A role of tyrosine phosphatase in acetylcholine receptor cluster dispersal and formation

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor-coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot-associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.     

Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.     

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.     

Disassembly of the cholinergic postsynaptic apparatus induced by axotomy in mouse sympathetic neurons: the loss of dystrophin and beta-dystroglycan immunoreactivity precedes that of the acetylcholine receptor

In mouse sympathetic superior cervical ganglion (SCG), cortical cytoskeletal proteins such as dystrophin (Dys) and beta1sigma2 spectrin colocalize with beta-dystroglycan (beta-DG), a transmembrane dystrophin-associated protein, and the acetylcholine receptor (AChR) at the postsynaptic specialization. The function of the dystrophin-dystroglycan complex in the organization of the neuronal cholinergic postsynaptic apparatus was studied following changes in the immunoreactivity of these proteins during the disassembly and subsequent reassembly of the postsynaptic specializations induced by axotomy of the ganglionic neurons. After axotomy, a decrease in the number of intraganglionic synapses was observed (t1/2 8 h 45'), preceded by a rapid decline of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3 AChR subunit (alpha3AChR) (t1/2 3 h 45', 4 h 30' and 6 h, respectively). In contrast, the percentage of postsynaptic densities immunopositive for beta1sigma2 spectrin remained unaltered. When the axotomized neurons began to regenerate their axons, the number of intraganglionic synapses increased, as did that of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3AChR. The latter number increased more slowly than that of Dys and beta-DG. These observations suggest that in SCG neurons, the dystrophin-dystroglycan complex might play a role in the assembly-disassembly of the postsynaptic apparatus, and is probably involved in the stabilization of AChR clusters.     

Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling

Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.     

Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK

Nerve-induced clustering of the nicotinic acetylcholine receptor (AChR) requires rapsyn, a synaptic peripheral membrane protein, as well as protein-tyrosine kinase activity. Here, we show that rapsyn induces the clustering of the synapse-specific receptor-tyrosine kinase MuSK in transfected QT-6 fibroblasts. Furthermore, rapsyn stimulates the autophosphorylation of MuSK, leading to a subsequent MuSK-dependent increase in cellular tyrosine phosphorylation. Moreover, rapsyn-activated MuSK specifically phosphorylated the AChR beta subunit, the same subunit that is tyrosine phosphorylated during innervation or agrin treatment of muscle. These results suggest rapsyn may mediate the synaptic localization of MuSK in muscle and that MuSK may play an important role in the agrin-induced clustering of the AChR.     

Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse

Tyrosine phosphorylation has been shown to be an important modulator of synaptic transmission in both vertebrates and invertebrates. Such findings hint toward the existence of extracellular ligands capable of activating this widely represented signaling mechanism at or close to the synapse. Examples of such ligands are the peptide growth factors which, on binding, activate receptor tyrosine kinases. To gain insight into the physiological consequences of receptor tyrosine kinase activation in squid giant synapse, a series of growth factors was tested in this preparation. Electrophysiological, pharmacological, and biochemical analysis demonstrated that nerve growth factor (NGF) triggers an acute and specific reduction of the postsynaptic potential amplitude, without affecting the presynaptic spike generation or presynaptic calcium current. The NGF target is localized at a postsynaptic site and involves a new TrkA-like receptor. The squid receptor crossreacts with antibodies generated against mammalian TrkA, is tyrosine phosphorylated in response to NGF stimulation, and is blocked by specific pharmacological inhibitors. The modulation described emphasizes the important role of growth factors on invertebrate synaptic transmission.     

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

Intracellular calcium regulates agrin-induced acetylcholine receptor clustering

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.     

Neurotrophins regulate agrin-induced postsynaptic differentiation

The precise orchestration of synaptic differentiation is critical for efficient information exchange in the nervous system. The nerve-muscle synapse forms in response to agrin, which is secreted from the motor nerve terminal and induces the clustering of acetylcholine receptors (AChRs) and other elements of the postsynaptic apparatus on the subjacent muscle cell surface. In view of the highly restricted spatial localization and the plasticity of neuromuscular junctions, it seems likely that synapse formation and maintenance are regulated by additional, as-yet-unidentified factors. Here, we tested whether neurotrophins modulate the agrin-induced differentiation of postsynaptic specializations. We show that both brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) inhibit agrin-induced AChR clustering on cultured myotubes. Nerve growth factor and NT-3 are without effect. Muscle cells express full-length TrkB, the cognate receptor for BDNF and NT-4. Direct activation of this receptor by anti-TrkB antibodies mimicked the BDNF/NT-4 inhibition of agrin-induced AChR clustering. This BDNF/NT-4 inhibition is likely to be an intrinsic mechanism for regulating AChR clustering, because neutralization of endogenous TrkB ligands resulted in elevated levels of AChR clustering even in the absence of added agrin. Finally, high concentrations of agrin can occlude the BDNF/NT-4 inhibition of AChR clustering. These results indicate that an interplay between agrin and neurotrophins can regulate the formation of postsynaptic specializations. They also suggest a mechanism for the suppression of postsynaptic specializations at nonjunctional regions.     

Differential localization of delta glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses

The delta 2 glutamate receptors are prominently expressed in Purkinje cells and are thought to play a key role in the induction of cerebellar long-term depression. The synaptic and subsynaptic localization of delta receptors in rat cerebellar cortex was investigated with sensitive and high-resolution immunogold procedures. After postembedding incubation with an antibody raised to a C-terminal peptide of delta 2, high gold particle densities occurred in all parallel fiber synapses with Purkinje cell dendritic spines, whereas other synapses were consistently devoid of labeling. Among the types of immunonegative synapse were climbing fiber synapses with spines and parallel fiber synapses with dendritic stems of interneurons. At the parallel fiber-spine synapse, gold particles signaling delta receptors were restricted to the postsynaptic specialization. By the use of double labeling with two different gold particle sizes, it was shown that delta and AMPA GluR2/3 receptors were colocalized along the entire extent of the postsynaptic specialization without forming separate domains. The distribution of gold particles representing delta receptors was consistent with a cytoplasmic localization of the C terminus and an absence of a significant presynaptic pool of receptor molecules. The present data suggest that the delta 2 receptors are targeted selectively to a subset of Purkinje cell spines and that they are coexpressed with ionotropic receptors in the postsynaptic specialization. This arrangement could allow for a direct interaction between the two classes of receptor.     

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.     

Association of phosphotyrosine with rapsyn expression in Xenopus embryonic cells

The postsynaptic membrane of the neuromuscular junction is highly enriched in rapsyn, which is thought to interact directly with nicotinic acetylcholine receptors (AChR) and anchor them at the synapse. We expressed rapsyn with or without AChRs in Xenopus embryos by mRNA injection. Co-expression of AChR and rapsyn caused the clustering of these two proteins in cultured cells isolated from the injected embryos. When rapsyn was expressed alone, it also became clustered at the substratum-facing membrane in cultured cells and at cell-cell contacts in whole mount embryos. No clusters were observed in cells that expressed AChRs alone. In rapsyn-expressing cells, proteins that are tyrosine phosphorylated as shown by anti-phosphotyrosine antibody labeling were concentrated at rapsyn clusters. Rapsyn itself does not appear to be a substrate for tyrosine kinase. This suggests that other phosphotyrosine-containing proteins are co-clustered with rapsyn in these cells.     

The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development

Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.     

Axon withdrawal during synapse elimination at the neuromuscular junction is accompanied by disassembly of the postsynaptic specialization and withdrawal of Schwann cell processes

Nerve terminal withdrawal is accompanied by a loss of acetylcholine receptors (AChRs) at corresponding postsynaptic sites during the process of synapse elimination at developing () and reinnervated adult () neuromuscular junctions. Aside from AChR and nerve terminal loss, however, the molecular and cellular alterations that occur at sites of elimination are unknown. To gain a better understanding of the cascade of events that leads to the disassembly of synaptic sites during the synapse elimination process, we surveyed the distribution of molecular elements of the postsynaptic specialization, the basal lamina, and supporting Schwann cells during the process of synapse elimination that occurs after reinnervation. In addition, quantitative techniques were used to determine the temporal order of disappearance of molecules that were lost relative to the loss of postsynaptic AChRs. We found that the dismantling of the postsynaptic specialization was inhomogeneous, with evidence of rapid dissolution of some aspects of the postsynaptic apparatus and slower loss of others. We also observed a loss of Schwann cell processes from sites of synapse elimination, with a time course similar to that seen for nerve terminal retraction. In contrast, all of the extracellular markers that we examined were lost slowly from sites of synapse loss. We therefore conclude that the synapse elimination process is synapse-wide, removing not only nerve terminals but also Schwann cells and many aspects of the postsynaptic apparatus. The disassembly occurs in a stereotyped sequence with some synaptic elements appearing much more stable than others.     

mu-Opioids activate tyrosine kinase focal adhesion kinase and regulate cortical cytoskeleton proteins cortactin and vinculin in chick embryonic neurons

We have investigated the signal transduction pathway of the G-protein mu-opioid receptor upstream of phospholipase D (PLD) and protein kinase C-epsilon (PKC-epsilon) activation in postmitotic E6CH chick embryo cortical neurons. The mu-opioid receptor and PLD-PKC-epsilon functional coupling depends on upstream tyrosine kinase activation. We now report that the mu-opioid agonists specifically stimulated tyrosine phosphorylation and activation of the focal adhesion kinase (FAK) in a time-dependent manner. We also demonstrate that met-enkephalin, a mu-opioid agonist in E6CH cultures, significantly increases tyrosine phosphorylation of another Src kinase substrate, the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin led to drastic changes in subcellular localization, an estimated 2-fold enrichment in the cytosol. Similarly, opioids stimulated a sustained tyrosine phosphorylation of vinculin, a protein enriched in focal adhesion sites. These data provide novel evidence that opioid receptor intracellular signaling engages the specific activation of tyrosine kinase FAK and regulates the neuronal cytoskeleton during central nervous system morphogenesis.     

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.     

Postsynaptic nicotinic receptors on dopaminergic neurons in the substantia nigra pars compacta of the rat

Previous studies have shown that application of nicotinic agonists in the substantia nigra pars compacta increases the firing rate of dopaminergic neurons. We have used intracellular recordings to show that the response of these neurons to nicotine is postsynaptic, since it persists in the presence of low-calcium buffer containing tetrodotoxin. Burst firing in the presence of nicotine was not observed. The presence of postsynaptic nicotinic receptors was confirmed by immunohistochemical localization of the alpha4 nicotinic receptor subunit on dendrites in the substantia nigra pars compacta. The majority of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta were also immunopositive for the alpha4 subunit. Immunohistochemical localization of the alpha4 and beta2 subunits in adjacent brain sections produced similar patterns of staining. Electron micrographs clearly indicated the presence of alpha4 subunit at postsynaptic densities. The predominant role of nicotinic receptors in the central nervous system has been suggested to be the presynaptic modulation of neurotransmitter release [McGehee D. S. and Role L. W. (1995) A. Rev. Physiol. 57, 521-546]. Although several postsynaptic nicotinic responses have also been reported in the literature, it is unclear as to whether the postsynaptic nicotinic receptors mediating responses to exogenously applied agonists are involved in synaptic transmission. From our electrophysiological and immunohistochemical results, we conclude that alpha4-containing nicotinic receptors are found at synapses on dopaminergic neurons. These synapses are similar to the cholinergic synapses described at these neurons, suggesting that nicotinic receptors are important in modulating the excitability of dopaminergic neurons by direct synaptic transmission.