these three nuclei may link the SCN with wake-regulatory neuronal groups. Biotinylated dextran-amine with or without cholera toxin B subunit was injected into selected main targets of SCN efferents; the retrograde labeling in the SCN was previously analyzed. Here, anterograde labeling was analyzed in immunohistochemically identified cholinergic, orexin/hypocretin-containing and aminergic cell groups. Tracer injections into the MPA, SPVZ and DMH resulted in moderate to dense anterograde labeling of varicose fibers in the orexin field and the tuberomammillary nucleus. The locus coeruleus, particularly the dendritic field, contained moderate anterograde labeling from the MPA and DMH. The ventral tegmental area, dorsal raphe nucleus, and laterodorsal tegmental nucleus all showed moderate anterograde labeling from the DMH. The substantia innominata showed moderate anterograde labeling from the MPA. These results suggest that the MPA, SPVZ and DMH are possible relay nuclei for indirect SCN projections not only to sleep-promoting preoptic nuclei as previously shown, but also to wake-regulatory cell groups throughout the brain. In the absence of major direct SCN projections to most of these sleep/wake-regulatory regions, indirect neuronal pathways probably play an important role in the circadian control of sleep-wake cycles and other physiological functions.

Arousal Effect of Caffeine Depends on Adenosine A2A Receptors in the Shell of the Nucleus Accumbens

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A2A receptors (A2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A2ARs were focally removed from the NAc core or other A2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

The role of basal forebrain neurons in tonic and phasic activation of the cerebral cortex

The basal forebrain and in particular its cholinergic projections to the cerebral cortex have long been implicated in the maintenance of cortical activation. This review summarizes evidence supporting a close link between basal forebrain neuronal activity and the cortical electroencephalogram (EEG). The anatomy of basal forebrain projections and effects of acetylcholine on cortical and thalamic neurons are discussed along with the modulatory inputs to basal forebrain neurons. As both cholinergic and GABAergic basal forebrain neurons project to the cortex, identification of the transmitter specificity of basal forebrain neurons is critical for correlating their activity with the activity of cortical neurons and the EEG. Characteristics of the different basal forebrain neurons from in vitro and in vivo studies are summarized which might make it possible to identify different neuronal types. Recent evidence suggests that basal forebrain neurons activate the cortex not only tonically, as previously shown, but also phasically. Data on basal forebrain neuronal activity are presented, clearly showing that there are strong tonic and phasic correlations between the firing of individual basal forebrain cells and the cortical activity. Close analysis of temporal correlation indicates that changes in basal forebrain neuronal activity precede those in the cortex. While correlational, these data, together with the anatomical and pharmacological findings, suggest that the basal forebrain has an important role in regulating both the tonic and the phasic functioning of the cortex.

Extent of colocalization of serotonin and GABA in the neurons of the rat raphe nuclei

Previous investigations of the distribution of neurons containing both serotonin and GABA in the brainstem raphe nuclei have yielded discrepant results amongst different authors. This study attempted to clarify the distribution as well as the proportions of raphe and other brainstem neurons that contain both neurotransmitters. All the nine serotonergic cell groups known to be present in the brainstem were examined with an indirect immunofluorescence method using antibodies against serotonin and glutamic acid decarboxylase in colchicine-treated rats. Sections were incubated either simultaneously or sequentially for the two immunolabels. Brainstem neurons that were labelled for both markers were generally infrequent. Of all the serotonin cell groups in the brainstem, the nucleus raphe magnus contained the most double-labelled cells (a mean of 3.6% of a total of 625-1155 serotonin-immunoreactive cells counted in this nucleus), followed by the nucleus raphe obscurus (1.5% of a total of 220-550 serotonin-immunoreactive neurons counted). The dorsal, median and pontine raphe nuclei as well as the supralemniscal nucleus (the B9 group) contained very few double-labelled cells, which comprised a mean of 0.1-0.7% of all serotonin-immunoreactive cells in each of these nuclei. No double labelled cells were present in the caudal linear raphe nucleus or the nucleus raphe pallidus, nor in the B4 group. These results suggest that only a very small percentage of serotonergic neurons in the medullary raphe nuclei (raphe magnus and raphe obscurus) also contain GABA, whereas such cells are virtually absent in the midbrain raphe nuclei or in the non-raphe serotonergic cell groups in the brainstem.

Dual projections of single cholinergic and aminergic brainstem neurons to the thalamus and basal forebrain in the rat

Compelling evidence indicates that cholinergic basal forebrain neurons are strongly activated during waking, and concurrently thalamic spindle activity is suppressed and thalamocortical sensory transmission is facilitated. Both thalamus and basal forebrain are known to receive projections from brainstem cholinergic and aminergic neuronal pools that are involved in wake/sleep regulation. The present study addressed the question of whether single cholinergic and aminergic neurons contributed to both of these ascending projections, by using two fluorescent retrograde tracers combined with immunofluorescence. Cholinergic neurons projecting to both the basal forebrain and thalamus were found in the pedunculopontine and laterodorsal tegmental nuclei, representing an average of 8.0% of the total cholinergic cell population in these nuclei. Serotonergic neurons with dual projections were observed in the dorsal, median and caudal linear raphe nuclei, accounting for a mean of 4.7% of total serotonergic neurons in these nuclei. Relatively few noradrenergic neurons (2.0%) in the locus ceruleus projected to both target structures, and a very small subpopulation of histaminergic neurons (1.5%) in the tuberomammillary hypothalamic nucleus had dual projections. Of all brainstem neurons with dual projections, cholinergic and serotonergic neurons accounted for an overwhelming majority, with noradrenergic followed by histaminergic neurons representing the remaining minority. These data suggest that through dual projections, cholinergic and aminergic brainstem neurons can concurrently modulate the activity of neurons in the thalamus and basal forebrain during cortical arousal.

A direct retinal projection to the dorsal raphe nucleus in the rat

A direct projection from the retina to the dorsal raphe nucleus at the pontomesencephalic junction was demonstrated with both antero- and retrograde tracing techniques in the rat. Following intravitreous injections of choleratoxin subunit B (CTB), horseradish peroxidase (HRP) and CTB-conjugated HRP, varicose fibers were labeled in the lateral region of the dorsal raphe nucleus, predominantly contralateral to the injection. Many of these labeled fibers were intermingled with serotonin-immunoreactive neurons, but some fibers were also found further laterally, beyond the boundary of dorsal raphe nucleus but within the periaqueductal gray. Following injections of the retrograde tracers Fluoro-Gold and CTB into the dorsal raphe nucleus and adjacent periaqueductal gray (without contamination of previously known targets of retinal projections), a small population of ganglion cells was labeled in the retina. These data provide evidence for the existence of a direct retinal projection to the lateral region of the dorsal raphe nucleus and the adjacent mesopontine periaqueductal gray in the rat. This projection may have a role in sensorimotor coordination and the regulation of circadian rhythm as well as sleep and wakefulness.

Distribution of pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in the hypothalamus and extended amygdala of the rat

Pituitary adenylate cyclase activating polypeptide (PACAP) is found in two forms of 27 and 38 amino acids (PACAP‐27 and PACAP‐38 respectively) in the mammalian central nervous system. Using antibodies to these two forms of PACAP, we examined the distribution of PACAP immunoreactivity in the rat hypothalamus and a number of extrahypothalamic areas. The patterns of immunostaining for PACAP‐27 and PACAP‐38 were similar: prominent terminal labelling was present in the retrochiasmatic area, median eminence, and posterior periventricular nucleus of the hypothalamus as well as the bed nucleus of the stria terminalis and amygdaloid complex. After colchicine treatment, immunopositive cell bodies were found in the preoptic region of the periventricular zone of the hypothalamus, the suprachiasmatic and paraventricular hypothalamic nuclei, neural structures adjacent to the median eminence (including the retrochiasmatic area, arcuate nucleus, ventromedial hypothalamus, and tuber cinereum), and the lateral mammillary and supramammillary nuclei. In all these areas, immunolabelling appeared specific since it was abolished by preabsorption of primary antisera with the appropriate PACAP peptide. However, the number of immunopositive cells in the suprachiasmatic nucleus was also reduced by preabsorption of PACAP‐27/38 antisera with vasoactive intestinal polypeptide, suggesting that a subpopulation of cells in the suprachiasmatic nucleus express a peptide which has significant sequence homology with both PACAP‐27/38 and vasoactive intestinal polypeptide. The distribution of PACAP immunoreactivity throughout the hypothalamus, bed nucleus of the stria terminalis, and amygdala suggests the involvement of PACAP in a number of processes including limbic, autonomic, and neuroendocrine functions as well as regulation of the circadian pacemaker.

Serotonergic synaptic input to cholinergic neurons in the rat mesopontine tegmentum.

Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.

Effects of Ibotenate and 192IgG-Saporin Lesions of the Nucleus Basalis Magnocellularis/Substantia Innominata on Spontaneous Sleep and Wake States and on Recovery Sleep after Sleep Deprivation in Rats

The basal forebrain (BF) is known for its role in cortical and behavioral activation, and has been postulated to have a role in compensatory mechanisms after sleep loss. However, specific neuronal phenotypes responsible for these roles are unclear. We investigated the effects of ibotenate (IBO) and 192IgG-saporin (SAP) lesions of the caudal BF on spontaneous sleep–waking and electroencephalogram (EEG), and recovery sleep and EEG after 6 h of sleep deprivation (SD). Relative to artificial CSF (ACSF) controls, IBO injections decreased parvalbumin and cholinergic neurons in the caudal BF by 43 and 21%, respectively, and cortical acetylcholinesterase staining by 41%. SAP injections nonsignificantly decreased parvalbumin neurons by 11%, but significantly decreased cholinergic neurons by 69% and cortical acetylcholinesterase by 84%. IBO lesions had no effect on sleep–wake states but increased baseline delta power in all states [up to 62% increase during non-rapid eye movement (NREM) sleep]. SAP lesions transiently increased NREM sleep by 13%, predominantly during the dark phase, with no effect on EEG. During the first 12 h after SD, animals with IBO and SAP lesions showed lesser rebound NREM sleep (32 and 77% less, respectively) and delta power (78 and 53% less) relative to ACSF controls. These results suggest that noncholinergic BF neurons promote cortical activation by inhibiting delta waves, whereas cholinergic BF neurons play a nonexclusive role in promoting wake. Intriguingly, these results also suggest that both types of BF neurons play important roles, probably through different mechanisms, in increased NREM sleep and EEG delta power after sleep loss.