Classical conditioning reduces amplitude and duration of calcium-dependent afterhyperpolarization in rabbit hippocampal pyramidal cells.

1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal pyramidal cells from classically conditioned and control rabbits. Measurements of the AHP were obtained with intracellular recordings from CA1 cells within hippocampal slices.

Specific protein changes during memory acquisition and storage.

Changes in several distinct types of neuronal proteins are now known to be associated with learning. In this review, we will summarize the properties of these proteins and relate these properties to prominent theories of the biochemical basis of memory.

Learning-induced activation of protein kinase C. A molecular memory trace.

PKC activation has been shown to mimic the biophysical consequences of classical conditioning in both rabbit hippocampus and Hermissenda type B cells. Furthermore, conditioning in rabbits results in the 24 h translocation of PKC from cytosol to membrane, which is probably responsible for mediating the biophysical consequences of conditioning. A model has been presented that suggests that long-term translocation of PKC occurs via the synergistic activation of a DG dependent pathway that activates PKC and a calcium dependent pathway that activates CaM kinase.

The nature of memory.

Memory, and its failings, are surely the most important factors in determining human behaviour. In humans and the higher animals the memory process is manifestly so complex as to defy direct experimental investigation. However, the snail Hermissenda crassicornis, with a nervous system many orders of magnitude simpler, has been shown to be capable of associative learning, and investigation of its cellular basis throws light on the mechanisms governing the memory process generally, and, more specifically, as now being revealed in the rabbit hippocampus and cerebellum.

Sequential modification of membrane currents with classical conditioning.

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985).

AHP reductions in rabbit hippocampal neurons during conditioning correlate with acquisition of the learned response.

Young adult male albino rabbits were conditioned using a free field auditory conditioned stimulus (CS) and periorbital shock unconditioned stimulus (US) in a short delay eye blink paradigm. All rabbits received two 80-trial training sessions. Intracellular recordings were made from hippocampal CA1 pyramidal neurons within brain slices prepared 24 h following the second training session.

Prolonged RNA changes in the Hermissenda eye induced by classical conditioning.

The incorporation of 32P into mRNA and the total amount of mRNA were increased 3- to 4-fold in eyes isolated from Hermissenda crassicornis trained to associate light with rotation on a turntable compared with animals trained with equal numbers of light and rotation events presented randomly and with naive animals. Incorporation of 32P into poly(A)- RNA was reduced by as much as 60%. The RNA changes were strongly correlated with the degree of learning and could not be accounted for by changes in [32P]ATP content.

Regulation of Hermissenda K+ channels by cytoplasmic and membrane-associated C-kinase.

Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low-molecular-weight protein was changed only within the eyes of conditioned Hermissenda.

Transient and persistent depolarization-induced changes of protein phosphorylation in a molluscan nervous system.

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea-water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g.

Classical conditioning induces long-term translocation of protein kinase C in rabbit hippocampal CA1 cells.

The role of the Ca2+/phospholipid-dependent, diacylglycerol-activated enzyme protein kinase C (PKC) in rabbit eyelid conditioning was examined. PKC was partially purified from the CA1 region of hippocampal slices from naive, pseudoconditioned, and conditioned rabbits 24 hr after the rabbits were well conditioned. Crude membrane and cytosol fractions were prepared.

Enhancement of synaptic potentials in rabbit CA1 pyramidal neurons following classical conditioning.

A synaptic potential elicited by high-frequency stimulation of the Schaffer collaterals was enhanced in hippocampal CA1 pyramidal cells from rabbits that were classically conditioned relative to cells from control rabbits. In addition, confirming previous reports, the after-hyperpolarization was reduced in cells from conditioned animals. We suggest that reduced after-hyperpolarization and enhanced synaptic responsiveness in cells from conditioned animals work in concert to contribute to the functioning of hippocampal CA1 pyramidal cells during classical conditioning.

A spatial-temporal model of cell activation.

A spatial-temporal model of calcium messenger function is proposed to account for sustained cellular responses to sustained stimuli, as well as for the persistent enhancement of cell responsiveness after removal of a stimulus, that is, cellular memory. According to this model, spatial separation of calcium function contributes to temporal separation of distinct phases of the cellular response. At different cellular sites, within successive temporal domains, the calcium messenger is generated by different mechanisms and has distinct molecular targets.

Molecular mechanisms of associative learning in mammal and mollusc.

Recent work in this laboratory has begun to cast light on the biochemical mechanisms by which a cell stores associatively acquired information. This appears to occur principally via two general pathways. The first seems to be a long-term activation of protein kinase C (resulting in long-term alterations in protein phosphorylation) while the second involves changes in RNA synthesis.

Inhibition of protein synthesis prolongs Ca2+-mediated reduction of K+ currents in molluscan neurons.

Elevated intracellular Ca2+ concentration within the Hermissenda type B cell has previously been shown to cause transient reduction of both the early K+ current IA and the delayed, Ca2+-dependent K+ current ICa2+-K+, a reduction that is more permanent with classical conditioning. Other earlier experiments suggested that Ca2+-mediated reduction of K+ currents initially involves the dual activation of Ca2+/calmodulin-dependent and Ca2+/lipid-dependent protein kinases. In the present study, voltage-clamp conditions that cause substantial increases in intracellular Ca2+ concentration (i.

In vitro associative conditioning of Hermissenda: cumulative depolarization of type B photoreceptors and short-term associative behavioral changes.

Cumulative depolarization of Hermissenda type B photoreceptors, a short-term neural correlate of associative learning, was produced by simulating associative training in the isolated nervous system (in vitro conditioning). This simulation entailed stimulation and recording from three classes of neurons normally affected by the associative training procedure: a type B photoreceptor, the silent/excitatory (S/E) optic ganglion cell, and a statocyst caudal hair cell. Exposure of the isolated nervous system to five simultaneous pairings of light and current-induced impulse activity of the caudal hair cell resulted in an average 10-mV depolarization of type B cells.

Associatively reduced withdrawal from shadows in Hermissenda: a direct behavioral analog of photoreceptor responses to brief light steps.

When the nudibranch Hermissenda crassicornis encounters a shadow in an otherwise uniformly illuminated field, it stops and turns back into the light within seconds. Associative conditioning, with paired light and rotation stimuli, produces learned modifications of phototaxis in illumination gradients. This same training procedure significantly reduced the ability of paired, but not random or naive control animals, to withdraw from shadows.

Effects of alpha 2-adrenergic agonists and antagonists on photoreceptor membrane currents.

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs.

Implicating causal relations between cellular function and learning behavior.

Learning in the nudibranch mollusc Hermissenda shows many features of vertebrate associative conditioning. Pairings of light and rotation produce conditioned suppression of phototaxis, which is retained for days, shows savings, extinction, contingency sensitivity, and, recently, temporal specificity. In addition, specific features of the behavior have been shown to undergo classical Pavlovian conditioning.

Ca2+/diacylglycerol-activated, phospholipid-dependent protein kinase in the Hermissenda CNS.

In mammalian systems, Ca2+/diacylglycerol-activated phospholipid-dependent protein kinase (C-kinase) appears to play an important role in regulating physiological responses that outlast the transient rise in cytosolic Ca2+. Electrophysiological experiments in neurons of the nudibranch mollusc, Hermissenda crassicornis, have suggested a role for C-kinase in the long-lasting reductions in early and late K+ currents that have been observed following associative learning. Accordingly, we have investigated the catalytic properties of C-kinase in Hermissenda CNS.

Inositol trisphosphate regulation of photoreceptor membrane currents.

In previous studies elevation of intracellular Ca2+ was shown to cause prolonged reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the membrane of the isolated Hermissenda photoreceptor, the type B cell (Alkon et al., 1982b; Alkon and Sakakibara, 1985). Here we show that iontophoretic injection of inositol trisphosphate (IP3), but not inositol monophosphate, also caused prolonged reduction of IA and ICa2+-K+.

Autoradiographic measurement of tritiated agmatine as an indicator of physiologic activity in Hermissenda visual and vestibular neurons.

[3H]Agmatine (amino-4-guanidobutane) has been shown to be potentially useful for identifying and assessing the ACh sensitivity of specific neurons. Small cationic amines are able to permeate ACh-activated ion channels in sympathetic neurons and vertebrate endplates. Sensory neurons of the photic pathway in the nudibranch mollusc Hermissenda crassicornis are cholinergic and the synaptic interactions between the photic and vestibular systems have been well characterized electrophysiologically.

The role of neurochemical modulation in learning.

Tsukahara creatively exploited the advantages of a "simple system" approach in a vertebrate context to gain cellular insights into the learning process. The molluscs Aplysia and Hermissenda have provided useful invertebrate examples of this approach. For classical conditioning of Hermissenda a temporal sequence of cellular transformations has been found to correspond to and to substantially account for a learning-specific behavioral transformation.

Modulation of calcium-mediated inactivation of ionic currents by Ca2+/calmodulin-dependent protein kinase II.

Iontophoretic injection of Ca2+ causes reduction of I0A (an early rapidly activating and inactivating K+ current) and I0C (a late Ca2+-dependent K+ current) measured across the isolated type B soma membrane (Alkon et al., 1984, 1985; Alkon and Sakakibara, 1984, 1985). Similarly, voltage-clamp conditions which cause elevation of [Ca2+]i are followed by reduction of I0A and I0C lasting 1-3 min.