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Papers by David Glanzman

Target-Dependent Structural Changes Accompanying Long-Term Synaptic Facilitation in Aplysia Neurons

Science, 1990

The mechanisms underlying structural changes that accompany learning and memory have been difficu... more The mechanisms underlying structural changes that accompany learning and memory have been difficult to investigate in the intact nervous system. In order to make these changes more accessible for experimental analysis, dissociated cell culture and low-light-level video microscopy were used to examine Aplysia sensory neurons in the presence or absence of their target cells. Repeated applications of serotonin, a facilitating transmitter important in behavioral dishabituation and sensitization, produced growth of the sensory neurons that paralleled the long-term enhancement of synaptic strength. This growth required the presence of the postsynaptic motor neuron. Thus, both the structural changes and the synaptic facilitation of Aplysia sensorimotor synapses accompanying long-term behavioral sensitization can be produced in vitro by applying a single facilitating transmitter repeatedly. These structural changes depend on an interaction of the presynaptic neuron with an appropriate postsynaptic target.

PLOS ONE, 2019

Zebrafish larvae have several biological features that make them useful for cellular investigatio... more Zebrafish larvae have several biological features that make them useful for cellular investigations of the mechanisms underlying learning and memory. Of particular interest in this regard is a rapid escape, or startle, reflex possessed by zebrafish larvae; this reflex, the Cstart, is mediated by a relatively simple neuronal circuit and exhibits habituation, a non-associative form of learning. Here we demonstrate a rapid form of habituation of the C-start to touch that resembles the previously reported rapid habituation induced by auditory or vibrational stimuli. We also show that touch-induced habituation exhibits input specificity. This work sets the stage for in vivo optical investigations of the cellular sites of plasticity that mediate habituation of the C-start in the larval zebrafish.

Journal of Neurophysiology, 1998

Bedi, Supinder S., Ali Salim, Shanping Chen, and David L. Glanzman. Long-term effects of axotomy ... more Bedi, Supinder S., Ali Salim, Shanping Chen, and David L. Glanzman. Long-term effects of axotomy on excitability and growth of isolated Aplysia sensory neurons in cell culture: potential role of cAMP. J. Neurophysiol. 79: 1371–1383, 1998. Crushing nerves, which contain the axons of central sensory neurons, in Aplysia causes the neurons to become hyperexcitable and to sprout new processes. Previous experiments that examined the effects of axonal injury on Aplysia sensory neurons have been performed in the intact animal or in the semi-intact CNS of Aplysia. It therefore has been unclear to what extent the long-term neuronal consequences of injury are due to intrinsic or extrinsic cellular signals. To determine whether injury-induced changes in Aplysia sensory neurons are due to intrinsic or extrinsic signals, we have developed an in vitro model of axonal injury. Isolated central sensory neurons grown for 2 days in cell culture were axotomized. Approximately 24 h after axotomy, sensory...

The Journal of Neuroscience, 2001

Injury ofAplysiasensory neurons, both in the CNS and in dissociated cell culture, produces long-t... more Injury ofAplysiasensory neurons, both in the CNS and in dissociated cell culture, produces long-term changes in these cells, among which are hyperexcitability and enhanced neuritic outgrowth (hypermorphogenesis). These long-term, injury-induced changes are attributable, in part, to the generation of new intrinsic cellular signals. Little is known, however, about the signals that maintain homeostasis within sensory neurons. To elucidate the role of homeostatic signals inAplysiasensory neurons, we investigated how axonal rejoining alters the cellular consequences of axotomy. Sensory neurons in dissociated cell culture were axotomized. In some cases, the distal segment of the severed axon was then removed; in other cases, the proximal and distal segments of the severed axon were permitted to rejoin. If the severed distal segment was left unmolested, then axonal rejoining invariably occurred within 7 hr. Surprisingly, we found that the characteristic long-term cellular consequences of a...

Neurobiology of learning and memory, Oct 1, 2016

The cellular and molecular basis of long-term memory in vertebrates remains poorly understood. Kn... more The cellular and molecular basis of long-term memory in vertebrates remains poorly understood. Knowledge regarding long-term memory has been impeded by the enormous complexity of the vertebrate brain, particularly the mammalian brain, as well as by the relative complexity of the behavioral alterations examined in most studies of long-term memory in vertebrates. Here, we demonstrate a long-term form of nonassociative learning-specifically, long-term habituation (LTH)-of a simple reflexive escape response, the C-start, in zebrafish larvae. The C-start is triggered by the activation of one of a pair of giant neurons in the zebrafish's hindbrain, the Mauthner cells. We show that LTH of the C-start requires the activity of NMDA receptors and involves macromolecular synthesis. We further show that the long-term habituated reflex can by rapidly dishabituated by a brief tactile stimulus. Our results set the stage for rigorous, mechanistic investigations of the long-term memory for habit...

eLife, Jan 9, 2017

Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (pa... more Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report thatLTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and ...

The Journal of Neuroscience, 2012

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-l... more A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist inAplysia californicadespite the fact that inhibitors of PKMζ erase memory inAplysiain a fashion similar to rodents. We have previously shown that, inAplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron proces...

Journal of Neurophysiology, 2008

To facilitate an understanding of injury-induced changes within the nervous system, we used a sin... more To facilitate an understanding of injury-induced changes within the nervous system, we used a single-cell, in vitro model of axonal injury. Sensory neurons were individually dissociated from the CNS of Aplysia and placed into cell culture. The major neurite of some neurons was then transected (axotomized neurons). Axotomy in hemolymph-containing culture medium produced long-term hyperexcitability (LTH-E) and enhanced neuritic sprouting (long-term hypermorphogenesis [LTH-M]). Axotomy in the absence of hemolymph induced LTH-E, but not LTH-M. Hemolymph-derived growth factors may activate tyrosine receptor kinase (Trk) receptors in sensory neurons. To examine this possibility, we treated uninjured (control) and axotomized sensory neurons with K252a, an inhibitor of Trk receptor activity. K252a depressed the excitability of both axotomized and control neurons. K252a also produced a distinct pattern of arborizing outgrowth of neurites in both axotomized and control neurons. Protein kinase...

Journal of Neurophysiology, 2008

Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitizat... more Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT–mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture ...

eneuro, 2018

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it i... more The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system ofAplysiagiven long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) inAplysiacan be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS inAplysiaand are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage inAplysia.

eneuro, 2020

Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological ana... more Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus (allyl isothiocyanate) and that persists for ≥ 30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 (Chen et al., 2013); due to the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of allyl isothiocyanate; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory. Significance Statement We have discovered a form of short-term behavioral sensitization in zebrafish larvae. Because the larvae are translucent, neural activity related to sensitization memory can be optically monitored in the intact and, in some cases behaving, fish using a genetically encoded ratiometric calcium indicator, GCaMP6. Taking advantage of this capability, we succeeded in identifying a region in the hindbrain that may mediate, at least in part, the memory for sensitization in the 5 zebrafish larva. These findings initiate an understanding of how activity in this region mediates a simple form of nonassociative memory in a relatively simple vertebrate animal.

Journal of Neurophysiology, 1997

Lin, Xiang Y. and David L. Glanzman. Effect of interstimulus interval on pairing-induced LTP of A... more Lin, Xiang Y. and David L. Glanzman. Effect of interstimulus interval on pairing-induced LTP of Aplysia sensorimotor synapses in cell culture. J. Neurophysiol. 77: 667–674, 1997. Long-term potentiation of Aplysia sensorimotor synapses (apLTP) can be induced in Hebbian fashion by pairing brief tetanic stimulation of the sensory neuron with depolarization of the motor neuron. It has been proposed that Hebbian apLTP plays a significant role in classical conditioning of the defensive withdrawal reflex of Aplysia. However, as originally demonstrated, Hebbian apLTP is induced by simultaneous pairing of sensory neuron stimulation and motor neuron depolarization, whereas in the Aplysia classical conditioning paradigm the onset of the conditioned stimulus (CS) precedes the onset of the unconditioned stimulus (US) by 0.5 s. Therefore, if Hebbian apLTP does indeed mediate classical conditioning in Aplysia, temporally offset delivery of presynaptic stimulation and postsynaptic depolarization mu...

The Journal of Neuroscience, 2003

Despite representing perhaps the simplest form of memory, habituation is not yet well understood ... more Despite representing perhaps the simplest form of memory, habituation is not yet well understood mechanistically. We used a reduced preparation to analyze the neurobiological mechanisms of persistent habituation of a simple behavior, the defensive withdrawal reflex of the marine snail Aplysia californica. This preparation permits direct infusion of drugs into the abdominal ganglion during training via a cannula in the abdominal artery. Using siphon-elicited gill withdrawal, we demonstrate habituation of withdrawal that persists for 1-6 hr after repeated, spaced blocks of habituating stimulation. This form of habituation exhibits site specificity and requires protein synthesis because it is blocked by the presence of anisomycin, a protein synthesis inhibitor. We also find that habituation of gill withdrawal requires protein phosphatase activity, because it is blocked by okadaic acid, an inhibitor of protein phosphatase. Finally, habituation of gill withdrawal requires activation of NMDA-type and AMPA-type postsynaptic receptors within the abdominal ganglion, because it is blocked by infusion of DL-2-amino-5-phosphonovaleric acid or 6,7-dinitroquinoxaline-2,3-dione. The requirement for activation of postsynaptic glutamatergic receptors indicates that homosynaptic depression, an exclusively presynaptic mechanism that has been implicated previously in habituation in Aplysia, does not play a significant role in persistent habituation of the withdrawal reflex. Our results indicate that postsynaptic mechanisms, possibly including modulation of glutamate receptor function, play a major, heretofore unsuspected, role in habituation in Aplysia.

The Journal of Neuroscience, 1986

DHT injections, to Zeiss for supplying the microscopic facilities, and to Phil Presley of Zeiss f... more DHT injections, to Zeiss for supplying the microscopic facilities, and to Phil Presley of Zeiss for expert technical assistance. We also thank Terri Teshiba and Drs. Joan Gunther and Sun-Hee Lee for their assistance with some ofthe experiments, Marvin Nalick for helo in oreuarine the fieures. and Harriet Avers for tvuina the manuscriot.

Handbook of Behavioral Neuroscience, 2013

The marine snail Aplysia californica exhibits a simple defensive withdrawal reflex that can under... more The marine snail Aplysia californica exhibits a simple defensive withdrawal reflex that can undergo several forms of learning. In particular, the reflex can exhibit long-term sensitization (LTS), a form of nonassociative memory. LTS is mediated by long-term facilitation (LTF) of the monosynaptic connection between the sensory and motor neurons that mediate the withdrawal reflex. LTS and LTF represent one of the bestunderstood model systems of long-term memory extent. Furthermore, discoveries from work on this system have provided fundamental insights into the cellular and molecular mechanisms that mediate the induction and maintenance of long-term memory. The present chapter reviews this work; it concludes with a discussion of recent studies of the role of protein kinase M in the persistence of the long-term memory and of memory reconsolidation in Aplysia. It is suggested that the study of LTS and LTF can provide important mechanistic information these two intriguing memory phenomena.

Chapter 6 Convergence of small molecule and peptide transmitters on a common molecular cascade

Progress in Brain Research, 1986

Publisher Summary This chapter summarizes the evidence that a conventional transmitter, serotonin... more Publisher Summary This chapter summarizes the evidence that a conventional transmitter, serotonin, and the small cardioactive peptides—SCP A and SCP B —can modulate a specific K + channel by means of cyclic adenosine monophosphate. Although the information is still fragmentary, there is the possibility that a third class of transmitter, yet unidentified, works by the same mechanism. In modulating this K + channel, each of these transmitters also regulates transmitter release at a specific set of synapses involved in two short-term forms of memory, each lasting minutes to hours: the memory for short-term behavioral sensitization and for classical conditioning. The specific set of connections modulated during short-term sensitization also undergoes a prolonged modulation lasting days and weeks, which contributes to long-term memory for sensitization. The ion channel gated by transmitters consists of at least three functional components: (1) a receptor or recognition site, (2) a channel, and (3) a gate. The receptor recognizes the transmitter and instructs the gate to open or close the channel. The channel conducts ions only when it is gated open.

eLife, Jan 17, 2014

Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. ... more Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated. Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin, which induces long-term facilitation. Serotonin (5HT) triggered growth of new presynaptic varicosities, a synaptic mechanism of long-term sensitization. Following 5HT training, two antimnemonic treatments-reconsolidation blockade and inhibition of PKM--caused the number of presynaptic varicosities to revert to the original, pretraining value. Surprisingly, the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but, rather, by an apparently arbitrary retraction of both 5HT-induced and original synapses. In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synapti...

Frontiers in behavioral neuroscience, 2010

Although habituation is possibly the simplest form of learning, we still do not fully understand ... more Although habituation is possibly the simplest form of learning, we still do not fully understand the neurobiological basis of habituation in any organism. To advance the goal of a comprehensive understanding of habituation, we have studied long-term habituation (LTH) of the gill-withdrawal reflex (GWR) in the marine snail Aplysia californica. Previously, we showed that habituation of the GWR in a reduced preparation lasts for up to 12 h, and depends on protein synthesis, as well as activation of protein phosphatases 1 and 2A and postsynaptic glutamate receptors. Here, we have used the reduced preparation to further analyze the mechanisms of LTH in Aplysia. We found that LTH of the GWR depends on RNA synthesis because it was blocked by both the irreversible transcriptional inhibitor actinomycin-D and the reversible transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside (DRB). In addition, LTH requires activation of protein phosphatase 2B (calcineurin), because it was disrupted...

Progress in brain research, 2008

The marine snail Aplysia has served for more than four decades as an important model system for n... more The marine snail Aplysia has served for more than four decades as an important model system for neurobiological analyses of learning and memory. Until recently, it has been believed that learning and memory in Aplysia were due predominately, if not exclusively, to presynaptic mechanisms. For example, two nonassociative forms of learning exhibited by Aplysia, sensitization and dishabituation of its defensive withdrawal reflex, have been previously ascribed to presynaptic facilitation of the connections between sensory and motor neurons that mediate the reflex. Recent evidence, however, indicates that postsynaptic mechanisms play a far more important role in learning and memory in Aplysia than formerly appreciated. In particular, dishabituation and sensitization depend on a rise in intracellular Ca(2+) in the postsynaptic motor neuron, postsynaptic exocytosis, and modulation of the functional expression of postsynaptic AMPA-type glutamate receptors. In addition, the expression of the ...

Frontiers in Neural Circuits, 2013

Larval zebrafish possess several experimental advantages for investigating the molecular and neur... more Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory.

Target-Dependent Structural Changes Accompanying Long-Term Synaptic Facilitation in Aplysia Neurons

Science, 1990

The mechanisms underlying structural changes that accompany learning and memory have been difficu... more The mechanisms underlying structural changes that accompany learning and memory have been difficult to investigate in the intact nervous system. In order to make these changes more accessible for experimental analysis, dissociated cell culture and low-light-level video microscopy were used to examine Aplysia sensory neurons in the presence or absence of their target cells. Repeated applications of serotonin, a facilitating transmitter important in behavioral dishabituation and sensitization, produced growth of the sensory neurons that paralleled the long-term enhancement of synaptic strength. This growth required the presence of the postsynaptic motor neuron. Thus, both the structural changes and the synaptic facilitation of Aplysia sensorimotor synapses accompanying long-term behavioral sensitization can be produced in vitro by applying a single facilitating transmitter repeatedly. These structural changes depend on an interaction of the presynaptic neuron with an appropriate postsynaptic target.

PLOS ONE, 2019

Zebrafish larvae have several biological features that make them useful for cellular investigatio... more Zebrafish larvae have several biological features that make them useful for cellular investigations of the mechanisms underlying learning and memory. Of particular interest in this regard is a rapid escape, or startle, reflex possessed by zebrafish larvae; this reflex, the Cstart, is mediated by a relatively simple neuronal circuit and exhibits habituation, a non-associative form of learning. Here we demonstrate a rapid form of habituation of the C-start to touch that resembles the previously reported rapid habituation induced by auditory or vibrational stimuli. We also show that touch-induced habituation exhibits input specificity. This work sets the stage for in vivo optical investigations of the cellular sites of plasticity that mediate habituation of the C-start in the larval zebrafish.

Journal of Neurophysiology, 1998

Bedi, Supinder S., Ali Salim, Shanping Chen, and David L. Glanzman. Long-term effects of axotomy ... more Bedi, Supinder S., Ali Salim, Shanping Chen, and David L. Glanzman. Long-term effects of axotomy on excitability and growth of isolated Aplysia sensory neurons in cell culture: potential role of cAMP. J. Neurophysiol. 79: 1371–1383, 1998. Crushing nerves, which contain the axons of central sensory neurons, in Aplysia causes the neurons to become hyperexcitable and to sprout new processes. Previous experiments that examined the effects of axonal injury on Aplysia sensory neurons have been performed in the intact animal or in the semi-intact CNS of Aplysia. It therefore has been unclear to what extent the long-term neuronal consequences of injury are due to intrinsic or extrinsic cellular signals. To determine whether injury-induced changes in Aplysia sensory neurons are due to intrinsic or extrinsic signals, we have developed an in vitro model of axonal injury. Isolated central sensory neurons grown for 2 days in cell culture were axotomized. Approximately 24 h after axotomy, sensory...

The Journal of Neuroscience, 2001

Injury ofAplysiasensory neurons, both in the CNS and in dissociated cell culture, produces long-t... more Injury ofAplysiasensory neurons, both in the CNS and in dissociated cell culture, produces long-term changes in these cells, among which are hyperexcitability and enhanced neuritic outgrowth (hypermorphogenesis). These long-term, injury-induced changes are attributable, in part, to the generation of new intrinsic cellular signals. Little is known, however, about the signals that maintain homeostasis within sensory neurons. To elucidate the role of homeostatic signals inAplysiasensory neurons, we investigated how axonal rejoining alters the cellular consequences of axotomy. Sensory neurons in dissociated cell culture were axotomized. In some cases, the distal segment of the severed axon was then removed; in other cases, the proximal and distal segments of the severed axon were permitted to rejoin. If the severed distal segment was left unmolested, then axonal rejoining invariably occurred within 7 hr. Surprisingly, we found that the characteristic long-term cellular consequences of a...

Neurobiology of learning and memory, Oct 1, 2016

The cellular and molecular basis of long-term memory in vertebrates remains poorly understood. Kn... more The cellular and molecular basis of long-term memory in vertebrates remains poorly understood. Knowledge regarding long-term memory has been impeded by the enormous complexity of the vertebrate brain, particularly the mammalian brain, as well as by the relative complexity of the behavioral alterations examined in most studies of long-term memory in vertebrates. Here, we demonstrate a long-term form of nonassociative learning-specifically, long-term habituation (LTH)-of a simple reflexive escape response, the C-start, in zebrafish larvae. The C-start is triggered by the activation of one of a pair of giant neurons in the zebrafish's hindbrain, the Mauthner cells. We show that LTH of the C-start requires the activity of NMDA receptors and involves macromolecular synthesis. We further show that the long-term habituated reflex can by rapidly dishabituated by a brief tactile stimulus. Our results set the stage for rigorous, mechanistic investigations of the long-term memory for habit...

eLife, Jan 9, 2017

Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (pa... more Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report thatLTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and ...

The Journal of Neuroscience, 2012

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-l... more A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist inAplysia californicadespite the fact that inhibitors of PKMζ erase memory inAplysiain a fashion similar to rodents. We have previously shown that, inAplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron proces...

Journal of Neurophysiology, 2008

To facilitate an understanding of injury-induced changes within the nervous system, we used a sin... more To facilitate an understanding of injury-induced changes within the nervous system, we used a single-cell, in vitro model of axonal injury. Sensory neurons were individually dissociated from the CNS of Aplysia and placed into cell culture. The major neurite of some neurons was then transected (axotomized neurons). Axotomy in hemolymph-containing culture medium produced long-term hyperexcitability (LTH-E) and enhanced neuritic sprouting (long-term hypermorphogenesis [LTH-M]). Axotomy in the absence of hemolymph induced LTH-E, but not LTH-M. Hemolymph-derived growth factors may activate tyrosine receptor kinase (Trk) receptors in sensory neurons. To examine this possibility, we treated uninjured (control) and axotomized sensory neurons with K252a, an inhibitor of Trk receptor activity. K252a depressed the excitability of both axotomized and control neurons. K252a also produced a distinct pattern of arborizing outgrowth of neurites in both axotomized and control neurons. Protein kinase...

Journal of Neurophysiology, 2008

Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitizat... more Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT–mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture ...

eneuro, 2018

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it i... more The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system ofAplysiagiven long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) inAplysiacan be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS inAplysiaand are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage inAplysia.

eneuro, 2020

Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological ana... more Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus (allyl isothiocyanate) and that persists for ≥ 30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 (Chen et al., 2013); due to the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of allyl isothiocyanate; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory. Significance Statement We have discovered a form of short-term behavioral sensitization in zebrafish larvae. Because the larvae are translucent, neural activity related to sensitization memory can be optically monitored in the intact and, in some cases behaving, fish using a genetically encoded ratiometric calcium indicator, GCaMP6. Taking advantage of this capability, we succeeded in identifying a region in the hindbrain that may mediate, at least in part, the memory for sensitization in the 5 zebrafish larva. These findings initiate an understanding of how activity in this region mediates a simple form of nonassociative memory in a relatively simple vertebrate animal.

Journal of Neurophysiology, 1997

Lin, Xiang Y. and David L. Glanzman. Effect of interstimulus interval on pairing-induced LTP of A... more Lin, Xiang Y. and David L. Glanzman. Effect of interstimulus interval on pairing-induced LTP of Aplysia sensorimotor synapses in cell culture. J. Neurophysiol. 77: 667–674, 1997. Long-term potentiation of Aplysia sensorimotor synapses (apLTP) can be induced in Hebbian fashion by pairing brief tetanic stimulation of the sensory neuron with depolarization of the motor neuron. It has been proposed that Hebbian apLTP plays a significant role in classical conditioning of the defensive withdrawal reflex of Aplysia. However, as originally demonstrated, Hebbian apLTP is induced by simultaneous pairing of sensory neuron stimulation and motor neuron depolarization, whereas in the Aplysia classical conditioning paradigm the onset of the conditioned stimulus (CS) precedes the onset of the unconditioned stimulus (US) by 0.5 s. Therefore, if Hebbian apLTP does indeed mediate classical conditioning in Aplysia, temporally offset delivery of presynaptic stimulation and postsynaptic depolarization mu...

The Journal of Neuroscience, 2003

Despite representing perhaps the simplest form of memory, habituation is not yet well understood ... more Despite representing perhaps the simplest form of memory, habituation is not yet well understood mechanistically. We used a reduced preparation to analyze the neurobiological mechanisms of persistent habituation of a simple behavior, the defensive withdrawal reflex of the marine snail Aplysia californica. This preparation permits direct infusion of drugs into the abdominal ganglion during training via a cannula in the abdominal artery. Using siphon-elicited gill withdrawal, we demonstrate habituation of withdrawal that persists for 1-6 hr after repeated, spaced blocks of habituating stimulation. This form of habituation exhibits site specificity and requires protein synthesis because it is blocked by the presence of anisomycin, a protein synthesis inhibitor. We also find that habituation of gill withdrawal requires protein phosphatase activity, because it is blocked by okadaic acid, an inhibitor of protein phosphatase. Finally, habituation of gill withdrawal requires activation of NMDA-type and AMPA-type postsynaptic receptors within the abdominal ganglion, because it is blocked by infusion of DL-2-amino-5-phosphonovaleric acid or 6,7-dinitroquinoxaline-2,3-dione. The requirement for activation of postsynaptic glutamatergic receptors indicates that homosynaptic depression, an exclusively presynaptic mechanism that has been implicated previously in habituation in Aplysia, does not play a significant role in persistent habituation of the withdrawal reflex. Our results indicate that postsynaptic mechanisms, possibly including modulation of glutamate receptor function, play a major, heretofore unsuspected, role in habituation in Aplysia.

The Journal of Neuroscience, 1986

DHT injections, to Zeiss for supplying the microscopic facilities, and to Phil Presley of Zeiss f... more DHT injections, to Zeiss for supplying the microscopic facilities, and to Phil Presley of Zeiss for expert technical assistance. We also thank Terri Teshiba and Drs. Joan Gunther and Sun-Hee Lee for their assistance with some ofthe experiments, Marvin Nalick for helo in oreuarine the fieures. and Harriet Avers for tvuina the manuscriot.

Handbook of Behavioral Neuroscience, 2013

The marine snail Aplysia californica exhibits a simple defensive withdrawal reflex that can under... more The marine snail Aplysia californica exhibits a simple defensive withdrawal reflex that can undergo several forms of learning. In particular, the reflex can exhibit long-term sensitization (LTS), a form of nonassociative memory. LTS is mediated by long-term facilitation (LTF) of the monosynaptic connection between the sensory and motor neurons that mediate the withdrawal reflex. LTS and LTF represent one of the bestunderstood model systems of long-term memory extent. Furthermore, discoveries from work on this system have provided fundamental insights into the cellular and molecular mechanisms that mediate the induction and maintenance of long-term memory. The present chapter reviews this work; it concludes with a discussion of recent studies of the role of protein kinase M in the persistence of the long-term memory and of memory reconsolidation in Aplysia. It is suggested that the study of LTS and LTF can provide important mechanistic information these two intriguing memory phenomena.

Chapter 6 Convergence of small molecule and peptide transmitters on a common molecular cascade

Progress in Brain Research, 1986

Publisher Summary This chapter summarizes the evidence that a conventional transmitter, serotonin... more Publisher Summary This chapter summarizes the evidence that a conventional transmitter, serotonin, and the small cardioactive peptides—SCP A and SCP B —can modulate a specific K + channel by means of cyclic adenosine monophosphate. Although the information is still fragmentary, there is the possibility that a third class of transmitter, yet unidentified, works by the same mechanism. In modulating this K + channel, each of these transmitters also regulates transmitter release at a specific set of synapses involved in two short-term forms of memory, each lasting minutes to hours: the memory for short-term behavioral sensitization and for classical conditioning. The specific set of connections modulated during short-term sensitization also undergoes a prolonged modulation lasting days and weeks, which contributes to long-term memory for sensitization. The ion channel gated by transmitters consists of at least three functional components: (1) a receptor or recognition site, (2) a channel, and (3) a gate. The receptor recognizes the transmitter and instructs the gate to open or close the channel. The channel conducts ions only when it is gated open.

eLife, Jan 17, 2014

Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. ... more Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated. Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin, which induces long-term facilitation. Serotonin (5HT) triggered growth of new presynaptic varicosities, a synaptic mechanism of long-term sensitization. Following 5HT training, two antimnemonic treatments-reconsolidation blockade and inhibition of PKM--caused the number of presynaptic varicosities to revert to the original, pretraining value. Surprisingly, the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but, rather, by an apparently arbitrary retraction of both 5HT-induced and original synapses. In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synapti...

Frontiers in behavioral neuroscience, 2010

Although habituation is possibly the simplest form of learning, we still do not fully understand ... more Although habituation is possibly the simplest form of learning, we still do not fully understand the neurobiological basis of habituation in any organism. To advance the goal of a comprehensive understanding of habituation, we have studied long-term habituation (LTH) of the gill-withdrawal reflex (GWR) in the marine snail Aplysia californica. Previously, we showed that habituation of the GWR in a reduced preparation lasts for up to 12 h, and depends on protein synthesis, as well as activation of protein phosphatases 1 and 2A and postsynaptic glutamate receptors. Here, we have used the reduced preparation to further analyze the mechanisms of LTH in Aplysia. We found that LTH of the GWR depends on RNA synthesis because it was blocked by both the irreversible transcriptional inhibitor actinomycin-D and the reversible transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside (DRB). In addition, LTH requires activation of protein phosphatase 2B (calcineurin), because it was disrupted...

Progress in brain research, 2008

The marine snail Aplysia has served for more than four decades as an important model system for n... more The marine snail Aplysia has served for more than four decades as an important model system for neurobiological analyses of learning and memory. Until recently, it has been believed that learning and memory in Aplysia were due predominately, if not exclusively, to presynaptic mechanisms. For example, two nonassociative forms of learning exhibited by Aplysia, sensitization and dishabituation of its defensive withdrawal reflex, have been previously ascribed to presynaptic facilitation of the connections between sensory and motor neurons that mediate the reflex. Recent evidence, however, indicates that postsynaptic mechanisms play a far more important role in learning and memory in Aplysia than formerly appreciated. In particular, dishabituation and sensitization depend on a rise in intracellular Ca(2+) in the postsynaptic motor neuron, postsynaptic exocytosis, and modulation of the functional expression of postsynaptic AMPA-type glutamate receptors. In addition, the expression of the ...

Frontiers in Neural Circuits, 2013

Larval zebrafish possess several experimental advantages for investigating the molecular and neur... more Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory.