Receptor-Stimulated Phospholipase A2 Liberates Arachidonic Acid and Regulates Neuronal Excitability Through Protein Kinase C

Isabel A. Muzzio,3 Chetan C. Gandhi,1 Upendra Manyam,2 Aarron Pesnell,1 and Louis D. Matzel1

 1Department of Psychology, Program in Biopsychology and Behavioral Neuroscience, Rutgers University, Piscataway 08854;  2Department of Ceramic Engineering, Rutgers University, New Brunswick, New Jersey 08903; and  3Center for Neurobiology and Behavior, Columbia University, New York, New York 10032


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muzzio, Isabel A., Chetan C. Gandhi, Upendra Manyam, Aarron Pesnell, and Louis D. Matzel. Receptor-Stimulated Phospholipase A2 Liberates Arachidonic Acid and Regulates Neuronal Excitability Through Protein Kinase C. J. Neurophysiol. 85: 1639-1647, 2001. Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca2+ increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca2+, co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A2 (PLA2), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA2 stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 µM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA2 inhibitor arachidonyltrifluoromethyl ketone (50 µM). Furthermore, direct stimulation of PLA2 by melittin (10 µM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 µM) and light (to elevate intracellular Ca2+), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 µM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for >= 90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 µm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA2 signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca2+ to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca2+ may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Native light-elicited behaviors of Hermissenda crassicornis can be modified using an associative learning paradigm in which a discrete light (conditioned stimulus; CS) is paired with rotation (unconditioned stimulus; US). At the behavioral level, learning is manifest as the emergence of novel responses to light (i.e., conditioned responses), including reduced phototaxis and contraction of the animal's foot (Crow and Alkon 1978; Lederhendler et al. 1986). At the cellular level, associative conditioning induces neuronal and synaptic facilitation at an initial point of convergence between the visual and vestibular pathways, the type B photoreceptors of the animal's eye (Alkon 1984; Alkon et al. 1982; Crow and Alkon 1980). This form of facilitation can be studied using an in vitro conditioning procedure that mimics training in the intact animal, in which isolated nervous systems are exposed to pairings of light and mechanical stimulation of the vestibular hair cells that project monosynaptically onto B photoreceptors. Both behavioral and in vitro training produce comparable increases in cellular excitability that are mediated by a reduction of voltage- (IA) and Ca2+-dependent (IK-Ca) K+ currents across the B cell membrane (Alkon et al. 1985). This reduction of K+ conductance results in an increase in the B cell's input resistance (Matzel et al. 1992; West et al. 1982), evoked spike rate (Muzzio et al. 1998; Rogers and Matzel 1995a), and action potential duration (Gandhi and Matzel 2000). As a consequence, the synaptic connections of the B photoreceptors with postsynaptic sensory (Frysztak and Crow 1994; Schuman and Clark 1994) and motor cells (Goh and Alkon 1984; Goh et al. 1985) are potentiated, effects that have been proposed to contribute to expression of the learned modifications of light-elicited behavior.

Stimulation of B photoreceptors with light (CS) initiates a phospholipase C (PLC)-mediated cascade that generates inositol triphosphate (IP3) and diacylglycerol (DAG) and a concomitant elevation of intracellular Ca2+. This Ca2+ elevation is in part due to depolarization-dependent Ca2+ influx but more significantly, from a release of Ca2+ from intracellular stores through the IP3 and ryanodine receptor channels (Blackwell and Alkon 1999; Connor and Alkon 1984; Muzzio et al. 1998; Sakakibara et al. 1994; Talk and Matzel 1996). Vestibular stimulation activates an independent cascade of events, triggered by binding of GABA [and potentially serotonin (5-HT)] onto postsynaptic G-protein-coupled receptors on type B cells (Rogers and Matzel 1995b; Rogers et al. 1994). Pharmacologic manipulations suggest that these receptors are linked to phospholipase A2 (PLA2) and promote the liberation of arachidonic acid (AA) (Talk et al. 1997).

While the signaling cascades in the B cell that are initiated by light and transmitter binding have been partially delineated, the nature of the biochemical cascade that is uniquely activated by the temporal convergence of light and rotation during associative conditioning is not fully understood. Despite this incomplete understanding, evidence indicates that the ultimate point of convergence of the two cascades is on protein kinase C (PKC), which regulates many of the biophysical changes in the B cell following associative training (Alkon et al. 1988; Crow and Forrester 1993a,b; Farley and Auerbach 1986; Farley and Schuman 1991; Lester et al. 1991; Matzel et al. 1990, McPhie et al. 1993; Muzzio et al. 1998).

The principle co-factors necessary for the activation of conventional PKC isoforms, DAG and Ca2+, are generated in the B photoreceptors in response to light. Paradoxically, light-alone or unpaired presentations of light and rotation are not sufficient to induce a sustained activation of PKC or to produce the biophysical changes in the B cell soma that accompany conditioning (Muzzio et al. 1998). However, the activation of many PKC isoforms is dependent on (or is potentiated by) the interaction of multiple cofactors, including phospholipids and/or fatty acids (Axelrod 1990; Axelrod et al. 1988), and evidence from a variety of cell types indicates that AA and conventional PKC activators have a synergistic influence on the enzyme (Chen and Murakami 1992; Seifert et al. 1987; Shearman et al. 1991; Verkest et al. 1988). Furthermore, AA alone may preferentially activate PKC subspecies that are not sensitive to conventional activators (Khan et al. 1995; Shearman et al. 1989). This raises the possibility that in the Hermissenda B cell, PKC activation during associative conditioning may be dependent on high levels of free AA, which may stimulate (or potentiate the stimulation of) isoforms of the enzyme that are not maximally activated by DAG and Ca2+ alone. In support of this hypothesis, it has been reported that the combined application of DAG and AA to the B photoreceptors produces a decrease in membrane K+ conductance that is larger than that which results from exposure to either agent alone (Lester et al. 1991). Moreover, antagonism of PLA2 or inhibition of AA metabolism blocks the induction of neuronal facilitation in the B cells during in vitro associative conditioning (Talk et al. 1997).

The purpose of the present series of experiments was to directly assess whether fatty acids (e.g., AA) were generated in Hermissenda B cells as a consequence of the stimulation of surface receptors. To this end, we monitored emission from the fluorescent fatty acid probe acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB) in response to direct stimulation of PLA2 by GABA, an endogenous neurotransmitter that mediates the synaptic response between vestibular hair cells and postsynaptic B cells (Alkon et al. 1993; Rogers et al. 1994). Furthermore the effects of GABA on ADIFAB emission were assessed in the presence of selective inhibitors of PLA2, which is believed to mediate the liberation of AA by stimulation of the G-protein-coupled GABA receptor. Finally, in parallel experiments, we determined the effects and time course of AA-induced neuronal facilitation in the B cell during in vitro associative conditioning.

Some of this work comprised the doctoral dissertation of I. A. Muzzio.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

H. crassicornis (0.80-1.4 g) were obtained from Sea Life Supply (Sand City, CA) and housed in a tank filled with refrigerated (12°C) artificial sea water (ASW). The animals were maintained on a 10 h/ 14 h light/dark cycle. The light was filtered through yellow acetate and had an intensity of 20 µW × cm-2 at the water's surface. Animals were fed portions of TetraMarine Flakes on alternating days. All experimental manipulations were conducted during the middle 8 h of the light cycle.

Tissue preparation for fluorescent assay

The eyes (containing 5 photoreceptors) and their synaptic endings (hereafter referred to as "eyes") were surgically isolated from the Hermissenda nervous system and suspended in ASW [containing (in mM) 430 NaCl, 10 CaCl2, 50 MgCl2, and 10 KCl, buffered with Tris to a pH of 7.4-7.5] with 40 mM glucose at 4°C. The synaptic endings were included because previous reports have demonstrated the presence of GABA receptors at the B cell synaptic terminals (Alkon et al. 1993; Rogers et al. 1994). Samples containing 30 eyes were used for all fluorescent assays.

Free fatty acid (FFA) determination with ADIFAB

Each sample of eyes was transferred to a 45-µl microcuvette (Hellma Cells, Forest Hills, NY) containing ASW (4°C) and 3.6 µM ADIFAB (Molecular Probes, Eugene, OR). ADIFAB responds to FFA binding by producing a shift in fluorescence emission from 432 nm in the apo form to 505 nm in the holo form of the dye. Increasing amounts of FFA decrease the fluorescence at 432 nm and increase it at 505 nm. Thus the concentration of FFA is proportional to the ratio of fluorescence at 505 and 432 nm, with a higher ratio indicative of an increasing concentration of FFA (Richieri and Kleinfeld 1995; Richieri et al. 1994). Fluorescence was captured with a Perkin Elmer MPF66 fluorometer using the photon counting mode with excitation at 386 nm, excitation slits set at 4 nm and emission slits at 10 nm.

On addition of ADIFAB to the cuvette containing the eyes, a baseline measure of fluorescence was recorded. Subsequently, independent samples were treated by the addition of either GABA (Sigma, St Louis, MO) dissolved in 5 µl of ASW or 5 µl of ASW alone (a control condition). It should be noted that ADIFAB exhibits only weak selectivity for AA and, in fact, binds other fatty acids including linoleate, oleate, and palmitate. Thus although ADIFAB is commonly described as an indicator of free AA, results based solely on ADIFAB emissions can be difficult to interpret, that is, convergent evidence implicating AA is often required before definitive conclusions can be reached. For instance, AA is most commonly regulated by PLA2, so ADIFAB's selectivity for AA can be better inferred if changes in ADIFAB emission are blocked by PLA2 inhibition or mimicked by PLA2 stimulation. With that in mind, a third sample was exposed to the PLA2 activator melittin (Gonzalez et al. 1997) (Calbiochem, La Jolla, CA), with the expectation that any effect of GABA on AA levels might be mimicked by the direct stimulation of PLA2. The final concentration of GABA and melittin in the sample cuvettes was 12.5 and 10 µM, respectively. Likewise, in a second experiment, the GABA and ASW conditions described in the preceding text were replicated, and a third sample was included in which GABA incubation was performed in the presence of the PLA2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3, Calbiochem). This inhibitor specifically blocks PLA2 (Riendeau et al. 1994; Street et al. 1993) with a higher affinity for the Ca2+-dependent isoenzyme (Ackermann et al. 1995; Alzola et al. 1998). AACOCF3 was dissolved in DMSO and redissolved in ASW to a final concentration of 50 µM (0.004% DMSO). This concentration of AACOCF3 has been shown to block PLA2 in central neurons (Phillis and O'Regan 1996). Small concentrations of DMSO, such as the one reported here, do not have observable effects on physiological processes in the B photoreceptors (Talk et al. 1997). However, to rule out the possibility that DMSO could affect the biochemical reactions studied in this experiment, equal concentrations of DMSO were also added to the GABA and ASW samples. In all instances, fluorescence was measured immediately and 5 and 10 min after the treatment of the sample. For the baseline measures and each of the three posttreatment measures, the fluorometer was set to average three scans (0.8 scans/s) between 420 and 515 nm.

Richieri and Kleinfeld (1995) demonstrated that the concentration of FFA for a single molecular species of FFA can be determined using the following formulas
[FFA]=<IT>K</IT><SUB><IT>d</IT></SUB><IT>Q</IT>(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)<IT>/</IT>(<IT>R</IT><SUB><IT>max</IT></SUB><IT>−</IT><IT>R</IT>) (1)

[ADIFABb]=[ADIFABtotal]<IT>Q</IT>(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)<IT>/</IT>(<IT>R</IT><SUB><IT>max</IT></SUB><IT>−</IT><IT>R</IT><IT>+</IT><IT>Q</IT>(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)) (2)
Equation 1 represents the aqueous-phase concentration of the fatty acids and Eq. 2 is the concentration of fatty acid bound to ADIFAB determined from the ratio of 505 to 432 nm fluorescence. The total amount of fatty acids in the sample is obtained by adding Eqs. 1 and 2. R is the measured ratio of 505 to 432 nm intensities and Ro is the ratio with no FFA. In the present study, Ro was the ratio calculated during baseline before the onset of any treatment. Rmax is the value when ADIFAB is saturated and Q = Iu(432)/Ib(432) where Iu(432) and Ib(432) are the ADIFAB intensities with zero and saturating concentrations of FFA, respectively. Values for these two empirical constants and the equilibrium constant (Kd) were obtained from Richieri et al. (1994). The Kd value used here (1.63 µM) corresponds to the equilibrium constant for arachidonate at 37°C and pH 7.4. Introducing the values of all the constants into Eqs. 1 and 2
[FFA]=1.63 &mgr;M 19.5(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)<IT>/</IT>(<IT>11.5−</IT><IT>R</IT>) (3)

[ADIFABb]=[3.6 &mgr;M] 19.5 (<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)<IT>/</IT>(<IT>11.5−</IT><IT>R</IT><IT>+19.5 </IT>(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>o</IT></SUB>)) (4)

Electrophysiology

Hermissenda nervous systems were dissected, pinned to strips of grease on a glass slide, subjected to proteolysis (Protease type IX, Sigma Chemical; 10 mg/ml) for 8 min at 22°C, and rinsed with ASW at 5°C. For intracellular recording, glass microelectrodes were pulled to a tip resistance of 20-35 MOmega and filled with 3.0 M KAc. The microelectrodes were connected by a chloridized wire to the input stage of a high-impedance amplifier (Axoclamp 2A). Responses were recorded on a storage oscilloscope and on a Brush Pen Recorder. During baseline and posttraining measurements, a small negative current was applied through the recording electrode to hold the membrane potential at -60 mV. Input resistance and evoked spike rate were assessed by passing small negative and positive current pulses (0.6 nA) using a balanced-bridge circuit. Light responses were induced in the B photoreceptors through a fiber optic bundle that projected an unfiltered white light (600 µW × cm-2) onto the nervous system. Preparations were continuously perfused with ASW at a rate of 1 ml/min.

Pharmacological manipulations of in vitro associative training

After successful impalement of a medial B cell, the photoreceptors received 8 min of dark adaptation. All training was conducted from an initial holding potential of -60 mV. In the first electrophysiology experiment, four groups of isolated nervous systems (n = 9) received one of four combinations of bath-applied oleic acid, light, and chelerythrine (Ch; a specific catalytic inhibitor of PKC): oleic acid and light (OA + L), oleic acid alone (OA), light alone (L), and oleic acid, light, and Ch (OA + L + Ch). The free acid form of oleic acid (Sigma) was dissolved in ethyl alcohol, aliquoted, and frozen at -20°C for final dilution in ASW (20 µM; final ethanol concentration, 0.03%). Equal concentrations of ethanol were added to the light alone control condition. Ch (Sigma) was dissolved in water, aliquoted, and stored at -20°C for final dilution in ASW (20 µM). All drugs were bath applied after the cells were dark adapted, and the baseline measures were taken. OA was perfused for 8 min and Ch for 16 min (beginning 8 min prior to OA application). Immediately after application of OA to the bath or after recording of baseline measures in the light alone group, the nervous systems were exposed to five presentations of a 5-s light. The light stimulation was provided by an unfiltered white light (600 µW × cm-2) that was focused onto the nervous systems through a fiber optic bundle. Resistance and spike rate activity were taken prior to the drug treatment (baseline) and 5, 10, and 45 min after training on injection of positive and negative (0.2, 0.4, 0.6 nA) brief (400 ms) current pulses.

Following associative learning, the maintenance of neuronal facilitation in Hermissenda B cells for periods 90 min requires new protein synthesis (Crow et al. 1999; Ramirez et al. 1998). It is of interest whether the AA-dependent stimulation of PKC in the B cell promotes a protein synthesis-dependent form of neuronal facilitation or whether the facilitation remains PKC dependent within 90 min of initial induction. However, in this preparation, stable intracellular recordings lasting 90 min or more are difficult to obtain. Thus to obtain electrophysiological data after the 90 min interval at which neuronal facilitation may become PKC independent, a between-groups analysis of cell excitability was used where recordings were made in different nervous systems 45 and 90 min after in vitro conditioning. For this procedure, independent groups of isolated Hermissenda nervous systems were exposed to in vitro training protocols analogous to that of the prior experiment, and electrophysiological measurements of B cell excitability were obtained at designated times (45 or 90 min) after training.

Nervous systems were dissected and subjected to proteolysis as described in the preceding text. Six groups were included in this experiment (n = 7). The slides on which the nervous systems were mounted were placed in a petri dish inside a light- and sound-proof training incubator for 60 min before training. The temperature inside the training chamber was maintained at 12°C. Sixty centimeters above the platform was a 40 W (nominal at 130 V) light that served as the CS. This light intensity was adjusted so that it illuminated the dish with a uniform intensity of 50 fc. During the 60 min prior to training, the nervous systems were bathed in ASW or anisomycin (ANI, 1 µM, Sigma), each of which was supplemented with 40 mM glucose. We have previously found that this ANI treatment inhibited the synthesis of new proteins by >80% (Ramirez et al. 1998). At the end of this period, ASW- or ANI-treated nervous systems were exposed to one of three different drug/training conditions. Two of the groups bathed in ASW were incubated for 8 min in OA (20 µM) and received five 5-s light presentations (group OA + L) with an interstimulus interval (ISI) of 2 min, and two groups were exposed to OA in the absence of light presentations (group OA). Immediately after the fifth light presentation (or at the comparable time for group OA), the bathing solution containing OA was replaced with ASW, after which the nervous systems were kept in the darkened incubator. Nervous systems bathed in ANI received treatment similar to group OA + L, i.e., exposure to OA and light except that ANI was present in the bathing medium (group OA + L + ANI). In all cases, the slides containing the nervous systems were removed from the incubator either 30 or 75 min after treatment and were transferred to the electrophysiology recording stage. In this manner, 15 min was allowed for electrode impalement and subsequent dark adaptation before electrophysiological recordings were obtained (i.e., 45- or 90-min post treatment). To ensure that the conditions were comparable across preparations, cells were not included in the experiment if impalement required >5 min. Indices of cell excitability were recorded as described in the preceding text.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA-mediated release of FFA

ADIFAB fluorescence was used to quantify changes in FFA levels induced by synaptic stimulation with GABA or by the PLA2 activator melittin. During the 10 min prior to treatment with these agents, baseline ratios were calculated for each of the groups represented in an experiment. These baseline ratios remained relatively constant across time and exhibited little variability across groups within an experiment, ranging from 0.343 to 0.347 in our first experiment (Fig. 1A) and from 0.385 to 0.387 in the second experiment (Fig. 1B). Thus while the baseline ratios were comparable across groups within an experiment, relatively higher variability in these values was observed between experiments. This was likely due to the fact that the isolated eyes were intrinsically fluorescent, and the signal could vary as a function of total tissue volume. In the individual experiments, the volume of the tissue was equated for each of the groups but varied across experiments.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Free fatty acid assessment using the fluorescent probe acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). A: ratio of ADIFAB fluorescence at 505 to 432 nm as function of time. An increase in ratio of 432 to 502 nm was observed in the groups that were incubated in GABA or melittin but not in the group incubated in ASW. Brackets indicate SE of measurement. B: ratio of ADIFAB fluorescence at 505 to 432 nm as function of time in a partial replication of the experiment described in A. An increase in the ratios of 505 to 432 nm was again observed following exposure to GABA, but this effect was blocked if the exposure to GABA occurred in the presence of the PLA2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3).

Figure 1A illustrates the change in ADIFAB fluorescence ratio in response to ASW (control solution), GABA, or melittin. Three scans were obtained and averaged immediately and 5 and 10 min after exposure to the treatment solution, and the ratios of 505 to 432 nm intensities were calculated. Exposure of the isolated eyes to GABA or melittin induced an increase in the fluorescence ratios (indicative of an increase in FFA concentration), presumably owing to lipid hydrolysis mediated by the stimulation of PLA2. In melittin-treated cells, there was an initially larger increase in the fluorescence relative to GABA treatment. However, 5 and 10 min after exposure to either melittin or GABA, the ratios were comparable and significantly elevated relative to cells treated with ASW. The ASW treatment had no significant effect on the fluorescence ratio throughout the 10 min of recording. These results indicate that GABA binding stimulates the liberation of FFA in the B photoreceptors and that this effect of GABA on FFA may be mediated by PLA2-stimulated lipid hydrolysis. This later possibility is evaluated in the following text.

Fatty acid liberation through activation of PLA2

PLA2 can mediate direct receptor-induced AA liberation through the hydrolysis of phospholipids (Burch et al. 1986; Farooqui et al. 1997; Jelsema 1987; Nakashima et al. 1988). Indeed, in some cell types, such as cerebellar granule cells, AA is purported to be liberated exclusively through the stimulation of PLA2 signaling pathways (Lazarewicz et al. 1990). Alternatively, in neurons such as dorsal root ganglion cells, the liberation of AA can result from PLC-stimulated diglyceride lipase (Allen et al. 1992; Bell et al. 1979). We attempted to further elucidate the endogenous signaling pathway through which GABA liberates AA in the Hermissenda B cell. To this end, we conducted a partial replication of the prior experiment (excluding the melittin manipulation) with the addition of one sample in which AACOCF3, a specific inhibitor of PLA2, was applied concomitantly with GABA.

Figure 1B summarizes the results of this experiment. Addition of ASW (a control solution) or GABA produced changes like those reported in the prior experiment, i.e., GABA induced a fast and persistent rise in ADIFAB emission ratios while ASW had no significant effect. However, when AACOCF3 was present during GABA incubation, the rise in the fluorescence ratio was severely inhibited such that ratios did not differ significantly from those recorded in response to ASW. As in the prior experiment, these results indicate that GABA liberates fatty acids in the B photoreceptors, but further, confirms that this liberation is mediated by PLA2 stimulation.

AA-mediated facilitation of neuronal excitability

A B photoreceptor was impaled with an intracellular recording electrode in isolated nervous systems. After a period of darkness, the nervous systems were then exposed to combinations of OA (a stable analogue of AA), light, and the inhibitor of PKC catalytic activity, Ch. Prior to these treatments, comparable neurophysiological responses (i.e., input resistance and evoked spike rates) were observed across the four treatment groups (n = 9), and these baseline measures of resistance and evoked spike rate were not significantly different, F(3, 21) = 0.59 and F(3, 21) = 1.41, respectively. Figure 2, top, illustrates the mean percent change in the hyperpolarizing voltage responses (indicative of input resistance) of the B photoreceptor in response to -0.6 nA current injections (from a -60 mV holding potential) 5, 15, and 45 min following exposure to OA paired with light (OA + L), OA alone (OA), light alone (L), or OA paired with light in the presence of Ch (OA + L + Ch). Representative voltage records obtained during baseline recording and 45 min after treatment are provided in Fig. 3. Treatment with OA + L induced an increase in input resistance that remained significantly elevated for the 45 min recording period. The OA alone, L alone, and OA + L + Ch treatments were associated with small decreases in input resistance that remained relatively constant throughout the 45-min recording period. A two-way repeated-measures ANOVA of these voltage responses, with type of treatment and time as factors, revealed a significant effect of type of treatment, F(3, 42) = 6.56; P < 0.003, but no effect of time. Multiple comparisons using the Student's Newman-Keuls' method indicated that the group exposed to OA and light displayed a significant increase in resistance in comparison to exposure to either OA or light alone or to OA and light in the presence of Ch, Ps < 0.05. These results indicate that OA + L promotes an increase in membrane resistance that is sustained for >= 45 min and that this increase in excitability is mediated by PKC since Ch completely blocked the effect. Given that OA alone did promote any increase in excitability, we can conclude that the facilitatory effect of OA is dependent on light (i.e., an increase in intracellular Ca2+ levels).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Contribution of oleic acid to excitability increases in the B cell. Top: percent change in voltage responses (input resistance) to -0.6 nA relative to premeasure values. Two groups received five 5-s lights (ITI = 2 min) in the presence of oleic acid (OA + L) or OA and chelerythrine (Ch), a specific protein kinase C (PKC) inhibitor (OA + L + Ch). A 3rd group was incubated in OA in the dark (OA). Only the group receiving light presentations in the presence of OA (OA + L) exhibited an increase in input resistance that was sustained for 45 min. Brackets indicate standard errors. Bottom: difference in the number of evoked spikes elicited by +0.6-nA current injections between premeasure and measures taken 5, 15, and 45 min after each of the three treatment conditions. Only the group trained with discrete lights in the presence of OA (OA + L) exhibited an increase in evoked spike rate that was sustained for 45 min.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 3. Representative voltage records from 4 B cells that contributed to the mean values summarized in Fig. 2. Responses to hyperpolarizing (left) and depolarizing (right) current injections were obtained during a baseline period (BL) and again 45 min after 1 of the 4 treatments described in Fig. 2. Concomitant exposure of the B cell to OA and light (OA + L) resulted in an increase in the hyperpolarizing voltage response (indicative of an increase in membrane resistance) and an increase in the rate of spike discharge during depolarization.

Figure 2, bottom, depicts the change in number of evoked spikes in relation to baseline values, and representative voltage records obtained during baseline recording and 45 min after treatment are provided in Fig. 3. The results of this analysis of evoked spikes is in agreement with the data on voltage responses presented in the preceding text. A two-way ANOVA with repeated measures revealed an effect of type of training, F(3, 42) = 7.34, P < 0.002, but no effect of time. Multiple comparisons (Student's Newman-Keuls' method) indicated that exposure to OA + light induced a significant increase in number of evoked spikes relative to exposure to either OA alone or L alone, and relative to exposure to OA + light + Ch, Ps < 0.05. In total, these results demonstrate that in the B photoreceptors, light (and its influence on intracellular Ca2+ levels and DAG production) interacts with FFAs (that can be generated by transmitter binding induced by presynaptic activity) to induce a persistent increase in cell excitability that is mediated by PKC.

Protein synthesis-dependence of AA-mediated increases in cell excitability

A characteristic feature of memory storage is that protein synthesis and transcription inhibitors block long-term memories while having no effect on initial memory induction (Castellucci et al. 1989; Tully et al. 1994; for reviews, see Davis and Squire 1984; Miller and Matzel 2000). In Hermissenda B cells, Crow and Forrester (1990) found that inhibition of protein synthesis during pairings of light and 5-HT (a facilitatory transmitter polysynaptically released onto B cells during hair cell stimulation) impairs the long-term (but not the initial) enhancement of light-induced generator potentials (another index of neuronal facilitation). More recently, Ramirez et al. (1998) found that in intact animals as well as in the isolated nervous system, associative training initiates protein synthesis-dependent processes within 90 min of the completion of training, i.e., both behavioral and neurophysiological evidence of learning was absent 90 min after training when training was conducted in the presence of the protein synthesis inhibitor anisomycin (also see Crow et al. 1999).

The effect of in vitro exposure of Hermissenda nervous systems to OA, OA + light, or OA + light in the presence of ANI (OA + L + ANI) on the B cell's input resistance and evoked spike rate is summarized in Fig. 4. Mean voltage responses to hyperpolarizing current (indicative of membrane resistance) are illustrated in the Fig. 4, top, and these responses were found to differ significantly across groups, F(5, 36) = 7, 79, P < 0.0001. Multiple comparisons (Student's Newman-Keuls' method) indicated that relative to exposure to OA alone, paired exposure to OA and light in the presence of either ASW (OA + L) or ANI (OA + L + ANI) induced a significant elevation of membrane resistance that persisted for >= 90 min, Ps < 0.05. The mean number of evoked spikes in response to +0.6-nA current injections are illustrated in Fig. 4, bottom. Again, ANOVA indicated a significant effect of treatment, F(5, 36) = 8.66, P < 0.0001. Multiple comparisons confirmed that paired exposure to OA and light in ASW (OA + L) or ANI (OA + L + A) produced an increase in evoked spike rates relative to cells exposed to OA alone, and this increase was apparent >= 90 min after treatment, Ps < 0.05. In total, these results suggest that OA paired with light induces an increase in cell excitability that can persist for >= 90 min in the absence of new protein synthesis.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Protein synthesis dependence of OA-mediated excitability increases. Top: voltage responses to -0.6 nA measured at 45 and 90 min. The groups exposed to light in the presence of OA (OA + L) or OA and the protein synthesis inhibitor anisomycin (OA + L + ANI) exhibited significantly elevated input resistances that persisted for >= 90 min. Brackets indicate SE. Bottom: number of evoked spikes observed 45 and 90 min after training elicited by +0.6-nA current injections. The groups exposed to light presentations in the presence of OA (OA + L and OA + L + ANI) exhibited significantly greater numbers of evoked spikes when they were trained in the normal bath or anisomycin (ANI).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present series of experiments, we used the fluorescent probe ADIFAB to demonstrate that GABA, a neurotransmitter released from hair cells onto postsynaptic photoreceptors in the Hermissenda eye, induced a liberation of fatty acid in the photoreceptors that was could be blocked by the PLA2 inhibitor AACOCF3 or mimicked by the PLA2 activator melittin. Although ADIFAB exhibits only weak selectivity for AA (binding other fatty acids including palmitate, linoleate, and oleate), the inhibition of the GABA-induced shift in ADIFAB emission by AACOCF3 and its having been mimicked by melittin suggests that the increase in FFA was in fact indicative of a liberation of AA. While this conclusion cannot be made with complete certainty, the regulation of ADIFAB emission by PLA2 signaling pathways strongly suggests that the shift in ADIFAB emission that we observed reflected increased levels of free AA. This conclusion is strengthened by previous pharmacological evidence indicating that AA liberation is consequent to transmitter binding during presynaptic hair cell stimulation (Talk et al. 1997).

In an additional set of experiments, we also observed that discrete light presentations (i.e., intracellular Ca2+ elevation) in the presence of OA, the nonhydrolyzable analogue of AA, induced a sustained (>90 min) increase in the excitability (membrane resistance and evoked spike rates) of the B cell. The synergistic action of OA and Ca2+ on B cell excitability was blocked by the PKC inhibitor Ch and was independent of new protein synthesis for >= 90 min. These results are in essential agreement with a molecular model of short- to intermediate-term learning-related neuronal facilitation in the Hermissenda B cell (Matzel et al. 1998). In this framework, it is assumed that the activation of PKC isoforms or the insertion of the enzyme in the cell membrane is potentiated by the convergence of a light-induced intracellular Ca2+ elevation and free AA liberated by transmitter binding owing to presynaptic activity. This general framework suggests a central role for G-protein-coupled receptors in the mediation of activity-dependent plasticity related to memory storage.

In the present studies, we demonstrate that GABA liberates fatty acids through a PLA2-dependent signaling pathway. Previous studies have shown that receptors analogous to the mammalian GABAB type are present on the photoreceptors at the synapse from the vestibular hair cells (Matzel et al. 1995; Rogers et al. 1994). However, it has also been observed that G-protein-coupled GABAB receptors are located in the soma membrane (Yamoah and Crow 1998). The occurrence of biochemical convergence among different signaling pathways requires that those signals arrive in a temporally and spatially contiguous manner; thus the presence of metabotropic GABA receptors in the soma could explain how the light-induced rise in somatic Ca2+ interacts with synaptically liberated AA to stimulate PKC and to promote increases in neuronal excitability.

The contribution of AA to facilitation in Hermissenda B cells may reflect several underlying influences. First, AA may promote a more efficient translocation of PKC to the neuronal membrane through the modification of the properties of the membrane bilayer (Lester et al. 1991). This enhanced coupling of the kinase to the membrane may produce a longer-lasting insertion of the enzyme in the membrane. Second, AA may reduce the Ca2+ dependency of Ca2+-dependent isoforms of PKC, making it possible to activate these isoenzymes at basal Ca2+ levels and thus promoting the persistent stimulation of the enzyme after the termination of a conditioning trial (Lester et al. 1991). Third, AA may impinge on a different subset of PKC isoforms than those targeted by conventional activators of the enzyme. For example, AA may activate atypical isoforms of PKC that have been shown to respond preferentially to fatty-acid stimulation (Khan et al. 1995; McGlynn et al. 1992). In the Hermissenda B cell, these atypical PKC isoforms may have different substrate proteins than those targeted by conventional isoforms activated by light alone. Relatedly, AA may activate an isoform of PKC involved in the regulation of an independent and unique biochemical cascade (McGahon and Lynch 1998). The data presented here do not allow us to distinguish between these potential roles for AA.

In this study, we measured AA release by adding GABA at basal Ca2+ levels. Thus it is possible that we underestimated the amount of AA that is released when presynaptic hair cell activity is paired with postsynaptic depolarization of the B photoreceptor. It has been shown that the Ca2+-dependent isoforms of PLA2 can be stimulated at intracellular concentrations of Ca2+ as low as 10 nM (Farooqui et al. 1997); however, it is likely that higher concentrations of Ca2+ potentiate that stimulation. Alternatively, it is possible that both Ca2+-dependent and -independent isoforms of PLA2 exist in the B photoreceptors. Nonetheless at this point, we cannot draw any conclusions regarding the possible role of Ca2+-independent subtypes of PLA2 in these cells because the specific PLA2 inhibitor (AACOCF3) used in the present experiments has been shown to block some Ca2+-independent subtypes but not others (Farooqui et al. 1997). It will be interesting to replicate these results with varying Ca2+ concentrations to determine maximum levels of AA release and the potential contribution of the different PLA2 isoforms in the B photoreceptors.

PKC-mediated effects of fatty acids on B cell excitability were assessed with Ch, a PKC inhibitor that blocks the enzyme at the catalytic domain by targeting the substrate binding site (Herbert et al. 1990; Sossin 1997). Because the catalytic domain is preserved in all isoforms of PKC, this drug is an effective inhibitor of Ca2+-dependent (Herbert et al. 1990), Ca2+-independent (Padua et al. 1998), and atypical isoforms (Hrabetova and Sacktor 1996; Lausanne et al. 1998). In addition, Ch has been shown to be an effective inhibitor of PKC in Aplysia (Manseau et al. 1998), also blocking the autonomous kinase activity that requires protein synthesis (Sossin 1997). Even though we do not know which PKC isoforms are expressed in Hermissenda B cells, we can speculate that Ca2+-independent forms, which are more sensitive to fatty acids, might play a role in the expression of neuronal facilitation related to associative learning.

Evidence in support of an intermediate period of memory consolidation that is independent of protein synthesis has been observed in other invertebrates (Daises and Tully 1995; Grunbaum and Muller 1998) as well as in hippocampal long-term potentiation, a putative memory model in mammals (Winder et al. 1998). For example, a recent study with honeybees showed that an olfactory memory leads to a sustained activation of constitutively active PKC that consists of an early phase, lasting 2-4 h after training, that was proteolysis-dependent and a second phase, lasting <= 3 days, that required RNA and protein synthesis. It has been proposed that persistent activation of the enzyme during the early phase could be achieved through proteolytic formation of the catalytic fragment PKM (Sacktor et al. 1993). It will be interesting to test whether a similar mechanism underlies the sustained increase in excitability observed in Hermissenda. Accordingly, we propose that in Hermissenda B photoreceptors convergence of fatty acids, arising from activation of the US pathway, with a rise in intracellular Ca2+ and release of DAG, generated by the CS pathway, induces an intermediate phase of memory consolidation that is dependent on PKC activation and does not require protein synthesis.

In summary, we have demonstrated that in B photoreceptors, PKC activation is induced by the molecular convergence of CS (light) and US (rotation) signaling pathways, i.e., the concomitant elevation of intracellular Ca2+ and free fatty acids. In total, these results suggest that in Hermissenda B photoreceptors, the liberation of cis-unsaturated fatty acids by presynaptic hair stimulation paired with postsynaptic, light-induced elevation of intracellular Ca2+ supports increases in neuronal excitability thought to subserve early stages of memory formation.


    ACKNOWLEDGMENTS

We thank Drs. Todd Sacktor, Andrew Talk, George Wagner, and Mark West for helpful comments during the development of these experiments and on an earlier version of the manuscript.

This work was supported by National Institute of Mental Health Grant MH-48387, the Charles and Johanna Busch Memorial Fund, and the James McKeen Cattell Fund (all to L. D. Matzel) and an American Psychological Association predoctoral minority fellowship to I. A. Muzzio.


    FOOTNOTES

Address for reprint requests: L. D. Matzel (E-mail: matzel{at}rci.rutgers.edu).

Received 24 May 2000; accepted in final form 21 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society