Identified neurons from the abdominal ganglion of the marine mollusc Aplysia californica make spe... more Identified neurons from the abdominal ganglion of the marine mollusc Aplysia californica make specific transmitter-mediated synapses in dissociated cell culture. The cholinergic interneuron L10 makes synapses in vitro with one group of its follower cells, the left upper quadrant cells, and these connections exhibit the features of these synapses in vivo when the postsynaptic cells are plated with their initial axon segments. Furthermore, L10 will avoid making synapses with right upper quadrant cells, which contain cholinergic receptors but do not synapse with L10 in vivo. This in vitro system can therefore be used as a model in which to study the development of specific neuronal connections.
The development of the cellular substrates underlying habituation and sensitization, two simple f... more The development of the cellular substrates underlying habituation and sensitization, two simple forms of learning, was examined at a polysynaptic sensory-to-motor connection in the neural circuit mediating defensive mucus release in the marine mollusc, Aplysia californica. Animals were studied throughout juvenile life, stages 9 (40 days of development) to 12 (95 days), and into adulthood, stage 13 (120 days), starting just after metamorphosis when mucus release first becomes evident. Homosynaptic depression, which mediates habituation, was already present in its adult form in stage 9. Heterosynaptic facilitation, which mediates sensitization, appeared in stage 10 and reached maturity during stages 11 and 12. Thus, the development of synaptic plasticity in this circuit occurs in discrete phases in which the gradual emergence of heterosynaptic facilitation occurs only after homosynaptic depression is well established.
Current Opinion in Nephrology and Hypertension, 1995
Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allogr... more Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allograft rejection therapy. This paper reviews its in-vitro biological properties, the current state of knowledge concerning its mechanism of action, and its therapeutic applications.
The effects of procainamide on strength-interval relations were evaluated in 18 patients. At plas... more The effects of procainamide on strength-interval relations were evaluated in 18 patients. At plasma concentrations of 4.3 to 13.6 micrograms/ml procainamide had minimal effects on threshold current in late diastole, but in early diastole it shifted the strength-interval curve to the right. The basic strength-interval relation (that is, decreasing refractory period as current is increased) was not altered. The control refractory period decreased by a mean of 44 ms as the current was increased from threshold to 10 mA, whereas a mean decrease of 42 ms was observed after procainamide. However, the steep portion of the strength-interval curve(absolute refractory period) was shifted to longer coupling intervals by a mean value of 24 ms. These findings suggest that procainamide may primarily affect active membrane properties, but exert little net effect on passive membrane properties late in diastole.
Using single-channel recording, we have been able to obtain some insight into the molecular mecha... more Using single-channel recording, we have been able to obtain some insight into the molecular mechanism of a modulatory transmitter action in Aplysia sensory neurones. Our results show that serotonin produces a slow EPSP and increases action potential duration in the sensory neurones by producing prolonged closures of the S potassium channel. Such closures appear to be mediated by cyclic AMPdependent phosphorylation of a membrane protein which may be the channel. Modulation of S channels by serotonin also occurs in sensory neurone growth cones. This provides the first direct evidence that channel modulation occurs in nerve processes and increases the likelihood of channel modulation at the nerve terminal.
Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdr... more Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdrawal reflex in Aplysia californica undergo heterosynaptic facilitation of transmitter release during sensitization. As in the siphon sensory neurons, the transmitter serotonin produces facilitation and also elicits a slow, decreased conductance excitatory postsynaptic potential (EPSP) in these neurons. Using voltage clamp and biochemical analyses, we have found that the slow EPSP in the pleural sensory neurons is due to a decrease in a potassium conductance identical to the S potassium current characterized in siphon sensory neurons. Like the S current, the current modulated by serotonin in the pleural sensory neurons is a non- inactivating potassium current, and it contributes to both the resting and action potentials. The current reverses in 120 mM external K+ at - 20 mV, close to the predicted Nernst equilibrium potential. Intracellular cesium blocks the serotonin response, but the cu...
Publisher Summary One of the best characterized second messenger systems is the cyclic 5',3&#... more Publisher Summary One of the best characterized second messenger systems is the cyclic 5',3'-adenosine monophosphate (cAMP) cascade that results in the activation of cAMP-dependent protein kinase (cAMPdPK) according to the following scheme: Neurotransmitter (the first messenger) binds to a receptor coupled to the enzyme adenylate cyclase and leads to a rise in intracellular levels of cAMP (the second messenger); cAMP binds to the regulatory subunit of cAMPdPK and activates the enzyme by causing the dissociation of the regulatory from the catalytic subunit; and free catalytic subunit then phosphorylates target substrate proteins. cAMP-dependent protein phosphorylation has been shown to affect several voltage-gated conductances in neurons and the heart muscle. Evidence for the role of cAMP in neurotransmission has been obtained in a variety of ways. These include the demonstration that a neurotransmitter's effect is mimicked by intracellular injection of cAMP or bath application of membrane-permeable analogs of cAMP by the application of drugs that stimulate adenylate cyclase, or by direct injection of the catalytic subunit of cAMPdPK. The blockade of cAMP effects have also been demonstrated using a specific protein inhibitor of the catalytic subunit of cAMPdPK.
Serotonin (5-HT) produces a slow depolarizing postsynaptic potential in the mechanoreceptor senso... more Serotonin (5-HT) produces a slow depolarizing postsynaptic potential in the mechanoreceptor sensory neurons of the abdominal ganglia of the marine snail Aplysia. This slow EPSP is accompanied by a decrease in resting membrane conductance, a broadening of the action potential duration, and an increase in transmitter release from the sensory neuron terminals. The increase in transmitter release is thought to underlie behavioral sensitization of the gill-withdrawal reflex (Kandel and Schwartz 1982). Voltage clamp experiments have shown that the primary effect of serotonin in the sensory neurons is to decrease a specific outward potassium membrane current (S current) that is distinct from the previously identified K+ currents in molluscan neurons (Klein et al. 1982).
1. Single sodium channel currents were recorded from canine ventricular myocytes in cell‐attached... more 1. Single sodium channel currents were recorded from canine ventricular myocytes in cell‐attached patches. The relative rates of single‐channel activation vs. inactivation as well as the voltage dependence of the rate of open‐channel inactivation were studied. 2. Ensemble‐averaged sodium currents showed relatively normal activation and inactivation kinetics, although the mid‐point of the steady‐state inactivation (h infinity) curve was shifted by 20‐30 mV in the hyperpolarizing direction. This shift was due to the bath solution, which contained isotonic KCl to depolarize the cell to 0 mV. 3. Steady‐state activation showed less of a voltage shift. The threshold for eliciting channel opening was around ‐70 mV and the mid‐point of activation occurred near ‐50 mV. 4. The decline of the ensemble‐averaged sodium current during a maintained depolarization was fitted by a single exponential function characterizing the apparent time constant of inactivation (tau h). The apparent rate of inac...
Proceedings of the National Academy of Sciences, 1984
We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA ... more We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA and SCPB, facilitate synaptic transmission from siphon mechano-sensory neurons and enhance the defensive withdrawal reflex that these sensory neurons mediate. Single-channel recording revealed that these peptides close a specific K+ channel, the S channel, which is sensitive to cAMP. Moreover, the peptides increase cAMP levels in these sensory neurons. This reduction in K+ current slows the repolarization of the action potential in these cells, which increases transmitter release. In these actions, the SCPs resemble both noxious sensitizing stimuli, which enhance the reflex, and serotonin. Bioassay of HPLC fractions of abdominal ganglion extracts and immunocytochemistry indicate that both the SCPs and serotonin are present in the ganglion and are found in processes close to the siphon sensory neurons, suggesting that these transmitters may be involved in behavioral sensitization. Recent ...
Proceedings of the National Academy of Sciences, 1982
Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by de... more Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by decreasing outward K+ current. The depression of outward current delays repolarization of the action potential and extends the duration of Ca2+ influx into the presynaptic terminals, thereby contributing to the facilitation of transmitter release that underlies behavioral sensitization. We have extended the analysis of serotonin's action and find that it acts on a specific serotonin-sensitive K+ current (S current), which is different from the early K+ current (IA), the delayed K+ current (IK), the Ca2+-dependent K+ current (IC), and the muscarine-sensitive M current. The serotonin-sensitive current in these cells persists when IA and IK are reduced by conditioning depolarization or channel-blocking agents. The S current is not activated by intracellular injection of Ca2+, nor is it affected by substitution of Ba2+ for Ca2+, a treatment that reduces IC. Moreover, intracellular injectio...
Cold Spring Harbor Symposia on Quantitative Biology, 1983
One of the important developments in the study of synaptic transmission has been the discovery th... more One of the important developments in the study of synaptic transmission has been the discovery that neu-rotransmitters produce a wide range of electrical signals by interacting with different types of ionic chan-nels. Since 1950, it has been well established that chemical transmitters elicit short-lived postsynaptic potentials, such as the end-plate potential produced by acetylcholine (ACh) in skeletal muscle (Katz 1966; Sakmann et al., this volume). Here, ACh activates a channel that opens only in the presence of the transmit-ter and therefore does not normally contribute to the resting or active electrical properties of the cell (Neher and Sakmann 1976). Recently, however, transmitters have been found to exert long-lasting effects on some neurons by modulating channels that contribute to the
Sensory neurons in the pleural ganglion of Aplysia mediate the afferent portion of the tail withd... more Sensory neurons in the pleural ganglion of Aplysia mediate the afferent portion of the tail withdrawal reflex. Previous work has shown that in these neurons and in the siphon sensory neurons of Aplysia, serotonin modulates a steady-state non-inactivating potassium current called the S current. Using the technique of patch clamping, we have examined the kinetics of single potassium channels and found that they share the properties of the S potassium channel of the siphon sensory neurons. This channel has an elementary slope conductance of 73 + 9.98 pS (~ + S.E.M.) and shows Goldman rectification. It is active at the resting potential and does not inactivate with maintained depolarization. Bath application of serotonin in a majority of experiments decreased the functional number of channels in the patch.
The preclinical and the pharmacology studies described herein were supported by a grant from the ... more The preclinical and the pharmacology studies described herein were supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK 38016-14) and the clinical trials were supported by Wyeth-Ayerst Research.
Identified neurons from the abdominal ganglion of the marine mollusc Aplysia californica make spe... more Identified neurons from the abdominal ganglion of the marine mollusc Aplysia californica make specific transmitter-mediated synapses in dissociated cell culture. The cholinergic interneuron L10 makes synapses in vitro with one group of its follower cells, the left upper quadrant cells, and these connections exhibit the features of these synapses in vivo when the postsynaptic cells are plated with their initial axon segments. Furthermore, L10 will avoid making synapses with right upper quadrant cells, which contain cholinergic receptors but do not synapse with L10 in vivo. This in vitro system can therefore be used as a model in which to study the development of specific neuronal connections.
The development of the cellular substrates underlying habituation and sensitization, two simple f... more The development of the cellular substrates underlying habituation and sensitization, two simple forms of learning, was examined at a polysynaptic sensory-to-motor connection in the neural circuit mediating defensive mucus release in the marine mollusc, Aplysia californica. Animals were studied throughout juvenile life, stages 9 (40 days of development) to 12 (95 days), and into adulthood, stage 13 (120 days), starting just after metamorphosis when mucus release first becomes evident. Homosynaptic depression, which mediates habituation, was already present in its adult form in stage 9. Heterosynaptic facilitation, which mediates sensitization, appeared in stage 10 and reached maturity during stages 11 and 12. Thus, the development of synaptic plasticity in this circuit occurs in discrete phases in which the gradual emergence of heterosynaptic facilitation occurs only after homosynaptic depression is well established.
Current Opinion in Nephrology and Hypertension, 1995
Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allogr... more Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allograft rejection therapy. This paper reviews its in-vitro biological properties, the current state of knowledge concerning its mechanism of action, and its therapeutic applications.
The effects of procainamide on strength-interval relations were evaluated in 18 patients. At plas... more The effects of procainamide on strength-interval relations were evaluated in 18 patients. At plasma concentrations of 4.3 to 13.6 micrograms/ml procainamide had minimal effects on threshold current in late diastole, but in early diastole it shifted the strength-interval curve to the right. The basic strength-interval relation (that is, decreasing refractory period as current is increased) was not altered. The control refractory period decreased by a mean of 44 ms as the current was increased from threshold to 10 mA, whereas a mean decrease of 42 ms was observed after procainamide. However, the steep portion of the strength-interval curve(absolute refractory period) was shifted to longer coupling intervals by a mean value of 24 ms. These findings suggest that procainamide may primarily affect active membrane properties, but exert little net effect on passive membrane properties late in diastole.
Using single-channel recording, we have been able to obtain some insight into the molecular mecha... more Using single-channel recording, we have been able to obtain some insight into the molecular mechanism of a modulatory transmitter action in Aplysia sensory neurones. Our results show that serotonin produces a slow EPSP and increases action potential duration in the sensory neurones by producing prolonged closures of the S potassium channel. Such closures appear to be mediated by cyclic AMPdependent phosphorylation of a membrane protein which may be the channel. Modulation of S channels by serotonin also occurs in sensory neurone growth cones. This provides the first direct evidence that channel modulation occurs in nerve processes and increases the likelihood of channel modulation at the nerve terminal.
Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdr... more Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdrawal reflex in Aplysia californica undergo heterosynaptic facilitation of transmitter release during sensitization. As in the siphon sensory neurons, the transmitter serotonin produces facilitation and also elicits a slow, decreased conductance excitatory postsynaptic potential (EPSP) in these neurons. Using voltage clamp and biochemical analyses, we have found that the slow EPSP in the pleural sensory neurons is due to a decrease in a potassium conductance identical to the S potassium current characterized in siphon sensory neurons. Like the S current, the current modulated by serotonin in the pleural sensory neurons is a non- inactivating potassium current, and it contributes to both the resting and action potentials. The current reverses in 120 mM external K+ at - 20 mV, close to the predicted Nernst equilibrium potential. Intracellular cesium blocks the serotonin response, but the cu...
Publisher Summary One of the best characterized second messenger systems is the cyclic 5',3&#... more Publisher Summary One of the best characterized second messenger systems is the cyclic 5',3'-adenosine monophosphate (cAMP) cascade that results in the activation of cAMP-dependent protein kinase (cAMPdPK) according to the following scheme: Neurotransmitter (the first messenger) binds to a receptor coupled to the enzyme adenylate cyclase and leads to a rise in intracellular levels of cAMP (the second messenger); cAMP binds to the regulatory subunit of cAMPdPK and activates the enzyme by causing the dissociation of the regulatory from the catalytic subunit; and free catalytic subunit then phosphorylates target substrate proteins. cAMP-dependent protein phosphorylation has been shown to affect several voltage-gated conductances in neurons and the heart muscle. Evidence for the role of cAMP in neurotransmission has been obtained in a variety of ways. These include the demonstration that a neurotransmitter's effect is mimicked by intracellular injection of cAMP or bath application of membrane-permeable analogs of cAMP by the application of drugs that stimulate adenylate cyclase, or by direct injection of the catalytic subunit of cAMPdPK. The blockade of cAMP effects have also been demonstrated using a specific protein inhibitor of the catalytic subunit of cAMPdPK.
Serotonin (5-HT) produces a slow depolarizing postsynaptic potential in the mechanoreceptor senso... more Serotonin (5-HT) produces a slow depolarizing postsynaptic potential in the mechanoreceptor sensory neurons of the abdominal ganglia of the marine snail Aplysia. This slow EPSP is accompanied by a decrease in resting membrane conductance, a broadening of the action potential duration, and an increase in transmitter release from the sensory neuron terminals. The increase in transmitter release is thought to underlie behavioral sensitization of the gill-withdrawal reflex (Kandel and Schwartz 1982). Voltage clamp experiments have shown that the primary effect of serotonin in the sensory neurons is to decrease a specific outward potassium membrane current (S current) that is distinct from the previously identified K+ currents in molluscan neurons (Klein et al. 1982).
1. Single sodium channel currents were recorded from canine ventricular myocytes in cell‐attached... more 1. Single sodium channel currents were recorded from canine ventricular myocytes in cell‐attached patches. The relative rates of single‐channel activation vs. inactivation as well as the voltage dependence of the rate of open‐channel inactivation were studied. 2. Ensemble‐averaged sodium currents showed relatively normal activation and inactivation kinetics, although the mid‐point of the steady‐state inactivation (h infinity) curve was shifted by 20‐30 mV in the hyperpolarizing direction. This shift was due to the bath solution, which contained isotonic KCl to depolarize the cell to 0 mV. 3. Steady‐state activation showed less of a voltage shift. The threshold for eliciting channel opening was around ‐70 mV and the mid‐point of activation occurred near ‐50 mV. 4. The decline of the ensemble‐averaged sodium current during a maintained depolarization was fitted by a single exponential function characterizing the apparent time constant of inactivation (tau h). The apparent rate of inac...
Proceedings of the National Academy of Sciences, 1984
We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA ... more We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA and SCPB, facilitate synaptic transmission from siphon mechano-sensory neurons and enhance the defensive withdrawal reflex that these sensory neurons mediate. Single-channel recording revealed that these peptides close a specific K+ channel, the S channel, which is sensitive to cAMP. Moreover, the peptides increase cAMP levels in these sensory neurons. This reduction in K+ current slows the repolarization of the action potential in these cells, which increases transmitter release. In these actions, the SCPs resemble both noxious sensitizing stimuli, which enhance the reflex, and serotonin. Bioassay of HPLC fractions of abdominal ganglion extracts and immunocytochemistry indicate that both the SCPs and serotonin are present in the ganglion and are found in processes close to the siphon sensory neurons, suggesting that these transmitters may be involved in behavioral sensitization. Recent ...
Proceedings of the National Academy of Sciences, 1982
Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by de... more Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by decreasing outward K+ current. The depression of outward current delays repolarization of the action potential and extends the duration of Ca2+ influx into the presynaptic terminals, thereby contributing to the facilitation of transmitter release that underlies behavioral sensitization. We have extended the analysis of serotonin's action and find that it acts on a specific serotonin-sensitive K+ current (S current), which is different from the early K+ current (IA), the delayed K+ current (IK), the Ca2+-dependent K+ current (IC), and the muscarine-sensitive M current. The serotonin-sensitive current in these cells persists when IA and IK are reduced by conditioning depolarization or channel-blocking agents. The S current is not activated by intracellular injection of Ca2+, nor is it affected by substitution of Ba2+ for Ca2+, a treatment that reduces IC. Moreover, intracellular injectio...
Cold Spring Harbor Symposia on Quantitative Biology, 1983
One of the important developments in the study of synaptic transmission has been the discovery th... more One of the important developments in the study of synaptic transmission has been the discovery that neu-rotransmitters produce a wide range of electrical signals by interacting with different types of ionic chan-nels. Since 1950, it has been well established that chemical transmitters elicit short-lived postsynaptic potentials, such as the end-plate potential produced by acetylcholine (ACh) in skeletal muscle (Katz 1966; Sakmann et al., this volume). Here, ACh activates a channel that opens only in the presence of the transmit-ter and therefore does not normally contribute to the resting or active electrical properties of the cell (Neher and Sakmann 1976). Recently, however, transmitters have been found to exert long-lasting effects on some neurons by modulating channels that contribute to the
Sensory neurons in the pleural ganglion of Aplysia mediate the afferent portion of the tail withd... more Sensory neurons in the pleural ganglion of Aplysia mediate the afferent portion of the tail withdrawal reflex. Previous work has shown that in these neurons and in the siphon sensory neurons of Aplysia, serotonin modulates a steady-state non-inactivating potassium current called the S current. Using the technique of patch clamping, we have examined the kinetics of single potassium channels and found that they share the properties of the S potassium channel of the siphon sensory neurons. This channel has an elementary slope conductance of 73 + 9.98 pS (~ + S.E.M.) and shows Goldman rectification. It is active at the resting potential and does not inactivate with maintained depolarization. Bath application of serotonin in a majority of experiments decreased the functional number of channels in the patch.
The preclinical and the pharmacology studies described herein were supported by a grant from the ... more The preclinical and the pharmacology studies described herein were supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK 38016-14) and the clinical trials were supported by Wyeth-Ayerst Research.
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Papers by J. Camardo