Elsevier

Neuroscience

Volume 107, Issue 2, 16 November 2001, Pages 249-263
Neuroscience

Evidence for glutamate, in addition to acetylcholine and GABA, neurotransmitter synthesis in basal forebrain neurons projecting to the entorhinal cortex

https://doi.org/10.1016/S0306-4522(01)00302-5Get rights and content

Abstract

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 (∼80%) of the retrogradely labeled cells was found to be immunopositive for phosphate-activated glutaminase. Equal minor proportions (∼40%) were immunopositive for choline acetyltransferase and glutamic acid decarboxylase. In other material dual-immunostained for neurotransmitter enzymes, ∼95% of choline acetyltransferase- and ∼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.

Section snippets

Experimental procedures

Twenty-four male Wistar rats (Charles River Canada, St. Constant, QC, Canada) weighing between 200 and 250 g were employed in these studies. All procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. Surgery was performed under barbiturate anesthesia (somnotol, 50–65 mg/kg, i.p.). In 17 rats, injections of CT followed by colchicine treatment were performed, and in five rats colchicine treatment alone was used. In the first operation, 100

PAG immunostaining in basal forebrain neurons

Viewed in immunofluorescent stained material, many cells in the basal forebrain appeared to be immunoreactive for PAG (Fig. 1A). The PAG immunostaining was granular and present within the soma and proximal dendrites of neurons. Punctate immunostaining was also apparent in the neuropil where it was presumably contained within nerve varicosities or terminals. The staining appeared to vary in intensity over neuronal cell bodies. As evident at high magnification, unambiguously PAG-immunostained

Discussion

The present results reveal that a major proportion of basal forebrain neurons projecting to the entorhinal cortex contain the enzyme PAG that is responsible for the synthesis of the transmitter pool of glutamate. The cortically projecting neurons containing PAG were co-distributed with those containing ChAT or GAD across the cholinergic cell nuclei of the basal forebrain. In other material, a vast majority of ChAT-immunopositive and a significant proportion of GAD-immunopositive neurons were

Acknowledgements

This research was supported by the Canadian Medical Research Council (MRC, MT-13458), and the National Institute of Mental Health (NIMH, RO1 MH60119-01A1). I.D.M. held a graduate student fellowship from the Canadian Natural Science and Engineering Research Council (NSERC). We thank Angel Alonso for his comments on the manuscript. We are most grateful to Dr. Takeshi Kaneko (Kyoto, Japan) for generously supplying the antibody to PAG.

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