Department of Physiology and Biophysics, Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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Magoski, Neil S. and
Andrew G. M. Bulloch.
Dopamine activates two different receptors to produce variability in
sign at an identified synapse. Chemical synaptic transmission was investigated at a central synapse between identified neurons in the
freshwater snail, Lymnaea stagnalis. The presynaptic neuron was the dopaminergic cell, Right Pedal Dorsal one (RPeD1). The postsynaptic neuron was Visceral Dorsal four (VD4). These neurons are
components of the respiratory central pattern generator. The synapse
from RPeD1 to VD4 showed variability of sign, i.e., it was either
inhibitory (monophasic and hyperpolarizing), biphasic (depolarizing
followed by hyperpolarizing phases), or undetectable. Both the
inhibitory and biphasic synapse were eliminated by low Ca2+/high Mg2+ saline and maintained in high
Ca2+/high Mg2+ saline, indicating that these
two types of connections were chemical and monosynaptic. The latency of
the inhibitory postsynaptic potential (IPSP) in high
Ca2+/high Mg2+ saline was ~43 ms, whereas the
biphasic postsynaptic potential (BPSP) had ~12-ms latency in either
normal or high Ca2+/high Mg2+ saline. For a
given preparation, when dopamine was pressured applied to the soma of
VD4, it always elicited the same response as the synaptic input from
RPeD1. Thus, for a VD4 neuron receiving an IPSP from RPeD1, pressure
application of dopamine to the soma of VD4 produced an inhibitory
response similar to the IPSP. The reversal potentials of the IPSP and
the inhibitory dopamine response were both approximately 90 mV. For a
VD4 neuron with a biphasic input from RPeD1, pressure-applied dopamine
produced a biphasic response similar to the BPSP. The reversal
potentials of the depolarizing phase of the BPSP and the biphasic
dopamine response were both approximately
44 mV, whereas the reversal
potentials for the hyperpolarizing phases were both approximately
90
mV. The hyperpolarizing but not the depolarizing phase of the BPSP and
the biphasic dopamine response was blocked by the D-2
dopaminergic antagonist (±) sulpiride. Previously, our laboratory
demonstrated that both IPSP and the inhibitory dopamine response are
blocked by (±) sulpiride. Conversely, the depolarizing phase of both
the BPSP and the biphasic dopamine response was blocked by the
Cl
channel antagonist picrotoxin. Finally, both phases of
the BPSP and the biphasic dopamine response were desensitized by
continuous bath application of dopamine. These results indicate that
the biphasic RPeD1
VD4 synapse is dopaminergic. Collectively, these data suggest that the variability in sign (inhibitory vs. biphasic) at
the RPeD1
VD4 synapse is due to activation of two different dopamine receptors on the postsynaptic neuron VD4. This demonstrates that two populations of receptors can produce two different forms of
transmission, i.e., the inhibitory and biphasic forms of the single
RPeD1
VD4 synapse.
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INTRODUCTION |
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The sign of transmission at a chemical synapse is usually
considered invariant, although there are reports that the sign of a
connection can vary between preparations (Park and Winlow
1993; Spencer and Winlow 1994
). However,
preparation-to-preparation variability in the sign of transmission at a
single, identified synapse was not documented. Differences in the types
of connections between the same neurons can be considered an extension
of the "polymorphic network" concept. First proposed by Getting
(1989)
, the polymorphic network theory suggests that physically defined circuits of neurons can produce more than one type of output. By
changing the sign of a specific synapse, the functional output of the
circuit that contains the neurons in question can be modified. There
are instances where it may be advantageous for a presynaptic neuron to
alter the excitability of a postsynaptic neuron in a specific manner,
for example, Cl
-dependent inhibition versus
K+-dependent inhibition. The levels of second messengers
may also be affected, depending on the types of synaptic connection,
providing access to different forms of neuromodulation and
neuroplasticity. We examine the physiological and pharmacological basis
of different synaptic potentials at a single, identified molluscan
synapse. The connection in question displays the unusual property of
variability in the type of synaptic potential between preparations.
The focus here is a well-characterized, identified dopaminergic neuron
known as Right Pedal Dorsal one (RPeD1) from the CNS of the freshwater
snail, Lymnaea stagnalis (Audesirk 1985;
Benjamin 1984
; Cottrell et al. 1979
;
Elekes et al. 1991
; Magoski and Bulloch 1997
; Magoski et al. 1995
; McCaman et al.
1979
; Werkman et al. 1991
; Winlow and
Benjamin 1977
; Winlow et al.1981
). One of
RPeD1's many postsynaptic cells is the cardiorespiratory interneuron
Visceral Dorsal four (VD4) (Benjamin 1984
;
Buckett et al. 1990
; Skingsley et al.
1993
; Syed and Winlow 1991
). Neurons RPeD1 and
VD4 are components of the central pattern generator responsible for
aerial respiration (Moroz and Winlow 1992
; Syed
and Winlow 1991
; Syed et al. 1990
, 1992
).
The synapse from RPeD1 to VD4 was observed in one of two forms. It is
either inhibitory (monophasic and hyperpolarizing) (Syed and
Winlow 1991; Syed et al. 1990
) or biphasic
(depolarizing followed by hyperpolarizing phases) (Benjamin
1984
). Beyond these reports documenting the synapse, there is
no information regarding physiological parameters such as latency or
reversal potential; as well, tests for a chemical or monosynaptic
connection were not carried out on the RPeD1
VD4 synapse.
Furthermore, although Magoski et al. (1995)
provided detail regarding
the pharmacology of the inhibitory form of the synapse (as expected it
is dopaminergic), there is no information on the pharmacology of the
biphasic form. Interestingly, all previous reports on the RPeD1
VD4
synapse implied that the connection is either inhibitory or biphasic.
By thoroughly surveying the sign of transmission at the RPeD1
VD4
synapse in many preparations this study will show that this connection
can be either inhibitory or biphasic, i.e., the sign of transmission is
variable between animals. The physiology and pharmacology of both forms
of the RPeD1
VD4 synapse will then be examined. We then provide
evidence that the differences in sign at this synapse are due to the
selective activation of two different postsynaptic dopamine receptors.
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METHODS |
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Animals, dissection, and salines
A stock of the mollusk, Lymnaea stagnalis (Gastropoda, Pulmonata, Basommatophora, Lymnaeidae), raised and maintained in a large scale aquaculture at the University of Calgary was used. Animals had shell lengths of 15-25 mm (age ~1-4 mo). The CNS was removed and pinned dorsal surface up to the silicone rubber base (General Electric RTV 616) of a ~500-µl recording chamber. The cerebral commissure was cut so that the CNS lay flat. Dissection, pinning of the CNS, and some electrophysiology were performed in normal Lymnaea saline (composition in mM was 51.3 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5 MgCl2, and 5.0 HEPES, adjusted to pH 7.9 with 1 N NaOH). To reduce the probability of polysynaptic effects, most electrophysiology was performed in high Ca2+/high Mg2+ saline (composition in mM was 51.3 NaCl, 1.7 KCl, 24.6 CaCl2, 1.5 MgCl2, 7.5 MgSO4, and 5.0 HEPES, pH 7.9). To test for chemical synapses, a low Ca2+/high Mg2+ saline was used (composition in mM was 51.3 NaCl, 1.7 KCl, 0.01 CaCl2, 1.5 MgCl2, 7.5 MgSO4, and 5.0 HEPES, pH 7.9). Salts were obtained from Sigma. Experiments were performed at room temperature (18-20°C).
Electrophysiology
Current-clamp recordings were made with single-barrel
borosilicate micropipettes filled with 2 M potassium acetate and having a resistance of 20-30 M. Data were collected with a dual-channel intracellular amplifier equipped with a bridge balance. The voltage was
displayed on a storage oscilloscope and recorded on a chart recorder as
well as a digital storage oscilloscope. Current was injected into the
neurons via the DC injection function on the amplifier. In some
instances, to facilitate microelectrode penetration of neurons, the
sheath surrounding the CNS was exposed to a small pronase crystal
(Sigma type XIV), held by forceps. The CNS was then rinsed in cold
(~4°C) normal saline to remove excess enzyme.
Identification of neurons
Neurons RPeD1 and VD4 are identifiable from preparation to
preparation with a high degree of reliability. Neuron RPeD1 is the only
large (>100 µm) neuron in the right pedal ganglion and easily
recognized on the basis of size, location, color, and relatively infrequent firing pattern (Benjamin and Winlow 1981).
Neuron VD4 is a small (20-30 µm), very white cell whose location in
the visceral ganglion can differ between preparations. To ensure that
the cell in question was VD4, one or more of three independent criteria were used: 1) VD4 always displayed a characteristic
discharge of steadily broadening action potentials immediately after
impalement; 2) VD4 very often displayed regenerative firing
properties; and 3) VD4 always made an excitatory synapse
with neurons Right Pedal Dorsal two or three (Nesic et al.
1996
; RPeD2/3; Syed and Winlow 1989
). We are
confident that the variability of synapses described in this study were
not due to discrepancies in neuronal identification.
Application of pharmacological agents
The chamber was perfused thoroughly at ~3 ml/min with a
peristaltic pump. For bath application, a compound was dissolved in high Ca2+/high Mg2+ saline containing 0.01%
(wt/vol) Fast Green (Sigma F7258), and the solution was introduced into
the bath via a three-way valve system. For dopamine (Sigma H8502),
which is prone to oxidation, 0.1% (wt/vol) sodium metabisulfite (Sigma
S1516) was also included. When Fast Green and sodium metabisulfite were
applied as a control, no discernible effect on membrane potential,
action potential wave form, firing pattern, or synaptic transmission
was observed. Sulpiride (Research Biochemicals International S116), a
dopaminergic antagonist, was first dissolved in a small volume of 80%
ethanol and then added to the saline. The final concentration of
ethanol was 0.4% (vol/vol). When 0.4% ethanol was applied as a
control, no discernable effect on neuronal physiology was observed. The only other drug bath applied was the Cl channel blocker
picrotoxin (Sigma P167).
Dopamine was also pressure applied. Dopamine was dissolved in high Ca2+/high Mg2+ saline, containing 0.01% Fast Green and 0.1% sodium metabisulfite. This solution was loaded into a wide-bore, fire-polished pipette connected to a pneumatic pressure unit. Dopamine was applied directly to the soma or was pressure ejected slightly adjacent to the soma and allowed to rapidly pass over the cell body; this did not affect the type of response. The Fast Green that was coejected with dopamine allowed us to consistently observe were dopamine was being applied. In almost all cases, the bolus of dopamine spread over an area that was two to three times the diameter of the soma.
Data analysis
The mean and SE of the mean are given either in the text or graphically. The program Inplot 4.01 (ISI Software) was used to plot data and fit regression lines (least-squares method).
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RESULTS |
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As outlined in the INTRODUCTION, the sign of the RPeD1
VD4 synapse was reported as either inhibitory or biphasic. We
examined whether the sign of this synapse in fact varied among a large number of CNS preparations. Neuron RPeD1 inhibited VD4 in 39% of the
preparations and in 48% of the cases made a biphasic synapse onto VD4,
and in the remaining 13% of preparations the synapse was undetectable
(Table 1). The possibility that seasonal
or environmental changes were related to the differences seen in the
sign of transmission was examined. No obvious correlation between the
month, day of the week, time of day, or feeding schedule and the sign
of the RPeD1
VD4 synapse was observed (data not shown). For the two
detectable forms of the synapse, i.e., inhibitory and biphasic, we
first sought to determine if the connections were indeed chemical and
monosynaptic. We then compared the physiological properties of the
synaptic response to the response evoked by applied dopamine (RPeD1's
transmitter). Importantly, the response of VD4 to applied dopamine
always mimicked the endogenous synaptic input from RPeD1. Finally, we
examined the pharmacology of the biphasic synapse to test whether it is
mediated by the activation of two different dopamine receptors.
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Physiology of the inhibitory RPeD1 VD4 synapse
The RPeD1 VD4 inhibitory synapse was first documented by Syed
et al. (1990)
and Syed and Winlow (1991)
. These studies demonstrated a
one-to-one, action potential-to-inhibitory postsynaptic potential (IPSP) ratio in normal saline. For this study, the chemical and monosynaptic nature of this synapse was more rigorously examined. This
included the standard criteria outlined by Austin et al. (1967)
and
Berry and Pentreath (1976)
: testing the Ca2+ dependence of
transmission, observing the effects of elevated divalent ion
concentrations (which raises the action potential threshold of any
intervening interneurons) on transmission, and quantifying the
consistency and magnitude of synaptic latency. The RPeD1
VD4
inhibitory connection was eliminated in a low Ca2+/high
Mg2+ saline and maintained in a high Ca2+/high
Mg2+ saline (n = 5 and 5, respectively;
Fig. 1A). In high
Ca2+/high Mg2+ saline, the inhibitory synapse
had a consistent one-to-one action potential to IPSP ratio and
displayed an action potential peak-to-IPSP inflection latency of
42.9 ± 1.0 ms (n = 32 IPSPs from 11 synapses; Fig. 1B).
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To further characterize the physiology of this synapse, we tested the
influence of VD4 membrane potential on IPSP magnitude. As VD4 was
hyperpolarized from 40 to
90 mV, the IPSP decreased in a linear
fashion (Fig. 2A). Previous
pharmacological work by Magoski et al. (1995)
indicated that the RPeD1
VD4 inhibitory synapse was dopaminergic. To determine if the
dopamine response in VD4 was similar to the IPSP, the effect of VD4
membrane potential on the response to exogenously applied dopamine was
examined. When VD4 was held at
40 mV, pressure-applied dopamine (0.1 M in the pipette) produced a large hyperpolarization; holding the cell
at more negative membrane potentials resulted in a steadily smaller
hyperpolarization, which reversed at approximately
90 mV (Fig.
2B). The relationship between the membrane potential of VD4
and both the IPSP and the dopamine response are plotted together in
Fig. 3. Linear regression of both
relationships provided similar extrapolated reversal potentials of
90.5 mV for the IPSP (n = 13) and
90.4 mV for the
dopamine response (n = 7). Note that the monophasic,
inhibitory pressure-applied dopamine responses were always observed on
VD4 neurons that received an inhibitory synaptic input from RPeD1.
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Physiology of the biphasic RPeD1 VD4 synapse
Benjamin (1984) provided the first documentation of the RPeD1
VD4 biphasic synapse. For the current work, the biphasic synapse was
subjected to the same tests for chemical and monosynaptic connections
as the inhibitory form. The RPeD1
VD4 biphasic connection was
eliminated in low Ca2+/high Mg2+ saline and
maintained in high Ca2+/high Mg2+ saline
(n = 4 and 4, respectively; Fig.
4A). The biphasic synapse consistently displayed a one-to-one action potential-to-biphasic postsynaptic potential (BPSP) ratio. Because there is evidence that
high cation concentrations can sometimes produce multiphased postsynaptic potentials (PSPs) in other molluscan neurons
(Getting 1981
), latency was demonstrated in both normal
and high Ca2+/high Mg2+ saline. The action
potential peak-to-depolarizing phase inflection latency in normal
saline was 12.0 ± 0.5 ms (n = 36 BPSPs from 8 synapses), and similarly in high Ca2+/high Mg2+
saline it was 12.1 ± 0.4 ms (n = 27 BPSPs from 8 synapses; Fig. 4B).
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Neuron RPeD1 is thought to exclusively use dopamine as a
neurotransmitter (see INTRODUCTION). Thus we sought to
determine if the BPSP and the VD4 biphasic dopamine response showed a
similar dependence on VD4 membrane potential, the assumption being that if RPeD1 uses dopamine to produce the BPSP the BPSP should be mimicked
by exogenously applied dopamine. For a biphasic connection, the effect
of RPeD1 stimulation on VD4 at a range of postsynaptic membrane
potentials can be seen in Fig.
5A. As VD4 was hyperpolarized, the depolarizing phase increased, and the hyperpolarizing phase decreased. Biphasic dopamine responses were always observed on VD4
neurons that received a biphasic synaptic input from RPeD1. Pressure-applied dopamine (0.1 M in the pipette) to the soma of VD4
produced a biphasic response (n = 7; Fig.
5B). Figure 6 shows a plot of
both phases of the RPeD1 VD4 BPSP and the VD4 biphasic pressure-applied dopamine response at various postsynaptic membrane potentials. The depolarizing phase of both the BPSP and the biphasic VD4 dopamine response had similar extrapolated reversal potentials of
44.4 and
43.6 mV, respectively. The hyperpolarizing phase of the
BPSP and the biphasic VD4 dopamine response had also similar extrapolated reversal potentials of
91.9 and
88.5 mV, respectively.
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In a few preparations, the depolarizing phase of the BPSP could be
reversed, i.e., when VD4 was held at 40 mV the depolarizing phase of
the BPSP was observed as a rapid hyperpolarization followed by a slow
hyperpolarization. When the membrane potential of VD4 was held at
50
mV or greater, the initial phase was now the more typical, rapid
depolarization (Fig. 7). This result
shows that both phases of the biphasic synapse are functionally
inhibitory.
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Pharmacology of the biphasic RPeD1 VD4 synapse
To determine if two separate receptors mediate the RPeD1 VD4 BPSP, a pharmacological investigation of both BPSP and biphasic VD4
pressure-applied dopamine response was undertaken. Previously, we
determined that of many dopaminergic antagonists, the only effective
drug at a number of RPeD1 synapses, including the monophasic inhibitory
connection with VD4, was (±) sulpiride (Magoski et al.
1995
). When (±) sulpiride (100 µM) was bath applied to a
biphasic synapse, the hyperpolarizing phase but not the depolarizing
phase of both the BPSP and the biphasic dopamine response was blocked (n = 5; Fig. 8).
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Because an effective dopaminergic antagonist was not available for the
depolarizing phase, a different approach was taken. The reversal
potential of the depolarizing phase suggested the involvement of a
Cl conductance, and therefore the Cl
channel blocker picrotoxin was tested. The depolarizing phase but not
the hyperpolarizing phase of both the BPSP and the biphasic response
was reversibly blocked by picrotoxin (100 µM; n = 4; Fig. 9).
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As a more conclusive test of whether the depolarizing phase was
dopaminergic, the ability of exogenous dopamine to desensitize the
receptor(s) on VD4 was examined. With continuous bath application of
dopamine (100 µM), both phases of the BPSP and the biphasic pressure-applied VD4 dopamine response were effectively desensitized (n = 5; Fig. 10). The
synaptic and pressure-applied dopamine responses were both desensitized
1 min after bath application of dopamine. To be certain that the
membrane of VD4 possessed adequate resistance to carry synaptic input
during bath-applied dopamine, a separate experiment was undertaken in
which the input resistance of VD4 was measured during bath application
of 100 µM dopamine. During dopamine exposure, the input resistance of
VD4 decreased by only 32.5 ± 9.7% (n = 6). This
would indicate that the response to RPeD1 stimulation and to
pressure-applied dopamine was desensitized rather than shunted.
Collectively, these data suggest that the RPeD1
VD4 biphasic
synapse is mediated by dopamine acting on two different receptors.
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DISCUSSION |
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By examining many preparations, we determined that the sign of
synaptic transmission at the RPeD1 VD4 synapse varies in that it is
either inhibitory or biphasic. In a minority of preparations, the
connection was undetectable. We investigated the physiology and
pharmacology of both the inhibitory and biphasic forms of the RPeD1
VD4 synapse. Both types of synapses appear to be chemical and
monosynaptic, suggesting that different synaptic responses are not due
to polysynaptic effects. Importantly, dopamine (RPeD1's transmitter)
mediates both the monophasic as well as the biphasic synapse. These
data indicate that the different synaptic responses at this connection
are due to the activation of two different postsynaptic dopamine
receptors. We have shown that variability in synaptic sign
at a single identified synapse is achieved by a transmitter
activating two different receptors. The uniqueness of our study is that
the same synapse between the same identified neurons (RPeD1
VD4)
can manifest itself differently in different preparations, i.e.,
inhibitory versus biphasic.
Both forms of the RPeD1 VD4 synapse are likely chemical and
monosynaptic. Elimination of transmission by a low
Ca2+/high Mg2+ saline indicates a chemical
rather than an electrical connection for the two types of synapse
(Figs. 1 and 4) (Berry and Pentreath 1976
). Furthermore,
the ability of RPeD1 to evoke single IPSPs or BPSPs in high
Ca2+/high Mg2+ suggests that transmission is
monosynaptic (Figs. 1 and 4). Elevated Ca2+ increases
action potential threshold and reduces the likelihood of spiking by
putative interneurons (Austin et al. 1967
; Berry and Pentreath 1976
; Elliot and Benjamin 1989
).
The constant and rapid latency of both the inhibitory and biphasic
synapses is consistent with a monosynaptic connection (Berry and
Pentreath 1976
). The difference in latencies between the
inhibitory and the biphasic synapses (~43 vs. ~12 ms) presumably
reflects different rates of postsynaptic transduction, or the
depolarizing phase of the BPSP may be initiated electrotonically closer
to the soma (the recording site) than the IPSP. There is good evidence
to suggest that hyperpolarizing, K+-dependent transmitter
responses in Aplysia neurons are mediated by the relatively
slow process of G-protein-coupled receptors (Brezina
1988
; Bolshakov et al. 1993
; Sasaki et
al. 1997
). A similar mechanism may be involved in producing the
long latency of the hyperpolarizing PSP at the RPeD1
VD4 synapse.
Incidentally, previous reports, from both Lymnaea
(Winlow et al. 1981
) and the related pulmonate mollusk,
Planorbis corneus (Berry and Cottrell 1975
),
show that the latency of inhibitory connections made by the giant
dopamine cell (RPeD1 in Lymnaea or GDC in
Planorbis) are usually 4-10 times slower than excitatory
and biphasic connections. Although we cannot completely rule out the
possibility that the inhibitory synapse is the result of a polysynaptic
pathway, the high concentration of divalent cations would make such a
possibility very remote. Both ourselves (Magoski and Bulloch
1997
) and other investigators (Winlow et al.
1981
) never reported a single spike in RPeD1 eliciting a spike
in a follower cell to which RPeD1 made an excitatory connection while
in the presence of high divalent cations. This would have to be the
case if the inhibitory RPeD1
VD4 synapse were polysynaptic.
Parenthetically, both the inhibitory (Syed et al. 1990
)
and the biphasic (O. Nesic, personal communication) synapses form in
vitro when the RPeD1 and VD4 are isolated and plated in culture.
Previous work shows that the soma of RPeD1 contains dopamine
(Audesirk 1985; Cottrell et al. 1979
;
Elekes et al. 1991
; Magoski et al. 1995
;
McCaman et al. 1979
; Werkman et al.
1991
). Also, Magoski et al. (1995)
and Magoski and Bulloch
(1997)
provided pharmacological evidence that RPeD1 uses dopamine at a
number of synapses, including its inhibitory synapse with VD4. This is supported by the observation that the inhibitory synapse and the inhibitory dopamine response of VD4 have similar reversal potentials (Figs. 2 and 3), indicating that RPeD1 and applied dopamine both activate a similar conductance. The approximately
90 mV reversal potential suggests that the conductance is K+ selective.
Similar inhibitory responses to dopamine were reported for identified
neurons in Achatina (Emaduddin et al. 1995
),
Aplysia (Ascher 1972
), Helix
(Nesic and Pasic 1992
), Lymnaea
(Audesirk 1989
; De Vlieger et al. 1986
),
Planorbis (Berry and Cottrell 1979
), and
Planorbarius (Bolshakov et al. 1993
).
It was important to determine that dopamine was involved in both phases
of the RPeD1 VD4 biphasic synapse. This would reinforce the
conclusion that variability in sign at this connection is due to
activation of either one or both of two receptors. It would also rule
out the unlikely possibility that the depolarizing phase was a
polysynaptic effect. At a biphasic RPeD1
VD4 synapse, pressure-applied dopamine produced a biphasic response in VD4 (Fig. 5).
The reversal potentials of the BPSP and the biphasic dopamine response
were essentially the same (Fig. 6), indicating that RPeD1 input and
dopamine activate a similar set of conductances. Both hyperpolarizing
phases reversed at approximately
90 mV, implicating a K+
conductance, and were blocked by 100 µM (±) sulpiride (Fig. 8). This
concentration of (±) sulpiride was previously found to be effective at
blocking both the RPeD1
VD4 inhibitory synapse as well as other
synapses of RPeD1 (Magoski et al. 1995
). The reversal
potentials of both depolarizing phases (approximately
44 mV)
implicated a Cl
conductance. Thomas (1977)
showed that
the Cl
Nernst potential for certain Helix
neurons is approximately
50 mV. Consistent with this, both
depolarizing phases were blocked by the Cl
channel
blocker picrotoxin (Fig. 9). Picrotoxin, at similar concentrations, blocks Cl
-dependent responses to GABA in locust neurons
(Jackel et al. 1994
) as well as histamine
(Hashemzadeh-Gargari and Freschi 1992
) and glutamate responses
(Cleland and Selverston 1995
) in lobster neurons.
Picrotoxin is thought to either directly block the pore of the
Cl
ionotropic receptor or bind to an associated,
nonreceptor site on the protein (Barker et al. 1983
).
Both the BPSP and the biphasic dopamine response desensitized when
dopamine was bath applied (Fig. 10). This suggests that bath-applied
dopamine competes with dopamine released at the synapse. It is unlikely
that the input resistance of VD4 was reduced during the response to
bath-applied dopamine to such an extent that the BPSP was shunted
rather than desensitized, given that in separate experiments there was
only a one-third reduction of input resistance during bath-applied
dopamine responses. Furthermore, on a number of occasions we observed
what is likely nondopaminergic synaptic input occurring in VD4 during
the peak of a response to bath-applied dopamine (e.g., see Fig. 15 of
Magoski et al. 1995). Finally, we also noted that both
the RPeD1
VD4 inhibitory and biphasic synapses are maintained in
the presence of 10 µM GDPFLRFamide, a Lymnaea peptide that
hyperpolarizes VD4 (Magoski and Bulloch, unpublished observations).
Thus it is likely that the lack of a response of VD4 to RPeD1
stimulation in the presence of 100 µM dopamine is due to
desensitization rather than shunting. Bath-applied dopamine
desensitizes ionophoretic dopamine responses in Aplysia (Ascher 1972
); furthermore, repeated or prolonged
agonist application to Aplysia (Matsumoto et al.
1987
) and mammalian dopamine receptors (Seeman and Van
Tol 1994
) causes desensitization.
Biphasic dopaminergic synapses and/or dopamine responses were observed
in identified neurons from Aplysia (Ascher
1972), Helisoma (Syed et al. 1993
),
Lymnaea (i.e., other types of neurons in Lymnaea) (Magoski et al. 1995
; Winlow and Benjamin
1977
; Winlow et al. 1981
), and
Planorbis (Berry and Cottrell 1975
, 1979
) as
well as neurons from the dorsal root ganglion of the rat
(Molokanova and Tamarova 1995
). Similarly, cholinergic
(Gardner and Kandel 1972
; Kehoe 1969
,
1972a
,b
,c
; Wachtel and Kandel 1967
) and
histaminergic (McCaman and Weinreich 1982
, 1985
)
biphasic synapses were described in identified Aplysia
neurons. The inferior ventricular nerve interneurons of the lobster
stomatogastric ganglion also make biphasic synapses with some follower
neurons (Sigvardt and Mulloney 1982
). However, in none
of these cases did the synapses or transmitter responses change, i.e.,
they consistently gave the same response. There are some examples in
the literature (Kehoe 1972a
,b
,c
; McCaman and
Weinreich 1985
) showing that the multicomponent nature of a
small number of cholinergic and histaminergic responses is not always
evident but requires pharmacological separation to demonstrate different components. This is not the situation here, where we are
investigating physiological differences in the sign of transmission that do not require pharmacological manipulations to be revealed. Furthermore, the actual number of inhibitory and biphasic synapses we
observed between RPeD1 and VD4 was substantial, suggesting that we are
not studying some subset of synapses that on rare occasions shows
differences in the sign of transmission.
We conclude that RPeD1 uses dopamine as a neurotransmitter at its
synapse with VD4, whether that synapse is inhibitory or biphasic. The
biphasic synapse is mediated by dopamine acting on two different
receptors, one that is (±) sulpiride sensitive and likely activates a
K+ conductance and a second that is (±) sulpiride
insensitive and probably activates a Cl conductance,
whereas the inhibitory RPeD1
VD4 synapse is likely mediated
exclusively by the (±) sulpiride-sensitive, K+
conductance-coupled receptor.
Variability in the sign of transmission at the RPeD1 VD4 connection
could be achieved by altering the functional expression of receptors.
Colocalization of different types of dopamine receptors is not unusual.
For example, D-1 and D-2 receptors are
colocalized in approximately one-half of medium spiny projection
neurons in the neostriatum (Surmeier et al. 1996
). The
receptors may be localized on different axon collaterals of VD4. Spike
propagation blockade of a particular innervating RPeD1 axon might cause
only certain receptors to be activated, resulting in the loss of the
depolarizing phase. Also, the depolarizing phase could elude detection
if its portion of the PSP decayed before reaching VD4's soma (the
recording site). For instance, there were preparations in which strong
stimulation of RPeD1 could overwhelm the depolarizing phase of a
biphasic synapse, resulting in only monophasic hyperpolarization (data not shown). However, we would contend that, whereas the two phases of
the BPSP can vary in amplitude at the biphasic synapse, the difference
between the inhibitory and biphasic synapse is likely not due to the
depolarizing component being obscured. At the inhibitory synapse, a
depolarizing component does not present itself at any time, even when
VD4 was held at hyperpolarizing voltages, or when only one or two
presynaptic spikes were elicited
conditions that assure detection of a
depolarizing phase. Furthermore, we have shown previously
(Magoski et al. 1995
) that a depolarizing component does
not appear when the inhibitory RPeD1
VD4 synapse is exposed to
sulpiride nor does a depolarizing component appear when a VD4 neuron,
responding to pressure-applied dopamine with only hyperpolarization, is
also exposed to sulpiride (Magoski 1996
).
It is not possible to be absolutely certain that a particular PSP
or transmitter response does not possess additional, masked components.
Pressure pipette position was known to affect the type of response
elicited by exogenous transmitters (see Acsher and Kehoe
1975 for review). In this study, however, positioning the
pipette directly over the soma or just adjacent to the soma (see
METHODS) did not alter a given response nor did it affect the correlation between dopamine response and the type of PSP. Accordingly, we feel that the application technique is sufficient to
distinguish between inhibitory and biphasic responses. Receptor localization combined with electrotonic distance from the recording site or the presence of barriers to transmitter access such as the
ganglion's inner sheath could also confound the results of the
pressure-applied dopamine experiments. The soma of VD4 in our
preparations is quite small (~20-30 µm) and its major axonal arbor
is located close by (see Benjamin 1984
or Syed
and Winlow 1991
for morphological details). It is likely that a
portion of the applied dopamine reached this arbor in the neuropile,
which is the presumed location of receptors mediating transmission. Thus the dopamine responses we observed probably reflect the activation of receptors on both the soma and on the adjacent axons and axon collaterals. Although we cannot rule out the inner sheath as a barrier
to transmitter access, there is no obvious indication from this or
previous work involving pressure- and bath-applied transmitters
(Hermann et al. 1997
; Magoski et al.
1995
; Nesic et al. 1996
) that the
Lymnaea inner sheath is a significant obstacle to
transmitters. Additional support for this conclusion comes from the
observation that a number of Lymnaea neurons respond in the
same manner to applied transmitter (dopamine or glutamate) whether they
are in the brain and covered by the inner sheath or isolated in culture
(Magoski et al. 1995
; Nesic et al. 1996
). Given these arguments and the consistent correlation between dopamine response and synaptic input in VD4 we would suggest that, rather than
experimental manipulation, differential localization or possibly differential expression of the two dopamine receptors more likely underlies variability. Cloning of Lymnaea dopamine receptors
and the production of antibodies may offer tools to address the
localization and expression issues.
To our knowledge, the RPeD1 VD4 synapse provides the first example
of a connection between two identified neurons where variability in
synaptic sign is achieved by a transmitter activating two different
receptors. Although an inhibitory or biphasic synapse mediated by a
single transmitter in not new, we have shown that this single,
identified synapse can vary between inhibitory and biphasic from animal
to animal. As well, we provided an explanation for this variability,
i.e., VD4 displays two distinct responses to dopamine (the presynaptic
transmitter), and from preparation-to-preparation this correlates
exactly with the form of endogenous synaptic response evoked by RPeD1
stimulation. Variability aside, the functional differences between the
inhibitory and biphasic synapses remain to be determined. For both
synapses, the overall effect of the input from RPeD1 is to inhibit VD4.
The initial depolarization in the biphasic synapses could serve one or
more purposes. The Cl
-dependent phase is rapid in
comparison with the K+-dependent phase. The biphasic
synapse could offer short-duration inhibition at a low frequency of
input and long-duration inhibition at a high frequency of input.
Additionally, a voltage-dependent phenomenon may be triggered by the
depolarizing phase of the biphasic synapse. For example, dendritic
Ca2+ levels could be elevated during the depolarizing
phase, which might modulate the response to subsequent
hyperpolarization or some other input. As well, the activity of
adenylate cyclase can be synergistically enhanced by depolarization
(Reddy et al. 1995
). Facilitation at the crayfish
neuromuscular junction is believed to be the result of activating a
presynaptic adenylate cyclase via the voltage change during tetanus
(Wojtowicz and Atwood 1988
). Finally, there is a recent
report that intracellular Cl
concentration can affect
G-protein-mediated conductances (Lenz et al. 1997
). The
changes in intracellular Cl
that would occur during the
depolarizing phase could modulate G-protein-dependent responses in
VD4. Collectively, mechanisms such as these could play a role in
regulating the excitability of VD4.
The documentation of two synaptic responses at the RPeD1 VD4
synapse indicates a level of complexity not previously recognized at
this connection and may represent a means by which the CNS synaptically
configures neural networks in different ways. This complexity also does
not support an organizational principle proposed by Segal (1983)
, i.e.,
that ".. synapses with the same neurotransmitter will all produce the
same synaptic action on any particular nerve cell." Rather individual
synaptic connections may display different responses, depending on the
specific receptors that are activated.
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ACKNOWLEDGMENTS |
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The authors thank G. C. Hauser for technical support and N. M. Magoski for critiquing earlier drafts of the manuscript and providing assistance with the construction of figures.
This work was supported by grants from the Medical Research Council (MRC) of Canada to A.G.M. Bulloch. N. S. Magoski was a recipient of studentships from the MRC, the Alberta Heritage Foundation for Medical Research (AHFMR), and the NeuroScience Network (Canadian National Centres of Excellence). A.G.M. Bulloch is an AHFMR Senior Scientist.
Present address of N. S. Magoski: Dept. of Pharmacology, Yale School of Medicine, New Haven, CT 06520.
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FOOTNOTES |
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Address for reprint requests: A.G.M. Bulloch, Dept. of Physiology & Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 May 1998; accepted in final form 3 November 1998.
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REFERENCES |
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