192IgG‐saporin immunotoxin‐induced loss of cholinergic cells differentially activates microglia in rat basal forebrain nuclei

Abstract

To characterize the specificity of a novel cholinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the low‐affinity nerve growth factor receptor with the cytotoxic protein saporin), coronal sections through the basal forebrain of adult rats, that received a single intracerebro‐ventricular injection of 4 pg of 192IgG‐saporin conjugate, were subjected to histochemical and immunocytochemical procedures to evaluate cholinergic (choline acetyltransferase (ChAT)‐immunoreactive, acetylcholinesterase‐positive, NADPH‐diaphorase‐positive) and GABAergic structures (parvalbumin‐immunoreactive, labeling of perineuronal nets with Wisteria floribunda agglutinin) as well as microglia (visualized with Griffonia simplicifolia agglutinin) and astrocytes (immunostaining for glial fibrillary acidic protein). Seven days following injection of the immunotoxin, ChAT‐immunoreactive cells nearly completely disappeared throughout the magnocellular basal forebrain complex, including globus pallidus, as compared to vehicle‐injected controls. However, there was no significant difference in the number of ChAT‐positive cells in the adjacent ventral pallidum and in the caudate‐putamen of immunolesioned and control animals. NADPH‐diaphorase‐containing cells, including a significant subpopulation of cholinergic cells, also strikingly decreased in number by more than 90% in the magnocellular basal forebrain complex following immunolesion, and only a few noncholinergic diaphorase‐positive cells survived in the medial septum, vertical and horizontal diagonal band, and nucleus basalis of Meynert. In contrast, the number of parvalbumin‐containing GABAergic projection neurons in the septum‐diagonal band of Broca complex and nucleus basalis of Meynert from immunolesioned rats was not different from that of vehicle‐injected control animals. Immunolesioning also did not result in any change in either number or shape of cells surrounded by perineuronal nets, which are frequently associated with parvalbumin‐containing GABAergic neurons. Seven days following injection of the immunotoxin, a very strong activation of microglia with an identical distribution pattern was observed in all experimental animals. Large numbers of activated microglia were found in all magnocellular basal forebrain nuclei, corresponding to the distribution of degenerating cholinergic cells. Additionally, immunolesioning also resulted in a dramatic activation of microglia in the lateral septal nuclei, which are known to be almost free of cholinergic cells, but not of penetrating cholinergic dendrites in adjacent zones, and in the ventral pallidum, where there was no observed loss of cholinergic cells. There was no significant increase in microglia activation in striatum and cortical areas, and no astrocytic response in any of the basal forebrain nuclei at this particular time point of survival. These results suggest that 192IgG‐saporin specifically destroys basal forebrain cholinergic neurons and does not suppress their neuronal activity. Therefore, 192IgG‐saporin represents a powerful tool for producing cortical cholinergic dysfunction.