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C S Cohan

Publications and source records attributed to C S Cohan.

18 recordsLinked to original sources
PubMed1992

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons.

This study focuses on the effects of K+ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K+ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K+ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K+ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K+ solution. Identified neurons differed in the magnitude of their response to K+ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K+, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K+ application evoked a brief period (5-10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K+ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca2+ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca2+ change during depolarization, reflecting their different sensitivities to depolarization.

Action Potentials↗
PubMed1991

Developmental regulation of a neurite-promoting factor influencing statoacoustic neurons.

The present study investigated a target-derived, neurite-promoting factor (NPF) released by the developing chick otocyst and its effects on statoacoustic ganglia (SAG). SAG explants cultured in the absence of otocysts produced little neurite outgrowth at all stages of development examined (E4-E13). However, extensive neurite outgrowth was seen when E4-E6 SAG were cultured in the presence of otocysts of the same age. The amount of neurite outgrowth observed in cocultures steadily decreased at later developmental stages. E7-E9 cocultures produced less outgrowth and E10-E13 cocultures produced the least outgrowth compared to E4-E6 cocultures. Additionally, otocysts from older stages were unable to promote outgrowth of E4 SAG. Thus, the level of the factor released by the otocysts declined during development. In contrast, neurite outgrowth was promoted when E10-E15 SAG were cocultured in the presence of younger stage otocysts. Our data indicate that the release of NPF from chick otocysts decreased from E6 to E13, although the ability of SAG neurons to respond to the NPF was maintained throughout development.

Animals↗
PubMed1991

A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements.

The neurotransmitter 5-HT alters growth cone motility and neurite elongation in neuron B19, isolated from the buccal ganglion of Helisoma trivolvis (Haydon et al., 1984). The effects of 5-HT are mediated by increases in intracellular calcium levels within the growth cones (Cohan et al., 1987). 5-HT causes a receptor-mediated depolarization of the membrane, which results in the opening of voltage-sensitive calcium channels. The resulting calcium influx decreases both the elongation rate and the total outgrowth of neurites. However, the mechanism(s) mediating these calcium-dependent changes is unclear. As many of the intracellular effects of calcium in eukaryotic cells are mediated by the calcium-binding protein calmodulin, we tested the involvement of such an interaction in the regulation of neurite outgrowth. In these experiments, a new, potent calmodulin antagonist with increased selectivity, CGS 9343B (CGS; Norman et al., 1987), was used to inhibit calmodulin activity during the application of 5-HT to neuron B19. The addition of 100 microM 5-HT to the culture medium resulted in a significant decrease in the rate of neurite elongation and total neurite outgrowth. Administration of CGS to the culture medium at a concentration (1.8 microM) equivalent to its IC50 for calmodulin inhibition completely blocked the inhibitory effects of 100 microM 5-HT, on both neurite elongation and total neurite outgrowth. CGS alone caused a slight decrease in elongation rate but had no significant effect on total outgrowth. CGS did not block 5-HT-induced electrical activity, indicating that it was not acting as a 5-HT receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Formation of electrical connections between cultured identified neurons and muscle fibers of the snail Helisoma.

We studied the formation of connections between identified neurons removed from the buccal ganglion of the snail Helisoma and muscle fibers dissociated from the buccal mass. Three types of identified neurons--B19, B5, and B4--were placed into cell culture and muscle fibers from the supralateral tensor muscle (SLT), normally innervated by B19, were subsequently plated adjacent to the neuronal cell bodies. Growth cones from the neurons contacted the muscle fibers within 6-12 h after isolation. Simultaneous intracellular recordings from the neuronal cell bodies and muscle fibers after 4 days in culture indicated that the neurons had formed electrical connections with the fibers. All 3 types of neurons coupled to the muscle fibers but displayed differing probabilities and strengths of connections. The role of growth cone contact in the formation of these connections was tested by plating muscle fibers onto fields of neurites after neuronal growth had stopped. Under these conditions, neurons still became electrically coupled to the muscle fibers, but the strength of these connections differed from those formed by neurons and fibers that were plated simultaneously. Thus, quantitative characteristics of electrical connections formed between cultured Helisoma neurons and dissociated muscle fibers are influenced by neuronal identity and the timing of neuronal contacts.

Animals↗
PubMed1990

Frequency-dependent and cell-specific effects of electrical activity on growth cone movements of cultured Helisoma neurons.

The present experiments address the question of how stimulation parameters, which evoke action potentials in neuronal cell bodies, influence growth cone movements of different identified neurons. The motility of growth cones of Helisoma buccal neurons B19 and B4 was monitored while somata were stimulated simultaneously via an intracellular microelectrode. The findings show that the responses of growth cones of B19 and B4 contain components that are common as well as unique to each neuron. Whereas rates of growth cone advance were suppressed in a graded fashion by stimulus frequencies beyond a threshold of 2 s-1 for both neurons, B4 was more sensitive to electrical stimulation and exhibited a new response, namely, growth rates were enhanced during the poststimulation recovery period after stimulation at specific frequencies. Thus, electrical activity can result in enhancement as well as in inhibition of growth cone movement. Changes in number of filopodia on B19 and B4 were graded also, with B4 again displaying greater sensitivity. The frequency dependence of filopodia compared to growth rate changes was different and suggests a possible dissociation between filopodial activity and growth cone motility. Patterned electrical activity produced effects similar to constant stimulation for B19 growth cones, whereas it decreased the threshold frequency and eliminated the growth enhancement effect for B4. Taken together, these data demonstrate that the quantitative features of electrical activity as well as intrinsic properties of neurons both determine the growth cone response to changes in neuronal activity.

Action Potentials↗
PubMed1989

Sealing cultured invertebrate neurons to embedded dish electrodes facilitates long-term stimulation and recording.

Recently it has become possible to form small networks of synaptically connected identified invertebrate neurons in culture. Using conventional saline-filled glass electrodes, it is difficult to simultaneously stimulate and record from more than 2 or 3 cultured neurons and to perform experiments lasting longer than several hours. We demonstrate that it is possible to overcome these limitations by using planar arrays of electrodes embedded in the bottom of a culture dish. The arrays employ conductive leads and insulation that are transparent, making the dishes compatible with voltage-sensitive dyes and inverted microscopy. Identified neurons from leech Hirudo medicinalis, slug Aplysia californica, and snail Helisoma trivolvis, have been grown on these arrays. Due to their large size (soma diameter 40-200 microns) these neurons form seals over the dish electrodes. Individual electrodes can then be used to stimulate and to record action potentials in the associated neuron. With sealing, action potentials have been recorded simultaneously from many neurons for up to two weeks, with signal-to-noise ratios as large as 500:1. We developed and tested a simple model that describes the voltage waveforms measured with array electrodes. Potentials measured from electrodes under cell bodies were primarily derivatives of the intracellular potential, while those measured from electrodes under axon stumps were primarily proportional to local inward Na+ currents. While it is relatively easy to record action potentials, it is difficult to record postsynaptic potentials because of their small size and slow rate of rise.

Action Potentials↗
PubMed1988

Interactive effects of serotonin and acetylcholine on neurite elongation.

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.

Acetylcholine↗
PubMed1987

Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ.

Previous work with identified Helisoma neurons has characterized an array of neuroplastic responses to axotomy that include the generation of new neuritic outgrowth, the reinnervation of target organs, and the formation of new electrical synapses. These responses are not random, but rather occur in a precise, predictable manner under a variety of culture conditions. The present investigation demonstrates that specific identified neurons display similar neuroplastic "behavior" within the living animal. In response to in situ nerve crushes, neurons B4 and B5 generate new neuritic outgrowth, neuron B4 functionally reinnervates the salivary glands, and new electrical synapses form between the left and right neurons B5. The in situ paradigm employed in the present experiments made it possible to examine responses to axotomy over longer periods than in earlier studies with organ cultures. New B5R-B5L connections, previously found to be stable over the short term in culture, gradually decreased in strength in situ, and the cells effectively uncoupled by 8 weeks after axotomy. This uncoupling did not depend upon target reinnervation and occurred in the continued presence of neurites in the buccal commissure. It is suggested that the stability of new connections is related to whether the connection previously existed in the unperturbed nervous system. The similarities between the ability of identified neurons to grow and to form synaptic connections in situ and in culture suggests that neurons are endowed with a specific program of regenerative responses that can be expressed reliably in a wide variety of environmental conditions.

Animals↗
PubMed1987

Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones.

In the present report we used the calcium indicator fura-2 to compare intracellular levels of free calcium in growth cones of isolated Helisoma neurons under a variety of experimental conditions. We tested whether 2 different signals that inhibit growth cone motility--action potentials and serotonin--changed calcium levels in growth cones. Electrical stimulation of the cell body caused a rise in calcium levels at the growth cone. After brief stimulation, calcium levels quickly recovered to normal values, whereas longer stimulation periods required longer recovery times. The application of serotonin to growth cones caused an increase in calcium levels that was selective for growth cones of neurons whose outgrowth was inhibited by serotonin, but not for neurons whose outgrowth was not affected. We also found that motile growth cones had higher free calcium levels than growth cones that had spontaneously stopped growing. Furthermore, the distribution of calcium in neurons that contained motile growth cones was heterogeneous; calcium levels were always higher in the growth cone than in the neurite or soma. These data indicate that calcium levels in growth cones vary in different states of outgrowth and that calcium levels can be modulated by both electrical and chemical signals.

Action Potentials↗
PubMed1986

Suppression of neurite elongation and growth cone motility by electrical activity.

Electrical activity may regulate a number of neuronal functions in addition to its role in transmitting signals along nerve cells. The hypothesis that electrical activity affects neurite elongation in sprouting neurons was tested by stimulating individual snail neurons isolated in cell culture. The findings demonstrated that growth cone advance, and thus neurite elongation, is reversibly stopped during periods when action potentials are experimentally evoked. A decrease in filopodial number and growth cone area was also observed. Thus, action potentials can mediate the cessation of neurite outgrowth and thereby may influence structure and connectivity within the nervous system.

Action Potentials↗
PubMed1986

Discriminative behavior and Pavlovian conditioning in the mollusc Pleurobranchaea.

The buccal motor system in the sea slug Pleurobranchaea californica is multifunctional; similar sets of neurons and muscles generate different behaviors through similar electrophysiological motor patterns. Such multifunctional systems compromise the traditional practice of identifying a motor pattern and then using that pattern to indicate the behavior in reduced preparations. We address this issue in a series of experiments leading to the comparison of differential Pavlovian conditioning in whole animals with the conditioned behavior of the same animals during electrophysiological recording. Because differential conditioning requires two conditioned stimuli (CSs), we show here that each of two CSs activated the conditioned response from animals after they received the stimulus (CS+) paired with an unconditioned stimulus (UCS). Conditioning sessions consisted of 5 training trials with a 2-h intertrial interval. In one study, experimental animals received a 60-s CS+, derived from beer (Sbr), paired with a 50-s electrical shock UCS whose onset occurred 10 s after the CS+ onset; control animals received the Sbr and UCS explicitly unpaired. In a second study, animals received similar procedures as in the first but with a CS+ consisting of squid homogenate (Ssq). Tests with both CSs showed that animals did not discriminate between Sbr and Ssq before beginning conditioning, but did so afterward. Experimental animals exhibited robust food aversion (withdrawal and suppressed feeding) to the CS+, but retained strong appetitive responses to the CS they did not receive in training; response thresholds to the CS+ changed as much as 1000-fold by comparison to the preconditioning values. Control animals exhibited similar though significantly smaller behavioral changes as the experimental animals. Both stimuli evoked associatively learned responses, but Sbr produced greater experimental-control differences than Ssq did. Two accompanying papers show the results of using both CSs in differential conditioning, and describe the behavioral/electrophysiological comparisons.

Animals↗
PubMed1986

Differential Pavlovian conditioning in the mollusc Pleurobranchaea.

The present differential Pavlovian conditioning experiments on the sea slug Pleurobranchaea californica extend conditioning described in a preceding paper and provide the conditioning foundation for studies reported in another accompanying paper comparing learned behavior in whole animals with the behavior and motor patterns of electrophysiological preparations. All animals received two appetitive-conditioned stimuli (CSs), one derived from beer (Sbr) and the other derived from squid muscle (Ssq), in different temporal relationships to an electric shock unconditioned stimulus (UCS). Two groups of animals were run concurrently. One group (n = 19) received Sbr as the CS+ in close temporal pairing with the UCS, and Ssq as the CS- explicitly unpaired with the UCS (Sbr +/Ssq-). The second group (n = 20) received the opposite contingencies (Sbr-/Ssq+). All animals received only one day of conditioning involving 5 trials with an intertrial interval of 2 h. There were two replicate experiments, each involving about half of the total n, and each yielding similar results as the sum we report here. Before conditioning, animals exhibited feeding behavior (extension of the proboscis and bite-strike responses) to both stimuli at similar low thresholds. Conditioning produced long-term behavioral changes in all animals throughout the 4.5-day postconditioning observation period. However, only the Sbr+/Ssq- animals consistently exhibited the appropriate differentially conditioned food-aversion behavior which consisted of strong withdrawal and high-threshold feeding responses to Sbr, and low-threshold feeding responses to Ssq. We discuss the possibility that such differences between Sbr+/Ssq- and Sbr-/Ssq+ conditioning may arise either from inherent differences in the responses of the animals to Sbr and Ssq, or, as seems more likely to us, from training and testing effects produced by differences in the compositions of the two stimuli.

Animals↗
PubMed1986

Comparison of differential Pavlovian conditioning in whole animals and physiological preparations of Pleurobranchaea: implications of motor pattern variability.

The present study compares differential Pavlovian conditioning in whole animals with the behavior of the same animals during electrophysiological recording. Untrained specimens of the sea slug Pleurobranchaea did not discriminate between two appetitive stimuli, one derived from an extract of beer (Sbr) and the other from a homogenate of squid muscle (Ssq). When animals received Sbr as the CS+ and Ssq as the CS- in a single day of five-trial, differential Pavlovian conditioning they learned to avoid selectively the Sbr but continued to exhibit appetitive responses to Ssq. Quantitative measures show that there was over a 1000-fold increase in the thresholds of the proboscis extension and bite-strike responses, many animals ceased all feeding behavior, and exhibited withdrawal responses to Sbr. We examined the behavior of the same trained animals immediately before preparing them for physiological recording and during the recording session. There was a close one-to-one relationship between these behavioral observations, showing that the qualitative and quantitative features of whole-animal Pavlovian conditioning persist into the physiological preparations. Unexpectedly, motor patterns from untrained preparations showed considerable variability both within the same preparation at different times and between preparations; conditioning appeared to increase such variability. Thus, it was not possible to state unequivocally the behavior of the animal by examining the electromyogram recording alone. Many of the trained preparations not only exhibited suppressed feeding behavior and withdrawal responses to Sbr, but, as a consequence of the multifunctional nature of the Pleurobranchaea buccal-oral system, also regurgitated previously ingested Ssq or squid meat when they were stimulated with Sbr. We discuss the findings with respect to self-organizing mechanisms that may establish motor patterns in multifunctional systems, and suggest that such mechanisms may lead to the generation of behaviors that are not specifically encoded by the conditioned cellular changes.

Animals↗
PubMed1986

Convergence in a distributed nervous system: parallel processing and self-organization.

The present findings show that the motor system of the carnivorous sea slug Pleurobranchaea californica consists of parallel, distributed, and interconnected neuronal channels by which motor activity may emerge from the dynamics of the system rather than from "switchboard" circuitry. The findings are shown primarily through the properties of the buccal-cerebral neurons (BCNs) that extensively converge and diverge monosynaptically and polysynaptically onto brain motoneurons, providing them with drive and patterned activity. The motoneurons, some of which are electrically coupled, feed back onto the BCNs. The BCNs are functionally heterogeneous both as a group and individually. Many are multifunctional in that they take part in the generation of different behaviors, and some also appear to change their timing with respect to the phase of the pattern generator in the different motor patterns. In the buccal ganglion, the BCNs affect the characteristics of the pattern generator and may be part of the pattern generator itself. By sending axons to buccal roots and to the brain, some BCNs may act as motoneurons and also integrate the activity of brain motoneurons. Because of the effects produced by the extensive interconnections among such functionally heterogeneous and nonlinear elements, and because the "history" of activity in the system can bias subsequent activity, there is ambiguity in assessing the response properties of neurons by examining them individually or in pairs. Such an assessment requires, first, an understanding of the context of activity in which a neuron becomes coactive, and, second, because of inherent variability in the system, it is necessary to consider the temporal, nonlinear computations of the system as a whole. We discuss the findings with regard to the attractor theory that has been used to study complex mammalian systems but that does not rely on modeling of any neuronal activity. The Pleurobranchaea nervous system may provide the means for studying individual neurons within such analyses of global activity.

Animals↗
PubMed1985

Neuron-specific growth cone properties as seen in identified neurons of Helisoma.

By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth cones of different neurons are intrinsically different from one another in terms of their structure, behavior, and response to environmental signals. Structurally neuron 5 has a greater number of filopodia per growth cone, has shorter filopodia, and has a smaller interfilopodial distance than neuron 19. Behaviorally, the growth cones of neuron 5 advance over the substratum at a faster rate than those of neuron 19; and the growth cones of neuron 19, but not of neuron 5, respond to the presence of serotonin in their environment by retracting their filopodia. In addition to such intrinsic differences between the growth cones of different neurons, we also demonstrate that the separate growth cones of a single neuron, while having identical properties, can act independently of one another. Focal application of serotonin to a growth cone causes only a localized retraction of that growth cones' filopodia. Other growth cones that are attached to the same neuron but that are not exposed to serotonin retain their normal structural features.

Animals↗
PubMed1985

Single channel activity differs in growing and nongrowing growth cones of isolated identified neurons of Helisoma.

When plated in cell culture, Helisoma neurons extend new neurites with large growth cones. These growth cones progress from actively growing organelles to stable structures over a period of several days. The ability to distinguish morphologically between these growth states has provided the opportunity to test whether changes in growth status are correlated with changes in the ionic properties of the growth cone membrane. This investigation has begun to study these changes by examining single channel ionic currents using patch clamp recording techniques and has demonstrated the presence of at least one ion channel having a conductance of 70 pS in the membrane of Helisoma growth cones. Recordings from cell-attached patches show that this channel is normally active in growing growth cones, whereas channel activity is absent in recordings from stable growth cones. The absence of channel activity in growth cones that have reached the stable state is not due to the loss of channels from these growth cones. Rather, the channel is present in an inactivated state. The activity of this channel is not dependent on the presence of Ca, as channel activity persists when Ca is removed from the solution that bathes the internal surface of the membrane. These data demonstrate a correlation between channel activity and the growth state of neuronal growth cones.

Action Potentials↗