GABAergic input to cholinergic forebrain neurons: an ultrastructural study using retrograde tracing of HRP and double immunolabeling

Amygdalopetal cholinergic neurons in the ventral pallidum were identified by combining choline acetyltransferase (ChAT) immunohistochemistry with retrograde tracing of horseradish peroxidase (HRP) following injections of the tracer in the basolateral amygdaloid nucleus. Although ChAT-positive terminals were identified in the ventral pallidum, they were never seen in contact with either immunonegative or ChAT-positive amygdalopetal neurons. In material, in which immunostaining against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA was combined with retrograde tracing of HRP from the basolateral amygdaloid nucleus, GAD-positive terminals were seen to contact immunonegative amygdalopetal neurons. In addition, when sections of the rostral forebrain were processed, first to preserve and identify the transported HRP, and then were sequentially tested for both ChAT and GAD immunohistochemistry with the immunoperoxidase reaction for both tissue antigens, GAD-immunopositive terminals were seen to make synaptic contacts with cholinergic amygdalopetal neurons. These results suggest that amygdalopetal, presumably cholinergic, neurons receive GAD-positive terminals. In separate experiments using immunoperoxidase for ChAT and ferritin-avidin for GAD labeling, we confirmed the presence of GAD-containing terminals on cholinergic neurons. In addition, cholinergic terminals were seen in synaptic contact with GAD-positive cell bodies. These morphological studies suggest that direct GABAergic-cholinergic and cholinergic-GABAergic interactions take place in the rostral forebrain.