Protein scaffolds are essential for specific and efficient downstream signaling at synapses. Though nicotinic receptors are widely expressed in the nervous system and influence numerous cellular events due in part to their calcium permeability, no scaffolds have yet been identified for the receptors in neurons. Here we show that specific members of the PSD-95 family of PDZ-containing proteins are associated with specific nicotinic receptor subtypes. At postsynaptic sites, the PDZ scaffolds are essential for maturation of functional nicotinic synapses on neurons. They also help mediate downstream signaling as exemplified by activation of transcription factors. By tethering components to postsynaptic nicotinic receptors, PDZ scaffolds can organize synaptic structure and determine which calcium-dependent processes will be subject to nicotinic modulation.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the pontine nucleus locus coeruleus (LC). Previous studies indicate that norepinephrine (NE) modulates the strength of GABAergic inhibition in MOB. However, the nature of this modulation and the NE receptors involved remain controversial. The goal of this study was to investigate the role of NE receptor subtypes in modulating the GABAergic inhibition of mitral cells using patch-clamp electrophysiology in rat MOB slices. NE concentration dependently and bi-directionally modulated GABA(A) receptor-mediated spontaneous and miniature inhibitory postsynaptic currents (sIPSCs/mIPSCs) recorded in mitral cells. Low doses of NE suppressed sIPSCs and mIPSCs because of activation of alpha2 receptors. Intermediate concentrations of NE increased sIPSCs and mIPSCs primarily because of activation of alpha1 receptors. In contrast, activation of beta receptors increased sIPSCs but not mIPSCs. These results indicate that NE release regulates the strength of GABAergic inhibition of mitral cells depending on the NE receptor subtype activated. Functionally, the differing affinity of noradrenergic receptor subtypes seems to allow for dynamic modulation of GABAergic inhibition in MOB as function of the extracellular NE concentration, which in turn, is regulated by behavioral state.

Linster C, Nai Q, Ennis M. Nonlinear effects of noradrenergic modulation of olfactory bulb function in adult rodents.

The mammalian main olfactory bulb (MOB) receives a dense noradrenergic innervation from the pontine nucleus locus coeruleus that is important for neonatal odor preference learning and odor processing in mature animals. Modulation of GABAergic granule cells (GCs) is thought to play a key role in the net functional impact of norepinephrine (NE) release in the MOB, yet there are few direct studies of the influence of NE on these cells. In the present study we investigated noradrenergic modulation of GC excitability using electrophysiological approaches in rat MOB slices. A moderate concentration of NE (10 µM) and the α1 receptor agonist phenylephrine (10 µM) depolarized and increased spontaneous or current injection-evoked spiking in GCs. By contrast, low NE concentrations (0.1–1.0 µM) or the α2 receptor agonist clonidine (10 µM) hyperpolarized and decreased the discharge of GCs. The effects of NE (10 µM) were blocked by antagonism of α1 and α2 receptors. Inhibitory effects of low NE concentrations were blocked or converted to excitatory responses by α2 receptor blockade, whereas excitatory effects of the moderate NE concentration were converted to inhibitory responses after α1 receptor blockade. NE (10 µM) and phenylephrine elicited inward currents that reversed near the potassium equilibrium potential. The effects of NE and phenylephrine were associated with increased membrane input resistance. Clonidine elicited an outward current associated with decreased membrane input resistance that reversed near the potassium equilibrium potential. These results indicate that α1 and α2 receptor activation exert opposing effects on GC excitability. Low concentrations of NE acting via α2 receptors suppress GC excitability, while higher concentrations of NE acting at α1 receptors increase GC excitability. These findings are consistent with recent findings that α1 and α2 receptor activation increase and decrease, respectively, GABAergic inhibition of mitral cells. The differential affinities of α1 and α2 noradrenergic receptor subtypes may allow for differential modulation of GABA release and olfactory processing as a function of the level of NE release, which in turn, is regulated by behavioral state.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widespread, diverse ion channels involved in synaptic signaling, addiction, and disease. Despite their importance, the relationship between native nAChR subunit composition and function remains poorly defined. Chick ciliary ganglion neurons express two major nAChR types: those recognized by ␣-bungarotoxin (␣Bgt), nearly all of which contain only ␣7 subunits (␣7-nAChRs) and those insensitive to ␣Bgt, which contain ␣3, ␣5, ␤4, and, in some cases, ␤2 subunits (␣3*-nAChRs). We explored the relationship between nAChR composition and channel function using toxins recognizing ␣7 subunits (␣Bgt), and ␣3/␤4 (␣-conotoxin-AuIB), or ␣3/␤2 (␣-conotoxin-MII) subunit interfaces to perturb responses induced by nicotine, ␣7-, or ␣3-selective agonists (GTS-21 or epibatidine, respectively). Using these reagents, fast-decaying whole-cell current components were attributed solely to ␣7-nAChRs, and slow-decaying components mostly to ␣3*-nAChRs. In outside-out patches, nicotine activated brief 60-and 80-pS single nAChR channel events, and mixed-duration 25-and 40-pS nAChR events. Subsequently, 60-and 80-pS nAChR events and most brief 25-and 40-pS events were attributed to ␣7-nAChRs, and long 25-and 40-pS events to ␣3*-nAChRs. ␣3*-nAChRs lacking ␤2 subunits seemed responsible for long 25 pS nAChR events, whereas those containing ␤2 subunits mediated the long 40 pS nAChR events that dominate single-channel records. These results reveal greater functional heterogeneity for ␣7-nAChRs than previously expected and indicate that ␤2 subunits contribute importantly to ␣3*-nAChR function. By linking structural to functional nAChR subtypes, the findings also illustrate a useful pharmacological strategy for selectively targeting nAChRs.

Neuroligins are cell adhesion molecules that interact with neurexins on adjacent cells to promote glutamatergic and GABAergic synapse formation in culture. We show here that neuroligin enhances nicotinic synapses on neurons in culture, increasing synaptic input. When neuroligin is overexpressed in neurons, the extracellular domain induces presynaptic specializations in adjacent cholinergic neurons as visualized by SV2 puncta. The intracellular domain is required to translate the SV2 puncta into synaptic input as reflected by increases in the frequency of spontaneous mini-synaptic currents. The PDZ-binding motif of neuroligin is not needed for these effects. Together, the extracellular and proximal intracellular domains of neuroligin are sufficient to induce presynaptic specializations, align them over postsynaptic receptor clusters, and increase synaptic function. Manipulation of endogenous neuroligin with beta-neurexin-expressing cells confirms its presence; repressing function with dominant negative constructs and inhibitory shRNA shows that endogenous neuroligin helps confer functionality on existing nicotinic synaptic contacts. Endogenous neuroligin does not appear to be required, however, for initial formation of the contacts, suggesting that other components under these conditions can also initiate synapse formation. The results indicate that postsynaptic neuroligin is important for functional nicotinic synapses on neurons and that the effects achieved will likely depend on neuroligin levels.

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Parasympathetic neurons do not require neurotrophins for survival and are thought to lack high-affinity neurotrophin receptors (i.e., trks). We report here, however, that mRNAs encoding both brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (trkB) are expressed in the parasympathetic chick ciliary ganglion (CG) and that BDNF-like protein is present in the ganglion and in the iris, an important peripheral target of ciliary neurons. Moreover, CG neurons express surface trkB and exogenous BDNF not only initiates trk-dependent signaling, but also alters nicotinic acetylcholine receptor (nAChR) expression and synaptic transmission. In particular, BDNF applied to CG neurons rapidly activates cAMP-dependent response element-binding protein (CREB), and over the long-term selectively upregulates expression of ␣7-subunit-containing, homomeric nAChRs (␣7-nAChRs), increasing ␣7-subunit mRNA levels, ␣7-nAChR surface sites, and ␣7-nAChR-mediated whole-cell currents. At nicotinic synapses formed on CG neurons in culture, brief and long-term BDNF treatments also increase the frequency of spontaneous EPSCs, most of which are mediated by heteromeric nAChRs containing ␣3, ␣5, ␤4, and ␤2 subunits (␣3*-nAChRs) with a minor contribution from ␣7-nAChRs. Our findings demonstrate unexpected roles for BDNF-induced, trk-dependent signaling in CG neurons, both in regulating expression of ␣7-nAChRs and in enhancing transmission at ␣3*-nAChR-mediated synapses. The presence of BDNF-like protein in CG and iris target coupled with that of functional trkB on CG neurons raise the possibility that signals generated by endogenous BDNF similarly influence ␣7-nAChRs and nicotinic synapses in vivo.