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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 × cm2 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
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
![]() |
(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 M 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|