Spatial and intracellular relationships between the alpha7 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 (alpha7nAChR) 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 alpha7nAChR 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 alpha7nAChR and VAChT. In both species, the alpha7nAChR-immunoreactivity ((-)ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable alpha7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of alpha7nAChR-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 alpha7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic alpha7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed alpha7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the alpha7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.

Fig. 1

Light micrographs showing coronal hemisections through (A) rat and (B) mouse PFC. These sections were cut with a vibratome at rostrocaudal levels defined anterior to Bregma as +2.80 mm in rat (Paxinos and Watson, 1986) and +2.20 mm in mouse (Paxinos and Franklin, 2001) from the mouse. The tissue was processed for immunoperoxidase labeling of the α7nAChR. The trapezoids show the portion of the PFC that was sampled for electron microscopy. Arrows indicate the dorsal (D) and medial (M) brain surfaces. ac = anterior commissure, cc = corpus callosum, pu = identifying hole punched in mouse. Scale bars = 500 μm.

Fig. 2

Western blots for characterization of the antibody against the α7nAChR using cortical protein lysates from (A) rat and (B) mouse. A single dominant band is detected in both the rat and mouse near the 50 kDa molecular weight marker which is comparable with the 54 kDa molecular weight of the α7nAChR (Seguela et al., 1993) indicating the specificity of the antibody to the α7nAChR.

Fig. 3

Light micrographs showing the regional distribution of immunoperoxidase labeling for the α7nAChR in coronal sections through the PFC in the (A and B) rat (+2.8 mm from Bregma) and (C and D) mouse (+2.2 mm from Bregma). The α7nAChR labeling is seen in cells throughout layers I–VI of the PFC. The majority of the somatic α7nAChR labeling (α7-s) is in layer III–V, the area sampled for electron microscopic analysis and enlarged in (B) and (D). Arrows indicate the dorsal (D) and medial (M) brain surfaces. bv = blood vessel, cc = corpus callosum. Scale bars = 50 μm.

Fig. 4

Electron micrographs showing the presynaptic location of the α7nAChR and VAChT in (A) single-labeled and (B) dual-labeled rat PFC. (A) Immunoperoxidase labeling (block arrow) of the α7nAChR is seen along the presynaptic membrane of an axon terminal (α7-ax) forming an asymmetric synapse (curved arrow) with an unlabeled dendritic spine (u-sp). (B) Immunoperoxidase labeling for the VAChT in vesicle filled axonal profiles, one of which shows immunogold labeling (small arrows) for the α7nAChR (α7V-ax). This terminal is apposed to a somata (α7-s) showing a single immunogold particle on the plasma membrane in contact with the dual-labeled terminal. u-t = unlabeled terminal. Scale bars = 500 nm.

Fig. 5

Electron microscopic immunoperoxidase and immunogold labeling for the α7nAChR in the cytoplasm of neuronal somata (α7-s) in (A) single-labeled rat and (B) dual-labeled mouse PFC. (A) The α7nAChR immunoperoxidase labeling is in the perinuclear cytoplasm of the soma (α7-s). A α7nAChR-labeled dendrite (α7-d) is also seen within the adjacent neuropil. (B) Immunogold labeling of the α7nAChR has a prominent location in the perinuclear cytoplasm, where the immunogold particles are associated with outer Golgi lamella (G). Immunoperoxidase-labeling for VAChT is in an axonal profile (V-ax) apposing the α7-s. mit = mitochondria, nu = nucleus, u-sp = unlabeled spine, u-t = unlabeled terminal. Scale bars = 500 nm.

Fig. 6

Single immunogold (A) and immunoperoxidase labeling (B) of the α7nAChR in dendritic spines in electron micrographs from mouse and rat PFC (A and B, respectively). (A) An α7nAChR-labeled dendrite (α7-d) with a spine (α7-sp) that has a single gold particle (small arrows) identifying α7nAChR. The spine receives an asymmetric synapse (curved arrow) from an unlabeled axon terminal (u-t). The peroxidase density can be compared with that seen in other spines that are unlabeled (u-sp) and also received asymmetric synapses from unlabeled terminals. (u-t). Scale bars = 500 nm.

Fig. 7

Dendritic distribution of α7nAChR immunogold labeling in relation to axon terminals containing the VAChT as seen by dense immunoperoxidase reaction product in rat (A) and mouse (B) PFC. (A) A large longitudinally oriented dendrite with cytoplasmic α7nAChR immunogold labeling (α7-d, black arrows), is seen in contact with a VAChT-labeled axon (V-ax; white arrow), < 0.2 μm (double head arrow) from a V-ax and third V-ax is > 0.2 μm from the α7-d. In the (B) mouse, longitudinally orientated α7-d, in which the immunogold particles are associated with endomembranes (em) near a VAChT-immunoperoxidase labeled axon terminal (V-ax). Scale bars = 500 nm.

Fig. 8

Schematic diagram showing the subcellular distributions of the α7nAChR in relation to the VAChT in the rodent prefrontal cortex (PFC). (A) The α7nAChR (black chevrons) is shown as having a presynaptic distribution in axon terminals (at), forming excitatory (+) axospinous synapses and co-localized in VAChT-labeled (black circles) axonal processes, in the vicinity of α7nAChR-labeled spines (sp). (B) The α7nAChR has a somatodendritic distribution in dendrites (d) and somata (s), where it is attached to endomembranes (em), Golgi lamella (G) and on plasmalemmal membrane (purple line) of spiny neurons (light blue). nu = nucleus.