Cholinergic modulation of an acetylcholine receptor-like antigen on the surface of chick ciliary ganglion neurons in cell culture

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the "main immunogenic region" of the alpha subunits in nicotinic ACh receptors from skeletal muscle and electric organ. Ultrastructural studies on antibody binding in the ganglion have shown that the cross-reacting antigen on the neuron surface is located predominantly in synaptic membrane. Biochemical studies have shown that the cross-reacting component has a number of other properties expected for the ganglionic nicotonic ACh receptor and that it is distinct from the alpha-bungarotoxin binding component in the tissue. Here we show that ciliary ganglion neurons grown in dissociated cell culture express a similar component that cross-reacts with monoclonal antibodies to ACh receptors, and that the number of antibody-binding sites on the neurons can be modulated by exposure to cholinergic agonists and a protein neurotoxin that reversibly inhibits ACh receptors on the neurons. In most, though not all, cases, levels of ACh sensitivity associated with the neurons are specifically comodulated in parallel with the changes in number of antibody binding sites. The results suggest that at least a portion of the cross-reacting sites on the surface of ciliary ganglion neurons is likely to represent nicotinic ACh receptors. The fact that in some instances levels of ACh sensitivity can be altered without changing the number of cross-reacting sites, however, leaves open the possibility that not all of the sites are associated with receptors or that the neurons can alter the proportion of receptors that is functional.     

Bouton ultrastructure and synaptic growth in a frog autonomic ganglion

Postmetamorphic growth in the frog, Xenopus laevis, is accompanied by an increase both in the size of autonomic neurons in the heart and in the number of synaptic boutons that contact their surface. To determine whether the properties of individual boutons change as their number increases, serial-section electron microscopy was used to examine bouton ultrastructure at the end of metamorphosis and in the adult. The area of bouton contact, number of active zones per bouton, active zone size, percent of bouton area occupied by active zone, and vesicle density were examined. No differences were found between the two bouton populations for any of the parameters examined. These results support the hypothesis that boutons are structural units of synaptic growth, whereby the total area of synaptic contact increases through the addition of boutons without a change in their morphological properties.     

Acetylcholine sensitivity in sensory neurons dissociated from the cat petrosal ganglion

The petrosal ganglia contain the somata of the sensory fibers of the glossopharyngeal nerves, innervating structures of the tongue, pharynx, carotid sinus and carotid body. Petrosal ganglia were excised from adult cats and their neurons were dissociated and kept in tissue culture for 7-12 days. Intracellular recordings were obtained through conventional microelectrodes. In response to depolarizing pulses, most cells (41/60) presented a 'hump' in the falling phase of their action potentials (H-type), while the remaining neurons lack such hump (F-type). The two types of cells had no differences in resting membrane potential or action potential amplitude. Acetylcholine (ACh) applied locally elicited responses in nearly two thirds of both H-type and F-type neurons tested. Most H-type neurons (17/19) responded with a slow long lasting depolarization, while the remaining (2) did so by generating spikes. In contrast, half of F-type neurons (6/12) responded with one or more spikes and the other half only with a slow depolarization. These results indicate that ACh receptors are present in the soma of many petrosal ganglion neurons subjected to tissue culture, thus supporting the idea that - under normal conditions - their peripheral sensory processes may be excited by ACh.     

Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion

The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin 3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular recording techniques. Scatchard analysis on homogenates indicates that cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a high-affinity site with an apparent dissociation constant (Kd,app) of 1.7 nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a low-affinity site with a Kd,app of 12 microM and a Bmax of 14 pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the binding of 125I-n-BuTX to either site. The high-affinity binding site is likely to be the functional nicotinic ACh receptor (AChR), given the similarity between its affinity for 125I-n-BuTX and the concentration of n-BuTX required to block AChR function. Light microscopic autoradiographic analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that toxin binding is concentrated at synaptic sites, which were identified using a synaptic vesicle-specific antibody. Scatchard analysis of autoradiographic data reveals that 125I-n-BuTX binding to the neuronal surface is saturable and has a Kd,app similar to that of the high-affinity binding site characterized in homogenates. Surface binding of 125I-n-BuTX is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20 microM), but not by atropine (IC50 greater than 10 mM). Denervation of the heart increases the ACh sensitivity of cardiac ganglion cells but has no effect upon the number of high-affinity binding sites for 125I-n-BuTX in tissue homogenates. Moreover, autoradiographic analysis indicates that denervation does not alter the number of 125I-n-BuTX binding sites on the ganglion cell surface. n-BuTX is as effective in reducing ganglion cell responses to ACh in denervated ganglia as it is in normally innervated ganglia. These results suggest that denervation alters neither the total number of nicotinic AChRs in the cardiac ganglion nor the number found on the surface of ganglion cells. These autonomic neurons thus respond differently to denervation than do skeletal myofibers. The increase in ACh sensitivity displayed by cardiac ganglion cells upon denervation cannot be explained by changes in AChR number.     

Disulfide bonds in the cholinoreceptors of frog sympathetic ganglion neurons

The effect of dithiotreitol (disulfide bonds reducing agent) and potassium ferricyanide (oxidative agent) on the nicotinic and muscarinic receptors of frog's sympathetic ganglion neurons were studied using intracellular electrodes. Dithiotreitol inhibited the responses evoked by carbachol, suberildicholine and 5-methylfurmethide. Ferricyanide if applied after dithiotreitol increased the inhibited responses to suberildicholine and decreased those to 5-methylfurmethide. Using protectors it was shown that both acetylcholine and tetramethylammonium protected nicotinic and muscarinic receptors from the action of dithiotreitol, the acetylcholine was more effective than tetramethylammonium in the case of nicotinic receptor. It was suggested that there are disulfide bonds in the active centres of nicotinic and muscarinic receptors located in the anionic centres as well as outside of those centres.     

Opposing roles of synaptic and extrasynaptic NMDA receptor signaling in cocultured striatal and cortical neurons

The NMDAR plays a unique and vital role in subcellular signaling. Calcium influx initiates signaling cascades important for both synaptic plasticity and survival; however, overactivation of the receptor leads to toxicity and cell death. This dichotomy is partially explained by the subcellular location of the receptor. NMDARs located at the synapse stimulate cell survival pathways, while extrasynaptic receptors signal for cell death. Thus far, this interplay between synaptic and extrasynaptic NMDARs has been studied exclusively in cortical (CTX) and hippocampal neurons. It was unknown whether other cell types, such as GABAergic medium-sized spiny projection neurons of the striatum (MSNs), which bear the brunt of neurodegeneration in Huntington's disease, follow the same pattern. Here we report synaptic versus extrasynaptic NMDAR signaling in striatal MSNs and resultant activation of cAMP response element binding protein (CREB), in rat primary corticostriatal cocultures. Similarly to CTX, we found in striatal MSNs that synaptic NMDARs activate CREB, whereas extrasynaptic NMDARs dominantly oppose CREB activation. However, MSNs are much less susceptible to NMDA-mediated toxicity than CTX cells and show differences in subcellular GluN2B distribution. Blocking NMDARs with memantine (30 μm) or GluN2B-containing receptors with ifenprodil (3 μm) prevents CREB shutoff effectively in CTX and MSNs, and also rescues both neuronal types from NMDA-mediated toxicity. This work may provide cell and NMDAR subtype-specific targets for treatment of diseases with putative NMDAR involvement, including neurodegenerative disorders and ischemia.     

Abundant distribution of TARP gamma-8 in synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor expression on spines and dendrites

Transmembrane alpha-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP gamma-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of gamma-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of gamma-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, gamma-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of gamma-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the gamma-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, gamma-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.     

Nicotinic acetylcholine receptor-like molecules in the retina, retinotectal pathway, and optic tectum of the frog

Forty-two monoclonal antibodies (mAbs) generated against nicotinic acetylcholine receptors (AChRs) from electric organ were tested for their ability to cross-react in the optic tectum of the frog Rana pipiens. Twenty-eight of the mAbs tested (67%) bound to the optic neuropil of the tectum as revealed by immunoperoxidase cytochemistry. The pattern of peroxidase stain for cross-reacting mAbs corresponded in position to a subset of the retinotectal projections. Electron microscopic examination revealed that peroxidase reaction product was associated with the surface of vesicle-containing profiles but not with synaptic sites. Removal of one retina resulted in the loss of immunoreactivity in the contralateral tectum. AChR-like immunoreactivity was also associated with the optic tract and optic nerve and with retinal ganglion cells. These results indicate that some classes of retinal ganglion cells bear AChR-like molecules on their surface. The existence of these molecules on ganglion cell axons and terminals seems the most likely explanation for the AChR-like immunoreactivity present in the tectum.     

Association of gephyrin with synaptic and extrasynaptic GABAA receptors varies during development in cultured hippocampal neurons

Several studies have reported extrasynaptic clusters of GABAA receptors in hippocampal neurons. Yet their functional relevance as well as their evolution in relation with gephyrin during synaptogenesis remain unknown. We have analyzed the expression pattern of the main proteins of the GABAergic synapses during synaptogenesis in cultured hippocampal neurons. We found that GABAergic terminals, characterized by VIAAT and GAD-65 expression, differentiated 3 to 7 days after the glutamatergic endings. At the postsynaptic side, the GABAAR- beta3 subunit was first diffuse and then clustered when GABAergic terminals differentiated and gephyrin formed large clusters. Colocalization of these proteins was high and increased with development. At later stages, GABAAR beta3 clusters colocalized with gephyrin at synaptic but also at extrasynaptic sites. GABAAR gamma2 subunits were directly expressed as clusters which were first extrasynaptic and not associated with gephyrin. Subsequently, the GABAAR gamma2 subunits associated with gephyrin at synaptic and/or extrasynaptic sites. Our data indicate that formation of GABAAR gamma2 subunit clusters is gephyrin independent.     

Excitatory synaptic potentials and morphological classification of tectal neurons of the frog

Under electrical stimulation of the optic nerve, intracellular recording and staining techniques were applied to tectal neurons of the frog. In the first part of this study, two response types in 25 cells were examined. One type (type I; 21 cells) was composed of an EPSP followed by an IPSP. The other type (type II; 4 cells) was composed of two successive EPSPs followed by an IPSP. The initial EPSP of type II response was concluded to be monosynaptic, while the other ones were assumed to be disynaptic. These responses were produced by myelinated fibers. Under stronger stimulus intensities, a pair of depolarization and hyperpolarization appeared at longer latencies.In the later part of this study, 22 cells were stained with Procion Yellow. Neurons were classified into three morphological types, i.e. vertical type (V-type; 10 cells), multipolar type (MP-type; 11 cells) and small neuron (S-neuron; one cell). All V-type neurons gave rise to type I response, while either type I or type II responses were recorded from MP-type neurons.     

Rapid swelling of neurons during synaptic transmission in the bullfrog sympathetic ganglion

Neurons in the lumbar sympathetic ganglion of the bullfrog were found to respond to presynaptic stimulation with rapid mechanical changes. These changes represent swelling of the presynaptic nerve terminals, followed by swelling of the somas of the postsynaptic neurons. After complete blockade of impulse transmission with D-tubocurarine, swelling of the neurons associated with the excitatory postsynaptic potential is observed.     

Pharmacological aspects of excitatory synaptic transmission to second-order vestibular neurons in the frog

Synaptic excitation of second-order vestibular neurons is mediated by two principal afferents: vestibular afferents projecting into the brain via the VIIIth cranial nerve and commissural afferents from the contralateral vestibular nuclear complex. The shape of the excitatory postsynaptic potentials (EPSPs) generated by selectively activating these two inputs differs qualitatively, such that ipsilateral VIIIth nerve afferents generate a faster-rising EPSP than do the commissural afferents. We have investigated the synaptic pharmacology of these two inputs in the isolated, intact medulla of the frog in order to determine the nature of the transmitter substances released by the afferents and the nature of the subsynaptic receptors with which these transmitters interact. Electrical stimulation of the ipsilateral VIIIth cranial nerve evokes in the region of the vestibular nuclear complex a field potential that exhibits a presynaptic (afferent volley) and a postsynaptic (slow negativity) component. Bath application of glutamate receptor antagonists, such as kynurenic acid (KENYA), blocks the postsynaptic component of this field potential in a dose-dependent manner, without affecting the presynaptic volley, suggesting that the VIIIth nerve afferent releases glutamate and/or similar substances as its neurotransmitter. A comparison of the actions of various glutamate receptor antagonists to block this postsynaptic negativity gives a rank order of effectiveness such that KENYA greater than gamma-D-glutamylglycine (gamma DGG) = gamma-D-glutamylaminomethylsulfonic acid (GAMS) greater than gamma-D-glutamyltaurine (gamma DGT) much greater than gamma-D-glutamylaminomethylphosphonic acid (GAMP) greater than D-2-amino-5-phosphonovaleric acid (D-APV) greater than D,L-APV greater than D-2-amino-7-phosphonoheptanoic acid (APH). This rank order of effectiveness suggests that the VIIIth nerve transmitter activates second-order neurons through kainate (KA)/quisqualate (QUIS) synaptic receptors. Intracellular studies support these conclusions. Chemically mediated EPSPs evoked from ipsilateral VIIIth nerve stimulation are completely blocked by high concentrations of KENYA (greater than or equal to 1 mM). Occasionally an extremely short-latency, probably electrically mediated, component to these EPSPs persists in the presence of KENYA. The slower-rising EPSPs evoked from contralateral VIIIth nerve or contralateral vestibular nuclear complex stimulation are also completely blocked by KENYA, suggesting that the transmitter released by the commissural afferents is also glutamate and/or related compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrophysiologic study of the properties of the cholinoreceptors of frog sympathetic ganglion neurons

The effect of the perfusion with cholinomimetics and cholinolytics on the membrane potential and membrane conduction of the isolated frog sympathetic ganglion neurons was studied using the intracellular microelectrodes. The activation of nicotinic cholinoreceptors with carbachol, suberildicholine and tetramethylammonium evoked depolarization with an increase in membrane conduction. The activation of muscarinic receptors with carbachol and 5-methylfurmethide evoked depolarization accompanied with a decrease or less frequently with an increase in the membrane conduction. It is found that atropine caused no changes in the membrane electric parameters when used as the perfused first drug, but it caused a short-lasting depolarization with the membrane conduction increase when used after carbachol.     

The effects of volatile anesthetics on synaptic and extrasynaptic GABA-induced neurotransmission

Examination of volatile anesthetic actions at single synapses provides more direct information by reducing interference by surrounding tissue and extrasynaptic modulation. We examined how volatile anesthetics modulate GABA release by measuring spontaneous or miniature GABA-induced inhibitory postsynaptic currents (mIPSCs, sIPSCs) or by measuring action potential-evoked IPSCs (eIPSCs) at individual synapses. Halothane increased both the amplitude and frequency of sIPSCs. Isoflurane and enflurane increased mIPSC frequency while sevoflurane had no effect. These anesthetics did not alter mIPSC amplitudes. Halothane increased the amplitude of eIPSCs, with a decrease in failure rate (Rf) and paired-pulse ratio. In contrast, isoflurane and enflurane decreased the eIPSC amplitude and increased Rf, while sevoflurane decreased the eIPSC amplitude without affecting Rf. Volatile anesthetics did not change kinetics except for sevoflurane, suggesting that presynaptic mechanisms dominate changes in neurotransmission. Each anesthetic showed somewhat different GABA-induced response and these results suggest that GABA-induced synaptic transmission cannot have a uniformly common site of action as suggested for volatile anesthetics. In contrast, all volatile anesthetics concentration-dependently enhanced the GABA-induced extrasynaptic currents. Extrasynaptic receptors containing α4 and α5 subunits are reported to have high sensitivities to volatile anesthetics. Also, inhibition of GABA uptake by volatile anesthetics results in higher extracellular GABA concentration, which may lead to prolonged activation of extrasynaptic GABA_A receptors. The extrasynaptic GABA-induced receptors may be major site of volatile anesthetic-induced neurotransmission.This article is part of a Special Issue entitled ‘Extrasynaptic ionotropic receptors’.     

Mode of synaptic transmission between vestibular afferents and neurons of the vestibular nucleus in the frog

Excitatory postsynaptic potentials (EPSPs) were evoked in vestibular neurons of the isolated frog brainstem by stimulation of the ipsilateral vestibular nerve or by direct intra-axonal activation of single vestibular fibers. Composite as well as single-fiber EPSPs usually displayed two components. A Ca2+-deficient, Mn2+-containing solution abolished the delayed chemical components of the EPSPs, but did not affect the short latency responses suggesting a dual (electrical-chemical) mode of transmission between some vestibular afferents and vestibular neurons.     

Asymmetry in the structure of average and large neurons of the ganglion layer of the frog retina

Morphometric analysis of the symmetry of middle and large ganglionic cells was performed on silver-impregnated retinal wholemounts of the frog. The nucleolus and the axis passing through the nucleolus in direction to optic disk were chosen as elements of symmetry characterizing the radial symmetry and bilateral one, respectively. It is demonstrated that the dendritic ramification angles of all cell types are smaller than 360 degrees and the angles of middle-type-GC are smaller than 180 degrees. In addition, their somata do not lie in the centre of the dendritic field, thus ganglionic cells have no radial symmetry. Directions of the axon and dendrites are opposite each other in the most of ganglionic cells, the terminals of dendrites being oriented from retinal centre to periphery in all quadrants of the retinal map. For estimation of bilateral symmetry the distance from the greatest remoted dendritic terminals to cell axis on the left and on the right from it was measured. Besides, the numbers of ramification knots and basal dendrites were counted. Most of ganglionic cells are asymmetrical in 2-3 mentioned structural parameters. Thus, the asymmetry in the structure of frog retinal neurons is rather norm than exception. Correlation between the asymmetry in ganglionic cell structure and functional asymmetry of their receptive fields is discussed.     

Anatomical remodelling of the supraoptic nucleus: changes in synaptic and extrasynaptic transmission

The adult hypothalamic-neurohypophysial system undergoes activity-dependent morphological plasticity that modifies the astrocytic enwrapping of its magnocellular neurones. For a long time, the functional consequences of such changes have remained hypothetical. Modifications in the glial environment of neurones are expected to have important physiological repercussions in view of the various functions played by astrocytes in the central nervous system. In particular, glial cells are essential for uptake of neurotransmitters, including glutamate, and for physically and functionally restricting diffusion of neuroactive substances within the extracellular space. Recent studies performed in the supraoptic nucleus of lactating and chronically dehydrated animals, in conditions where astrocytic coverage of neurones is reduced, have revealed a significant impairment of glutamate clearance. The resulting accumulation of the excitatory amino acid in the extracellular space around glutamatergic inputs causes an enhanced activation of presynaptic metabotropic glutamate receptors that inhibit transmitter release. In the supraoptic nucleus of lactating rats, neuroglial remodelling is accompanied by modification of the geometry, size and diffusion properties of the extracellular space. The latter observations suggest that, in the activated supraoptic nucleus, the range of action and the concentration of released neuroactive substances may be significantly enhanced. Overall, our observations indicate that the glial environment of supraoptic neurones influences synaptic glutamatergic transmission, as well as extrasynaptic forms of communication.     

Mechanisms regulating spill‐over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons

N‐Methyl‐d ‐aspartate glutamate receptors (NMDARs) contribute to neural development, plasticity and survival, but they are also linked with neurodegeneration. NMDARs at synapses are activated by coincident glutamate release and depolarization. NMDARs distal to synapses can sometimes be recruited by ‘spill‐over’ of glutamate during high‐frequency synaptic stimulation or when glutamate uptake is compromised, and this influences the shape of NMDAR‐mediated postsynaptic responses. In substantia nigra dopamine neurons, activation of NMDARs beyond the synapse during different frequencies of presynaptic stimulation has not been explored, even though excitatory afferents from the subthalamic nucleus show a range of firing frequencies, and these frequencies change in human and experimental Parkinson's disease. This study reports that high‐frequency stimulation (80 Hz/200 ms) evoked NMDAR‐excitatory postsynaptic currents (EPSCs) that were larger and longer lasting than those evoked by single stimuli at low frequency (0.1 Hz). MK‐801, which irreversibly blocked NMDAR‐EPSCs activated during 0.1‐Hz stimulation, left a proportion of NMDAR‐EPSCs that could be activated by 80‐Hz stimulation and that may represent activity of NMDARs distal to synapses. TBOA, which blocks glutamate transporters, significantly increased NMDAR‐EPSCs in response to 80‐Hz stimulation, particularly when metabotropic glutamate receptors (mGluRs) were also blocked, indicating that recruitment of NMDARs distal to synapses is regulated by glutamate transporters and mGluRs. These regulatory mechanisms may be essential in the substantia nigra for restricting glutamate diffusion from synaptic sites and keeping NMDAR‐EPSCs in dopamine neurons relatively small and fast. Failure of glutamate transporters may contribute to the declining health of dopamine neurons during pathological conditions.     

Discharges of neurons of the frog tectum during electric stimulation of individual retinal ganglion cells

In the frog optic tectum, at the depth of 200-270 micron extracellular monosynaptic PSPS of one axon were recorded under threshold electrical stimulation of the retina by a short train of stimuli. The latency of presynaptic spike was 8-12 ms. At a 5-25 ms interval between successive stimuli a negative spike was observed on the top of the testing EEG quanta enlarged 1.5-2.5 times. This spike is considered to be a population discharge evoked by the firing of a single fibre of the optic tract. This fibre terminates in layer F or G, while the population spike is generated by the tectum units of layers 6-8. A possibility that the firing of the 3d-5th class detectors excites tectal ganglionic cells whose axons form the main part of the tectobulbospinal tract is discussed.