Distribution of cholinergic, gabaergic and serotonergic neurons in the medial medullary reticular formation and their projections studied by cytotoxic lesions in the cat

Abstract

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 ∼9080 in total), through the medial medullary reticular formation, and are present in all tegmental fields, including the gigantocellular (∼3700) and magnocellular (∼1760) rostrally and the ventral (∼3240) and paramedian (∼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 ∼18,720) through the medial medullary reticular formation, being present in the gigantocellular (∼5960), magnocellular (∼8260), ventral (∼2280) and paramedian (∼2220) tegmental fields, and are also numerous within the raphe magnus and pallidus-obscurus nuclei (∼3880). Serotonin-immunoreactive cells are sparse in the medial medullary reticular formation (estimated to total ∼1540), where they are mainly located in the magnocellular tegmental field (∼1340), and are concentrated in larger numbers within the raphe nuclei (∼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 ∼55% of the cholinergic neurons in the medial medullary reticular formation was associated with a minor decrease (∼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. GABAergic terminals were highly densely distributed through the medullary reticular formation, as well as through distant brainstem and upper spinal cord regions. With destruction of$˛60% of the medial medullary GABAergic cells, there was a dramatic loss ($˛50% in optical density measures) of GABAergic terminals in the magnocellular and gigantocellular tegmental fields, that was correlated with the number of surviving GABAergic neurons in the region. No reduction of GABAergic varicosities was evident outside the zone of GABAergic cell destruction, suggesting that these cells provide primarily a local innervation to the medial medullary reticular formation and raphe nuclei. Destruction of a minority (∼30%) of the serotonergic cells that lie within the reticular formation (and variable related destruction of those within the midline raphe nuclei) was associated with reductions (≤50%) of the moderately dense local serotonergic varicose fibres and of the similarly dense distant fibres (in the medullary and pontine lateral tegmental fields, the pontine gigantocellular tegmental field, the subcoeruleus, the spinal trigeminal nucleus and ventral horn) that were correlated with the number of medullary serotonergic cells, establishing the existence of both local and distant projections of the serotonergic neurons. These results demonstrate the presence of a significant population of cholinergic neurons in the medial medullary reticular formation, which like medullary serotonergic neurons, provide a proportion of the local and distant brainstem and upper spinal cord innervation. In contrast, a large population of GABAergic neurons provides the major local GABAergic innervation to the medial medullary reticular formation and raphe, terminating around GABAergic as well as non-GABAergic cells, and may thus serve primarily to control, by disinhibition as well as inhibition, other projection neurons of the medullary reticular formation and raphe.