Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4340
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ABSTRACT |
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Horning, Michelle S. and
Paul Q. Trombley.
Zinc and Copper Influence Excitability of Rat Olfactory Bulb
Neurons by Multiple Mechanisms.
J. Neurophysiol. 86: 1652-1660, 2001.
Zinc and copper are highly concentrated in
several mammalian brain regions, including the olfactory bulb and
hippocampus. Whole cell electrophysiological recordings were made from
rat olfactory bulb neurons in primary culture to compare the effects of
zinc and copper on synaptic transmission and voltage-gated ion
channels. Application of either zinc or copper eliminated GABA-mediated
spontaneous inhibitory postsynaptic potentials. However, in contrast to
the similarity of their effects on inhibitory transmission, spontaneous
glutamate-mediated excitatory synaptic activity was completely blocked
by copper but only inhibited by zinc. Among voltage-gated ion channels,
zinc or copper inhibited TTX-sensitive sodium channels and delayed
rectifier-type potassium channels but did not prevent the firing of
evoked single action potentials or dramatically alter their kinetics.
Zinc and copper had distinct effects on transient A-type potassium
currents. Whereas copper only inhibited the A-type current, zinc
modulation of A-type currents resulted in either potentiation or
inhibition of the current depending on the membrane potential. The
effects of zinc and copper on potassium channels likely underlie their
effects on repetitive firing in response to long-duration step
depolarizations. Copper reduced repetitive firing independent of the
initial membrane voltage. In contrast, whereas zinc reduced repetitive
firing at membrane potentials associated with zinc-mediated enhancement of the A-type current (50 mV), in a significant proportion of neurons, zinc increased repetitive firing at membrane potentials associated with zinc-mediated inhibition of the A-type current (
90
mV). Application of zinc or copper also inhibited voltage-gated Ca2+ channels, suggesting a possible role for
presynaptic modulation of neurotransmitter release. Despite
similarities between the effects of zinc and copper on some ligand- and
voltage-gated ion channels, these data suggest that their net effects
likely contribute to differential modulation of neuronal excitability.
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INTRODUCTION |
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Zinc and copper are important
trace metals that are differentially distributed throughout the
mammalian CNS (Donaldson et al. 1973; Ono and
Cherian 1999
; Slomianka 1992
). A variety of chemical and anatomical techniques have shown that pools of zinc and/or
copper are stored in synaptic vesicles and the terminals of some,
mostly glutamatergic, neurons (Friedman and Price 1984
; Holm et al. 1988
; Ibata and Otsuka 1969
;
Kardos et al. 1989
; Perez-Clausell and Danscher
1985
; Sato et al. 1994
; Schrder et al.
2000
; Slomianka et al. 1990
). Such pools of zinc
and/or copper can be released following membrane depolarization or
neural activity in a calcium-dependent manner (Assaf and Chung
1984
; Hartter and Barnea 1988
; Howell et
al. 1984
; Kardos et al. 1989
).
The mammalian olfactory bulb has one of the highest concentrations of
zinc and copper in the CNS (Donaldson et al. 1973;
Ono and Cherian 1999
). In the olfactory bulb, zinc, as
identified by a variety of histological methods, is contained primarily
in olfactory sensory neuron terminals in the glomerular layer and neuron terminals of unknown origin in the granule cell layer
(Friedman and Price 1984
; Jo et al. 2000
;
Perez-Clausell and Danscher 1985
). Furthermore, it has
been recently reported that zinc-containing neurons are also
immunoreactive for ZnT-3, a putative zinc transporter localized to
synaptic vesicles (Jo et al. 2000
; Palmiter et
al. 1996
; Wenzel et al. 1997
), and for
metallothionen-3 (MT3), a zinc binding metalloprotein (Cole et
al. 2000
; Masters et al. 1994
) that can also
bind copper (Chen et al. 1996
; Sewell et al.
1995
; Winge et al. 1994
). In contrast to zinc,
little is known about the precise location of copper in the olfactory
bulb. However, many of the histological techniques used to identify
zinc (e.g., Timm's stain) cross-react with copper and, therefore may
reflect both zinc and copper. Furthermore, it remains unclear whether ZnT-3 can transport copper in addition to zinc. Therefore as is the
case for zinc, some of the copper in the olfactory bulb may be in
synaptically releasable pools as occurs in other brain regions (Assaf and Chung 1984
; Hartter and Barnea
1988
; Howell et al. 1984
; Kardos et al.
1989
).
We have already shown that physiologically relevant concentrations of
zinc or copper can modulate amino acid receptors and synaptic
transmission and, in some cases, with differential effects (Trombley and Shepherd 1996; Trombley et al.
1998
). However, the neuromodulatory role(s) of zinc and copper
in the olfactory bulb have yet to be completely defined. In the present
report, we used whole cell electrophysiology and primary neuronal
cultures of olfactory bulb neurons to demonstrate that the net effects
of zinc and copper on neuronal excitability are distinct and therefore may contribute to selective modulation of synaptic circuits involved in
olfactory information processing.
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METHODS |
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Cell culture
Olfactory bulbs were prepared for primary dissociated cultures
using methods previously described in Trombley and Westbrook (1990) and Trombley and Blakemore (1999)
. The
olfactory bulbs were isolated from 0- to 3-day-old rat pups and cut
into small pieces after removing the meninges. The tissue was incubated
at 37°C for 60 min in a calcium-buffered solution containing 20 U/ml papain (Worthington, Freehold, NJ) and 1 mM cysteine. The tissue was
gently triturated with a fire-polished Pasteur pipette after inactivating the enzyme with excess media containing serum. The cell
suspension was plated at a density of approximately 260 cells/mm2 in 35-mm culture dishes (Corning,
Fisher) containing a confluent layer of astrocytes prepared previously.
The neuronal growth medium contained 93% minimal essential medium
(MEM, Gibco), 7% horse serum, 6 g/l glucose, and a nutrient supplement
(Serum Extender, Collaborative Research, Bedford, MA).
Electrophysiological recordings were conducted after the neurons were
in culture for 2-14 days.
Astrocyte feeder layers were prepared by plating olfactory bulb cells, as described in the preceding text, in a 75-cm2 poly-L-lysine-coated flask, containing MEM with 7% fetal calf serum and 6 g/l glucose. After reaching confluence, the astrocytes were removed with 0.125% trypsin, resuspended, and plated on 35-mm culture dishes coated with poly-L-lysine. After reaching confluence, the astrocytes were treated with a mitotic inhibitor, 5-fluoro-2'-deoxyuridine, and uridine (7 µg/ml) to prevent overgrowth.
Electrophysiology
All whole cell voltage-clamp recordings were made at room
temperature. The 35-mm culture dish was used as the recording chamber and perfused at 0.5-2.0 ml/min with a bath/control solution containing (mM) 162.5 NaCl, 2.5 KCl, 2 CaCl2, 10 HEPES, 10 glucose, and 1 or 0 MgCl2 and 1 µM glycine. The
pH was adjusted to 7.3 with NaOH, and the osmolarity was adjusted to
325 mosM. Patch electrodes were pulled from filamentous borosilicate
glass to a final electrode resistance of 4-6 M. Electrodes were
filled with a solution containing (mM): 145 KMeSO4 or CsCl, 1 MgCl2, 10 HEPES, and 1.1 EGTA. The final pH was adjusted to 7.2 with KOH or CsOH,
and the final osmolarity was 310 mOsm. Drugs were diluted in recording
solution and applied to the cultured neurons using a gravity-fed
flow-pipe perfusion system, assembled from an array of 600-µm-ID
square glass barrels. The barrels were positioned 100 µm from the
neuron using an electronic micromanipulator (Warner Instrument, Hamden,
CT), and the flow was controlled using pinch clamps. Complete solution
exchanges occurred within 100-200 ms of application. Neurons were
always perfused with a control solution via flow pipes except during drug application. Substances applied were ZnCl2,
CuCl2, CdCl2, and TTX (Sigma).
Experimental procedure
To examine membrane currents mediated by K+, Na+, and Ca2+ voltage-gated ion channels, whole cell voltage-clamp recordings were made using an AxoClamp 2B amplifier (Axon Instruments, Foster City, CA) in either discontinuous mode (switching frequency of 10-15 kHz) or continuous mode. Membrane currents were filtered at 1-3 kHz, digitized at 5-10 kHz, and analyzed using AxoData and AxoGraph software (Axon Instruments). Current-clamp data from studies designed to examine single action potentials, repetitive firing, or input resistance were collected unfiltered using the same amplifier and software.
Data analysis
Data are expressed as means ± SD. P values were
determined by paired t-tests, and values 0.05 were
considered significant.
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RESULTS |
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Zinc and copper inhibit spontaneous excitatory and inhibitory synaptic transmission
We have previously reported that zinc and copper can modulate
glutamate, GABA, and glycine receptors on olfactory bulb neurons (Trombley and Shepherd 1996). Here, we examined the
effects of zinc and copper on spontaneous excitatory and inhibitory
synaptic activity. To facilitate robust spontaneous activity, the
neurons were plated in 35-mm culture dishes at a density of
approximately 260 cells/mm2, and the recordings
were made after the neurons were in culture for approximately 1 wk.
Spontaneous inhibitory synaptic activity was recorded in current-clamp
mode from mitral/tufted cells, the primary targets of olfactory bulb
inhibitory interneurons. The large amplitude of the inhibitory synaptic
potentials in Fig. 1A was due
to a low intracellular cloride concentration. Flow-pipe application of
either metal resulted in complete suppression of inhibitory synaptic
transmission in all neurons examined (copper, n = 4;
zinc, n = 6; Fig. 1A). In these experiments,
the inhibitory postsynaptic potentials were mediated by
GABAA receptors, as confirmed by their inhibition
in response to application of 3 µM bicuculline (data not shown).
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In contrast to the similarity of the effects of zinc and copper on inhibitory transmission, zinc and copper had quantitatively different effects on excitatory transmission. Whereas application of 100 µM zinc only reduced glutamate-mediated excitatory transmission in mitral/tufted cells (n = 7), 30 µM copper completely eliminated all excitatory activity (n = 9; Fig. 1B). The effects of either metal on inhibitory or excitatory transmission were rapidly reversible on washing with control solution.
Zinc and copper inhibit inward Ca2+ currents
The modulatory effects of zinc and copper on voltage-gated
calcium-channel currents were examined to explore whether differential effects on these currents could contribute to their differential effects on excitatory transmission, possibly through effects on transmitter release. For ease of analysis, 2 mM calcium in the bath
solution was substituted with 10 mM Ba2+ to
increase the amplitude of the current. Calcium-channel currents were
evoked in voltage-clamp by holding the potential at 60 mV and
stepping to 0 mV for a duration of 50 ms. Data were collected in the
presence of 1 µM TTX and a CsCl-based electrode solution to block
Na+ and K+ currents, respectively.
Calcium-channel currents with kinetic profiles typical of high-threshold Ca currents were induced in 15 of 16 neurons. The peak amplitude of these currents ranged between 179.3 and 673.9 pA (mean = 293 ± 232 pA). Measurements of the currents were made at the peak and steady-state components of the current. Zinc application inhibited the Ca2+ channel currents at both time points by 63 ± 33 and 63 ± 13%, respectively (n = 16; P < 0.001; Fig. 2). Copper application reduced the inward current at both time points by an average of 52 ± 27 and 54 ± 22%, respectively (n = 12; P < 0.001; Fig. 2).
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Differential effects of zinc and copper on input resistance
We next hypothesized that the differential effects of zinc and
copper on basic membrane properties may have contributed to their
quantitatively different effects on excitatory transmission. In
current-clamp mode, the effects of zinc and copper on input resistance
were determined using intracellular injections of 250-ms hyperpolarizing steps of 50 pA. Hyperpolarizing pulses were used to
reduce any contribution from activation of voltage-gated ion channels
on input resistance. The prepulse membrane potential was maintained
near
50 mV with a small amount of current injection (±10 pA).
Measurements for analyses were made after the membrane voltage reached
steady-state. Analyses of neuronal input resistance in the presence of
30 µM copper indicate no significant difference compared with that of
the control solution (1 ± 15% increase, n = 13;
P > 0.5). However, application of 100 µM zinc
produced a modest (18 ± 11%) increase in input resistance (Fig.
3A, n = 10;
P < 0.001).
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Zinc and copper inhibit inward Na+ currents but do not prevent evoked single action potentials
We further hypothesized that the quantitatively different effects
of zinc and copper on the postsynaptic response to excitatory transmission may be mediated, in part, by distinct effects on voltage-gated ion channels. As an initial test of this hypothesis, we
examined the effects of zinc and copper on inward
Na+ currents, the major component of the upstroke
of an action potential. The Na+ currents were
evoked by 50-ms, 70- to
20-mV steps under voltage-clamp conditions.
The electrodes in this experiment were filled with a CsCl-based
solution to block K+ currents. Measurements for
analyses were made at the peak amplitudes of the evoked current.
Individual application of 100 µM zinc (n = 13;
P < 0.001) or 30 µM copper (n = 10;
P < 0.005) resulted in significant decreases in the
Na+ current by an average of 22 ± 17 and
20 ± 16%, respectively, compared with control currents (Fig.
3B). Current blockade with 1 µM TTX confirmed that these
currents were mediated by typical TTX-sensitive
Na+ channels.
Because zinc and copper reduced inward Na+
currents, we next examined whether zinc or copper could reduce the
ability of olfactory bulb (OB) neurons to fire single evoked
action potentials. Single action potentials were evoked by injecting
incremental steps of 0.05 nA, for a 5-ms duration, until the neuron
fired an action potential. The initial prestep resting potential was
approximately 60 mV.
Analyses of the action potential amplitudes following metal application show that neither copper (n = 8; P > 0.3) nor zinc (n = 10; P > 0.2) induced significant changes compared with control conditions. Furthermore, data analyses show that neither zinc nor copper caused significant differences in the half-widths of the action potentials (Cu: P > 0.3; Zn: P > 0.5) nor the amplitude of the current required to initiate an action potential (Cu: P > 0.3; Zn: P > 0.3) compared with control conditions (Fig. 3C). In 4 of 10 neurons, zinc application reduced the spike after-hyperpolarization by an average of 64 ± 69% (P < 0.02).
Zinc and copper inhibit delayed-outward K+ currents
Because the delayed outward K+ current
plays a major role in the downstroke and repolarization of an action
potential, we examined the modulatory actions of zinc and copper on
this current. Potassium currents were examined using a
KMeSO4-based electrode solution and 1 µM TTX to
block Na+ currents. Neurons were clamped at 50
mV and stepped to +20 mV for a duration of 100 ms. Neurons responded
with an outward current consistent with the kinetic profile
characteristic of delayed rectifier-type currents in these neurons
(Trombley and Westbrook 1991
). Measurements for data
analyses were made at peak and steady-state amplitudes.
Copper (30 µM) application inhibited the peak amplitudes by an average of 18 ± 9% (n = 5; P < 0.02) and the steady-state amplitudes by an average of 19 ± 9% (n = 5; P < 0.01; Fig. 4). Similarly, zinc (100 µM) application inhibited steady-state current amplitudes by an average of 21 ± 13% (n = 11; P < 0.003; Fig. 4). However, zinc potentiated the peak current amplitude in most neurons (n = 8) and had no effect in three others. In the eight neurons in which potentiation was observed, the potentiation of the peak current amplitude was due to the appearance of a transient A-type outward current component that was not present under control conditions. In these neurons, the peak current amplitude was potentiated by an average of 21 ± 16% (P < 0.007). Zinc had no significant effect on the peak current in the remaining three neurons. In these neurons, no A-type current component was apparent.
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Zinc and copper modulate transient-outward K+ current
The transient-outward K+ current plays
a role in mediating the interspike interval and is usually activated by
a depolarizing event following a hyperpolarizing event. To examine this
current, both the transient and delayed outward
K+ currents were activated by holding the neuron
at a hyperpolarizing potential of 90 mV and stepping the neuron to
+20 mV for a duration of 100 ms. As in the previous
experiments, these K+ currents were isolated
using 1 µM extracellular TTX and a KMeSO4-based intracellular solution. Measurements for analyses were made at peak and
steady-state amplitudes.
Zinc (n = 8; P < 0.001) and copper (n = 7; P < 0.003) application attenuated the steady-state outward K+ current in all of the neurons examined by an average of 17 ± 8 and 20 ± 11%, respectively (Fig. 5). Similarly, copper (n = 7; P < 0.001) application inhibited the transient outward K+ current in all neurons examined by an average of 17 ± 7%. Zinc application significantly inhibited the transient outward K+ current in most neurons (n = 8; P < 0.005) by an average of 15 ± 11%. Two other neurons showed no effect.
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Zinc and copper modulate repetitive firing
Although our results show that zinc or copper application did not
significantly reduce the ability of a neuron to fire single action
potentials, we hypothesized that the complex effects of these metals on
K+ currents may influence repetitive firing.
Repetitive firing was evoked by injecting 0.1-nA increments of 150-ms
duration until trains of action potentials were fired from holding
potentials of 50 and
90 mV. The application of 30 µM copper
reduced neuronal firing at a holding potential of
50 mV by an average
of 45 ± 28% (n = 29; P < 0.001;
Fig. 6). At a holding potentials of
90 mV, copper reduced the number of evoked action potentials by 46 ± 23% in five neurons (Fig. 7,
P < 0.001), but had no effect on repetitive firing in
four others. In contrast to copper, zinc modulation of repetitive
firing was voltage dependent in some neurons. The application of zinc
reduced neuronal firing in all neurons examined at a holding potential
of
50 mV by an average of 49 ± 41% (n = 9, P < 0.001; Fig. 6). At
90 mV, the majority (69%) of
neurons responded to zinc application with a reduction in the number of
action potentials fired (63 ± 16%; P < 0.001; n = 9). However, in contrast to copper, a significant
proportion of the neurons (31%, n = 4) increased
action potential frequency by 100-500% in response to application of
zinc (Fig. 7).
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DISCUSSION |
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The present study demonstrates that, in addition to their effects
on ligand-gated ion channels (Trombley and Shepherd
1996; Trombley et al. 1998
), physiologically
relevant concentrations of zinc and copper can influence the neuronal
excitability of olfactory bulb neurons by modulating voltage-gated ion
channels. Furthermore, our results indicate that zinc and copper have
distinct effects on voltage-gated K+ channels and
input resistance, effects that may alter a neuron's capacity to
repetitively fire action potentials.
Reportedly, 15% of brain zinc is contained in synaptic vesicles
(Frederickson et al. 1989
). Particularly high
concentrations of zinc are found in the vesicular compartments of the
olfactory bulb, where zinc is primarily localized to the glomerular and granule cell layers (Friedman and Price 1984
; Jo
et al. 2000
; Ono and Cherian 1999
;
Perez-Clausell and Danscher 1985
). Jo et al.
(2000)
have demonstrated that the pattern of zinc visualized with zinc autometallography in the olfactory bulb is correlated with
the pattern of immunoreactivity for ZnT3, a zinc transporter required
for vesicular accumulation of zinc (Cole et al. 1999
). Furthermore, metallothionein III, a zinc-binding protein that can also
bind copper (Chen et al. 1996
; Sewell et al.
1995
; Winge et al. 1994
), is expressed in
olfactory bulb neurons that sequester zinc in synaptic vesicles
(Masters et al. 1994
). In a subclass of vesicular
zinc-containing neurons, glutamate and zinc have been shown to be
co-localized to the same synaptic terminal from which they are
co-released in a Ca2+-dependent manner following
membrane depolarization or neural activity (Assaf and Chung
1984
; Beaulieu et al. 1992
; Crawford and
Connor 1973
; Frederickson and Moncrieff 1994
;
Holm et al. 1988
; Howell et al. 1984
;
Martinez-Guijarro et al. 1991
; Rubio and Juiz
1998
).
The olfactory bulb also contains a high concentration of copper (e.g.,
Donaldson et al. 1973; Ono and Cherian
1999
), although the precise location of copper in the OB has
yet to be determined. However, it has been shown in the hypothalamus
and cortex that, similar to zinc, copper can be released by membrane
depolarization in a Ca2+-dependent manner
(Hartter and Barnea 1988
; Kardos et al.
1989
). It is possible that the pattern of zinc staining in the
OB may also reflect the presence of copper because neither the Timm's nor the sodium selenide method is completely selective for zinc. It is
also unclear whether ZnT3 or metallothionein III in olfactory bulb
neurons could play a role in the use of copper as a neuromodulator, such as in vesicular compartmentalization.
Mounting evidence supports the notion that zinc and copper function as
neuromodulators, modulating excitatory amino acid receptors (Forsythe et al. 1988; Trombley and Shepherd
1996
; Trombley et al. 1998
; Vlachova et
al. 1996
; Weiser and Wienrich 1996
;
Westbrook and Mayer 1987
), inhibitory amino acid
receptors (Laube et al. 1995
; Mayer and Vyklicky
1989
; Sharonova et al. 1998
; Smart et al.
1994
; Trombley and Shepherd 1996
;
Trombley et al. 1998
), and purinergic receptors
(Acuna-Castillo et al. 2000
; Cloues et al. 1993
; Li et al. 1993
, 1996
). Zinc or copper can
alter the function of these receptors (Acuna-Castillo et al.
2000
; Forsythe et al. 1988
; Legendre and
Westbrook 1991
; Sharonova et al. 1998
;
Trombley and Shepherd 1996
; Westbrook and Mayer
1987
) at concentrations an order of magnitude lower than the
estimates of extracellular/synaptic concentrations (100-300 µM)
achieved during neuronal depolarization (Frederickson et al.
1983
; Hartter and Barnea 1988
; Kardos et al. 1989
; Xie and Smart 1991
).
Direct effects
The lack of an effect of applied copper on input resistance
supports the notion that copper ions do not directly activate ion
channels or have a significant effect on leakage channels. Our results
show modest increases in membrane resistance in response to zinc, which
appear to be due to blockade of leakage channels. This finding is
consistent with the effects of zinc on the input resistance of
hippocampal neurons, as reported by Mayer and Vyklicky (1988). In contrast, Zhou and Hablitz (1993)
showed that zinc application induced no effect on membrane resistance
in rat neocortical slices. The differences in these results may be due
to differences in the preparations (culture vs. slice) and experimental
approach. For example, the application techniques used in the present
culture experiments ensured that the neurons were enveloped in 100 µM zinc. The concentration of zinc reaching the cell in the slice preparation could be substantially lower than the 50- to 300-µM concentration that was applied. Differences in the expression and/or
sensitivity of leakage channels between these preparations have not
been determined but could also contribute to the observed differences.
Sodium currents and action potentials
We observed significant inhibition of Na+
currents with the application of either metal. In contrast,
Easaw et al. (1999) observed no significant change in
acutely dissociated neurons from the horizontal limb of the diagonal
band of Broca in response to application of 50 µM zinc. Acute
dissociations tend to lower the density of ion channels. This, combined
with the lower concentration of zinc used (50 vs. 100 µM), may have
contributed to the different results. Furthermore, our observation that
applied copper significantly inhibits Na+
currents is consistent with the previous findings of some investigators (Flonta et al. 1998
; Skulskii and Lapin
1992
).
Although application of either metal inhibited currents mediated by
voltage-gated TTX-sensitive Na+ channels, these
inhibitory effects were not sufficient to inhibit the firing of evoked
single action potentials nor to significantly alter their kinetics.
These results are similar to the effects of zinc on hippocampal neurons
reported by Mayer and Vyklicky (1989). However, they
reported that, at 50 µM, zinc caused a small reduction in action
potential half-width with a substantial effect at 1 mM. In olfactory
bulb neurons, we saw little effect of zinc at 100 µM (the highest
concentration used) on action potential half-widths. These subtle
differences may reflect differences in potassium channels between these
neuronal populations, which mediate repolarization, thus influence
action potential kinetics. The present experiments also suggest that
the effects of zinc and copper on voltage-gated
Na+ channels, at physiologically relevant
concentrations, are probably not a major contributor to the observed
significant reduction/elimination of spontaneous excitatory activity
between olfactory bulb neurons.
Zinc and copper have distinct effects on voltage-gated K+ channels
Results from the present experiments suggest that the effects of
zinc on K+ channels depend not only on the type
of K + channel (transient vs. delayed rectifier)
but are also voltage dependent. At all voltages examined, zinc
inhibited a delayed rectifier-type outward current. Delayed
rectifier-type channels play a significant role in the repolarization
phase of action potentials, thus facilitate recovery of
Na+ channels from voltage-dependent inactivation.
Their inhibition would likely lead to a reduction in the rate of
repetitive firing of action potentials. Our results support this
notion, as both zinc and copper reduced the amplitude of a delayed
rectifier-type K+ current and also reduced the
frequency of action potentials evoked from a membrane potential of 50
mV by long depolarizing current steps.
The effects of zinc and copper on the transient A-type current were
more complex. Copper inhibited the transient current at all membrane
potentials. However, zinc inhibited the current at hyperpolarized
potentials but potentiated the current at membrane potentials at, or
depolarized to, the typical resting potential. The effects of zinc on
transient A-type currents in OB neurons are consistent with the
reported effects of zinc on K+ currents in
hippocampal neurons (Harrison et al. 1993), cerebellar neurons (Bardoni and Belluzzi 1994
), and OB
periglomerular neurons (Puopolo and Belluzzi 1998
).
Although the A-type current is activated by depolarizations from
hyperpolarized potentials, in most neurons, including OB neurons (e.g.,
Puopolo and Belluzzi 1998
), it is largely inactivated at
the resting membrane potential. The A-type outward current slows the
rate of depolarization and, hence, reduces the rate of repetitive
action potential firing. The effects of zinc we observed, in a
significant proportion of neurons, suggests that zinc may modulate
repetitive firing in a voltage-dependent manner. That is, at
hyperpolarized potentials, zinc can inhibit the A-type current and
increase repetitive firing, whereas at depolarized potentials, zinc can
enhance the current and reduce repetitive firing.
In a significant proportion of neurons, zinc reduced repetitive firing
from a holding potential of 90 mV. Copper always reduced the
transient current but also reduced repetitive firing. We believe that
the relative magnitude of the effects of zinc and copper on the A-type
versus the delayed rectifier-type currents, as well as the variations
in the relative amplitudes of A-type currents versus delayed
rectifier-type currents, may explain the variation in the effects of
zinc at
90 mV. For example, in neurons in which the amplitude of the
transient current is low, a reduction in this current at
90 mV by
zinc (which should lead to an increase in repetitive firing) may be
overwhelmed by a reduction in the delayed rectifier current and thus
produce an overall net decrease in repetitive firing.
Copper has a higher affinity than zinc for most substrates, thus affinity differences between zinc and copper could contribute to differences in their effects. However, the differences in the effects of zinc and copper discussed in the preceding text are not likely to be due simply to differences in their binding affinities because either zinc had a greater effect or zinc and copper had similar effects.
Significance to olfactory bulb circuit function
Zinc and copper can modulate synaptic transmission postsynaptically via direct actions on glutamate, GABA, or glycine receptors. The present results also suggest that zinc and copper could have presynaptic effects on transmitter release via inhibition of voltage-gated calcium channels. In addition to these effects on neurotransmission, zinc and copper can influence neuronal excitability by modulating voltage-gated ion channels, particularly K+ channels. The voltage-dependent effects of zinc on the A-type current suggest that the net result (a decrease or an increase in the ability to repetitively fire) is dependent on whether the neuron has just experienced a depolarizing event (e.g., an EPSP) or a hyperpolarizing event (e.g., an IPSP).
Recent reports suggest that effects of zinc (and perhaps copper) on
A-type channels may have important implications for synaptic circuits
that involve the two primary populations of olfactory bulb neurons,
mitral/tufted cells and inhibitory interneurons (periglomerular cells
and granule cells). Puopolo and Belluzzi (1998) have
recently reported that periglomerular cells express prominent A-type
currents that are modulated by zinc. A subset of these neurons
expresses almost exclusively the A-type channel (in contrast to a
combination of A-type and delayed-rectifier-type channels in other
periglomerular cells). Chen and Shepherd (1997)
have
reported that mitral/tufted cells display a characteristic delay in the
onset of action potential firing, which they suggest is likely due to
A-type potassium currents. Because zinc-containing olfactory sensory
neurons make synaptic contact with both mitral/tufted cells and
periglomerular cells, zinc may have significant effects on these
neurons, hence, glomerular circuit activity. Furthermore, it has
recently been demonstrated that A-type currents generate a delay in
action potential firing in granule cells and play an important role in
the synaptic timing of reciprocal inhibition (Schoppa and
Westbrook 1999
). In light of the opposing action of A-type
currents, AMPA receptor-mediated events are attenuated, thus require
the longer-duration NMDA receptor-mediated events for effective
reciprocal inhibition.
The present results, in combination with other recent work, suggest that zinc and copper can influence neuronal excitability and synaptic transmission in the olfactory bulb by multiple mechanisms. Such modulation may contribute to odor information processing through effects on transmitter release, amino acid receptor function, and synaptic timing.
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ACKNOWLEDGMENTS |
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The authors thank Laura J. Blakemore, M.D. for conceptual discussions of this work and editing of the manuscript.
This work has been supported in part by the National Institute on Deafness and Other Communication Disorders (NIDCD) (National Institutes of Health). M. S. Horning was supported in part by NIDCD Chemosensory Training Grant T32 DC-00044.
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FOOTNOTES |
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Address for reprint requests: P. Q. Trombley (E-mail: trombley{at}neuro.fsu.edu).
Received 22 January 2001; accepted in final form 13 June 2001.
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