␣7 neuronal nicotinic acetylcholine receptors (nAChRs) constitute one of the predominant nAChR subtypes in the mammalian brain. Within the ventral tegmental area (VTA), nicotine application, paired with postsynaptic stimulation, contributes to a form of long-term potentiation, an effect attributed to presynaptic ␣7 nAChRs on glutamatergic afferents (Mansvelder and McGehee, 2000). The aim of this study was to examine the precise subcellular distribution of ␣7 nAChRs in the adult rat VTA to establish whether these receptors are indeed present on glutamatergic axon terminals and to determine their relationship with cholinergic afferents. The spatial relationship between ␣7 nAChRs, labeled using the ␣7 nAChR-specific antagonist ␣-bungarotoxin, and the local neurochemical environment was investigated by the application of multiple labeling strategies with antibodies against tyrosine hydroxylase, vesicular glutamate transporters (VGluTs), vesicular acetylcholine transporter, and glial fibrillary acidic protein. ␣7 nAChRs were localized at both somatodendritic and presynaptic loci within the VTA: on subpopulations 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 similar 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 not associated with cholinergic synapses, consistent with their activation by a paracrine mode of acetylcholine or choline delivery.
Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of antireceptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunitdirected antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the a3-, a4-, a7-, b2-, and b4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly
Nicotinic acetylcholine receptors (nAChR) are widely distributed in the central nervous system, where they exert a modulatory influence on synaptic transmission. For the striatum, pharmacological evidence supports the presence of presynaptic alpha3beta2* and alpha4beta2* nAChR that modulate dopamine release from nigrostriatal terminals. The objective of this study was to examine the precise subcellular distribution of the nAChR beta2 subunit in these neurones and its localisation at presynaptic sites. Double immunolabelling with tyrosine hydroxylase (TH) at the confocal level revealed that the cell bodies and axon terminals (synaptosomes) of nigrostriatal neurones were also immunoreactive for the nAChR beta2 subunit. Double-preembedding electron microscopy confirmed that beta2-immunogold labelling was enriched in TH-positive terminals in the dorsal striatum. Quantitative analysis of doubly immunogold-labelled sections in postembedding electron microscopy showed that 86% of TH-positive axonal boutons are also labelled for the nAChR beta2 subunit, whereas 45% of beta2 subunit-immunolabeled boutons do not contain TH. Thus the beta2 subunit is localised within at least two populations of axon terminals in the dorsal striatum. In these structures, 15% of beta2 subunit immunoreactivity was at the plasma membrane but was rarely associated with synapses. These findings are compatible with functional presynaptic beta2-containing nAChR that may be stimulated physiologically by acetylcholine that diffuses from synaptic or nonsynaptic sites of acetylcholine release. These results demonstrate the presynaptic localisation of an nAChR subunit in nigrostriatal dopaminergic neurones, providing morphological evidence for the presynaptic nicotinic modulation of dopamine release.
Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.
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