Spatial and intracellular relationships between the α7 nicotinic acetylcholine receptor and the vesicular acetylcholine transporter in the prefrontal cortex of rat and mouse

Abstract

The alpha-7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the α7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the α7nAChR and VAChT. In both species, the α7nAChR-immunoreactivity (-ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable α7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of α7nAChR labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n = 240). Of the α7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic α7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed α7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the α7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.

Dopamine D2 and acetylcholine α7 nicotinic receptors have subcellular distributions favoring mediation of convergent signaling in the mouse ventral tegmental area

“…The specificity of these antisera was described previously (Pickel et al, 2006; Duffy et al, 2009, 2011) and that of the D 2 R further confirmed in the dorsal striatum of MSN-D2R KO. Each receptor had a similar subcellular location when detected with either the immunogold or immunoperoxidase method, and reversal of markers assured that each antigen was optimally identified by methods having different sensitivities and resolution.…”

“…However, in dendrites smaller than 0.5-μm diameter and in small unmyelinated axons a single particle was considered positive immunogold-labeling, since almost no gold–silver deposits were seen over myelin and other tissue elements not known to express the receptors under examination. The validity of this approach was established previously for other receptors (Garzón et al, 1999; Garzón and Pickel, 2006) and has also been used for α7nAChR and D 2 R in the cerebral cortex (Duffy et al, 2009, 2011). In addition, however, we examined the number of gold particles over dendritic and axonal profiles in control experiments in which the tissue was processed for dual immunolabeling with the omission of one of the respective primary antisera to assess the level of background spurious gold–silver particles.…”

“…Epitope mapping has shown that mAb306 recognizes the sequence corresponding to the cytoplasmic loop residues 380–400 of the α7nAChR from rat and chicken (Schoepfer et al, 1990; McLane et al, 1992). This antibody was used previously to study the α7nAChR distribution, in (1) rat and mouse brain using bright field (Dominguez del Toro et al, 1994; Duffy et al, 2009) or electron microscopy (Fabian-Fine et al, 2001; Levy and Aoki, 2002; Duffy et al, 2009) and (2) macaque brain using fluorescent microscopy (Centeno et al, 2006). The α7nAChR antibody has been examined using Western blotting of cultured hippocampal neurons, mouse, rat and macaque brain extracts (Dominguez del Toro et al, 1994; Centeno et al, 2006; Arnaiz-Cot et al, 2008; Duffy et al, 2009).…”

Ultrastructure of Synapses in the Mammalian Brain

“…A host of other ligand-gated receptors and channels have been identified by pre-embedding methods, mainly concentrated along the spine plasma membrane, away from the PSD (but as noted above, these methods may fail to detect protein within the PSD), including dopamine and 5-HT receptors, nicotinic acetylcholine receptors, and EphA receptors (Miner et al 2000(Miner et al , 2003Riad et al 2000;Fabian-Fine et al 2001;Tremblay et al 2007;Duffy et al 2009). New data come from the "SDS-FRL" approach (sodium dodecyl sulfate-freeze-fracture replica labeling), in which freeze-fractured tissue is coated with an inert film of platinum and carbon (to protect proteins embedded in the membrane from subsequent digestion in hot SDS, which removes tissue from the back of the platinum-carbon replica) before immunolabeling (Fig.…”

Organization of the Basal Forebrain Cholinergic Projection System

“…Thus, cholinergic cells can promote cortical activation, which underlies arousal, sensory processing and attention. The effect of ACh released through BF cholinergic axons in the cortex is complex due to: (1) the differential regional and laminar distribution of cholinergic axons; (2) different effects on various neuronal types and cellular sites of action (cell bodies, dendrites, axons); (3) ACh effects are mediated by synaptic and volume transmission at different time scales; and (4) ACh acts through a variety of muscarinic and nicotinic receptors (Levey et al, 1991;Mrzljak et al, 1996;Palomero-Gallagher and Zilles, 2004;Disney et al, 2007;Duffy et al, 2009;Parikh et al, 2010;Howe et al, 2010;Bell et al, 2011;Bennett et al, 2012) that are expressed differentially in pyramidal cells and in the various interneurons (Yamasaki et al, 2010). It seems that the complex effects of ACh fall in line with each specific neuronal cell type's role in the information processing architecture of the cortex.…”