Formation and modulation of chemical connections: evoked acetylcholine release from growth cones and neurites of specific identified neurons.

The ability to release acetylcholine (ACh) from cultured neurons of Helisoma was assessed by micromanipulating ACh-sensitive somata into contact with presynaptic neurons. ACh release was reliably detected from neurites and growth cones of cholinergic neuron B5, but not neuron B19, as early as 3 s after contact with novel target neurons. The rapid onset of transmission correlates with the ability of neuron B5, but not neuron B19, to indiscriminately form chemical connections and may be related to the specificity of synaptogenesis. The neuropeptide FMRFamide reduces ACh release at early chemical connections. The rapid onset of functional transmission and the ability of FMRFamide to modulate chemical transmission at this early chemical connection suggest that neuron B5 acquires its presynaptic apparatus through an intrinsic program independently of target contact.

Target contact regulates the calcium responsiveness of the secretory machinery during synaptogenesis.

Neuron B19 of Helisoma is selective in synaptogenesis. Presynaptic mechanisms underlying this selectivity were tested. Acetylcholine-sensitive assay cells were micromanipulated into contact with B19 somata to assess its secretory state. Prior to appropriate muscle target contact, spontaneous synaptic currents were detected; however, action potential-evoked release of neurotransmitter was detected only following hours of muscle contact. Photolysis of a calcium cage, DM-nitrophen, accelerated the frequency of synaptic currents in muscle-contacted, but not novel neuron-contacted, B19 somata. These studies demonstrate that contact with appropriate target muscle enhances the responsiveness of this neuron's secretory machinery to internal calcium levels, thereby imparting the presynaptic cell with the ability to couple action potentials with neurotransmitter release.

The lateral giant fibers of the tubificid worm, Branchiura sowerbyi: structural and functional asymmetry in a paired interneuronal system.

Neuroanatomical and ultrastructural studies of the paired lateral giant nerve fibers (LGFs) in posterior segments of the tubificid worm, Branchiura sowerbyi, demonstrate that the fibers are 1) segmental in origin (two cell bodies per segment), 2) joined longitudinally and transversely to form an intersegmental syncytial network, and 3) surrounded by a myelinlike sheath. The LGFs are unique among paired giant fiber systems because of their extreme asymmetry, the diameter of the left fiber being several times greater than that of the right. Electrophysiological studies demonstrate that the small, right fiber has a high input resistance and, during mechanosensory stimulation, functions as the locus for LGF spike initiation. The larger, left fiber contributes by enhancing the speed of LGF spike conduction along the animal. One physiological benefit of this asymmetric arrangement may be optimization of escape reflex sensitivity to mechanosensory inputs.

Regulation and restoration of motoneuronal synaptic transmission during neuromuscular regeneration in the pulmonate snail Helisoma trivolvis.

Regeneration of motor systems involves reestablishment of central control networks, reinnervation of muscle targets by motoneurons, and reconnection of neuromodulatory circuits. Still, how these processes are integrated as motor function is restored during regeneration remains ill defined. Here, we examined the mechanisms underlying motoneuronal regeneration of neuromuscular synapses related to feeding movements in the pulmonate snail Helisoma trivolvis. Neurons B19 and B110, although activated during different phases of the feeding pattern, innervate similar sets of muscles. However, the percentage of muscle fibers innervated, the efficacy of excitatory junction potentials, and the strength of muscle contractions were different for each cell's specific connections. After peripheral nerve crush, a sequence of transient electrical and chemical connections formed centrally within the buccal ganglia. Neuromuscular synapse regeneration involved a three-phase process: the emergence of spontaneous synaptic transmission (P1), the acquisition of evoked potentials of weak efficacy (P2), and the establishment of functional reinnervation (P3). Differential synaptic efficacy at muscle contacts was recapitulated in cell culture. Differences in motoneuronal presynaptic properties (i.e., quantal content) were the basis of disparate neuromuscular synapse function, suggesting a role for retrograde target influences. We propose a homeostatic model of molluscan motor system regeneration. This model has three restoration events: (1) transient central synaptogenesis during axonal outgrowth, (2) intermotoneuronal inhibitory synaptogenesis during initial neuromuscular synapse formation, and (3) target-dependent regulation of neuromuscular junction formation.

Retrograde regulation of presynaptic development during synaptogenesis.

Major advances are occurring in our understanding of the events leading to synapse formation. Contact between the growth cone and target tissue leads to intercellular signaling which controls both pre- and postsynaptic development of the synapse. The identity of retrograde signals that regulate presynaptic development are beginning to emerge, and the signal transduction cascades that are activated presynaptically are being characterized. Recent studies have shown that both the resting calcium level and activation of presynaptic protein kinase A are critical in the development of the presynaptic terminal. An understanding of these regulatory mechanisms is beginning to provide insight into the molecular control of synaptic specificity.

Activity-dependent induction of functional secretory properties at cultured neuromuscular synapses of Helisoma.

1. The role of activity-dependent mechanisms in target-mediated induction of secretory properties was investigated at regenerating neuromuscular synapses of the American pond snail, Helisoma trivolvis, in cell culture. 2. Identified motoneurons were isolated into cell culture conditions that promoted neurite outgrowth. Buccal neurons 19 (B19) were cultured alone for 2 days, at which time dissociated muscle fibers were manipulated into contact with newly formed neurites. 3. Immediately before the plating of muscle fibers, the sodium channel blocker, tetrodotoxin (TTX), or the acetylcholine receptor antagonist, d-tubocurarine chloride (curare), was added to the culture dish. After 48 h of exposure, the inhibitors were removed by repeated dilution of the culture medium and electrophysiological analyses were performed. 4. Cholinoceptive assay cells were manipulated into contact with the presynaptic neurons to assess secretory properties along neuronal processes. Assay cells were used to control for variations in postsynaptic sensitivity that could result from long-term exposure to activity inhibitors. 5. These analyses demonstrated that inhibition of TTX-sensitive presynaptic activity and inhibition of curare-sensitive postsynaptic activation both blocked the induction of excitation-secretion coupling typically induced in these motoneurons by appropriate target contact. Neuron B5, which rapidly acquires functional synaptic properties in vitro, was unaffected in its secretory function by 48 h of activity inhibition. 6. Acquisition of secretory competence was not suppressed due to a reduction in the viability or long-term changes in excitability of the activity-inhibited neurons, as indicated by analyses of electrophysiological properties. 7. Although target-contact and activity both participated in the induction of secretory modifications in neuron B19, target-mediated changes did not involve retrograde effects on presynaptic neuronal excitability. 8. We hypothesize that contact-mediated mechanisms govern the initiation of presynaptic modifications in B19, however, our data indicate that the acquisition of functional excitation-secretion coupling also involves activity-dependent mechanisms. Although the mechanistic role of activity remains undefined, our results suggest that the activation of the target muscle plays a critical role in a retrograde signaling pathway underlying maturation of a functional secretory apparatus in target-contacted neuronal processes.

Aminergic and peptidergic modulation of motor function at an identified neuromuscular junction in Helisoma.

Electrophysiological studies suggest that motoneurone B19 in the buccal ganglia of Helisoma makes monosynaptic, cholinergic connections with the supralateral radular tensor (SLT) muscle of the buccal mass. Serotonin (5-HT) and small cardioactive peptide B (SCPB) were found to have peripheral modulatory effects on this motor pathway that are consistent with their previously described central facilitatory effects. Both neurotransmitters, when applied exogenously (10(-6) mol l-1) to isolated buccal ganglion-buccal muscle preparations, potentiated the magnitude of motoneurone B19-evoked muscle contractions (6.3 and 2.7 times, respectively) without affecting excitatory junctional potential (EJP) amplitudes. When applied to single dissociated SLT muscle fibres in cell culture, these modulators had similar effects on acetylcholine (ACh)-evoked muscle fibre shortening, demonstrating that these neuromodulators exert direct actions on the muscle cells. The cardioactive peptide FMRFamide (10(-6) mol l-1), although slightly potentiating muscle contractions in reduced neuromuscular preparations, significantly decreased both ACh-evoked muscle fibre shortening and depolarizing potentials in cultured SLT muscle cells. The differential effects of FMRFamide may, in part, be due to the elimination of interactive effects between multiple neurotransmitters that might exist in semi-intact preparations and in vivo. These results demonstrate that 5-HT, SCPB and FMRFamide in Helisoma can directly modulate the peripheral muscle targets of buccal motoneurones involved in the generation of cyclical feeding behaviour.

Target-dependent induction of secretory capabilities in an identified motoneuron during synaptogenesis.

Cholinergic neurons isolated from the buccal ganglia of Helisoma were plated into cell culture with a variety of defined target cells to study the specificity of synaptogenesis. Motoneuron B19 selectively formed chemical connections with single dissociated muscle fibers derived from its appropriate target, the supralateral radular tensor (SLT) muscle. B19 did not form such connections with novel neuronal targets. In contrast to neuron B19, cholinergic neuron B5 nonselectively formed chemical connections with novel muscle and neuronal targets. Target cells were micromanipulated into contact with presynaptic neurons to examine the latent period until the onset of functional synaptic transmission. Neuron B5 formed chemical connections within the first minutes of contact with ACh-sensitive neurons and muscle while B19 required sustained periods of muscle-specific contact to induce the acquisition of a functional excitation-secretion coupling mechanism. These different latent periods from the onset of target contact suggest that neuron B5 acquires presynaptic secretory function before target contact, while B19 must receive a specific signal(s) from its appropriate target to induce the transformation of its terminal into a secretory state.

Specific muscle contacts induce increased transmitter release and neuritic arborization in motoneuronal cultures.

Identified buccal motoneuron 19 (B19), isolated from the nervous system of Helisoma and plated into cell culture, is selective in synapse formation and requires contact with appropriate muscle targets before acquiring secretory competence. Action potential-regulated transmitter release was elevated specifically at neuritic arbors in contact with appropriate muscle fibers. In contrast, contact between B19 and inappropriate muscle targets failed to induce changes in presynaptic secretory properties. Spontaneous transmitter release rate was elevated globally across neuritic arbors of B19 in appropriate muscle cocultures, including arbors without direct muscle contact. In addition, dual contacts formed between B19 and two different muscle targets resulted in significant elevations in excitation-secretion coupling only at neuritic sites of appropriate muscle contact and not at sites of contact with mismatched targets. Image analyses of presynaptic neuronal architecture revealed that appropriate, but not novel, muscle targets elicited increased arborization of neurites at sites of neuron-muscle contact. In contrast to results with neuron B19, secretory properties of buccal neuron 5 (B5), a neuron capable of forming inappropriate chemical synapses with a number of novel targets in culture, were not enhanced by inappropriate muscle contact. We conclude that muscle targets vary in their ability to induce presynaptic modifications (e.g., excitation-secretion coupling, spontaneous transmitter release, and neuritic arborization) in identified motoneurons of Helisoma. These results imply that separate molecular pathways exist which control each of these events during synaptic differentiation. The differential potency of muscle targets for induction of presynaptic changes indicates the existence of a cellular mechanism of target recognition. We hypothesize that such a mechanism underlies the ability of neuron B19 to discriminate between potential postsynaptic partners in cell culture.

Daily and circadian variation in the electroretinogram of the domestic fowl: effects of melatonin.

Visual and circadian function are integrally related in birds, but the precise nature of their interaction is unknown. The present study determined whether visual sensitivity measured electroretinographically (ERG) in 7-week-old cockerels varies over the time of day, whether this rhythm persists in constant darkness (DD) and whether exogenous melatonin affects this ERG rhythmicity. ERG b-wave amplitude was rhythmic in LD and persisted in DD with peak amplitude during mid- to late afternoon in LD and mid-subjective day in DD, indicating that the ERG rhythm is endogenously generated. No daily or circadian variation in a-wave amplitude was observed, and ERG component latency and durations were not rhythmic. Intramuscular injection of 10 micrograms/kg melatonin at ZT10 in LD significantly decreased b-wave amplitude but had no effect on a-wave. Intraocular injection of 600 pg melatonin, however, had no effect on any aspect of the ERG. These data indicate that a circadian clock regulates ocular sensitivity to light and that melatonin may mediate some or all of this effect. The level at which melatonin modulates retinal sensitivity is not known, but the present data suggest a central site rather than a direct effect of the hormone in the eye.

Neuron-muscle contact changes presynaptic resting calcium set-point.

The role of retrograde signaling between target cells and their presynaptic partners during early cell-cell interactions was examined in cell culture. Using time-lapse video microscopy and fura-2 calcium analysis, we followed contacts between presynaptic neurites of Helisoma motoneurons and muscle fibers dissociated from identified partners in chemical synaptogenesis. Cytosolic calcium rose dramatically at the region of cell-cell contact and subsequently increased throughout the entire neuron. Changes in the calcium set-point of presynaptic neurons were maintained even when connections with muscle targets were severed. Changes in presynaptic calcium concentration were not detected following contact with partners in electrical synapse formation. These data, taken together with the fact that calcium changes coincide with the time course of chemical synaptogenesis in these neuron-muscle cultures, suggest that calcium changes may be involved in early stages of synapse formation.

A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels.

The mechanisms that underlie synaptic plasticity have been largely inferred from electrophysiological studies performed at sites remote from synaptic terminals. Thus the mechanisms involved in plasticity at the secretory sites have remained ill-defined. We have now used somatic synapses of cultured Helisoma neurones to directly assess presynaptic ion conductances and study the secretory apparatus. At these synapses we determined the actions of a modulatory neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFa), on the release of the neurotransmitter acetylcholine (ACh). Using voltage- and calcium-clamp techniques, we have demonstrated that FMRFa causes a presynaptic inhibition of ACh release by (1) reducing the magnitude of the voltage-dependent calcium current, and (2) regulating the secretory apparatus. The photolabile calcium cage, nitr-5 (refs 3-8), was dialysed into the presynaptic cell. In response to ultraviolet light, calcium was released from nitr-5 and ACh secretion was stimulated. Under conditions of constant internal calcium, FMRFa reduced the rate of ACh release. Thus we conclude that FMRFa reduces the influx of calcium during the action potential and decreases the sensitivity of the secretory apparatus to elevated internal calcium, thereby contributing to a presynaptic inhibition of transmitter release.

A relation between synaptic specificity and the acquisition of presynaptic properties.

1. The specificity of synaptogenesis of identified adult neurons of Helisoma was determined in cell culture. Cholinergic neuron B5 indiscriminately forms the presynaptic element of chemical connections with novel cholinoceptive target neurons and muscle. By contrast, cholinergic neuron B19 is selective and discriminates between novel and appropriate target cells. Neuron B19 forms chemical connections with appropriate muscle targets only. 2. The acquisition of presynaptic properties independent of target contact was studied for both identified neurons. Functional connections form between neuron B5 and novel targets within seconds of contact, indicating that this cell has synthesized the presynaptic apparatus before target contact. In contrast, neuron B19 showed no evidence of possessing the ability to release neurotransmitter. 3. To further study the development of presynaptic properties, a model system of giant synaptic terminals was developed. The soma of neuron B5, acutely isolated from the nervous system is non-secretory. In conditions that prevent the extension of neurites, somata gain the ability to release neurotransmitter. This experimentally tractable system was used to study the calcium currents of presynaptic neuron B5. Acutely-isolated non-secretory somata contain two types of calcium currents: low-voltage-activated (LVA) and high-voltage-activated (HVA). The types of calcium currents in the soma change when B5 gains its secretory capacity. Secretory somata contain HVA calcium current only. 4. Neuron B5 was also plated in conditions which permit the extension of neurites. LVA and HVA calcium currents were maintained in its soma (non-secretory) but HVA calcium current only was maintained in its growth cones (secretory). Thus, B5 differentially regulates the presence of specific calcium currents in its membrane in relation to local secretory capacity without target-derived cues. 5. These data suggest that neuron B5 has an intrinsic program which generates presynaptic calcium channels and secretory apparatus prior to target contact. This autonomy of initial presynaptic development may underlie the lack of target cell discrimination exhibited by B5 in synaptogenesis.