Experimental approach for studying structural changes in axonal membrane upon nerve excitation

The Hodgkin and Huxley (HH) model of action potential has become a central paradigm of neuroscience. Despite its ability to predict action potentials with remarkable accuracy, it fails to explain several biophysical findings related to the initiation and propagation of the nerve impulse. The isentropic heat release and optical phenomena demonstrated by various experiments suggest that action potential is accompanied by a transient phase change in the axonal membrane. In this study a method was developed for preparing a giant axon from the crayfish abdominal cord for studying the molecular mechanisms of action potential simultaneously by electrophysiological and optical methods. Also an alternative setup using a single-cell culture of an Aplysia sensory neuron is presented. In addition to the description of the method, the preliminary results on the effect of phloretin, a dipole potential lowering compound, on the excitability of a crayfish giant axon are presented.

The Role of Chemical Mechanisms in Neural Computation and Learning

Most computational models of neurons assume that their electrical characteristics are of paramount importance. However, all long-term changes in synaptic efficacy, as well as many short-term effects, are mediated by chemical mechanisms. This technical report explores the interaction between electrical and chemical mechanisms in neural learning and development. Two neural systems that exemplify this interaction are described and modelled. The first is the mechanisms underlying habituation, sensitization, and associative learning in the gill withdrawal reflex circuit in Aplysia, a marine snail. The second is the formation of retinotopic projections in the early visual pathway during embryonic development.

Identification of an α-peptide in Haliotis rubra with homology to the Lymnaea α-CDCP spawning peptide

It is now understood that a combination of molluscan reproductive peptides are commonly cleaved from a large preprohormone and influence different aspects of spawning behavior. One type of reproductive peptide, known in Lymnaea stagnalis as [alpha]-CDCP, and in Aplysia californica as [alpha]-BCP, acts in egg laying via temperature-dependent autoinhibition or autoexcitation of neuronal cells. In our study, the expression of [alpha]-CDCP-like peptide in the blacklip abalone, Haliotis rubra, was identified by Western blots and immunocytochemistry, using an antiserum developed against [alpha]-CDCP. Western blots of total protein isolated from the central nervous system, cerebral and pleuropedal ganglia, as well as gonad and heptopancreas tissues of sexually mature adults, identified a protein of approximately 100 kDa as well as a range of smaller reactive peptides. This finding suggests that a reproductive [alpha]-peptide is probably synthesized from a single larger precursor protein. The larger peptides were also identified in Western blots of several abalone tissues, lmmunocytochemistry using the same antiserum showed the presence of immunoreactive axons in all the tissues studied, indicating synthesis or transport of products. The function of the abalone [alpha]-CDCP-like peptide is yet to be determined.

Stimulatory effects of egg-laying hormone and gonadotropin-releasing hormone on reproduction of the tropical abalone, Haliotis asinina linnaeus

Egg-laying hormone (ELH) is a neuropeptide hormone that stimulates ovulation of gastropods, including Aplysia californica and Lymnaea stagnalis. Other neuropeptides, gonadotropin releasing hormones (GnRHs), also play important roles in controlling reproduction in both vertebrates and invertebrates. In the current study, the effects of abalone ELH (aELH) and several GnRHs on somatic growth, sex differentiation, gonad maturation, and spawning of Haliotis asinina were investigated in 3 experiments. In experiment 1, groups of 4-mo-old juveniles (11.8 ±  0.03 mm shell length (SL) and 0.33 ± 0.04 g body weight (BW)) were injected with aELH and GnRHs, including buserelin (mammalian GnRH analogue), octopus GnRH (octGnRH), and tunicate GnRH-I (tGnRH-I), at doses of 20 ng/g BW and 200 ng/g BW. The aELH induced early sex differentiation with a bias toward females, but with normal somatic growth, whereas the different isoforms of GnRH had no effect on sexual differentiation or somatic growth. In experiment 2, groups of 1-y-old-abalone (SL, 4.04 ± 0.02 cm; BW, 20.15 ± 0.25 g) were injected with aELH and the 3 isoforms of GnRH including buserelin, octGnRH, and lamprey GnRH (1GnRH-I) at doses of 500 ng/g BW and 1,000 ng/g BW, and all produced stimulatory effects. For each peptide treatment, the gonads reached full maturation within 5- 6 wk and spawning occurred, whereas control groups took 8 wk to reach maturity. In experiment 3, injections of ripe abalone with aELH stimulated spawning of both sexes in a dose-dependent manner. Buserelin had a lesser effect on inducing spawning, and octGnRH had no apparent effect. The gametes released from induced spawnings by aELH and GnRH showed normal fertilization and development of larvae. Altogether, these findings provide further knowledge on manipulating abalone reproduction, which is important in improving abalone aquaculture.

SPASMOGENIC NONCHOLINERGIC ACTIVITY OF A DIGESTIVE GLAND HOMOGENATE OF THE AMPHIBIAN SNAIL POMACEA-LINEATA SPIX-1827

The digestive gland of Pomacea lineata, a prosobranch gastropod mollusc inhabiting both fresh water and land, does not contain cholinomimetic compounds as do the glands of species of Aplysia, marine opisthobranch gastropods, in which both acetylcholine and urocanylcholine are present. The only pharmacological action detected for the digestive gland of Pomacea was spasmogenic activity of a crude homogenate containing 0.1 g tissue equivalents on the snail's own esophagus bathed in 10 ml of a physiological solution prepared on the basis of the animal's hemolymph composition. The spamodic activity was not blocked by atropine, bromlysergic acid diethylamide or anthazoline.

It's not what it looks like: molecular data fails to substantiate morphological differences in two sea hares (Mollusca, Heterobranchia, Aplysiidae) from southern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentielle Zellzykluskontrolle bei Spiraliern und ihre Bedeutung für die Determination der Blastomeren

Bei inäqual furchenden Spiraliern, wie Platynereis dumerilii, entstehen durch die ersten beiden Furchungen vier unterschiedlich große und auch bereits unterschiedlich determinierte Blastomeren. Im allgemeinen ist die größte der vier Blastomeren die Gründerzelle des D-Quadranten (Dorresteijn und Fischer 1988). Dieser Quadrant etabliert die dorsoventrale Körperachse im Keim, indem er das Schicksal benachbarter Blastomeren über Zell-Zell-Interaktionen positionsgerecht bestimmt (Damen und Dictus 1996). Der D-Quadrant erhält bei Platynereis einen überproportional großen Anteil (60%) des gesamten dotterfreien Zytoplasmas im Keim (Dorresteijn 1990). Sein Schicksal wird durch morphogenetische Faktoren innerhalb des dotterfreien Zytoplasmas bestimmt. Der Gehalt an dotterfreiem Zytoplasma bestimmt nicht nur das Schicksal der Blastomeren, sondern korreliert auch direkt mit den jeweils unterschiedlichen Zellzyklusgeschwindigkeiten der Blastomeren. Die plasmareichen Zellen des D-Quadranten, aber auch bereits die Vorläuferzelle CD, teilen sich im Vergleich mit den jeweils anderen Blastomeren im Keim besonders rasch (Dorresteijn 1990). In dieser Arbeit wurde unter verschiedenen Aspekten untersucht (a) inwieweit die Etablierung der dorsoventralen Körperachse von der raschen Proliferation der D-Zellinie abhängt, (b) inwieweit Zellzyklusregulatoren Bestandteil der oben genannten morphogenetischen Faktoren sein könnten und (c) wie die unterschiedlichen Zellzyklusgeschwindigkeiten auf molekularer Ebene reguliert werden.Zum einen wurden die frühen Furchungsstadien von Aplysia californica volumetrisch vermessen. Anders als bei den meisten inäqual furchenden Spiraliern wird bei Aplysia nicht der größte, sondern einer der kleineren embryonalen Quadranten als D-Quadrant determiniert. Ich konnte zeigen, daß die CD-Blastomere (27% des Eivolumens) dennoch, ähnlich wie bei Platynereis, bei der ersten Furchungsteilung überproportional viel dotterfreies Zytoplasma (40%) erhält und so als Vorläuferzelle des D-Quadranten determiniert wird. Bei der zweiten Furchung teilt sich die CD-Blastomere jedoch, anders als bei Platynereis, symmetrisch. Welche der beiden Tochterzellen von CD als definitiver D-Quadrant determiniert wird, erfordert also zusätzliche (induktive?) Mechanismen. Auch bei Aplysia sind die Zellzyklusgeschwindigkeiten der Blastomeren mit ihren jeweiligen Anteilen am dotterfreiem Zytoplasma korreliert. Das Postulat, daß die rasche Proliferation des D-Quadranten und seiner Vorläuferzelle CD für die Etablierung der dorsoventralen Körperachse und für die Determination der Blastomeren in Keimen inäqual furchender Spiralier erforderlich sind, konnte ich zusätzlich bestätigen, indem ich die Teilungsabfolge im Keim von Platynereis mit Hilfe des Cdc2-spezifischen Inhibitors Olomoucin experimentell abänderte. Durch pulse chase-Behandlung mit Olomoucin wurde erreicht, daß die Blastomeren die vierte Mitose, anders als im normalen Keim, synchron einleiteten. Die so behandelten Keime entwickelten sich zu Trochophorae, die keine oder nur eine stark reduzierte dorsoventrale Polarität erkennen ließen. Das dorsoventrale Muster entsteht in Keimen von Spiraliern durch die Organisatorwirkung der Blastomeren 3D und 4d und bei Platynereis eventuell auch 2d (Damen und Dictus 1996, Dorresteijn und Eich 1991). Der Teilungsvorsprung, den diese Blastomeren normalerweise gegenüber anderen Zellinien haben, war in den mit Olomoucin-behandelten Keimen stark vermindert. Dadurch haben diese Blastomeren ihre induktiven Kapazitäten möglicherweise nicht, oder nicht rechtzeitig erwerben können, um die benachbarten Zellen gemäß ihrer Position entlang der dorsoventralen Körperachse zu determinieren. Insofern ist die differentielle Zellzyklusregulation fest in das Determinationsgeschehen integriert. Das bedeutet auch, daß zellzyklusregulierende Faktoren Bestandteil der anfangs genannten

Differentielle Expression und molekulare Evolution von Mollusken-Hämocyanin

Molekularbiologische und biochemische Untersuchungen an den zwei Gastropoden-Arten Haliotis tuberculata und Haliotis asinina zeigten, dass diese jeweils zwei unterscheidbare Hämocyanin-Isoformen (HtH1/HaH1 und HtH2/HaH2) besitzen, die in unterschiedlichen Mengen in der Hämolymphe vorkommen. In situ-Hybridisierungsversuche an H. asinina ergaben, dass die beiden Hämocyanin-Isoformen sowohl entwicklungsspezifisch als auch gewebsspezifisch exprimiert werden. Die Transkription der Hämocyanin-Gene setzt bereits 9 Stunden nach der Befruchtung ein und ist von diesem Zeitpunkt an in allen Stadien der Larvalentwicklung nachweisbar. Während dieser Entwicklungsphase sind die Expressionsmuster der beiden Isoformen weitgehend überlappend, wohingegen in adulten Tieren in verschiedenen Geweben isoformspezifische Expressionsmuster auftreten. Diese Ergebnisse deuten auf funktionelle Unterschiede der beiden Hämocyanin-Isoformen hin, und somit darauf, dass Hämocyanin neben dem Transport von Sauerstoff noch weitere Funktionen ausüben könnte (Streit et al., 2005). Weiterhin wurden Untersuchungen zur Primär- und Sekundärstruktur der Hämocyanine aus H. tuberculata und zwei weiteren Arten (Megathura crenulata und Aplysia californica) durchgeführt. Von den Vetigastropoden M. crenulata und H. tuberculata konnten die für die beiden Hämocyanin-Isoformen kodierenden cDNA-Sequenzen vervollständigt werden. Von HtH1 und HtH2 wurden zudem die Gensequenzen komplettiert. Die Sequenzen des KLH1-Gens wurden bis auf 24 bp der 5’UTR und die für das Signalpeptid 1 kodierenden 33 bp ermittelt. Erstmals ist es gelungen, Promotorsequenzen von Mollusken-Hämocyanin-Genen zu sequenzieren. Für HtH2 wurden 181 bp und für KLH2 906 bp des Promotors analysiert. Beide Gensequenzen weisen das konservierte Sequenzmotiv der TATA-Box auf. Wie bei H. tuberculata treten auch bei M. crenulata die beiden Isoformen in unterschiedlichen Mengenverhältnissen in der Hämolymphe auf. In den bisher analysierten Sequenzen dieser beiden Gastropoden konnten keine regulatorischen Elemente identifiziert werden, welche die differentielle Expression bedingen könnten. Die Genstruktur des Hämocyanins von A. californica konnte ebenfalls aufgeklärt werden. Die kodierenden Bereiche des AcH-Gens werden durch insgesamt 45 interne Introns fragmentiert. Im Gen liegen neun Insertionspositionen vor, in denen paraloge Introns inserieren. Zudem sind neun Introns ortholog zu internen Introns anderer Mollusken-Hämocyanin-Gene. Im Fall der paralogen und orthologen Introns handelt es sich um sehr ursprüngliche Introns, die bereits vor der Radiation der Mollusken inserierten. Damit widerlegen diese Ergebnisse die bisherige Annahme („Intron late”-Hypothese), der zufolge die Insertion interner Introns erst nach der Trennung der Gastropoden und Cephalopoden eingesetzt haben soll. Im Zuge dieser Sequenzanalysen ergaben sich zudem Hinweise auf die Existenz einer weiteren AcH-Isoform, da 13 Fragmente ermittelt wurden, die in den kodierenden Bereichen Sequenzunterschiede von bis zu 20% zu AcH 1 aufweisen. Die detaillierten Studien der Haliotis-Hämocyanine deckten einen weitreichenden phylogenetischen Informationsgehalt der Hämocyanin-Sequenzen auf. In weiterführenden Analysen wurden Teilsequenzen der Hämocyanin-Gene von 12 verschiedenen Haliotis-Arten amplifiziert. Der daraus rekonstruierte Stammbaum liefert entsprechend spezifischer Indels eine deutliche Auftrennung der Haliotidae in eine nordpazifische und eine europäischaustralasische Abstammungslinie. Anhand dieser Analyse lassen sich der phylogeographische Ursprung der Haliotiden aufzeigen (Streit et al., 2006) und deren Wanderungsbewegungen nachvollziehen. Hämocyanin-Daten wurden des Weiteren für phylogenetische Analysen auf höherem taxonomischem Niveau eingesetzt. Innerhalb der Klasse der Polyplacophoren wurden interfamiliäre Verwandtschaftsverhältnisse rekonstruiert. Für diese Analyse wurden Teilsequenzen der Hämocyanin-Gene 17 unterschiedlicher Arten ermittelt. Die phylogenetische Untersuchung zeigt, dass sich die Polyplacophoren eindeutig in die beiden Ordnungen der Lepidopleurida und Chitonida auftrennen, da die Chitonida eine spezifische „Deletion” aufweisen. Anhand dieses Merkmals kann auch Callochiton bouveti, der diese „Deletion” besitzt und dessen phylogenetische Einordnung bisweilen umstritten war, eindeutig den Chitonida zugeordnet werden. Innerhalb der Chitonida bilden sowohl die Chitonina als auch die Acanthochitonina monophyletische Gruppen.

SNNAP: A Tool for Teaching Neuroscience

Simulation tools aid in learning neuroscience by providing the student with an interactive environment to carry out simulated experiments and test hypotheses. The field of neuroscience is well suited for the use of simulation tools since nerve cell signaling can be described by mathematical equations and solved by computer. Neural signaling entails the propagation of electrical current along nerve membrane and transmission to neighboring neurons through synaptic connections. Action potentials and synaptic transmission can be simulated and results displayed for visualization and analysis. The neurosimulator SNNAP (Simulator for Neural Networks and Action Potentials) is a simulation environment that provides users with editors for model building, simulator engine and visual display editor. This paper presents several modeling examples that illustrate some of the capabilities and features of SNNAP. First, the Hodgkin-Huxley (HH) model is presented and the threshold phenomenon is illustrated. Second, small neural networks are described with HH models using various synaptic connections available with SNNAP. Synaptic connections may be modulated through facilitation or depression with SNNAP. A study of vesicle pool dynamics is presented using an AMPA receptor model. Finally, a central pattern generator model of the Aplysia feeding circuit is illustrated as an example of a complex network that may be studied with SNNAP. Simulation code is provided for each case study described and tasks are suggested for further investigation.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

Long-lasting synaptic potentiation induced by depolarization under conditions that eliminate detectable Ca2+ signals.

Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca(2+) signals generated by depolarization. While it is widely assumed that Ca(2+) is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca(2+)-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca(2+) entry (using nominally Ca(2+)-free solutions containing EGTA, "0Ca/EGTA") or the absence of detectable Ca(2+) transients (adding BAPTA-AM, "0Ca/EGTA/BAPTA-AM"). The current study reports that depolarization of central ganglia to approximately 0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1-3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting > or = 1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca(2+) during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca(2+)-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.

Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

Molecular mechanisms underlying a cellular analog of operant reward learning.

Operant conditioning is a ubiquitous but mechanistically poorly understood form of associative learning in which an animal learns the consequences of its behavior. Using a single-cell analog of operant conditioning in neuron B51 of Aplysia, we examined second-messenger pathways engaged by activity and reward and how they may provide a biochemical association underlying operant learning. Conditioning was blocked by Rp-cAMP, a peptide inhibitor of PKA, a PKC inhibitor, and by expressing a dominant-negative isoform of Ca2+-dependent PKC (apl-I). Thus, both PKA and PKC were necessary for operant conditioning. Injection of cAMP into B51 mimicked the effects of operant conditioning. Activation of PKC also mimicked conditioning but was dependent on both cAMP and PKA, suggesting that PKC acted at some point upstream of PKA activation. Our results demonstrate how these molecules can interact to mediate operant conditioning in an individual neuron important for the expression of the conditioned behavior.