Departments of Cellular and Molecular Pharmacology and Physiology, University of California, San Francisco, California 94143
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ABSTRACT |
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Schnell, Eric and Roger A. Nicoll. Hippocampal Synaptic Transmission and Plasticity Are Preserved in Myosin Va Mutant Mice. J. Neurophysiol. 85: 1498-1501, 2001. Recent studies have identified myosin Va as an organelle motor that may have important functions in neurons. Abundantly expressed at the hippocampal postsynaptic density, it interacts with protein complexes involved in synaptic plasticity. It is also located in presynaptic terminals and may function to recruit vesicles in the reserve pool to the active zone. Dilute-lethal mice are spontaneous myosin Va mutants and have severe neurological symptoms. We studied hippocampal physiology at CA3-CA1 excitatory synapses in dilute-lethal mutant mice to test the hypothesis that myosin Va plays a role in pre- or postsynaptic elements of synaptic transmission. In all assays performed, the mutant synapses appeared to be functioning normally, both pre- and postsynaptically. These data suggest that myosin Va is not essential for the synaptic release machinery, postsynaptic receptor composition, or plasticity at this synapse, but does not exclude significant roles for myosin Va in other cell types nor potential compensation by other myosin V isoforms.
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INTRODUCTION |
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Myosin proteins are
actin-based motors with a wide variety of functions, including the
trafficking and localization of intracellular compartments. Membrane
trafficking events are critical to both pre- and postsynaptic
processes, and knowledge of how myosins contribute to these processes
will lead to a greater understanding of neuronal function. One
particular isoform, myosin Va, is highly enriched in brain tissue and
has been well characterized as an organelle motor (Bridgman
1999; Reck-Peterson et al. 2000
). Recent evidence has suggested that it may be essential for various aspects of
neuronal function.
Myosin Va may play a role in postsynaptic
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
trafficking. Myosin Va is highly expressed in hippocampal pyramidal
cells, localizing both to dendritic spines and shafts, and is an
abundant component of the postsynaptic density (PSD) (Espreafico
et al. 1992
; Walikonis et al. 2000
). During
long-term potentiation, AMPA receptors are rapidly translocated into
dendritic spines and added to the postsynaptic membrane, most likely
from receptor pools located in the dendritic shaft (Hayashi et
al. 2000
; Shi et al. 1999
). Although the exact
steps involved in AMPA receptor insertion are unknown, a myosin
Va-dependent translocation of AMPA receptor containing vesicles to
peri-synaptic sites of exocytosis provides a plausible mechanism.
Myosin Va is required for the delivery of intracellular organelles into
Purkinje cell spines (Takagishi et al. 1996
). If myosin
Va is performing a similar function in pyramidal cell spines, AMPA
receptor trafficking, and thus long-term potentiation, might be myosin
Va dependent.
Several additional studies provide data consistent with this
hypothesis. Human myosin V was recently identified as a binding partner
for guanylate kinase domain-associated protein (GKAP) (Naisbitt et al. 2000), which interacts with PSD-95, a
synaptic scaffolding protein implicated in long-term potentiation (LTP) (Migaud et al. 1998
). Myosin Va is also a major
phosphorylation target of CaMKII (Coelho and Larson
1993
), a critical enzyme involved in generating LTP, and itself
binds to CaMKII, possibly activating it by delivering calmodulin to the
enzyme (Costa et al. 1999
). Additionally, LTP is blocked
when actin is perturbed in the postsynaptic cell (Kim and Lisman
1999
).
Myosin Va may also function presynaptically, in mobilizing the reserve
pool of synaptic vesicles toward release sites. Myosin Va has been
identified as a synaptic-vesicle-associated protein, interacting with
synaptobrevin in a Ca2+-dependent manner
(Prekeris and Terrian 1997). Myosin inhibition blocks
reserve pool mobilization in cultured hippocampal cells (Ryan
1999
) and significantly reduces glutamate release from
hippocampal synaptosomes (Prekeris and Terrian 1997
). It
is believed that myosin may be necessary to quickly translocate reserve
vesicles to the active zone during high-frequency transmission, when
endo/exocytotic cycling is insufficient to sustain release.
Accordingly, sustained high-frequency release is actin dependent
(Wang et al. 1996
). Recent work has demonstrated a role
for myosin Va activity in vesicle movements within synaptic terminals
(Bridgman 1999
), and vesicles purified from brain can be
activated to catalyze myosin-V-dependent movement (Evans et al.
1998
). These data suggest that synaptic vesicle release
mechanisms may be modulated by, and are perhaps dependent on, myosin Va.
Dilute-lethal mice (referred to here as dilute
mice) carry a recessive functional null mutation in the myosin Va gene
(Mercer et al. 1991). Homozygous dilute mice
are characterized by a lightened coat color, seizures, and death at
approximately 3 wk of age. In humans, myosin Va mutations have been
identified as the cause of the recessively inherited Griscelli disease,
which involves abnormal pigmentation, immunodeficiency, and
neurological defects (Langford and Molyneaux 1998
). A
recent report found that dilute mice had alterations in
parallel fiber plasticity due to deficient Ca2+
store localization in Purkinje cells (Miyata et al.
2000
). We examined possible direct roles of myosin Va in
synaptic transmission by studying hippocampal physiology in
dilute mutant mice. Assays of both pre- and postsynaptic
function demonstrate that various elements of synaptic transmission
appear intact at the CA3-CA1 synapse, suggesting that myosin Va is not
essential to synaptic transmission and plasticity at this locus.
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METHODS |
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Homozygous dilute-lethal mice were obtained by breeding heterozygous pairs (Jackson labs, stock no. 000253), and were identified on the basis of their distinctive coat color. Transverse hippocampal slices (300-400 µm) were prepared from 12- to 19-day-old homozygous dilute mutants (n = 14 mice) and littermate controls (n = 18 mice). Slices were maintained in a submerged chamber for both recovery (1 h) and recording at room temperature (24-28°C). Perfusion medium contained (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose, and 0.1 picrotoxin, saturated with 95% O2-5% CO2. The perfusion rate was 1.5 ml/min. A cut was made between CA1 and CA3 to prevent the propagation of epileptiform activity.
Field recordings were made with 3 M artificial cerebrospinal fluid
(ACSF)-filled glass pipettes and an Axopatch-1D amplifier. Stimulation
electrodes consisted of bipolar tungsten electrodes (FHC). Stimulation
and recording electrodes were both placed in stratum radiatum of region
CA1 at approximately the same distance from stratum pyramidale. Data
were filtered at 2 kHz and digitized at 5 kHz. Baseline stimulation
rate was 0.1 Hz for all experiments, and field EPSP magnitude was
measured using the initial EPSP slope. LTP magnitude for each slice was
calculated by averaging the normalized responses from 56-60 min after tetani.
Somatic whole cell voltage-clamp recordings were obtained from visually
identified CA1 pyramidal cells using 3-5 M glass electrodes filled
with (in mM) 117.5 Cs-gluconate, 2.5 CsCl, 10 tetraethylammonium-Cl, 5 QX-314 (chloride salt; Precision Biochemicals, Vancouver, British
Columbia, Canada), 8 NaCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, and 0.3 Na3-GTP, pH 7.2, 280 mOsm.
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RESULTS |
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Assays for postsynaptic function
Extracellular field EPSPs in CA1 were easily elicited from dilute hippocampal slices, using stimulus strengths similar to those used in control slices. As a measure of excitatory synaptic efficacy, the magnitude of the field EPSP was compared with the magnitude of the presynaptic afferent fiber volley associated with Schaffer collateral stimulation. The ratio of the initial field EPSP slope to the fiber volley amplitude was unchanged in dilute versus control mice (control ratio, 1.63 ± 0.19, mean ± SE, n = 14 slices; dilute ratio, 1.83 ± 0.33, n = 12 slices). As the field EPSP is largely mediated by AMPA receptors, this implies that AMPA receptor delivery to synapses under basal conditions is normal.
To assess N-methyl-D-aspartate (NMDA) receptor
activity at synapses, the AMPA/NMDA ratios of excitatory synaptic
responses were determined using whole cell voltage-clamp techniques. We found no difference in the AMPA/NMDA ratio between control and dilute mice (Fig.
1A). AMPA and NMDA response
decay kinetics were also similar (at +40 mV, AMPA = 20.9 ± 1.5 ms control,
= 21.5 ± 2.8 ms dilute;
NMDA
= 112 ± 22 ms control,
= 85 ± 16 ms dilute).
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To assess possible changes in synaptic plasticity, LTP was induced in slices during field recording experiments. As shown in Fig. 1B, no significant changes in LTP induction or maintenance were observed (unpaired t-test, P > 0.3). Hippocampal long-term depression, induced using 600 pulses at 1 Hz, was also intact and of normal magnitude in dilute slices (data not shown).
Assays for presynaptic function
Presynaptic function was examined using assays to detect for altered vesicle release. Short-term plasticity assays included analyzing paired-pulse facilitation (PPF) and posttetanic potentiation (PTP). PPF was tested at intervals ranging from 20 to 250 ms, and a similar degree of facilitation for both control and dilute slices was found at all intervals tested (Fig. 2A). Additionally, both control and dilute mice had a similar PTP, with responses potentiating and decaying back to baseline levels with similar time courses (Fig. 2B). Although not examined in detail, synaptic transmission at mossy fiber synapses, including frequency-dependent facilitation, PPF, and LTP, all appeared normal (data not shown).
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We then tested reserve pool mobilization by recording responses to sustained repetitive stimulation at both moderate and high frequencies. A short 1-s/100-Hz tetanus produced similar field potential responses in both control and dilute slices (Fig. 3A). Both the amplitude of the field at the end of the tetanus as well as the integrated area of the field (normalized to the amplitude of the EPSP to the 1st stimulus) were similar (Fig. 3B), suggesting similar amounts of release during the train. A more prolonged (30-s) 10-Hz train was given to study reserve pool mobilization on a longer time scale. In both control and dilute slices, responses showed an initial facilitation, followed by similar degrees of synaptic depression (Fig. 3C), presumably due to reserve pool depletion. Recovery to baseline response magnitude was complete by the first posttrain stimulus in dilute mice (103 ± 15% of baseline 10 s after the train), suggesting normal vesicle replenishment.
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DISCUSSION |
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The goal of the present study was to assess the potential roles of myosin Va in synaptic transmission using the dilute-lethal mouse, a spontaneous myosin Va mutant. The lack of a phenotype in this study suggests several conclusions.
First, myosin Va is not required for postsynaptic assembly or
activity-dependent modification at the CA3-CA1 synapse. The implication
of myosin Va as a major CaMKII phosphorylation target at the
PSD had suggested a simple model for LTP, in which myosin Va
was activated by CaMKII phosphorylation and delivered AMPA receptors to
synaptic sites. However, as the dilute mouse has normal LTP,
a different protein must be the main target for activated CaMKII in
mediating LTP. Roles for other myosin isoforms in these processes
remain likely, as myosin molecules have been implicated in a variety of
membrane trafficking events (Mermall et al. 1998). For
instance, data suggesting that myosin II is responsible for membrane
trafficking from trans-golgi network (TGN)-derived structures (Müsch et al. 1997
) might support an involvement
for myosin II in the AMPA receptor trafficking underlying LTP.
Second, presynaptic vesicle trafficking at this synapse does not require myosin Va. Synapses in dilute mice had normal release probabilities as assessed by paired-pulse measurements and were able to sustain release during conditions that required reserve pool mobilization. Given the demonstrated roles for myosin in presynaptic function, further analyses of neuronal myosin isoforms and their localization at synaptic sites will hopefully identify candidates for future study.
An important caveat to these conclusions is that synaptic functions of
myosin V may be obscured from analysis in the dilute mouse
due to compensation by a second murine myosin V isoform, myr6. While
myr6 is highly expressed in the hippocampus, its expression is not
increased in the dilute mouse (Zhao et al.
1996), nor was it detected in PSD mass spectroscopy
(Walikonis et al. 2000
). It remains unclear whether
myosin Va and myr6 (or other currently unidentified myosin V isoforms)
are redundant proteins in the hippocampus or whether myosin V isoforms
in general do not play a significant role in hippocampal physiology.
The neurological disturbance underlying the convulsions observed in
dilute mice remains to be elucidated. Given the high level of expression at hippocampal synapses, as well as the frequent implication of hippocampal abnormalities in seizure generation, it
might have been expected that the neurological changes underlying the
dilute phenotype would have been associated with changes in hippocampal synaptic function. Studies have demonstrated cerebellar abnormalities in dilute mice (Miyata et al.
2000; Takagishi et al. 1996
), but as the
cerebellum is not typically implicated in seizure activity, presumably
other brain areas more centrally involved in seizure generation share
these abnormalities. An analysis of differential expression patterns of
myosin V isoforms could identify brain regions that express only the Va
isoform, thus providing for more focused physiological studies.
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ACKNOWLEDGMENTS |
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We thank L. Chen and R. Wilson for helpful comments on the manuscript.
E. Schnell is supported by the Medical Scientist Training Program. R. A. Nicoll is a member of the Keck Center for Integrative Neuroscience and the Silvio Conte Center for Neuroscience Research and is supported by grants from the National Institutes of Health and the Bristol-Myers Squibb Corporation.
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FOOTNOTES |
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Address for reprint requests: R. A. Nicoll, Dept. of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Ave., Box 0450, San Francisco, CA 94143-0450 (E-mail: nicoll{at}phy.ucsf.edu).
Received 2 November 2000; accepted in final form 21 December 2000.
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REFERENCES |
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