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The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.

The basal forebrain (BF) contains cholinergic neurons that stimulate cortical activation during waking. In addition, both the BF and adjacent preoptic area (POA) contain neurons that promote sleep. We examined c-Fos expression in cholinergic and GABAergic neurons in the BF and POA to determine whether they are differentially active following sleep deprivation versus recovery and whether the GABAergic neurons are active during sleep. Whereas the numbers of c-Fos+ cells and proportions of c-Fos+ cells that were cholinergic were decreased, the proportions that were GABAergic were increased following sleep recovery across BF and POA nuclei. Moreover, the sleep-active GABAergic neurons were immunostained for alpha2A-adrenergic receptors. We conclude that GABAergic neurons that commonly bear alpha2-adrenergic receptors comprise sleep-active cells of the BF and POA. These GABAergic cells would be inhibited by noradrenaline (NA) released from locus coeruleus neurons during waking; they would be disinhibited through diminished NA release during drowsiness and thus become active to promote sleep by inhibiting in turn wake-promoting neurons.

Acetylcholine has long been known to play an important role in the cortical activation that accompanies the states of wakefulness and paradoxical sleep (for review, see Refs 17, 21) when this neurotransmitter is released from the cerebral cortex at the highest rates. The major supply of acetylcholine to the cerebral cortex arises from the cholinergic neurons of Meynert's Basal-ganglion or nucleus basalis of the forebrain. Lying in the substantia innominata within the major ascending pathway from the brain stem reticular formation, magnocellular basalis neurons project upon the cerebral cortex as the important ventral, extrathalamic relay of the ascending reticular activating system. Although the cholinergic basalis nucleus neurons have been shown to be important for cortical activation, the precise manner in which they influence cortical activity has not as yet been elucidated, in part because the cholinergic cells of this nucleus have not been identified in electrophysiological studies. Using intracellular recording in guinea-pig brain slices, we were able to record and fill with biocytin nucleus basalis neurons which were subsequently revealed by immunohistochemical staining to be choline acetyltransferase-positive and thus cholinergic. The cholinergic cells displayed rhythmic bursting activity mediated by a low-threshold calcium spike in vitro, which would endow them with a capacity for phasic (in addition to tonic) firing in vivo. By virtue of these different modes, cholinergic basalis neurons may accordingly deter or facilitate the cortical response to sensory input and may furthermore modulate the major frequencies of cortical activity across the different states of the sleep-waking cycle.

Basal forebrain neurons play important parts in processes of cortical activation and memory that have been attributed to the cortically projecting, cholinergic neurons. Yet, non-cholinergic neurons also project to the cerebral cortex and also appear to participate in processes of cortical modulation and plasticity. GABAergic neurons compose a portion of the cortically projecting cell group, but do not fully account for the non-cholinergic cell contingent. In the present study in the rat, we investigated whether the non-cholinergic, non-GABAergic cell component might be composed of glutamatergic neurons. We examined afferents to the entorhinal cortex, which is known to be modulated by basal forebrain neurons and to be critically involved in memory. Dual immunofluorescent staining was performed for cholera toxin, as retrograde tracer, and phosphate-activated glutaminase, the synthetic enzyme for the neurotransmitter pool of glutamate. The retrogradely labeled cells were distributed across the basal forebrain through the medial septum, diagonal band, magnocellular preoptic area and substantia innominata. The major proportion (approximately 80%) of the retrogradely labeled cells was found to be immunopositive for phosphate-activated glutaminase. Equal minor proportions (approximately 40%) were immunopositive for choline acetyltransferase and glutamic acid decarboxylase. In other material dual-immunostained for neurotransmitter enzymes, approximately 95% of choline acetyltransferase- and approximately 60% of glutamic acid decarboxylase-immunopositive neurons were also immunopositive for phosphate-activated glutaminase. From these results it appears that a significant proportion of these cell groups, including their cortically projecting contingents, could synthesize glutamate together with acetylcholine or GABA as neurotransmitters and another proportion of cells could synthesize glutamate alone. Accordingly, as either co-transmitter or primary transmitter within basalocortical afferents, glutamate could have the capacity to modulate the entorhinal cortex and promote its role in memory.

As part of a larger study concerning the role of neurons in the medial medullary reticular formation in sleep-wake states, the distribution and projections of cholinergic, GABAergic and serotonergic neurons were studied within the lower brainstem of the cat. Cells were plotted with the aid of an image analysis system through the medullary reticular formation and raphe in adjacent sections immunostained for choline acetyltransferase, glutamic acid decarboxylase and serotonin. Immunostained fibres and varicosities were examined and quantified by microdensitometry in regions of the medulla, pons and upper spinal cord in normal and quisqualate-injected animals to assess the loss of local and distant projections following cytotoxic destruction of neurons in the medial medullary reticular formation. Choline acetyltransferase-immunoreactive neurons are unevenly and sparsely distributed, though none the less in significant numbers (estimated at approximately 9080 in total), through the medial medullary reticular formation, and are present in all tegmental fields, including the gigantocellular (approximately 3700) and magnocellular (approximately 1760) rostrally and the ventral (approximately 3240) and paramedian (approximately 380) caudally, and are absent in the midline raphe. Glutamic acid decarboxylase-immunoreactive neurons are more evenly and densely distributed in large numbers (estimated at approximately 18,720) through the medial medullary reticular formation, being present in the gigantocellular (approximately 5960), magnocellular (approximately 8260), ventral (approximately 2280) and paramedian (approximately 2220) tegmental fields, and are also numerous within the raphe magnus and pallidus-obscurus nuclei (approximately 3880). Serotonin-immunoreactive cells are sparse in the medial medullary reticular formation (estimated to total approximately 1540), where they are mainly located in the magnocellular tegmental field (approximately 1340), and are concentrated in larger numbers within the raphe nuclei (approximately 8060). Cholinergic varicose fibres were moderately densely distributed through the medial medullary reticular formation, as well as through more distant lateral, rostral and caudal brainstem and upper spinal regions. After cytotoxic lesions focussed in the gigantocellular and magnocellular tegmental fields, a loss of approximately 55% of the cholinergic neurons in the medial medullary reticular formation was associated with a minor decrease (approximately 35% in optical density measures) of local cholinergic fibres. Small and variable reductions in varicose fibres (and their optical density measures) were detected in distant structures (including the pontine lateral, gigantocellular and subcoerular tegmental fields and the caudal spinal trigeminal nucleus), that were none the less correlated with the number of intact medial medullary cholinergic cells, suggesting that these cells may project to distant brainstem targets, in addition to providing a minor proportion of the local cholinergic innervation of the medial medullary reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)