Cholinergic Innervation of Pyramidal Cells and Parvalbumin-Immunoreactive Interneurons in the Rat Basolateral Amygdala

The basal forebrain contains an array of cholinergic neurons that extends through a continuous region that includes the medial septal area, diagonal band, ventral pallidum, and substantia innominata. Different portions of this complex have connections with different forebrain regions, including the hippocampus, neocortex, and basolateral nuclear complex of the amygdala (BLC; Mesulam et al., 1983a,b; Zaborszky et al., 1999). The BLC in the rat, monkey, and human receives an especially dense cholinergic innervation from the ventral pallidum and substantia innominata, which is significantly reduced in Alzheimer’s disease (Mesulam et al., 1983a,b; Carlsen et al., 1985; Carlsen and Heimer 1986; Amaral and Bassett, 1989; Kordower et al., 1989; Emre et al., 1993). In fact, it has been suggested that the degeneration of the cholinergic projections to the amygdala in Alzheimer’s disease may be more important for the memory disturbances seen in this disorder than the cholinergic projections to the cortex (Power et al., 2003). Experiments in rats have demonstrated that cholinergic afferents to one specific BLC nucleus, the anterior subdivision of the basolateral nucleus (BLa), are primary mediators of the neuromodulation involved in memory consolidation of emotionally arousing experiences by the amygdala (McGaugh, 2004). Cholinergic projections to the BLC have also been implicated in fear conditioning (Vazdarjanova and McGaugh, 1999), reward devaluation learning (Salinas et al., 1997), conditioned place preference (McIntyre et al., 2002), and conditioned cue reinstatement of drug seeking (See, 2005). Knowledge of the cholinergic innervation of specific cell types in the BLC is critical for understanding the physiology and pathophysiology of these important inputs. Previous studies have shown that there are two major cell classes in the BLC, pyramidal neurons and non-pyramidal neurons. Although these cells do not exhibit a laminar or columnar organization, their morphology, synaptology, electrophysiology, and pharmacology are remarkably similar to those of their counterparts in the cerebral cortex (McDonald, 1982, 1984, 1992a,b; Carlsen and Heimer, 1988; Washburn and Moises, 1992; Rainnie et al., 1993; Pare, 2003; Sah et al., 2003; Muller et al., 2005, 2006, 2007). Thus, pyramidal neurons in the BLC are projection neurons with spiny dendrites that utilize glutamate as an excitatory neurotransmitter, whereas most nonpyramidal neurons are spine-sparse interneurons that utilize GABA as an inhibitory neurotransmitter. Recent dual-labeling immunohistochemical studies suggest that the BLC contains at least four distinct subpopulations of GABAergic interneurons that can be distinguished on the basis of their content of calcium-binding proteins and peptides. These subpopulations are: 1) parvalbumin+/calbindin+ neurons; 2) somatostatin+/calbindin+ neurons; 3) small bipolar and bitufted inter-neurons that exhibit extensive colocalization of vasoactive intestinal peptide, calretinin, and cholecystokinin; and 4) large multipolar cholecystokinin+ neurons that are often calbindin+ (Kemppainen and Pitkanen, 2000; McDonald and Betette, 2001; McDonald and Mascagni, 2001, 2002, Mascagni and McDonald, 2003). There is evidence from electrophysiological studies that basal forebrain cholinergic inputs activate both pyramidal projection neurons and GABAergic interneurons in the BLa by both muscarinic (Washburn and Moises, 1992; Yajeya et al., 1997; Pape et al., 2005; Power and Sah, 2008) and nicotinic (Zhu et al., 2005; Klein and Yakel, 2006) receptor-mediated mechanisms. Consistent with these findings, ultrastructural analyses of the BLa revealed synaptic contacts of cholinergic (i.e., choline acetyltransferase-positive) axons with both major neuronal classes (Carlsen and Heimer, 1986; Nitecka and Frotscher, 1989). Carlsen and Heimer (1986) found that some cholinergic axons formed synapses with cell bodies of BLa pyramidal neurons that were identified either morphologically or by virtue of their labeling by injections of retrograde tracers into the ventral striatum. However, most synaptic contacts were seen with unlabeled dendritic shafts and to a lesser extent with dendritic spines (Carlsen and Heimer, 1986). Cholinergic synaptic inputs were also seen targeting BLa interneurons that were identified either morphologically (Carlsen and Heimer, 1986) or by virtue of their immunohistochemical labeling for GAD (Nitecka and Frotscher, 1989) or choline acetyltransferase (Carlsen and Heimer, 1986). Because neither of these two ultrastructural analyses used quantitative methods, and because neither labeled the great majority of dendritic shafts, the major targets of cholinergic synaptic inputs to the BLa, there is currently no information regarding the postsynaptic targets of the majority of cholinergic inputs to this brain region. The present dual-labeling EM study addressed this issue by using antibodies to the vesicular acetylcholine transporter (VAChT) to label cholinergic axons (Weihe et al., 1996; Gilmor et al., 1996) and antibodies to cell-specific markers to identify different neuronal subpopulations in the BLa. An antibody to the alpha subunit of calcium/calmodulin kinase II (CaMK) was used to label pyramidal cell perikarya and dendrites (McDonald et al., 2002), and an antibody to parvalbumin (PV) was used to label this important subpopulation of interneurons. PV+ interneurons are the predominant subpopulation in the BLa, making up approximately 40% of all interneurons (McDonald and Mascagni, 2001; Mascagni and McDonald, 2003). In addition, the nonaccommodating, rapid firing pattern of many PV+ neurons (Rainnie et al., 2006; Woodruff and Sah, 2007a) indicates that they correspond to many of the interneurons identified in previous electrophysiological studies of cholinergic activation of the BLa (Washburn and Moises, 1992; Zhu et al., 2005).