Indented synapses in Aplysia

A new type of synaptic contact has been found in Aplysia californica, in which a post-synaptic spine extensively invaginates the pre-synaptic element. The post-synaptic spine, usually less than 0.25 micrometer in diameter, may protrude up to 2 micrometer into the pre-synaptic element. In some instances a larger post-synaptic element indents and forms multiple thin projections into the pre-synaptic varicosity. Along or at the end of these projections a zone occurs at which the surface membranes of the two apposed synaptic elements are rigidly parallel, and the extracellular gap is approximately 60% greater than normal and contains a small amount of electron-dense material. Synaptic vesicles are concentrated against the pre-synaptic membrane in these regions. There are twice as many vesicles per unit area positioned against the membrane at these zones than at similar active zones occurring in the alternative type of synapse, which has a flat, rather than indented, geometry. Single pre-synaptic varicosities have been found to form both flat and indented synapses. These findings raise the possibility that these two forms of synapse may be dynamic transformations of each other, having differing synaptic effectiveness.     

Electrotonic synapses between Aplysia neurons in situ and in culture: aspects of regulation and measurements of permeability

Properties of electrotonic synapses between L14 neurons in the abdominal ganglion of the marine mollusc Aplysia californica were examined in situ and between unidentified buccal neurons maintained in tissue culture. In culture, depolarizing postsynaptic potentials in response to a train of action potentials showed apparent facilitation with increasing spike number, which was attributable to the low-pass filter properties of electrotonic transmission via gap junctions and to network properties. Gap junctional conductance (gj), calculated from current-clamp data or measured directly under voltage clamp, indicated no significant dependence of gj on transjunctional or inside-outside potential in situ or in culture. Octanol, a local anesthetic agent that reduces gj in many other systems, had no effect on gj between Aplysia neurons. The effect of intracellular acidification, a treatment that rapidly and reversibly uncouples a variety of cell types, reduced gj between Aplysia neurons but did not completely abolish it. The relationship between intracellular pH (pHi), measured with ion-sensitive microelectrodes, and gj was steeper in cultured neurons than in situ and was maximally reduced by 70-80%, as compared to 50% or less in situ at the lowest pHi values tested. The coupling coefficient (k) was reduced less by low pHi than was gj, which could be explained by a simultaneous increase in nonjunctional membrane resistance. Permeability properties of Aplysia electrotonic synapses to a variety of tracer molecules were also examined between identified L 14 neurons in situ and in dissociated buccal, abdominal, and bag neurons in culture. The fluorescent dyes Lucifer yellow, 6-carboxyfluorescein, and dichlorofluorescein (1.2-1.4 nm maximal diameters) did not spread detectably from an injected neuron to its electrically coupled neighbors, regardless of the strength of electrotonic coupling. However, the smaller tetraalkylammonium ions TMA and TEA (diameters 0.66 and 0.8 nm, concentrations measured with ion-selective electrodes), could be detected in neighboring cells within minutes. In culture, transfer of the tetraalkylammonium ions was slow and not easily detectable in cell pairs where gj was low (less than 20 nS). The permeability was as high as 10(-10) cm3/sec in situ and 10(-12) cm3/sec in culture, and values were roughly correlated with simultaneously measured values of gj. Electrotonic synapses in the nervous system of Aplysia, therefore, have a quantitatively different spectrum of sensitivities than has been found for gap junctions of other systems and appear to possess reduced permeability to tracer molecules.     

Selective formation and modulation of electrical synapses between cultured Aplysia neurons

When dissociated neurons from the mollusc, Aplysia californica, are placed in primary cell culture, they form electrical synapses in a specific, yet alterable, manner. Pairs of neurons from the same ganglion ("homoganglionic" pairs) form electrical synapses with high coupling coefficients. This is due to relatively high macroscopic junctional conductance as determined directly by voltage clamping both neurons of each pair. By contrast, synapses between pairs of neurons from different ganglia ("heteroganglionic" pairs) exhibit lower coupling coefficients as a result of lower macroscopic junctional conductance. Both types of junction are nonrectifying, not gated by voltage, and resistant to uncoupling by octanol and heptanol. This dichotomy of synaptic efficacy is altered upon exposure of the neurons to the lectin, conacanavalin A (Con A). Acute treatment of heteroganglionic cell pairs with Con A increases their junctional conductance to the higher level characteristic of homoganglionic pairs within several hours. However, the higher junctional conductance of homoganglionic pairs is not modulated by Con A. The results presented here suggest that synaptic specificity among these regenerating neurons may be mediated at least in part by ganglion-specific cell-recognition molecules. Furthermore, these molecules may be, or may be linked to, lectin receptors that regulate gap junction channels.     

Chemical and electrotonic connections between Aplysia neurons in primary culture

Dissociated Aplysia neurons will regenerate neurites and form functional connections in primary cell cultures. The specificity of intercellular connectivity in these cultures was investigated by coculturing neurosecretory bag cells with neurons dissociated from the buccal ganglion. It was found that bag-bag and buccal-buccal electrotonic synapses form with high frequency, consistent with previous findings in pure bag and buccal cultures. There is specificity in the formation of these connections, since no bag-buccal electrotonic synapses were observed. Chemical interactions, on the other hand, are present between bag and buccal neurons. In a buccal-bag pair, injection of sufficient depolarizing current into the buccal cell to elicit a train of action potentials leads to a slow hyperpolarizing response in the bag cell. The bag cell hyperpolarization is accompanied by an increase in the cell's input conductance. This connection appears to be unidirectional, produces a voltage shift in the bag cell which is opposite in sign to that in the buccal cell, and is blocked by the removal of Ca2+ from the extracellular medium, indicating that it is mediated by a chemical neurotransmitter. The selective formation of electrotonic synapses in these mixed bag-buccal cultures, together with the presence of chemically mediated interactions, make this system particularly useful for investigating the establishment of intercellular connectivity.     

Development of synapses between chick retinal neurons in dispersed culture

Morphological criteria allow several kinds of synapse to be recognized in the vertebrate retina. It is, however, not presently known if, or how, these morphological differences reflect physiological distinctions. Since a proper investigation of synaptic physiology in the intact retina is compromised by technical difficulties, we have examined dispersed cultures to discover if they are likely to provide a more tractable physiological preparation. The chief question addressed here concerns the extent to which normal synaptic development takes place in the impoverished conditions of dispersed cell culture. Cultures were established from embryonic day 8 chick retina and fixed for microscopy on embryonic equivalent (E.E.) days 12, 14, 16, and 18. Neuronal processes appeared shortly after plating and continued to increase in number and extent through E.E. 16. Cone cells were recognizable by virtue of their distinctive oil droplets. Two classes of cone could be distinguished on the basis of the density of their cytoplasmic staining. Presynaptic ribbons could be observed in cone cells on E.E. 12, but characteristic dyad and triad postsynaptic organization was seldom present at this stage nor was it often observed at subsequent times. An increase in the number of ribbon synapses in culture was seen on E.E. 18. These synapses may represent those of bipolar cells. Conventional synapses were found at all times examined but the number of these increased greatly between E.E. 14 and 16. Of these conventional synapses, we found some whose anatomy was characteristic of synapses made by amacrine cells as well as some whose anatomy was characteristic of synapses made by bipolar cells.     

Modulation of peptide release from single identified Aplysia neurons in culture.

Aplysia neurons B1 and B2 contain large amounts of the neuropeptides SCPA and SCPB. When grown in culture, individual B1 and B2 cells incorporate 35S-methionine into the SCPs, which can be released in a stimulus- and calcium-dependent fashion (Lloyd et al., 1986). We now show that single cells can be stimulated in a manner to evoke release of the SCPs that declines only slightly with repeated stimulation. This has allowed us to examine the ability of several physiologically relevant agonists to modulate the stimulus-evoked release of the SCPs. Bath application of either FMRFamide or 5-HT resulted in a significant decrease in the amount of SCPs released by intracellular stimulation of B1 or B2. The action of 5-HT was dose dependent with an inhibition of release of approximately 70% at a concentration of 100 microM. SCPA did not significantly affect release. The bath application of several compounds that are expected to elevate intracellular levels of cAMP were also found to depress release. To investigate the possibility that the agonists inhibited the release of the SCPs via a hyperpolarization of membrane potential (and perhaps a loss of spikes in the neurites), we examined the actions of 5-HT, FMRFamide, and SCPA on several electrophysiological parameters intended to monitor the level of cell excitability. Surprisingly, even though 5-HT depressed the release of the SCPs from both cells, it depolarized and increased the excitability of B1, and hyperpolarized and decreased the excitability of B2. Furthermore, in contrast to the effects seen in culture, 5-HT depolarized both B1 and B2 in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.     

Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.     

Cholinergic transmission at newly formed synapses made by retinal neurons in culture

The purpose of this study was to investigate the properties of cholinergic transmission at nascent synapses formed by neurons from the embryonic chick retina. By using a cell culture system in which striated muscle cells served as postsynaptic targets for dissociated retinal neurons, it was possible to record synaptic activity soon after the establishment of a functional cholinergic synapse. Postsynaptic potentials ranged from a few hundred microvolts to greater than 10 mV. The high temperature dependence (Q10 of 10) of this cholinergic transmission indicated that the release of acetylcholine from the embryonic retinal neurons was not injury-related. The wide range in event amplitudes did not appear to be due to electrotonic conduction from adjacent myotubes. Rather, a lack of correlation between event amplitude and rise time indicated that the release of acetylcholine occurred over a confined area of contact. Amplitude histograms of these events always showed a unimodal distribution, with small events being most common. No pattern to the timing of the events was evident. In addition, the inward current blockers, tetrodotoxin and cadmium did not affect this activity. Taken together, our findings indicate that the embryonic retinal neurons studied in this culture system spontaneously release acetylcholine in a pulsatile manner by a mechanism that is stimulus-independent and highly temperature sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substance P immunoreactive boutons form synapses with feline sympathetic preganglionic neurons.

In this study, the relationship between substance P-immunoreactive boutons and antidromically activated sympathetic preganglionic neurons was examined by light and electron microscopy. Sympathetic preganglionic neurons in the T2-T4 spinal segments of the cat were identified by intracellular recording and antidromic activation from the corresponding white ramus. Neurons were filled with lucifer yellow and then stained to reveal, simultaneously, substance P and lucifer yellow immunoreactivity. All of the neurons examined with the light microscope (n = 13) received appositions from substance P-immunoreactive boutons. Appositions were found on all parts of the neuron, including the somata, dendrites, and axon initial segment. In most cases (11/13) few close appositions were seen; however, two neurons received large numbers of appositions from substance P-immunoreactive boutons. On one neuron, 16 substance P-immunoreactive varicosities that were identified as being closely apposed at the light microscope level were serially sectioned and examined with the electron microscope. Of these 16 varicosities, eight either directly contacted the neuron or formed morphologically identifiable synapses. The remaining eight varicosities were separated from the neuron by thin glial processes. Two other sympathetic preganglionic neurons that were examined ultrastructurally also received substance P-immunoreactive synapses and close contacts. These findings suggest that substance P-containing nerve fibres could affect all sympathetic preganglionic neurons but are likely to be important in regulating the activity of only a small proportion of these neurons.     

Identified FMRFamide-immunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons.

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.     

Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment

Neurons from the abdominal ganglion of the mollusc Aplysia californica regenerate neurite processes in dissociated cell culture. Both the nature of neurite outgrowth and the morphology of the cells are influenced by the presence of adult Aplysia hemolymph in the growth medium and the presence of a portion of a cell's original axonal process. Aplysia hemolymph enhances cell survival, the initiation of neurite outgrowth from multiple sites on the cell body surface, the linear growth of the processes, and the amount of branching by those processes. Hemolymph also decreases the diameter of the outgrowing neurite fascicles and the diameter of the individual neurites within the fascicles. The presence of a cell's original axon reduces the time required for the initiation of neurite outgrowth and restricts the formation of multipolar processes. In addition, the presence of an initial axonal segment is essential for neurite regeneration from large adult neurons.     

Presynaptic source of quantal size variability at GABAergic synapses in rat hippocampal neurons in culture

The variability of quantal size depends on both presynaptic (profile of the neurotransmitter concentration in the cleft) and postsynaptic (number and gating properties of postsynaptic receptors) factors. Here we have examined the possibility that at nonsaturated synapses in cultured hippocampal neurons, changes in both the transmitter concentration peak and its clearance from the synaptic cleft may influence the variability of spontaneous miniature synaptic GABAergic currents (mIPSCs). We found that, in contrast to the slow-off GABAA receptor antagonist bicuculline, fast-off competitive antagonists such as SR-95103 and TPMPA differentially blocked small and large mIPSCs. In the presence of flurazepam, a drug believed to increase the affinity of GABA for GABAAR, small mIPSCs were enhanced more efficiently than large events. Moreover, the addition of dextran, which increases the viscosity of the extracellular fluid, preferentially increased small mIPSCs with respect to large ones. These observations suggest that changes in the concentration peak and the speed of GABA clearance in the cleft may be an important source of synaptic variability. The study of the correlation between peak amplitude and kinetics of mIPSCs allowed determination of the relative contribution of transmitter peak concentration vs. time of GABA clearance. Small synaptic responses were associated with fast onset and decay kinetics while large amplitude currents were associated with slow kinetics, indicating a crucial role for GABA synaptic clearance in variability of mIPSCs. By using model simulations we were able to estimate the range of variability of both the concentration and the speed of clearance of the GABA transient in the synaptic cleft.     

Ontogeny of serotonergic neurons in Aplysia californica

Although the identity, projection patterns, and functions of serotonergic neurons in juvenile and adult Aplysiaare relatively well understood, little is known about the development of these cells. We have used light and electron microscopic immunocytochemistry to investigate the genesis, differentiation, identity, and fate of the serotonergic cells in the embryonic, larval, and metamorphic stages of the life cycle of Aplysia. The results indicate that the first serotonergic cells emerge at midembryogenesis and that a total of five cells makes up the entire serotonergic system by hatching. These cells are part of a newly discovered ganglion in Aplysia, called the apical ganglion. This serotonergic system of five cells remains essentially intact throughout larval development. The apical ganglion, together with its serotonergic cells, is resorbed at metamorphosis. A distinct set of serotonergic cells, which begins to emerge by the end of the larval period, is rapidly elaborated during the metamorphic and early juvenile periods to form the adult serotonergic system. These results support the view that the larval and adult forms of the Aplysia nervous system consist of entirely distinct sets of serotonergic cells, each adapted to the stage-specific morphological and behavioral characteristics of the animal. J. Comp. Neurol. 386:477-490, 1997. © 1997 Wiley-Liss, Inc.     

Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization

The gill withdrawal reflex of the marine mollusk, Aplysia californica, shows habituation and sensitization, two simple forms of learning. In order to extend the cellular studies on synaptic plasticity underlying the changes in the reflex behavior, and to explore further the development of synaptic plasticity during synapse formation, we have sought to establish the neural circuit of the gill withdrawal reflex in vitro. We report here the reconstruction of the elementary gill withdrawal circuit in cell culture and find that the cells show short-term homosynaptic depression and heterosynaptic facilitation, the cellular mechanisms of habituation and sensitization, respectively.     

A vasoactive intestinal peptide-like cotransmitter at cholinergic synapses between rat myenteric neurons in cell culture

Intracellular recording and immunochemical techniques were used to study synaptic transmission between individual pairs of rat myenteric plexus neurons in cell culture. This report describes the synaptic connections made by "dual function" presynaptic neurons that evoked slow postsynaptic depolarizations (slow EPSPs) in the same neurons in which they also evoked fast nicotinic cholinergic EPSPs. The slow EPSPs occurred only when presynaptic neurons were stimulated at frequencies of 5 Hz or higher. During the slow EPSPs, slope input resistance increased. The slow EPSPs were not detectably voltage-dependent, and they reversed sign at the estimated K+ equilibrium potential, suggesting that they resulted from a synaptically mediated decrease in resting K+ conductance. Several lines of evidence suggested that dual-function neurons evoke slow EPSPs by releasing a vasoactive intestinal peptide (VIP)-like cotransmitter. (1) Immunocytochemical staining revealed VIP-like immunoreactivity in all physiologically identified dual-function neurons. (2) Responses to exogenously applied VIP mimicked the slow EPSPs. (3) Superfusion of cultures with anti-VIP antisera blocked the slow EPSPs reversibly, as did application of desensitizing doses of VIP. These findings suggest that during periods of increased activity, subsets of cholinergic myenteric neurons release a VIP-like cotransmitter that enhances postsynaptic excitability. The effects of the cotransmitter may help to compensate for decreases in nicotinic EPSPs that occur during increased presynaptic activity.     

Antipsychotic drugs and dopamine-mediated responses in Aplysia neurons

Summary The effect of antipsychotic drugs was tested on responses to micro-electrophoretically applied dopamine, acetylcholine and 5-hydroxy-tryptamine in identified neurons of the marine gastropod Aplysia californica. Fluphenazine was able to depress the response to DA in concentration of 10M, with 100M DA-responses of many neurons were blocked completely. Thioridazine (10 and 100M) and haloperidol (50M) were also effective in depressing DA-responses, while the non-antipsychotic phenothiazines mepazine (10 and 100M) and promethazine (100M) had only a slight action on DA-receptors. ACh-and 5-HT-responses were slightly affected only by high concentrations after long lasting perfusion. The investigated drugs had no persistent or only an insignificant effect on resting membrane potential and amplitude of action potentials of the neurons. With haloperidol depolarizing afterpotentials leading to double discharges were observed in some neurons. In a few instances spontaneous EPSPs disappeared with the DA-response under the influence of anti-psychotic drugs.The results render a direct neurophysiological evidence for the blockade of DA-receptors by antipsychotic drugs in correspondence to their clinical efficacy and agree with data from clinical observations and obtained in neurochemical, behavioral and indirect neurophysiological experiments.Supported by the österreichischen Fonds zur Förderung der wissenschaftlichen Forschung. —A preliminary report of a part of the results was published in Experientia30, 1318–1320 (1974).     

Single cell glutamate analysis in Aplysia sensory neurons

Glutamate is the major excitatory neurotransmitter in the central nervous system. However, techniques and assays available for the determination and detection of glutamate are limited. Here we have applied an effective glutamate assay toward the high-throughput analysis of single neurons. Initial physiological studies and recent immunohistochemical data strongly suggested that mechanosensory neurons could use L-glutamate as a co-transmitter (in addition to sensorin) in the sensory-motor synapse of Aplysia californica. We have evaluated the levels of glutamate in these cells and compared them to other non-sensory Aplysia neurons. Since this is the first report of this assay in single cellular analysis, a series of chemical and cellular controls were also done. Based on our results, we were able to determine the concentration levels inside single Aplysia sensory neurons to be 29 mM, with significant heterogeneity between individual cells. In comparison to the pleural mechanosensory neurons, non-sensory abdominal neurons contained approximately 3 mM glutamate. These elevated levels in the sensory neurons confirm the earlier findings [Dale N, Kandel ER. L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons. Proc Natl Acad Sci USA 1993;90:7163-7.], suggesting that glutamate plays a role as neurotransmitter in these cells.