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The Journal of Neuroscience, June 1, 2001, 21(11):3721-3728
Presynaptic Ca2+ Channels and Neurotransmitter
Release at the Terminal of a Mouse Cortical Neuron
Jing
Qian and
Jeffrey L.
Noebels
Department of Neurology, Baylor College of Medicine, Houston, Texas
77030
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ABSTRACT |
Regional variation in synaptic efficacy is an important determinant
of associative processing as information flows through major circuits
of the brain. The perforant path is the principal route of entry from
cortex to the hippocampus and contains the first synapse in the
cortical-hippocampal projection pathway. We used optical imaging
techniques to analyze presynaptic Ca2+ entry and
neurotransmitter release at synapses in the medial perforant path
linking stellate neurons located in layer II of the entorhinal cortex
to granule cells in the dentate gyrus. Similar to other excitatory
central synapses, the relationship between neurotransmitter release and
the amount of Ca2+ influx can be best described by a
Hill equation with a Hill coefficient of 3.5. Our
Ca2+ channel toxin studies indicate that P/Q-type
channels are the predominant Ca2+ source triggering
neurotransmitter release in this pathway, as shown by a potent
inhibition of Ca2+ entry and synaptic transmission
by the P/Q-type channel blocker  -agatoxin IVA. However,
compared with the downstream hippocampal pyramidal neuron CA3-CA1
synapse, neurotransmitter release was less sensitive to the N-type
Ca2+ channel blocker -conotoxin GVIA,
although the amount of N-type Ca2+ current is
comparable. The contribution of N-type channels to neurotransmitter
release approximates that found at the CA3-CA1 synapse when tested
under lower [Ca2+]o, which
effectively reduces the size of the Ca2+ microdomain
surrounding each channel. These results suggest that P/Q-type channels
are more closely associated with release machinery then N-type channels
at this synapse and that cooperativity differences for each channel
subtype may characterize variations in signaling at central synapses.
Key words:
presynaptic calcium entry; corticohippocampal
synapse; medial perforant path; synaptic efficacy; power relationship; cooperativity; optical imaging.
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INTRODUCTION |
Synaptic efficacy is a critical
determinant of functional connectivity and plasticity within central
neural networks and may vary throughout development from zero
("silent synapses") to an optimal value for signaling. Recent study
of synaptic transmission between cortical neurons indicates that
different types of neurons release neurotransmitter with different
degrees of efficacy. Reliable quantal release of neurotransmitter with
a few failures was found at the synaptic connection between spiny
stellate cells in layer IV of rat somatosensory cortex, in contrast to
the low release probability observed at synapses between pyramidal
cells in layer V (Feldmeyer et al., 1999 ; Feldmeyer and Sakmann, 2000).
Although excitation-release coupling at terminals involves a complex
series of events, from the standpoint of
Ca2+ ion entry, the probability of release
is dependent on the type and density of presynaptic
Ca2+ channels expressed and their
individual proximity to and interaction with transmitter release
machinery. Because of the limitation of current
electrophysiological techniques, it has been difficult to directly
access presynaptic Ca2+ currents to
determine the mechanism for the distinct release probabilities at these
synapses and the role of specific calcium channel subtypes at terminals
of cortical neurons.
One major subcortical pathway that originates from cortical stellate
neurons is the synaptic projection from the entorhinal cortex to the
hippocampus, the medial perforant pathway. Spiny stellate neurons are
the most abundant cell type in layer IV of neocortex and layer II of
entorhinal cortex (Woolsey et al., 1975; Feldman and Peters, 1978;
Klink and Alonso, 1997). Their functional role is presently thought of
as an information amplifier that collects inputs destined to the cortex
and then relays this information both to other cortical layers and over
longer distances to subcortical brain networks. Spiny stellate neurons
located in the layer II of the medial entorhinal cortex extend their
axons subcortically to synapse on the dendrites of granule cells within
the outer two-thirds of the molecular layer of the dentate gyrus
(Steward and Scoville, 1976). Therefore, study of the presynaptic
Ca2+ currents and synaptic transmission in
the hippocampal medial perforant pathway could provide the first
insight into the characteristics of presynaptic
Ca2+ channels and neurotransmitter release
of a major cortical projection neuron.
The laminar synaptic organization in the molecular layer of the dentate
gyrus offers an anatomical advantage to selectively load and optically
isolate a defined population of presynaptic terminals, similar to that
found in the stratum radiatum of the hippocampal CA3-CA1 area in which
fluorescence Ca2+ imaging has been used to
access presynaptic Ca2+ currents in
several species (Wu and Saggau, 1994a; Qian et al., 1997; Qian and
Noebels, 2000). In this study, we use the same technique to explore the
types of presynaptic Ca2+ channels and
neurotransmitter release in the mouse medial perforant pathway. Our
study identifies a distinct difference in the cooperativity of
neurotransmitter release for each Ca2+
channel subtype at the presynaptic terminals of cortical stellate neurons compared with the hippocampal CA3-CA1 synapses that lie immediately downstream.
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MATERIALS AND METHODS |
Transverse brain slices (315 µm thickness) were prepared from
hippocampi of adult wild-type mice (between 4 and 9 months old) and
incubated at 25°C in artificial CSF containing (in
mM): 124 NaCl, 3.5 KCl, 2.5 CaCl2, 2 MgCl2, 22 NaHCO3, 1.25 NaH2PO4, and 10 D-glucose, gassed with 95% O2-5%
CO2 to maintain a constant pH of 7.4. The slices
were transferred into a recording chamber controlled at 30°C for the
loading of Ca2+ indicator and subsequent
fluorescence imaging.
The low-affinity Ca2+-sensitive
fluorescence indicator Magnesium Green-AM (Molecular Probes, Eugene,
OR) was loaded into presynaptic terminals of the medial
perforant pathway by a method similar to that used at the mouse
hippocampal CA3-CA1 synapse (Qian and Noebels, 2000). Figure
1 shows a schematic diagram for the
loading of Ca2+ indicator and the
recording of optical and electrophysiological signals. A small amount
of dye dissolved in DMSO solution was pressure injected into the middle
of the molecular layer of dentate gurus in which axons from the
projection neurons located in the layer II of entorhinal cortex
terminate and synapse with dendrites of granule cells. A small
recording area in the middle of the molecular layer with a diameter of
150 µm was excited at a wavelength of 488 ± 20 nm; the emitted
fluorescence was filtered using a bandpass filter of 535 ± 25 nm
and converted into an electrical signal with a single photodiode. A
bipolar tungsten electrode was positioned in the molecular layer,
~0.5-0.8 mm away from the recording area (as shown in Fig. 1) to
stimulate the medial perforant pathway. Stimuli were delivered every 20 sec using current pulses of 0.03-0.05 mA/0.1 msec to elicit a
submaximal response. The stimulation-induced presynaptic
Ca2+ transient
([Capre]t) and the evoked
field EPSP (fEPSP) were simultaneously sampled at 10 kHz. Three
successive traces were averaged to improve the signal-to-noise ratio.
The maximal slope of the fEPSP was taken as the measure of synaptic
transmission. For field recording, glass microelectrodes (1-5 M ,
filled with 2 M NaCl) were positioned in the center of the
optical recording area. At the beginning of each experiment, a pair of
stimulating pulses separated by an interval of 40 msec was given to
test for activation of the medial perforant pathway. The response of
the medial perforant pathway was judged by the presence of short-term
inhibition of synaptic transmission, as shown by the sample trace in
Figure 1. Ca2+ influx was measured by the
fluorescence ratio of  F/F. Autofluorescence of
the brain slice was subtracted from the total fluorescence signal. The
selective presynaptic loading of the Ca2+
indicator in the mouse hippocampal slice was verified by applying the
glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) and
D-amino-phosphonovalerate
(D-APV) (25 µM) at the
end of each experiment. As shown in Figure 1, the glutamate receptor
antagonists did not alter the optical signal,
F/F, but completely blocked the fEPSP,
indicating a pure presynaptic origin of optical signals. The signal
containing the fiber volley and stimulation artifact obtained after
application of CNQX and APV was then used as a template to subtract
these components from the raw recording trace of fEPSP before measuring
the slope of the response. Unless otherwise stated, data in each
experiment were normalized to the baseline before any drug application
and then pooled together and expressed as mean ± SD.
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Figure 1.
Recording presynaptic Ca2+
influx and postsynaptic response in the mouse hippocampal medial
perforant pathway. A, A schematic diagram for loading
Ca2+ indicator into presynaptic terminals of the
hippocampal medial perforant pathway. B, Sample traces
of [Capre]t and fEPSP in response to a pair
of stimuli.
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Drugs. Ca2+ channel toxin
-conotoxin GVIA (-CgTx GVIA), -agatoxin IVA (-Aga IVA), and
-CgTx MVIIC were purchased from Bachem AG (Bubendorf, Switzerland).
CNQX and D-APV purchased from Tocris Cookson
(Ballwin, MO).
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RESULTS |
Presynaptic Ca2+ channel types responsible for
neurotransmission in the hippocampal medial perforant pathway
The types of Ca2+ channels mediating
neurotransmitter release at the synaptic connection from entorhinal
cortex to hippocampal dentate gyrus were first evaluated by application
of selective Ca2+ channel toxins. Figure
2A shows the time
course of [Capre]t and fEPSP from a representative experiment before, during, and after bath
perfusion of 1 µM -CgTx GVIA, the selective
N-type Ca2+ channel blocker. As shown in
Figure 2A, the toxin substantially reduced
[Capre]t but had only a
minor effect on synaptic transmission. The mean inhibition of
[Capre]t and fEPSP by
-CgTx GVIA was ~31 ± 6 (n = 9) and 12 ± 3% (n = 9), respectively. Although the amount of
[Capre]t reduced by the
toxin at this synapse was comparable with that measured at the
hippocampal CA3-CA1 synapse of the same mouse strain (Qian and
Noebels, 2000), a much reduced effect of N-type
Ca2+ channel blockade on synaptic
transmission (12 ± 3 versus 45 ± 4% at the CA3-CA1
synapse) was observed here. The contribution of P/Q-type channels to
neurotransmitter release was measured by bath perfusion of 1 µM -Aga IVA, the selective P/Q-type
Ca2+ channel blocker. As revealed by the
time course shown in Figure 2B, application of the
toxin sharply reduced
[Capre]t and potently inhibited synaptic transmission. On average, -Aga IVA reduced [Capre]t by 53 ± 5% (n = 3) and inhibited the fEPSP by 86 ± 2% (n = 3). These data indicate that, similar to other
excitatory central synapses studied to date,
Ca2+ entering through P/Q-type channels is
the major source of Ca2+ triggering
neurotransmitter release in response to stimulation of the hippocampal
medial perforant pathway.
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Figure 2.
Types of presynaptic Ca2+
channels controlling neurotransmitter release in the hippocampal medial
perforant pathway. A, Time courses of
[Capre]t and fEPSP in response to 1 µM -CgTx GVIA, the N-type Ca2+
channel blocker, in the hippocampal medial perforant pathway. The toxin
substantially reduced [Capre]t but had only a
minor effect on synaptic transmission. The mean inhibition of
[Capre]t and fEPSP by -CgTx GVIA was
~31 ± 6 and 12 ± 3% (n = 9).
Although the amount of [Capre]t reduced by
the toxin at this synapse was comparable with that measured at the
hippocampal CA3-CA1 synapse of the same species, much less effect of
N-type Ca2+ channel blockade on synaptic
transmission was observed here. Inset shows sample
traces taken during steady-state periods in the control solution and
after application of -CgTx GVIA. B, Time courses of
[Capre]t and fEPSP in response to 1 µM -Aga IVA, the P/Q-type Ca2+
channel blocker. Application of the toxin strikingly reduced
[Capre]t and potently inhibited synaptic
transmission. On average, -Aga IVA reduced
[Capre]t by 53 ± 5%
(n = 3) and inhibited the fEPSP by 86 ± 2%
(n = 3). These data indicate that, similar to other
excitatory central synapses studied to date, Ca2+
entering through P/Q-type channels is the major source of
Ca2+ triggering release of neurotransmitter in the
hippocampal medial perforant pathway. Inset shows sample
traces taken during steady-state periods in the control solution and
after application of -Aga IVA.
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We also measured the
[Capre]t and fEPSP after
blocking both P/Q- and N-type Ca2+
channels to assess the remaining contribution of other
Ca2+ channel types. Figure
3A shows the time course of
the [Capre]t and fEPSP
during application of -CgTx GVIA after previous blockade of P/Q-type
channels with -Aga IVA. Combined application of both toxins reduced
[Capre]t to 24 ± 1% (n = 2) of baseline and almost completely
eliminated synaptic transmission. The average fEPSP was 6 ± 2%
of baseline (n = 2). The possible involvement of L-type Ca2+ channels in the residual release of
neurotransmitter at this synapse was also tested. Similar to the
findings at other central synapses (Wu and Saggau, 1994b; Mintz et al.,
1995; Wu et al., 1999), L-type Ca2+
channels did not contribute to Ca2+ influx
at presynaptic terminals in this pathway, as indicated by the lack of
significant effects of the L-type channel antagonist nifedipine (10 µM) on
[Capre]t and fEPSP (data
not shown). Figure 3B summarizes the results obtained from
experiments after the application of Ca2+
channel toxins.
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Figure 3.
Blocking both P/Q- and N-type channels eliminates
the release of neurotransmitter in the hippocampal medial perforant
pathway. A, Time courses of
[Capre]t and fEPSP in response to 1 µM -Aga IVA and 1 µM CgTx GVIA together.
Combined application of two toxins reduced
[Capre]t to 24 ± 1%
(n = 2) of baseline and almost completely
eliminated synaptic transmission. The average fEPSP was 6 ± 2%
of baseline (n = 2). Inset shows
sample traces taken during steady-state periods in the control solution
and after application of -Aga IVA and -CgTx GVIA.
B, Summary data comparing the inhibition of
[Capre]t and fEPSP in response to application
of -CgTx GVIA and -Aga IVA. P/Q- and N-type channels are the
major Ca2+ source controlling neurotransmitter
release at this synapse.
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Ca2+ cooperativity of neurotransmitter release
through P/Q- and N-type channels in the hippocampal medial perforant
pathway
In addition to the types of voltage-dependent
Ca2+ channels mediating
Ca2+ entry at the terminal, the
relationship between presynaptic Ca2+
influx and neurotransmitter release is a second critical aspect characterizing the Ca2+ induced-release of
neurotransmitter. Previous studies in the CNS show that
neurotransmitter release is a power function of presynaptic
Ca2+ current, with a power number between
3 and 4 (Wu and Saggau, 1994a; Mintz et al., 1995; Borst and Sakmann,
1996). Here, we investigated this power relationship in the
hippocampal medial perforant pathway by measuring
[Capre]t and fEPSP
amplitudes while varying extracellular
Ca2+ concentration
([Ca2+]o) from 2.5 mM under control conditions up to 4 mM and down
to 1.5, 1.0, 0.75, and 0.5 mM, respectively. In each
experiment, the extracellular Mg2+ level
was either reduced to 0.5 mM (in the case of 4 mM
[Ca2+]o) or raised
appropriately to maintain the size of the presynaptic fiber volley
constant. As shown in Figure
4A, at the end of each experiment, two glutamate receptor antagonists, CNQX and APV, were
routinely applied to isolate the stimulation artifact and presynaptic
fiber volley. There were two purposes for this experimental manipulation. One was to allow subtraction of the stimulation artifact
and presynaptic fiber volley from the raw recording trace for a more
accurate measurement of the fEPSP slope. Another was to determine the
presence of any postsynaptic contamination of Ca2+ indicator labeling, which would cause
an underestimation of the power relationship. As shown by the sample
trace in the inset of Figure 4A, the
Ca2+ signal measured in our experiments
was purely presynaptic, because there was no significant difference
between [Capre]t before
or after the combined application of CNQX and APV to block the
postsynaptic response (the mean difference was 3 ± 4%;
n = 25). Figure 4B summarizes the
[Capre]t and fEPSP
amplitudes obtained at steady state during reduction of
[Ca2+]o from
control levels to a particular test concentration.
[Capre]t was 75 ± 7 (n = 6), 60 ± 2 (n = 5), 50 ± 2 (n = 5), and 44 ± 2% (n = 2) of baseline when exposed to 1.5, 1.0, 0.75, and 0.5 mM [Ca2+]o,
respectively. The corresponding fEPSPs were 61 ± 7, 34 ± 2, 17 ± 2, and 2 ± 1% of baseline. To test whether
neurotransmitter release was saturated by the amount of
Ca2+ influx under control conditions, we
measured the [Capre]t and fEPSP under 4 mM
[Ca2+]o. In these
experiments, the average
[Capre]t increased to
151 ± 15% (n = 7) of baseline. The corresponding
mean fEPSP was 131 ± 11% (n = 7) of baseline, a
substantial increase, but much less than what would be obtained
if it adhered to the power law. This indicates that, although the basal
level of release is not saturated by the amount of
Ca2+ influx under control conditions, it
operates in the upper part of the synaptic transmission curve as shown
in Figure 4B. The synaptic transmission curve in
Figure 4B is a best fit of experimental data with the
following Hill equation: fEPSP = fEPSPmax[([Capre]t)n/([Capre]f)n + (Kd)n],
where n is the Hill coefficient and represents the degree of Ca2+ cooperativity in the process of
release, fEPSPmax is the maximal fEPSP
(normalized to control), and Kd is the
amount of [Capre]t when
fEPSP is 50% of the maximal amplitude. At this synapse, the Ca2+ cooperativity of neurotransmitter
release is n = 3.5. This is consistent with findings at
other central synapses (Wu and Saggau, 1994a; Mintz et al., 1995; Borst
and Sakmann, 1996). To compare the results with those obtained from the
CA3-CA1 synapse (Qian and Noebels, 2000), Figure 4C shows
the calculated apparent power numbers [defined as
log(fEPSP%)/log([Capre]t%)],
corresponding to each tested
[Ca2+]o. Data from
experiments using 0.5 mM
[Ca2+]o are not
included because the slope of the fEPSP at 0.5 mM
[Ca2+]o was very
small and comparable with noise level. The average apparent power
numbers were dependent on the tested
[Ca2+]o and were
0.7 ± 0.2 (n = 7), 1.7 ± 0.3 (n = 6), 2.1 ± 0.1 (n = 5), and
2.6 ± 0.3 (n = 5) for 4.0, 1.5, 1.0, and 0.75 mM, respectively. We also independently
calculated the power relationship for transmission at this synapse
while selectively reducing Ca2+ entry
through a particular type of Ca2+ channel.
The mean apparent power number obtained during blockade of P/Q channels
was 2.7 ± 0.5 (n = 3), and the mean apparent
power number obtained during N-type blockade was 0.4 ± 0.1 (n = 9). The very low power number obtained for N-type
channel coupling cannot be simply explained by the dose-response curve
of the apparent power number to the
[Capre]t, because
reduction of [Capre]t to 75% of baseline with 1.5 mM
[Ca2+]o produced a
greater power number. Rather, the high power number in the
relationship between
[Capre]t and
neurotransmitter release mediated by P/Q-type channels suggests that
P/Q channels are more tightly coupled with the vesicle release
machinery than N-type channels.
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Figure 4.
Relationship between Ca2+ entry
and the release of neurotransmitter in the hippocampal medial perforant
pathway. A, Time courses of
[Capre]t and fEPSP in response to the
manipulation of extracellular Ca2+ concentration
([Ca2+]o) in a typical
experiment. At the end of each experiment, two glutamate receptor
antagonists, CNQX and APV, were routinely applied to isolate the
stimulation artifact and presynaptic fiber volley. This allows
subtraction of the stimulation artifact and presynaptic fiber volley
from the raw recording trace for a more accurate measurement of the slope of fEPSP.
Inset shows sample traces taken at steady state in
solutions containing 2.5 mM
[Ca2+]o (control), 1.0 mM [Ca2+]o, and
after application of CNQX and APV. B, Summary data of
[Capre]t and fEPSP in response to each
[Ca2+]o tested. The solid
line is a best fit of experimental data with a Hill equation:
fEPSP = fEPSPmax[([Capre]t)n/([Capre]f)n + (Kd)n].
fEPSPmax = 150% of baseline fEPSP, and
Kd = 85% of baseline
[Capre]t; n = 3.5. C, Comparison of the apparent power number
[log(%fEPSP)/log(%[Capre]t)] as a
result of selective blockade of Ca2+ channels and
nonselective reduction of Ca2+ entry. The calculated
apparent power numbers for -Aga IVA and -CgTx GVIA are 2.7 ± 0.5 (n = 3) and 0.4 ± 0.1 (n = 9), respectively. The calculated apparent
power numbers for 4.0, 1.5, 1.0, and 0.75 mM
[Ca2+]o are 0.7 ± 0.2 (n = 7), 1.7 ± 0.3 (n = 6), 2.1 ± 0.1 (n = 5), and 2.6 ± 0.3 (n = 5), respectively. This result indicates that
P/Q-type channels interact with the release machinery more tightly than
N-type in the hippocampal medial perforant pathway.
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Colocalization of P/Q- and N-type Ca2+
channels at the release site in the hippocampal medial
perforant pathway
Our measured signal at perforant path synapses onto granule cell
dendrites reflects a population response of single corticofugal presynaptic terminals, and their release properties are not necessarily uniform. Application of -Aga IVA resulted in ~86% reduction of synaptic transmission, whereas -CgTx GVIA reduced synaptic
transmission by only 12%. If these values were superadditive (sum in
excess of 100%), the results would be taken as evidence for
colocalization of P/Q- and N-type channels at the same presynaptic
terminals and for an anatomically overlapping contribution of multiple
Ca2+ channel types in neurotransmitter
release (Wheeler et al., 1994; Wu and Saggau, 1994b; Mintz et al.,
1995). In the present case, the sum of both values was ~100%. This
result can be interpreted as reflecting a segregation of P/Q- and
N-type channels at different sets of presynaptic terminals or as
indicating that Ca2+ microdomains around
each channel do not overlap. Alternatively, the lack of superaddition
might be coincidental, and the minor effect on neurotransmitter release
observed by blocking N-type channels could be attributable to high
Ca2+ entry near the presynaptic release site.
To distinguish between these two hypotheses, we repeated the
Ca2+ channel toxin experiments shown in
Figure 2 under a condition of low
[Ca2+]o (1.0 mM), which would reduce Ca2+
currents and thereby effectively shrink the size of the functional Ca2+ microdomain. If P/Q- and N-type
Ca2+ channels are segregated in different
sets of presynaptic terminals or their microdomains are not
overlapping, the total reduction should remain at ~100% in 1.0 mM
[Ca2+]o. If not, a
superaddition in the fractional reduction of neurotransmitter release
by selectively blocking P/Q- and N-type channels would be expected.
Consistent with the latter hypothesis, as shown by the experiment in
Figure 5A, the inhibition of
synaptic transmission by -CgTx-GVIA was increased to 37 ± 3%
(n = 6) in 1.0 mM
[Ca2+]o. The mean
reduction of [Capre]t was
27 ± 7% (n = 5). Figure 5B shows
a representative experiment with -Aga IVA. On average, the
toxin reduced neurotransmitter release by 94 ± 1%
(n = 4) in 1.0 mM
[Ca2+]o. The mean
reduction of [Capre]t was
56 ± 6% (n = 2). The sum of reductions in
neurotransmitter release by selectively blocking both
P/Q- or N-type channels therefore exceeded 100%. Compared with control
saline, the relatively larger reductions of fEPSP in 1.0 mM
[Ca2+]o by these
two toxins indicate increased cooperativity of N- and P/Q-type channels
in controlling neurotransmitter release when the size of
Ca2+ microdomains around each channel was
reduced. These results clearly indicate that P/Q- and N-type
Ca2+ channels are colocalized at the
release site.
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Figure 5.
Increased cooperativity of N- and P/Q-type
Ca2+ channels in controlling neurotransmitter
release under lower [Ca2+]o.
A, B, Time course of neurotransmitter
release in response to N- and P/Q-type Ca2+ channel
blockers when tested under lower
[Ca2+]o. Insets show
sample traces of fEPSP and [Capre]t before
and after application of -CgTx GVIA and -Aga IVA under a condition of 1 mM
[Ca2+]o. C, Summary
data for the inhibition of synaptic transmission by 1 µM
-CgTx GVIA and 1 µM -Aga IVA under 1 mM
[Ca2+]o. The inhibition of fEPSP by
-CgTx GVIA was increased from 12 ± 3% (n = 9) under 2.5 mM [Ca2+]o
to 37 ± 3% (n = 5) under 1.0 mM
[Ca2+]o. Meanwhile, the inhibition of
synaptic transmission by -Aga IVA increased from 86 ± 2%
(n = 3) under 2.5 mM
[Ca2+]o to 94 ± 1%
(n = 4) under 1.0 mM
[Ca2+]o. These results are consistent
with the hypothesis that P/Q- and N-type Ca2+
channels are colocalized at the release site, and the insensitivity of
neurotransmitter release to -CgTx GVIA under control conditions is
attributable in part to high Ca2+ entry at this
synapse.
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DISCUSSION |
The neuroanatomical isolation of presynaptic terminals originating
from stellate cells in the entorhinal cortex and terminating in the
hippocampal formation facilitates the analysis of release properties at
cortical synapses. In this study, we have examined the subtypes of
Ca2+ channels and their cooperativity in
controlling the release of neurotransmitter at a mouse cortical synapse.
Anatomy of the corticohippocampal perforant path projection
Axons from the entorhinal cortex to the dentate gyrus constitute
the principal cortical input to the hippocampal formation. Two major
divisions, the medial and lateral perforant pathways, project to
different regions of the hippocampus. The medial pathway studied here
emanates from neurons located in layer II of the medial entorhinal
cortex. These axons terminate in a highly restricted band favorable for
optical imaging within the midportion of the molecular layer of the
dentate gyrus and form synaptic connections with dendrites of granule
cells (Steward and Scoville, 1976). Two distinct categories of
projection neurons in layer II of the entorhinal cortex have been
defined on the basis of their morphology and electrical membrane
properties (Steward and Scoville, 1976; Alonso and Klink, 1993). Over
two-thirds of these neurons belong to the category of spiny stellate
cells (Alonso and Klink, 1993), whereas pyramidal-like neurons
are much less abundant and constitute the remainder. In addition to
outnumbering pyramidal-like cells, stellate neurons also produce more
extensive patterns of recurrent axonal collaterization (Klink and
Alonso, 1997). Therefore, the majority of axons terminating in the
molecular layer of the dentate gyrus originate from stellate cells
located in layer II of the entorhinal cortex. Consistent with this
estimate, we observed a short-term depression of synaptic transmission
in our experiments when a paired-pulse stimulation was given. This is
in close agreement with the behavior of synaptic connections between
stellate cells in layer IVA of rat somatosensory cortex, in which a
prominent short-term depression of synaptic transmission resulting from high release probabilities at presynaptic terminals of stellate cells
was reported (Egger et al., 1999) and with earlier analysis of the
perforant pathway (McNaughton, 1980). Thus, our fluorescence imaging
and electrophysiological recordings at the terminal site of the
hippocampal medial perforant pathway primarily reflect the presynaptic
Ca2+ currents and neurotransmitter release
at presynaptic terminals of cortical spiny stellate neurons.
Selective Ca2+ channel subtype contributions to
release at presynaptic terminals of the medial perforant path
Our Ca2+ imaging data indicate that
Ca2+ current through P/Q-type channels
contributes to ~50% of the total Ca2+
influx at perforant path presynaptic terminals. The ensuing release of
neurotransmitter relies predominantly on P/Q-type channels, as
indicated by a potent inhibition of synaptic transmission with the
P/Q-type channel blocker -Aga IVA. This pattern is very similar to
that found at the presynaptic terminals of hippocampal pyramidal neurons, cerebellar granule cells, and the calyx synapse in brainstem in several different species (Wu and Saggau, 1994b; Mintz et al., 1995;
Wu et al., 1999). In contrast, Ca2+
current through N-type channels, although it contributes to ~30% of
the total presynaptic Ca2+ entry and is
comparable with the amount seen at hippocampal CA3-CA1 Shaffer
collateral terminals (Qian and Noebels, 2000), has only a minor impact
on neurotransmitter release at this synapse. By comparing the apparent
power numbers for each type of Ca2+
channel (2.7 for P/Q and 0.4 for N), it can be concluded that the
P/Q-type channel interacts more tightly with the release machinery than
does the N-type channel at this synapse. A low power number for the
N-type channel cannot be explained simply as the consequence of
[Capre]t-dependent
release, because reduction of
[Capre]t to ~75% of
baseline by lowering
[Ca2+]o (to 1.5 mM) produced a greater power number. This preference of
P/Q-type over N-type channels in the interaction with the release machinery has been observed at several central synapses (Mintz et al.,
1995; Wu et al., 1999; Qian and Noebels, 2000) and may reflect the
spatial arrangement of local channels near sites of neurotransmitter
release (Bertram et al., 1999).
Elevated presynaptic Ca2+ influx at the release
site ensures a high safety factor for synaptic transmission from
entorhinal cortex to the hippocampus
Reliable quantal release of neurotransmitter with few failures has
been found at synapses connecting stellate cells in layer IV of rat
somatosensory cortex, in contrast to low release probability at the
synaptic connection between layer V pyramidal cells (Feldmeyer et al.,
1999; Feldmeyer and Sakmann, 2000). In this study, two lines of
evidence point to a similar high release probability at the synapse
from entorhinal cortex stellate cells to dentate granule cells when
compared with hippocampal CA3-CA1 pyramidal neuron terminals. First,
relatively smaller apparent power numbers in the
Ca2+ cooperativity of neurotransmitter
release were measured compared with those obtained at the synapse
between hippocampal pyramidal neurons of the same mouse strain (Qian
and Noebels, 2000). This reduction in the apparent power number is
apparently not attributable to a decreased
Ca2+ cooperativity in the process of
vesicle exocytosis, because the relationship between
Ca2+ influx and neurotransmitter release
can still be described by a fourth power function (Wu and Saggau,
1994a; Mintz et al., 1995; Borst and Sakmann, 1996). Rather, the
smaller values indicate that the basal level of neurotransmitter
release is closer to saturation levels at the presynaptic terminals of
entorhinal cortical stellate cells than at those of hippocampal
pyramidal cells. A small apparent power number when release approaches
saturation is predicted by the Hill equation that describes the
relationship between the optically measured
Ca2+influx and neurotransmitter release
(Qian and Saggau, 1999). Second, the minor impact on neurotransmitter
release observed after blocking N-type channels in normal
[Ca2+]o and the
increased contribution of N-type channels to release in low
[Ca2+]o suggest
that the release machinery is exposed to high
Ca2+ entry under physiological conditions
at this synapse. The contribution of N-type channels to
neurotransmitter release only becomes evident when the degree in the
overlap of Ca2+ microdomains is decreased.
This is comparable with the observation made at the CA3-CA1 synapse,
in which blocking N-type channels substantially inhibited
neurotransmitter release under physiological conditions but had a much
smaller effect on synaptic transmission when presynaptic
Ca2+ entry was increased by broadening
action potential duration (Wheeler et al., 1996; Qian and
Saggau, 1999). Consequently, it is reasonable to conclude that the
release machinery at this synapse is exposed to a larger
Ca2+ influx than that present at the
hippocampal CA3-CA1 synapse and that the synapse therefore exhibits a
high release probability.
Potential molecular mechanisms underlying the different efficacies
of cortical stellate neuron synapses and hippocampal pyramidal neuron
synapses
Biophysical properties of voltage-gated
Ca2+ channels are determined not only by
the kinetics of the specific pore-forming  subunit but also by other
ancillary interacting subunits, especially the  subunit (Walker and
De Waard, 1998). Differential splicing and expression of
1A subunits may account for the observed
difference in efficacy between the cortical stellate neuron synapse and
hippocampal pyramidal neuron synapse. The 1Ab
isoform is highly expressed in CA3-CA1 pyramidal neurons in the
hippocampus (Bourinet et al., 1999). Although the
1A subunit isoform expressed in the spiny neurons of entorhinal cortex is not yet known, study of neostriatal spiny neurons indicates that they exclusively express the
1Aa isoform (Mermelstein et al., 1999). In the
Xenopus oocyte expression system, the
1Ab isoform displayed a significant 6 mV
positive shift in the current-voltage relationship compared with that
shown by the 1Aa isoform (Bourinet et al.,
1999). If layer II stellate cells in the entorhinal cortex also express
1Aa, then P/Q-type Ca currents evoked by
action potential invasion would be larger at the spiny neuron terminals
than at the pyramidal neuron terminals, given an equal density of
P/Q-type Ca2+ channels at both sites.
Alternatively, variability in the expression of ancillary
Ca2+ channel subunits, especially the subunit, provides another plausible mechanism for the difference
observed between these two synapses. Single-cell reverse
transcription-PCR analysis of Ca2+ channel
and subunit mRNAs indicates different subunit expression in
neostriatal spiny stellate cells compared with cortical pyramidal cells
(Mermelstein et al., 1999). Compared with cortical pyramidal cells,
neostriatal spiny neurons express significantly higher levels of
2 mRNA and lower levels of
1 mRNA. Interestingly, this difference
correlates well with the biophysical properties of
Ca2+ Q-type channels in these two types of
cell types. Q-type currents in the neostriatal spiny neurons display
little or no inactivation. If entorhinal spiny neurons are similar to
neostriatal spiny neurons and cortical pyramidal neurons are similar to
hippocampal pyramidal neurons, presynaptic Q-type currents at the
investigated synapse would be larger than those at the hippocampal
CA3-CA1 synapse because they are less inactivated. To test whether the
4 subunit is a potential candidate for this
mechanism, we measured presynaptic Ca2+
channels and neurotransmitter release in lethargic mutants
(lh), which lack functional 4
subunits (Burgess et al., 1997). Similar to our previous findings at
the hippocampal CA3-CA1 synapse (Qian and Noebels, 2000), presynaptic
Ca2+ influx was comparable with wild type
at the synaptic connection from entorhinal cortex to dentate gyrus in
lh mice (data not shown). Thus, variable expression of
4, if it exists in these two neurons, is
unlikely to account for different properties of the presynaptic Ca2+ channels at these two synapses.
Finally, the possibility that a higher density of
Ca2+ channels is present at the
presynaptic terminals of stellate neurons in the layer II of entorhinal
cortex than at those of hippocampal pyramidal neurons, although less
likely, cannot be completely ruled out. Therefore, detailed study of
Ca2+ channel subunit mRNA and the
expressed proteins in these two cell types will be helpful in
elucidating the basis for underlying differences in the biophysical
properties of presynaptic Ca2+ channels at
these two important synapses in the CNS.
| |
FOOTNOTES |
Received Dec. 11, 2000; revised March 13, 2001; accepted March 13, 2001.
This work was supported by an Eric Lothman Postdoctoral Fellowship from
the American Epilepsy Society/Milken Foundation (J.Q.) and National
Institute of Neurological Disorders and Stroke Grant NS29709
(J.L.N.).
Correspondence should be addressed to Dr. Jeffrey L. Noebels,
Department of Neurology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030. E-mail: jnoebels{at}bcm.tmc.edu.
| |
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ABSTRACT
INTRODUCTION
