Capabilities of neurexins in the chick ciliary ganglion

Transcellular interactions between neuroligins (NL) and beta-neurexin have been widely documented to promote maturation and function of both glutamatergic and GABAergic synapses. Recently it has been shown that neuroligin-1 plays a similar role at nicotinic synapses on chick ciliary ganglion neurons in culture, acting from the postsynaptic side to enhance transmitter release from adjacent cholinergic terminals and boost nicotinic input to the cells. We show here that the ciliary ganglion expresses three forms of neuroligin as well as two beta-neurexins and an alpha-neurexin. Overexpression of the beta-neurexins, but not the alpha-neurexin, can induce clustering of endogenous PSD-95 in adjacent neurons, presumably engaging neuroligin in the postsynaptic cell. The trans effects of beta-neurexins are selective; though both alpha3- and alpha7-containing nicotinic receptors are available on opposing cells, beta-neurexins induce coclustering of alpha3- but not alpha7-containing nicotinic receptors. Overexpression of other putative synaptogenic molecules, including SynCAM and L1, are ineffective at trans-clustering of PSD-95 on adjacent neurons. The beta-neurexins also exert a cis effect, coclustering presynaptic markers along with beta-neurexin in neurites juxtaposed to postsynaptic proteins, consistent with organizing presynaptic components as well. Striated muscle, the synaptic target of ciliary neurons in vivo, also expresses neuroligin. The results demonstrate that NL and neurexins are present at multiple sites in nicotinic cholinergic pathways and suggest the possibility of both cis- and trans-interactions to influence nicotinic signaling.     

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.     

In vivo imaging of presynaptic terminals and postsynaptic sites in the mouse submandibular ganglion

Much of what is currently known about the behavior of synapses in vivo has been learned at the mammalian neuromuscular junction, because it is large and accessible and also its postsynaptic acetylcholine receptors (AChRs) are readily labeled with a specific, high-affinity probe, alpha-bungarotoxin (BTX). Neuron-neuron synapses have thus far been much less accessible. We therefore developed techniques for imaging interneuronal synapses in an accessible ganglion in the peripheral nervous system. In the submandibular ganglion, individual preganglionic axons establish large numbers of axo-somatic synapses with postganglionic neurons. To visualize these sites of synaptic contact, presynaptic axons were imaged by using transgenic mice that express fluorescent protein in preganglionic neurons. The postsynaptic sites were visualized by labeling the acetylcholine receptor (AChR) alpha7 subunit with fluorescently tagged BTX. We developed in vivo methods to acquire three-dimensional image stacks of the axons and postsynaptic sites and then follow them over time. The submandibular ganglion is an ideal site to study the formation, elimination, and maintenance of synaptic connections between neurons in vivo.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.     

Formation of calyx-like contacts preferentially on appropriate target neurons in culture

The availability of culture systems for both Edinger Westphal and ciliary ganglion neurons has made it possible to examine the interactions in culture between two populations of vertebrate neurons that synapse in vivo. In the chick, Edinger Westphal neurons provide the sole presynaptic input to the ciliary ganglion and, through this projection, are responsible for the control of lens curvature (accommodation), iris constriction, and possibly smooth muscle function in the choroid layer of the eye. When embryonic chick Edinger Westphal and ciliary ganglion neurons were combined in culture and stained for enkephalin-like immunoreactivity to visualize Edinger Westphal terminals, stained calyx-like contacts were observed that resemble the calyciform terminals formed between Edinger Westphal processes and ciliary neurons in the ciliary ganglion in vivo. Although stained calyx-like contacts could also be found in Edinger Westphal-alone and ciliary ganglion-alone cultures, many more were observed when the two cell types were cultured together. The increase depended specifically on the ciliary ganglion neurons since substitution of either dorsal root ganglion or sympathetic ganglion neurons for them in the cocultures did not increase the number of calyx-like contacts staining positive for enkephalin over those present in cultures of Edinger Westphal neurons alone. When Edinger Westphal neurons were grown simultaneously with dorsal root and ciliary ganglion neurons, calyx-like contacts with enkephalin-like immunoreactivity were found to terminate preferentially on the latter. These findings suggest that vertebrate neurons can form morphologically specific contacts preferentially on appropriate target cells in culture in the absence of many of the potential cues present in the intact tissue.     

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.     

The alpha 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain.

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.     

In vivo activation of midbrain dopamine neurons via sensitized, high-affinity alpha 6 nicotinic acetylcholine receptors

Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.     

Tyrosine kinase inhibitors alter composition of nicotinic receptors on neurons

Protein tyrosine kinase (PTK) inhibitors were used to examine the roles of tyrosine phosphorylation in synaptic function. We show here that two different PTK inhibitors, herbimycin A and lavendustin A, both selectively downregulate a subpopulation of nicotinic acetylcholine receptors (AChRs) on chick ciliary ganglion neurons in culture. The downregulation requires a number of hours to occur and involves only those receptors containing the α3, α5, and β4 gene products. Not affected are AChRs that additionally contain the β2 gene product or AChRs that are made up of the α7 gene product. The downregulation preferentially targets receptors destined for the cell surface and has little effect on the large pool of intracellular receptors. The receptor loss is not additive with that seen in the presence of either cycloheximide or tunicamycin, two compounds that the block appearance of new receptors. The downregulation induced by herbimycin A in surface receptors is accompanied by a specific decrement in the amount of α3 protein in the cells. The results indicate that PTKs, either by phosphorylating AChR gene products directly or by acting through intermediary proteins, regulate the size and composition of the AChR pool maintained on the cell surface. Receptor regulation by PTKs may provide a mechanism for long-term control of synaptic signaling between neurons. © 1996 John Wiley & Sons, Inc.     

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide–Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.     

Regulation of nicotinic receptor expression by the ubiquitin-proteasome system

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.     

Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.     

Synergistic effects of muscarinic agonists and secretin or vasoactive intestinal peptide on the regulation of tyrosine hydroxylase activity in sympathetic neurons

Cholinergic agonists and certain peptides of the glucagon-secretin family acutely increase tyrosine hydroxylase activity in the superior cervical ganglion in vitro. The present study was designed to investigate possible interactions between these two classes of agonists in regulating catecholamine biosynthesis. Synergistic effects were found between carbachol and either secretin or vasoactive intestinal peptide in the regulation of DOPA (dihydroxyphenylalanine) synthesis. In addition, synergism was found at the level of the accumulation of cyclic adenosine monophosphate, the likely second messenger in the peptidergic regulation of tyrosine hydroxylase activity. The synergism seen with carbachol was blocked by a muscarinic, but not by a nicotinic, antagonist. Synergism was also found between bethanechol, a muscarinic agonist, and secretin, but not between secretin and dimethylphenylpiperazinium, a nicotinic agonist. Since previous immunohistochemical results suggest that vasoactive intestinal peptide and acetylcholine are colocalized in some preganglionic sympathetic neurons, the present data raise the possibility that the two might act synergistically in vivo in regulating catecholamine biosynthesis. Synergistic postsynaptic actions may be a common feature at synapses where peptides of the secretin-glucagon and acetylcholine are colocalized.     

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.