Elsevier

Brain Research

Volume 822, Issues 1–2, 20 March 1999, Pages 114-125
Brain Research

Research report
Ryanodine receptor modulation of in vitro associative learning in Hermissenda crassicornis

https://doi.org/10.1016/S0006-8993(99)01105-1Get rights and content

Abstract

Classical conditioning of the mollusc, Hermissenda crassicornis, is a model system used to study cellular correlates of associative learning. Paired presentation of light and turbulence, but not unpaired presentations, causes Hermissenda to contract its foot in response to light alone. Intracellular recordings from the type B photoreceptors of the Hermissenda eye reveal a learning specific increase of input resistance, and a reduction of voltage-dependent potassium currents, both of which depend on an elevation of intracellular calcium. Two previously demonstrated sources of calcium are influx through voltage-dependent channels, and release of calcium from intracellular stores through the IP3 receptor channel. Both modeling studies and identification of memory-related genes using RNA fingerprinting suggest that a third source of calcium, release from intracellular stores through the ryanodine receptor, may be involved in classical conditioning. We describe here an experiment suggesting that this third source of calcium is necessary for the cellular changes underlying associative memory storage. Paired presentations of a light stimulus with a turbulence stimulus resulted in a significant increase in input resistance. Unpaired presentations of light and turbulence did not produce a significant increase in input resistance. A third group of nervous systems first was incubated in dantrolene to block release of calcium through the ryanodine receptor, and then received paired training. There was no change in input resistance for this group. The effect of dantrolene on light adaptation of the photoreceptor was assessed by measuring the generator potential of a second light pulse presented some number of seconds after a first light pulse. The results show that at interpulse intervals of 5 s, 10 s and 20 s, the generator potential of the dantrolene group is significantly greater than that of the control group. These results suggest a role for the ryanodine receptor in both a cellular correlate of classical conditioning and light adaptation.

Introduction

Hermissenda crassicornis can be classically conditioned with light, the conditioned stimulus (CS), and turbulence, the unconditioned stimulus (US). Paired presentation of light and turbulence causes a reduction in phototactic behavior, and contraction of the snail's foot in response to light. Intracellular recordings of classically conditioned and control animals have revealed a number of cellular correlates of the behavior. These include an increase in input resistance, and an increase in the spikes evoked by extrinsic current of type B photoreceptors 15, 18, 23, 59. In addition, enhancement of the type B to type A synapses is seen in classically conditioned animals 22, 24, 51. The increased input resistance is mediated by a reduction of potassium currents 3, 4attributed to phosphorylation by protein kinase C 2, 19, 20, 22, 42, which is activated in part by calcium elevation 35, 36. Thus, an elevation in calcium is believed to initiate a chain of cellular events which leads to modification of cell properties underlying the behavior in associative conditioning.

An elevation in intracellular calcium concentration is a requirement for many types of activity-dependent forms of plasticity. Long-term potentiation (LTP), long-term depression (LTD), and associative conditioning of the Aplysia gill withdrawal reflex all require an elevation in intracellular calcium of which there are several sources. One source of calcium is influx through voltage-dependent channels that are activated by depolarization. LTD of the parallel fiber to Purkinje cell synapse [30]depends on calcium influx through voltage-dependent calcium channels 34, 50. LTP of the Aplysia siphon sensory to motor neuron synapses [27]requires calcium influx via NMDA or voltage-dependent calcium channels 6, 41.

A second source of intracellular calcium is release from intracellular stores through the IP3 receptor channel [7], which is required, though not necessarily sufficient, for all forms of hippocampal LTP 10, 39. For the NMDA-dependent form of LTP 1, 32, an additional requirement is influx through voltage-dependent channels, namely the NMDA channel. In contrast, the sole source of calcium for NMDA-independent LTP is release of calcium from intracellular stores through the IP3 receptor channel, initiated by activation of metabotropic glutamate receptors 5, 11, 31, 40, 46.

Similarly, two sources of calcium elevation have been identified in Hermissenda photoreceptors. One is influx through voltage-dependent channels activated during the light-induced depolarization. A second source of calcium in Hermissenda photoreceptors is release from intracellular stores. In invertebrate photoreceptors such as Limulus, Drosophila and squid, light activation of rhodopsin activates the phosphoinositide-specific phospholipase C (PI-PLC) pathway and produces IP38, 14, 26, 54, which produces an increase in intracellular calcium concentration 45, 58. Similarly, the PI-PLC pathway is involved in Hermissenda phototransduction. Antagonists of PI-PLC block the light-induced current [48], whereas injection of IP3 enhances the light-induced sodium current [49]. An early study [13]using the indicator Arsenazo III, which showed that hyperpolarization during the light or perfusion of the photoreceptors with cadmium blocks the light-induced calcium increase, suggested that voltage-dependent channels are the most important source of calcium during a light stimulus. But Fig. 4 from that study shows that light-induced depolarization produces a far greater elevation in intracellular calcium than current-induced depolarization. This is consistent with the more recent study using Fura-2 [43], which demonstrated that blocking voltage-dependent calcium channels does not block the light-induced calcium elevation, and concluded that release from intracellular stores is the most important source of calcium during a light stimulus.

Matzel and Rogers [37]and Talk and Matzel [55]used an in vitro classical conditioning paradigm to evaluate the contribution of these two sources of calcium to a cellular correlate of associative learning in Hermissenda. They showed that injection of hyperpolarizing current during pairings of light (the CS) and statocyst stimulation (the US) did not prevent the pairing-induced increase in input resistance. However, injection of either EGTA (a calcium buffer) or heparin, which blocks release from the ER through the IP3 receptor channel [25], prevented the pairing-induced increase in input resistance. These results suggest that calcium release, and not influx through voltage-dependent channels, is the source of calcium required for this in vitro correlate of associative learning in Hermissenda.

Another calcium release channel is the ryanodine receptor, which has been found in skeletal muscle, cardiac muscle, and nervous tissue [53]. Cavallaro et al. [12]showed ryanodine receptor type two was elevated in water maze trained rats, suggesting it may be involved in learning. Release of calcium through the ryanodine receptor also has been implicated in long-term depression of cerebellar Purkinje cells [33]. The possibility that release of calcium through the ryanodine receptor may be involved in classical conditioning of Hermissenda was suggested by modeling studies of the Hermissenda B photoreceptor [9]. In that study, a multi-compartment, channel level model of the B photoreceptor was developed. Equations for the voltage- and calcium-dependent currents were derived from voltage clamp data. Also included were equations describing buffers, diffusion, release from intracellular stores, and pumping out of the cytosol. Parameters describing these mechanisms were obtained from physiology experiments. Model simulations show that light stimulation causes an increase in the concentration of IP3 and calcium in the rhabdomere, but the concentration of IP3 in the soma does not increase to the same degree as in the rhabdomere, unless very bright lights are used. This leads to the hypothesis that calcium release through the ryanodine receptor, initiated by either influx through the voltage-dependent channel, or diffusion of calcium from the rhabdomere, may be instrumental in classical conditioning.

The present experiments were designed to test that hypothesis. An in vitro conditioning paradigm, similar to that used by Matzel and Rogers [37]and Talk and Matzel [55], was used to assess a cellular correlate of classical conditioning, namely the change in input resistance in response to paired or unpaired stimuli. In vitro conditioning was performed following incubation of the nervous system with Dantrolene, which blocks release of calcium through the ryanodine receptor 21, 44.

Section snippets

Animals

Hermissenda crassicornis were obtained from Sea Life supply. They were maintained in a chilled (14°C), artificial seawater aquarium. Light was provided using a fluorescent bulb with an orange acetate filter for 12 h of the day. Animals were fed three times a week with Hikari ciclid gold fish food.

Electrophysiology

The circumesophageal nervous system was dissected out and pinned to a glass slide. Fig. 1A illustrates a part of the nervous system containing the statocyst and eye. Connective tissue was dissolved by

Results

Fig. 2 illustrates the effect of treatment and time on spike count ratio and input resistance ratio. Paired training in ASW, but not unpaired training, causes an increase in input resistance ratio. However, paired training in 50 μM dantrolene does not cause an increase in input resistance ratio. As shown in Fig. 2A, dantrolene blocks the effect of pairing on input resistance ratio. This effect is seen as early as 5 min after the last paired stimulus, and lasts as long as 20 min, the duration of

Discussion

An experiment was performed to determine if calcium release through the ryanodine receptor was essential for a cellular correlate of classical conditioning in the marine snail, Hermissenda. Increase in input resistance was used as the cellular correlate of classical conditioning, in an in vitro preparation. Pairing of light with statocyst stimulation, but not unpaired light and statocyst stimulation, caused an increase in input resistance which persisted for at least 20 min, the duration of

Acknowledgements

K.T.B. was supported by the US Public Health Service (NIMH Grant K21-MH01141). We thank Lou Matzel for many helpful discussions, and the reviewer for suggesting the light adaptation experiment.

References (60)

  • D.S Lester et al.

    Arachidonic acid and diacylglycerol synergistically activate protein kinase C in vitro and in vivo

    Biochem. Biophys. Res. Commun.

    (1991)
  • K Mikoshiba

    Inositol 1,4,5-trisphosphate receptor

    Trends Pharmacol. Sci.

    (1993)
  • R Payne et al.

    Feedback inhibition by calcium limits the release of calcium by inositol trisphosphate in Limulus ventral photoreceptors

    Neuron

    (1990)
  • J.-P Pin et al.

    The metabotropic glutamate receptors: structure and function

    Neuropharmacology

    (1995)
  • M Sakakibara et al.

    Induction of photoresponse by the hydrolysis of polyphosphoinositides in the Hermissenda type B photoreceptor

    Biochem. Biophys. Res. Commun.

    (1994)
  • K Scott et al.

    Lights out: deactivation of the phototransduction cascade

    Trends Biol. Sci.

    (1997)
  • T Suzuki et al.

    Squid photoreceptor phospholipase C is stimulated by membrane Gqα but not by soluble Gqα

    FEBS Lett.

    (1995)
  • A.C Talk et al.

    Calcium influx and release from intracellular stores contribute differentially to activity-dependent neuronal facilitation in Hermissenda photoreceptors

    Neurobiol. Learn. Mem.

    (1996)
  • Y Tang et al.

    A model of calcium dynamics in cardiac myocytes based on the kinetics of ryanodine-sensitive calcium channels

    Biophys. J.

    (1994)
  • S Alford et al.

    Characterization of Ca2+ signals induced in hippocampal neurons by the synaptic activation of NMDA receptors

    J. Physiol. Lond.

    (1993)
  • D.L Alkon et al.

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

    J. Neurochem.

    (1988)
  • D.L Alkon et al.

    Primary changes of membrane currents during retention of associative learning

    Science

    (1982)
  • L Aniksztejn et al.

    Persistent current oscillations produced by activation of metabotropic glutamate receptors in immature rat CA3 hippocampal neurons

    J. Neurophysiol.

    (1995)
  • J.X Bao et al.

    Involvement of presynaptic and postsynaptic mechanisms in a cellular analog of classical conditioning at Aplysia sensory-motor neuron synapses in isolated cell culture

    J. Neurosci.

    (1998)
  • M.J Berridge

    Inositol trisphosphate and calcium signalling

    Nature Lond.

    (1993)
  • K.T. Blackwell, T.P. Vogl, D.L. Alkon, Cellular mechanisms of calcium elevation involved in long term memory, in: J.M....
  • T.V.P Bliss et al.

    A synaptic model of memory: long term potentiation in the hippocampus

    Nature

    (1993)
  • S Cavallaro et al.

    Late memory-related genes in the hippocampus revealed by RNA fingerprinting

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • J Connor et al.

    Light- and voltage-dependent increases of calcium ion concentration in molluscan photorecpetors

    J. Neurophysiol.

    (1984)
  • K Contzen et al.

    Selective inhibition of the phospholipse C pathway blocks one light-activated current component in Limulus photoreceptor

    J. Comp. Physiol.

    (1995)
  • Cited by (31)

    • The role of intracellular calcium stores in synaptic plasticity and memory consolidation

      2013, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      The role of RyRs in memory formation has been examined using pharmacological agents and genetic knockout techniques within a variety of behavioural paradigms. Although most work has been conducted with mammals and avians, invertebrate paradigms have also demonstrated that RyRs are involved in associative learning such as in the mollusc H. crassicornis (Blackwell and Alkon, 1999). Paired presentations of light and turbulence will in turn cause the contraction of the snail's foot in response to light alone.

    • Multisite Cellular and Synaptic Mechanisms in Hermissenda Pavlovian Conditioning

      2013, Handbook of Behavioral Neuroscience
      Citation Excerpt :

      IP3 opens rhabdomeric Na+ and Ca2+ channels, which results in a depolarizing generator potential and Ca2+ influx.72 IP3 also binds to its receptor (IP3R), which triggers Ca2+ release from the endoplasmic reticulum.118 The Ca2+ influx from the rhabdomere and the IP3R-gated storage compartment can cause Ca2+ release from the RyR-gated compartment.118

    • Inhibition of mGluR1 and IP<inf>3</inf>Rs impairs long-term memory formation in young chicks

      2008, Neurobiology of Learning and Memory
      Citation Excerpt :

      Calcium (Ca2+) is an ubiquitous signal transduction molecule, active in a diverse range of cellular functions in the brain including the regulation of neuronal excitability, neurotransmitter release and gene transcription (Berridge, 1998). Many studies have revealed that Ca2+ has an integral role in memory processing (Bauer, Schafe, & LeDoux, 2002; Blackwell & Alkon, 1999; Deyo, Nix, & Parker, 1992; Gibbs, Gibbs, & Ng, 1979; Quevedo et al., 1998; Woodside, Borroni, Hammonds, & Teyler, 2004). A vital role of Ca2+ can also be identified in long-term potentiation (LTP) and long-term depression (LTD), the putative cellular correlates of learning and memory (Bliss & Collingridge, 1993; Cavazzini, Bliss, & Emptage, 2005; Lynch, 2004).

    • Molecular mechanisms of associative learning in Hermissenda

      2007, Learning and Memory: A Comprehensive Reference
    View all citing articles on Scopus
    View full text