The sections were subsequently washed four times for 10 min each in PBS, mounted on microscope slides in Vectorshield mounting medium (Vector Laboratories, Burlingame, CA), and viewed in a Zeiss (Goettingen, Germany) LSM 510 confocal microscope using multitracking protocols to prevent fluorochrome cross talk. 7 nAChRs were localized in unfixed coronal VTA slices using either biotinylated Bgt (Molecular Probes) or 1.4 nm gold-conjugated Bgt (custom synthesized by Molecular Probes) (Jones et al., 2004). of dopaminergic and nondopaminergic neurons and glutamatergic and nonglutamatergic terminals. There was no detectable 7 nAChR expression within astrocytes in the VTA. Most 7 nAChRs were cytoplasmic (82%), and the remainder were associated with the plasma membrane. Most presynaptic receptors (75%) were on glutamatergic axon terminals, with comparable levels of -bungarotoxin binding present on both VGluT1- and VGluT2-immunoreactive boutons. Both preembedding and postembedding electron microscopy revealed that presynaptic 7 nAChRs are often located at extrasynaptic (27%) and perisynaptic (61%) loci. 7 nAChRs were Darifenacin not associated with cholinergic synapses, consistent with their activation by a paracrine mode of acetylcholine or choline delivery. microdialysis studies have suggested that systemic nicotine increases dopamine release in the NAcc by enhancing this excitatory input to the VTA via local 7 nicotinic acetylcholine receptors (nAChRs) (Schilstrom et al., 1998a,b). The demonstration that, in midbrain slice preparations, pairing presynaptic application of nicotine with postsynaptic depolarization results in NMDA receptor-dependent long-term potentiation (LTP) in VTA dopaminergic neurons (Mansvelder and McGehee, 2000) has led to the presumption that 7 nAChRs reside on glutamatergic terminals in the VTA, in which they may participate in synaptic changes that contribute to long-term enhancement of excitation of Darifenacin the brain reward system. 7 nAChRs are pentameric ligand-gated cation channels distinguished by a high relative permeability to Ca2+ (Seguela et al., 1993; Fucile, 2004), enabling 7 nAChRs Darifenacin to exert diverse modulatory influences via Darifenacin Ca2+-dependent cellular mechanisms (Dajas-Bailador and Wonnacott, 2004). 7 nAChRs have been implicated in the modulation of glutamate release in several brain regions, in addition to the VTA (Engelman and MacDermott, 2004), including the hippocampus (Gray et al., 1996), olfactory bulb (Alkondon et al., 1996), and sensory cortex (Aramakis and Metherate, 1998). In these studies, the Ca2+ dependence and tetrodotoxin insensitivity of nicotinic mechanisms implied that 7 nAChRs are located on glutamatergic axon terminals. In addition to their involvement in nicotine dependence, nAChRs in the VTA may also mediate a cholinergic component of addictive behavior in response to other drugs of abuse (Schoffelmeer et al., 2002; Fagen IFN-alphaA et al., 2003). Cholinergic inputs to the VTA arise from your lateral dorsal and pedunculopontine tegmental nuclei and synapse directly onto the somatodendritic membranes of local GABAergic and dopaminergic neurons (Oakman et al., 1995; Garzon et al., 1999). The elucidation of the relationship between these cholinergic terminals and presynaptic nAChRs is usually of important importance to understanding the mechanisms of cholinergic nicotinic signaling within the VTA, particularly because choline can also activate and/or desensitize 7 nAChRs (Alkondon et al., 1997). The specificity of current anti-7 subunit antibodies used to localize these receptors has been questioned recently (Herber et al., 2004). Therefore, we have developed an alternative strategy, based on the 7 nAChR-specific antagonist -bungarotoxin (Bgt), which targets put together 7 nAChRs. Using fluorescent, biotin, and platinum conjugates of Bgt, the aims of this study were (1) to Darifenacin define the precise subcellular localization of 7 nAChRs in the rat VTA and (2) to examine the spatial relationship between 7 nAChRs and cholinergic and glutamatergic inputs to the VTA. Materials and Methods All experiments were performed under a United Kingdom Home Office license and in accordance with the regulations of the United Kingdom Animals (Scientific Procedures) Take action of 1986. Efforts were made to minimize animal suffering and to use only the minimum quantity of animals required. All reagents were obtained from VWR International (West Chester, PA) unless stated normally. Adult male Sprague Dawley rats (300 gm; = 5) were deeply anesthetized with chloral hydrate (350 mg/kg, i.p.) and perfused transcardially with 30 ml of 0.1 m phosphate buffer (PB), pH 7.4, followed by 150.
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