Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Aizenman, Carlos D. and David J. Linden. Regulation of the Rebound Depolarization and Spontaneous Firing Patterns of Deep Nuclear Neurons in Slices of Rat Cerebellum. J. Neurophysiol. 82: 1697-1709, 1999. Current-clamp recordings were made from the deep cerebellar nuclei (DCN) of 12- to 15-day-old rats to understand the factors that mediate intrinsic spontaneous firing patterns. All of the cells recorded were spontaneously active with spiking patterns ranging continuously from regular spiking to spontaneous bursting with the former predominating. A robust rebound depolarization (RD) leading to a Na+ spike burst was elicited after the offset of hyperpolarizing current injection. The voltage and time dependence of the RD was consistent with mediation by low-threshold voltage-gated Ca2+ channels. In addition, induction of a RD also may be affected by activation of a hyperpolarization-activated cation current, Ih. A RD could be evoked efficiently after brief high-frequency bursts of inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons. IPSP-driven RDs were typically much larger and longer than those elicited by direct hyperpolarizing pulses of approximately matched amplitude and duration. Intracellular perfusion of the Ca2+ buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) dramatically enhanced the RD and its associated spiking, sometimes leading to a plateau potential that lasted several hundred milliseconds. The effects of BAPTA could be mimicked partly by application of apamin, a blocker of small conductance Ca2+-gated K+ channels, but not by paxilline, which blocks large conductance Ca2+-gated K+ channels. Application of both BAPTA and apamin, but not paxilline, caused cells that were regularly spiking to burst spontaneously. Taken together, our data suggest that there is a strong relationship between the ability of DCN cells to elicit a RD and their tendency burst spontaneously. The RD can be triggered by the opening of T-type Ca2+ channels with an additional contribution of hyperpolarization-activated current Ih. RD duration is regulated by small-conductance Ca2+-gated K+ channels. The RD also is modulated tonically by inhibitory inputs. All of these factors are in turn subject to alteration by extrinsic modulatory neurotransmitters and are, at least in part, responsible for determining the firing modes of DCN neurons.
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INTRODUCTION |
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The deep cerebellar nuclei (DCN) comprise the core
of the cerebellar circuitry, integrating a variety of converging
excitatory and inhibitory inputs representing several streams of
sensory-motor information. DCN neurons receive inhibitory projections
from Purkinje cells, which are the sole output of the cerebellar
cortex. In addition, the DCN also receive excitatory inputs from mossy
fibers and climbing fibers. These two afferent systems originate,
respectively, in various precerebellar nuclei and in the inferior
olive. As a consequence, the output of all the neural computations that are performed in the cerebellum is reflected in the firing patterns of
DCN neurons, which then are translated into different motor functions
through projections to a variety of premotor centers including the
thalamus, red nucleus, and superior colliculus. Moreover, the DCN now
are thought to play an increasingly important role in some forms of
cerebellar-mediated motor learning (see du Lac et al.
1995; Kim and Thompson 1997
for review). Several converging lines of evidence, based on lesion, inactivation, and extracellular recordings, have pointed to the DCN as a possible locus
for information storage during associative eyeblink conditioning, and
to their analogous nuclei, the vestibular nuclei, in adaptation of the
vestibuloocular reflex (Mauk 1997
; Raymond et al.
1996
). It is therefore important to bring to light some of the
factors that control and modulate the intrinsic spiking behavior of the DCN cells, so that we may begin to understand how its output is regulated, as well as for understanding the cellular substrates that
may underlie information storage at this site.
The intrinsic electrophysiological properties of DCN neurons have been
studied with intracellular recordings in a variety of different
preparations, including isolated brain stem (Llinás and
Mühlethaler 1988), cerebellar slices (Aizenman et
al. 1998
; Gardette et al. 1985b
; Jahnsen
1986a
,b
), and organotypic cultures (Mouginot and
Gähwiler 1995
; Muri and Knöpfel
1994
; see Sastry et al. 1997
for review). A
general characteristic of DCN neurons, which has been observed in each
preparation, is that they exhibit a pronounced rebound depolarization
(RD), often accompanied by a burst of Na+ spikes,
immediately after a hyperpolarizing current pulse. This has been
attributed to the opening of low-threshold, voltage-gated Ca2+ channels, which become deinactivated during
the hyperpolarizing pulse and open on return to more depolarized
membrane potentials at the offset of the pulse. The RD and its
associated spike burst have been shown to induce large intracellular
Ca2+ transients in DCN neurons; something that is
revealed by the use of Ca2+-sensitive dyes
(Aizenman et al. 1998
; Muri and Knöpfel
1994
). Hyperpolarizing inhibitory postsynaptic potentials
(IPSPs), originating from Purkinje cell inputs, can also transiently
hyperpolarize the DCN cells and thereby elicit a RD (Aizenman et
al. 1998
; Gardette et al. 1985a
;
Llinás and Mühlethaler 1988
). Therefore the
RD provides an efficient mechanism by which inhibitory Purkinje cell inputs can drive postsynaptic excitation and Ca2+
entry. This mechanism has a central role in some cerebellar learning models (Mauk and Donegan 1997
) and may be the basis for
use-dependent plasticity at the Purkinje cell to DCN synapse
(Aizenman et al. 1998
).
Little is known about the exact parameters necessary for induction of a RD or about the interplay between the various currents which underlie it. We performed microelectrode recordings in rat cerebellar slices to understand the factors that underlie the induction and modulation of the RD and the role the RD plays in determining the spontaneous spiking behavior of the DCN cells.
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METHODS |
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Rat pups (12-15 days old) were decapitated, and the brains
promptly removed and immersed in ice-cold standard artificial
cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 5 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 20 D-glucose, continuously bubbled with 95% O2-5%
CO2. Coronal slices (400-µm thick) from the
cerebellum were cut using a Vibratome and were incubated in ice-cold
ACSF. Although rats of this age still are considered juveniles, the intrinsic properties of these neurons are remarkably similar to that
previously reported in mature guinea pigs (Jahnsen
1986a; Llinás and Mühlethaler
1988
) and in slice cultures (Muri and Knöpfel
1994
), suggesting that at 2 wk the DCN neurons already have a
mature electrophysiological phenotype. In addition, there is evidence
in the literature that after the first postnatal week, DCN cells
already express both intrinsic and synaptic conductances that are
present in mature neurons (Gardette et al. 1985a
,b
). Slices then were incubated for
1 h at room temperature in a homemade interface chamber; after this recordings were performed in a Haas-style interface chamber at 33°C that was perfused with ACSF containing 2 mM
kynurenate to block ionotropic glutamate receptors. Borosilicate glass
microelectrodes (80-150 M
) were filled with 3 M KAc. Where indicated, 100 µM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA) or 100 µM
N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) were included in the electrode solution and
were allowed to passively diffuse into the cell until a consistent effect was seen (typically for 10 min.). To evoke IPSPs, concentric bipolar stimulating electrodes were placed in the white matter immediately adjacent to the nuclei. Recordings were made from either
the medial or lateral group of the DCN, and no obvious differences were
observed between the two nuclei. Membrane voltage was measured using an
Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) and analyzed
with a Macintosh computer using AxoGraph (Axon Instruments) and Igor
Pro software (WaveMetrics, Lake Oswego, WI). Bicuculline methiodide
(BMI), picrotoxin, and gabazine (SR-95531) were obtained from Research
Biochemicals International (Natick, MA). Paxilline and apamin were
obtained from Alomone Labs (Jerusalem, Israel). All other chemicals
were from Sigma.
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RESULTS |
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Spontaneous firing properties
Sharp electrode, current-clamp recordings from 68 DCN neurons in
coronal slices of rat cerebellum were included in the total data pool.
DCN cells had resting membrane potentials of 58 ± 1 (SE) mV,
and input resistances of 51 ± 3 M
. All cells were spontaneously active at rest. All experiments were performed with 2 mM
kynurenate in the bath to eliminate excitatory drive mediated by
ionotropic glutamate receptors. This afferent input has been shown in
different preparations of this tissue (isolated cerebellum/brainstem and cultured slice) to drive spiking in DCN cells (Llinás
and Mühlethaler 1988
; Mouginot and Gahwiler
1995
). Thus most of the spontaneous activity can be attributed
to the intrinsic properties of the DCN neurons. An example of a typical
cell is shown in Fig. 1A. At
rest, this neuron fired action potentials regularly at a fairly high
frequency (~35 spikes/s; top). On the application of tonic
hyperpolarizing current through the recording electrode, the firing
rate slowed in a manner proportional to the amount of current injected
(bottom traces), until firing finally subsided at
approximately
70 mV. This intrinsic, regular spiking behavior was
typical of most of the cells recorded. However, in a small subpopulation of neurons [9 of 136 cells, from all DCN cells ever recorded by our group, including those in this study as well as cells
from another study (Aizenman et al. 1998
)] a different spontaneous firing pattern was observed. An example of this pattern is shown in
Fig. 1B. This neuron also appears to fire regularly at rest (top). Yet when tonic hyperpolarizing current is injected,
the firing frequency not only slows but also shifts mode to reveal brief, spontaneously occurring bursts of spikes (2nd and
3rd traces). The bursts become clearly resolved with larger
amounts of hyperpolarizing current (4th trace). Although
these spontaneously bursting cells are a minority, the behavior of many
cells falls somewhere in between the two types shown here, forming a
continuum between regular spiking cells and spontaneously bursting
cells. There were no obvious differences in the resting membrane
potential and input resistance between the bursting (
60 ± 3 mV,
61 ± 20 M
, n = 9) and the regular spiking
cells.
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Intrinsic responses to current pulses
After application of a hyperpolarizing pulse, a prominent RD was
evoked that typically was accompanied by an associated burst of action
potentials. (Figs. 2A1,
3A1, and 4A1; see also Aizenman et al.
1998; Jahnsen 1986a
,b
; Llinás and
Mühlethaler 1988
; Muri and Knöpfel
1994
). In this manuscript, we use "rebound depolarization" as an operative term to describe the overall depolarization that follows a transient period of hyperpolarization and that may consist of
several underlying component currents (see following text). The RD and
associated spike burst are believed to be triggered by the opening of
low-threshold Ca2+ channels (T-type) and are
accompanied by a large intracellular Ca2+
transient (Aizenman et al. 1998
; Muri and
Knöpfel 1994
). To further characterize the stimulation
parameters that control the RD, we performed several experimental
manipulations shown in Figs. 2-4. It should be noted that there was
significant variability in the size, time course, and activation
threshold of the RD among different cells. Thus we have attempted to
portray this variability in our choice of sample voltage traces for
these figures while at the same time extracting common elements of the
RD that are present in all of the recorded DCN cells.
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The RD is dependent on the membrane potential of the cell, being more
strongly activated at more depolarized membrane potentials and usually
peaking between 60 and
70 mV, consistent with the activation range
of T-type Ca2+ channels (Carbone and Lux
1984
, 1987
). In Fig. 2A1, responses to a
0.5-nA
hyperpolarizing pulse are shown while the cell is being held at
different membrane potentials by tonic hyperpolarization. The peak
spiking frequency, as well as the number of RD-evoked spikes are used
as an index of RD strength (Fig. 2, A2 and C). Note that the RD is larger at more depolarized membrane potentials. The
average membrane potential at which the RD was most strongly activated,
as assessed by the number of evoked spikes and peak firing frequency
observed across a population of cells, was
61 ± 4 mV and
65 ± 4 mV (n = 4), respectively. The average
number of evoked spikes for a maximally elicited RD was 28 ± 16 spikes, and the average peak frequency was 77 ± 25 Hz
(n = 4).
The RD itself is not abolished by blockade of voltage-gated
Na+ channels by intracellular perfusion of QX-314
(Fig. 2B1). The RD observed in the presence of QX-314 is
nearly indistiguishable from that which is observed in the presence of
TTX (see Aizenman et al. 1998). In Fig. 2, B2
and D, the RD area and peak amplitude are used to assess RD
strength in the absence of spiking. Again, the RD is more strongly
evoked when the cell is held at more positive membrane potentials. The
average membrane potential at which the RD was most strongly activated
in QX-314-filled cells, as assessed by RD area and peak amplitude, was
58 ± 2 mV and
59 ± 3 mV (n = 8),
respectively. This value is slightly more positive than that measured
in cells without QX-314 and can perhaps be attributed to a mild
blockade of T-type channels by QX-314 (Talbot and Sayer 1996
). The average maximum RD area was 3 ± 1 mV/s, and
the peak amplitude was 15 ± 3 mV (n = 8).
A well-known property of T-type Ca2+ channels is
that they rapidly inactivate in a time- and voltage-dependent manner,
and to remove the inactivation, the cell first must be hyperpolarized. One prediction that derives from this is the observation that increasing hyperpolarization to remove more inactivation will elicit a
stronger RD. This is illustrated in Fig.
3A, where the cell was
tonically hyperpolarized below spike threshold (65 mV) to suppress
spontaneous spiking while increasingly larger hyperpolarizing steps are
applied. As expected, the RD is also increasingly stronger, as assessed
by the number and frequency of the associated spike burst (Fig. 3,
A, 1 and 2, and C). The same is
observed for a QX-314-filled cell held at
55 mV (Fig. 3B,
1 and 2, and D). Another way in which
deinactivation of T-type channels can be increased is by increasing the
duration of the hyperpolarizing pulse rather than the amplitude. In
Fig. 4, we can see that increasingly long hyperpolarizing pulses, applied while the cell was tonically
hyperpolarized below spike threshold, elicited a proportionally larger
RD in cells recorded both with (Fig. 4, B and D)
and without (Fig. 4, A and C) QX-314.
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In response to a hyperpolarizing current pulse, the membrane voltage
deflection had both a transient and a sustained component. The
sustained component appears as a "sag" in the voltage deflection (Fig. 5A) and increases in
amplitude as the cell is increasingly hyperpolarized (Fig. 5,
A and B). This hyperpolarization-activated sag
can be suppressed in part by extracellular application of 2 mM CsCl
(n = 4; Fig. 5C). At this concentration,
CsCl reduced the absolute amplitude of the sag by 86 ± 3%
(n = 3) and by 66 ± 6% if expressed as percent
of the total hyperpolarization-induced change in
Vm. Notice also that QX-314 perfusion
seems to almost completely abolish the sag (Figs. 2B1,
3B1, and 4B1), consistent with a previous report
where this drug was seen to block hyperpolarization-activated currents
(Perkins and Wong 1995). This observation, together with the strong voltage dependence of the sag, suggests that it is at least
partly mediated by Ih
(McCormick and Pape 1990
). The incomplete blockade by
CsCl, however, suggests that additional hyperpolarization-activated
currents also may be involved (see Williams et al.
1997
). When hyperpolarizing pulses were given when the cell was
held subthreshold to the activation range of T-type
Ca2+ channels, there still was a small RD,
presumably mediated by Ih (Fig. 5D, top), suggesting that it
may play a role in boosting the RD as has been observed previously in
thalamic neurons (see Pape 1996
for review).
Unfortunately, this is difficult to assess directly because application
of external CsCl has additional nonspecific effects (presumably
mediated via blockade of various K+
conductances) that potentiate the RD (Fig. 5D, bottom).
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In summary, these manipulations are consistent with the notion that the
RD is mediated, at least in large part, by opening of T-type
Ca2+ channels. First, the membrane potentials at
which the RD is activated maximally are consistent with the activation
range of these channels. Second, increasing the size and duration of
the hyperpolarizing pulse elicits a stronger RD, consistent with the
reported kinetics of T-channel deinactivation (Carbone and Lux
1984, 1987
). Third, the RD is still present when voltage-gated
Na+ channels are blocked. And fourth, the RD is a
transient event, similar to low-threshold spikes observed in other cell
types (Bal and McCormick 1993
; Llinás and
Yarom 1981
). Unfortunately, because of a lack of a specific and
potent antagonist of T-type Ca2+ channels, it has
not been possible to test the role of these channels directly.
Application of several putative specific T-channel antagonists
(mibefradil, 1 µM; nickel, 100 µM; ethosuximide, 1 mM) failed to
alter significantly the RD (data not shown). Activation of
Ih also potentially could contribute
to the RD induction by boosting the membrane potential when it is
subthreshold to T-channel activation. This is supported by the fact
there is still a small RD at hyperpolarized membrane potentials as well
as by the observation that when the hyperpolarization-induced sag is
blocked by QX-314 the induction threshold for the RD is more
depolarized. One possible factor that may complicate the interpretation
of the RD voltage dependence in these experiments is the observation
that QX-314 can have nonspecific effects on several voltage-gated
conductances, including T-type Ca2+ currents
(Talbot and Sayer 1996
). Nevertheless, the
voltage-dependent properties of the RD in QX-314-filled cells are
consistent with those observed in control neurons (Fig. 2, A
and B), in experiments performed with TTX (Aizenman
et al. 1998
) and with those reported by others
(Llinás and Mühlethaler 1988
). This suggests
that application of QX-314 should not significantly alter the main interpretation of these results.
Synaptically driven RDs
Because all experiments were performed in the presence of
kynurenate, stimulating the white matter adjacent to the DCN elicited a
pharmacologically isolated IPSP. This IPSP is mediated by
GABAA receptors (Aizenman et al.
1998), has a reversal potential of approximately
75 mV, and
is reported by others to be unaffected by GABAB
receptor antagonists (see Sastry et al. 1997
for
review). Although a single IPSP was usually not sufficient to evoke a
RD, a high-frequency train of IPSPs was quite effective (Fig.
6, A and C). Like a
hyperpolarizing pulse-driven RD, the IPSP-driven RD was larger at
depolarized membrane potentials and was attenuated by tonic
hyperpolarization (Fig. 6, B and D). The RD is
therefore a mechanism by which hyperpolarizing IPSPs can drive
subsequent postsynaptic excitation and their associated intracellular
Ca2+ transients, processes that have been shown
to be important for inducing use-dependent changes of inhibitory
synaptic strength in the DCN (Aizenman et al. 1998
).
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Interestingly, IPSP trains were much more effective at strongly
activating a RD than were hyperpolarizing current pulses of similar
amplitude and duration. An example is shown in Fig.
7A, where a train of 10 IPSPs
evokes a robust RD and an associated spike burst, whereas a current
pulse of similar amplitude and duration completely fails to elicit a RD
in the same neuron. This was a consistent observation and data are
summarized for three different cells in Fig. 7B. One
possible explanation is that because the synaptic input from inhibitory
fibers is widely distributed throughout a DCN cell (Chan-Palay
1977), the hyperpolarization produced thereby is more effective
in deinactivating T channels as compared with a hyperpolarization
delivered from a point source, such as an intracellular electrode.
Another possibility is that the synaptically induced RD also may be
facilitated by the activation of nonionotropic glutamate receptors or
by the tetanically induced release of additional modulatory
neurotransmitters. In fact, a slow depolarizing synaptic component is
observed even when the IPSP burst is nearly reversed, as in Fig.
6B, bottom. The identity of this component will prove to be
an important element in further studies that consider the nature of
excitatory synaptic inputs to the DCN.
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Modulation of the RD
Although the induction of the RD is consistent with the activation
of T channels, the majority of the intracellular
Ca2+ transient has been shown, with the aid of
Ca2+-sensitive dyes, to be dependent on
postsynaptic spiking (Aizenman et al. 1998; Muri
and Knöpfel 1994
). It is possible then that the time
course of the RD and associated spike burst may be mediated in part by
Ca2+-dependent processes. Intracellular perfusion
with the Ca2+ chelator BAPTA markedly increased
the excitability of the cells within 10-20 min of impalement
(n = 9). An example is shown in Fig.
8A. After 25 min of
intracellular perfusion of BAPTA, a depolarizing current pulse caused
the cell to fire a few spikes at a very high frequency, followed by a
plateau response (bottom left). A hyperpolarizing pulse
induced a RD that also elicited a plateau response lasting several
hundred milliseconds (bottom right). This suggests that Ca2+-dependent processes, particularly activation
of Ca2+-gated K+ channels,
may regulate the duration and amplitude of the RD. To test this
hypothesis, we bath applied apamin, a specific blocker of small
conductance (SK) Ca2+-gated
K+ channels (Blatz and Magleby
1986
) (n = 5). Figure 8B shows the effect of 1 µM apamin on the intrinsic properties of a DCN cell. Apamin caused the cell to fire action potentials at extremely high
frequencies (
300 spikes/s, ~4 times the control rate) during a
depolarizing step and during a hyperpolarizing-pulse evoked RD. In four
of five cells, apamin also caused the cell to exhibit plateau
potentials lasting for hundreds of milliseconds, similar to those seen
with BAPTA perfusion (data not shown). Bath application of paxilline
(100 nM, n = 3), a blocker of the large conductance (BK) Ca2+-gated K+ channels
(Sanchez and McManus 1996
) did not cause the cell to fire plateau potentials nor did it substantially increase the firing
rate elicited by either a depolarizing step or a RD (Fig. 8C).
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Another interesting effect of BAPTA and apamin is that they both induce spontaneous, regularly occurring spike bursts while the neuron is at rest. With tonically injected hyperpolarizing current, the interval between the bursts increases, until finally the cell stops firing (Fig. 9, A and B). This was observed in all the BAPTA-perfused cells (n = 8) as well as all of the cells in which apamin was applied (n = 5). Paxilline did not cause the cells to burst spontaneously (Fig. 9C, n = 3). Interestingly, all of the cells in these experiments were regularly spiking before BAPTA or apamin were applied, suggesting that any cell has the capability of bursting if it is subjected to the appropriate type of modulation. Moreover it shows that modulation of the RD affects the spontaneous firing properties of the cell and determines its propensity to fire spike bursts. It is interesting to consider the source of the interburst hyperpolarization when the Ca2+-gated K+ channels are blocked. One possibility is that both BAPTA and apamin fail to completely block these channels, resulting eventually in sufficient K+ flux. Alternatively, the cell could be hyperpolarized by slowly activating, voltage-gated K+ channels.
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Role of tonic inhibitory drive
The neurons of the DCN receive large amounts of inhibitory drive,
as they are the sole target of Purkinje neurons. In addition, they
receive inhibitory drive from local interneurons. It has been shown
that Purkinje cell inputs can exert a powerful control over DCN cell
activity and affect spontaneous firing properties (Mouginot and
Gähwiler 1995). We examined the role of inhibitory drive
in regulating the RD and consequently the propensity of the DCN cells
to fire spike bursts. Bath application of the
GABAA antagonist bicuculline methiodide (BMI, 20 µM, n = 7) caused a substantial increase in the
excitability of the cell (Fig.
10A). On average, BMI caused
an approximately threefold increase in the firing rate produced by a
depolarizing pulse, relative to control levels. Peak frequencies of
370 spikes/s were achieved in one particularly dramatic case. It also
caused a substantial enhancement of the RD, increasing the firing rate,
on average, by approximately sevenfold, achieving frequencies >200
spikes/s. In addition, BMI caused five of seven cells to fire
high-frequency plateaus much like those seen with apamin (compare Figs.
10A and 8B). Spontaneous bursts were seen in four
of seven BMI-treated cells (Fig. 10B). One might suspect
initially that tonic inhibition is playing a strong modulatory role on
the intrinsic firing modes of DCN cells. This spontaneous bursting
cannot be attributed to recurrent excitation because all experiments
were performed with an ionotropic glutamate receptor antagonist in the
bath. However, recent reports have indicated that quaternary salts of
bicuculline, such as BMI, in addition to blocking
GABAA receptors, also block SK channels
(Debarbieux et al. 1998
; Johnson and Seutin
1997
; Seutin et al. 1998
). In addition, it has
been reported that other compounds containing at least one quaternary
ammonium group are potent inhibitors of SK channels (Castle et
al. 1993
; Dunn et al. 1996
). This possibly could
explain the apamin-like effect caused by BMI application.. To address
this potential complication, we tested the effects of two other
GABAA antagonists, picrotoxin (200 µM,
n = 3) and gabazine (100 µM, n = 4).
Although both antagonists caused an increase in firing rate in response
to a depolarizing pulse (~1.5-fold) and a more substantial
enhancement of RD-driven spiking (approximately threefold), neither
drug induced plateaus nor spontaneous bursts (Fig. 10, C and
D). This suggests that although there is some degree of
tonic synaptic inhibition in the slice, blocking the inhibition does
not change the firing mode of DCN cells. Therefore the effect of BMI on
DCN cell firing properties can likely be attributed to blockade of SK
channels. In addition, this observation strengthens the view that DCN
cells can spontaneously burst using purely intrinsic mechanisms, as
these cells are isolated both from excitatory (kynurenate) and
inhibitory inputs (BMI).
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DISCUSSION |
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In summary, our data show that the spontaneous firing pattern of DCN neurons can range from regular spiking, in which the rate depends on the membrane potential of the cell, to spontaneously bursting. Most cells that we recorded from had characteristics that more closely resembled regular spiking. DCN cells also exhibited a prominent RD, typically associated with a Na+ spike burst, which could be elicited either by the offset of a hyperpolarizing pulse or by a train of IPSPs. IPSPs were markedly more effective in eliciting a RD than was a hyperpolarizing pulse of similar magnitude. The properties of RD induction, such as the voltage and time dependence, transient duration, and resistance to Na channel blockade, are consistent with mediation by T-type Ca2+ channels. In addition, there is some evidence that hyperpolarization-activated current Ih may play a role in boosting subthreshold RDs. There was a strong modulation of the amplitude and duration of the RD by SK channels. Blockade of these by apamin, or by intracellular Ca2+ chelation with BAPTA, resulted in a robust enhancement of the RD and caused regular spiking cells to display spontaneous bursts. Although an enhancement in firing frequency was observed after blockade of GABAA receptors, this did not cause the cells to burst spontaneously.
Are there multiple cell types in the present DCN recordings?
It is interesting to consider the question of whether the
variability we see in the RD and in the propensity to burst reflects a
difference in cell type or whether we are recording from a single cell
type but under different modulatory influences. Anatomic data suggest
that there are three populations of neurons in the DCN, the small
neurons that are GABAergic and send projections to the inferior olive,
small GABAergic local interneurons as well as somewhat larger
glutamatergic neurons, which project to premotor areas (see
Chan-Palay 1977; Voogd et al.
1996
for review). Because our recordings were performed
"blind," it is possible that we have recorded from multiple cell
types. The predominance of regular spiking cells in our sample may
reflect a bias toward the large cells, which we are presumably more
likely to successfully impale. At present, there is no evidence that
the different cell types of the DCN have different electrophysiological
characteristics. Another possibility is suggested by the present
observation that bath application of apamin or BMI, or intracellular
perfusion with BAPTA, could shift the firing mode of a regular spiking
cell into a spontaneous burst mode. This suggests that the variability we observed may depend on the different modulatory states of the cells
encountered rather than morphologically distinct populations of cells.
We currently are filling, staining, and reconstructing microelectrode-recorded DCN cells to address this issue directly.
Modulation of the RD
The present data suggest that there is a relationship between the
strength of an evoked RD and the tendency of the cells to burst
spontaneously. This suggests that factors that modulate the RD
therefore can play an important role in defining the spiking pattern of
the cell. We have identified several of these factors. First, we have
shown that trains of IPSPs can be highly effective in eliciting a RD.
Indeed, the anatomic distribution of Purkinje cell inputs, which have
been shown to densely innervate the cell soma and principal dendrites
of DCN neurons (Chan-Palay 1977), is highly favorable to
causing a cell-wide change in membrane potential in response to IPSPs.
Thus inhibitory drive can modulate the induction of a RD in several
ways. One way is illustrated by the fact that IPSP trains are
significantly more effective in driving a RD as compared with a
hyperpolarizing current pulse injected at a single point source through
the recording electrode (Fig. 7). Changes in the amplitude and duration
of inhibitory inputs will evoke RDs of different sizes by removing
different amounts of T-channel inactivation. In addition, tonic
inhibitory drive can regulate the membrane potential of the DCN cell,
making it more or less likely to exhibit a RD depending on its level of
depolarization. This is supported by the observation that
pharmacological blockade of GABAA receptors leads
to an increase in cell excitability (Fig. 10) (see also Mouginot
and Gähwiler 1995
). These observations, taken together
with evidence that inhibitory Purkinje cell synapses can undergo
activity-dependent long-term depression and long-term potentiation
(Aizenman et al. 1998
), suggest that plasticity at this
synapse may play a homeostatic role in controlling levels of cell
excitability, helping to normalize the output levels of the cell.
Previous work has shown that large amounts of postsynaptic spiking will
cause a sustained potentiation of Purkinje cell-DCN IPSPs
(Aizenman et al. 1998
), perhaps making the cell more
tonically inhibited and less likely to fire action potentials.
Conversely, smaller amounts of postsynaptic activity will induce a
sustained depression of IPSPs, presumably making the cell less
inhibited and more likely to spike. Thus the regulation of synaptic
efficacy by postsynaptic spiking may serve, in turn, to regulate the
spiking itself.
Another factor that can modulate the RD is activation of
Ca2+-gated K+ channels.
Intracellular perfusion of BAPTA caused previously regular-spiking
cells to exhibit spontaneous bursts. This effect was mimicked by
blockade of SK, apamin-sensitive K+ channels.
These channels underlie Iahp, which
follows an action potential and is critical in determining the firing
rate of a neuron (Sah 1996). Blockade of the
large-conductance (BK) channels does not have the same effect, probably
because they do not significantly contribute to the
afterhyperpolarizing potential that follows a Na+
spike (Sah 1996
). Thus our data suggest that the DCN
cells are under tonic modulation by a Ca2+-gated
K+ current, which is being intermittently but
frequently activated by an influx of Ca2+ ions,
either via RD-driven T-type Ca2+ channels or by
high-threshold voltage-gated Ca2+ channels activated by
spontaneous or RD-evoked action potentials. Ca2+-gated K+ channels have
been shown to be modulated by several different neurotransmitter types
via various second-messenger cascades (Müller et al.
1992
; Nicoll 1988
; Pedarzani and Storm
1993
). Thus they may be possible targets for modulation by
serotonergic, noradrenergic, dopaminergic, and peptidergic fibers
innervating the DCN (Voogd 1996
). In addition,
calmodulin, which is required for the Ca2+ gating
of SK channels (Xia et al. 1992
), may itself be subject to modulation by calmodulin-binding proteins such as MARCKS, GAP-43, and Ca2+/calmodulin-dependent protein kinases.
The observation that Ca2+-gated
K+ currents modulate spontaneous spiking also has
been observed in other cell types (Llinás and Suguimori
1980a
,b
; Llinás and Yarom 1981
). It is
important to note that analysis of the contribution of specific
Ca2+ channel subtypes to the RD, using the
present recording techniques, is complicated by the dual contribution
of Ca2+ to both the initiation and in the
termination of the RD. Pharmacological agents that partially reduce
Ca2+ flux into the cell will result on the one
hand in a reduced depolarizing current but also in an enhanced RD due
to reduced activation of Ca2+-gated
K+ channels.
A third factor that may affect the RD is modulation of the T-type
Ca2+ channels and the channels mediating
Ih that underlie its induction. T
channels have been shown to be modulated by acetylcholine, serotonin, dopamine, norepinephrine, and some peptides, as well as by protein kinase C (see Huguenard 1996; Linden and
Routtenberg 1989
). Ih has been
shown to be strongly regulated by
[Ca2+]i
(Lüthi and McCormick 1998
) as well as by various
neurotransmitters (Pape 1996
). Some evidence supporting
the presence of Ih in our recordings
is the fact that a sustained sag in the membrane voltage deflection was
observed during the administration of hyperpolarizing current pulses.
This sag was partially suppressed by Cs+ and by
QX-314, both of which are known to block
Ih. Interestingly, in the cells filled
with QX-314, the membrane voltage at which the RD was maximally evoked
was slightly shifted (from
65 ± 4 mV to
59 ± 4 mV),
suggesting that Ih may be boosting the
induction of the RD. Finally, the BAPTA- and apamin-induced plateaus
may be carried in part by noninactivating Na+
currents previously described in DCN cells (Gardette et al.
1985b
; Jahnsen 1986b
; Llinás and
Mühlethaler 1988
). This conductance also is subject
to modulation by various second messengers and G proteins (Crill
1996
).
Our results support the recent observations that BMI causes a
significant blockade of apamin-sensitive,
Ca2+-gated K+ channels
(Debarbieux et al. 1998; Johnson and Seutin
1997
; Seutin et al. 1998
). Because this highly
water soluble form of bicuculline often is used to block
GABAA receptors, it is very easy to confuse the
effects of reduced inhibition with the effects of blocking apamin-sensitive K+ channels, making any interpretation ambiguous because both will result in increased neuronal excitability. Thus we
suggest that BMI is not a useful drug for studying
GABAA receptor function and that other more
specific compounds should be considered (e.g., picrotoxin, gabazine,
bicuculline free-base).
Function of the RD
The RD endows the DCN neurons with several interesting
computational properties. For example, the RD might allow the DCN cells to distinguish between different types of inputs received by the cerebellum. Although the continuous barrage of parallel fiber inputs to
Purkinje cells, which is translated as simple spikes (see Ito
1984), may present itself to the DCN as a tonic inhibitory drive; a climbing fiber input arriving at the Purkinje cell will cause
it to fire a complex spike and be presented to the DCN as a brief,
high-frequency burst of IPSPs followed by a pause (see Ito
1984
), which then will elicit a RD. Thus because of its
intrinsic membrane properties, a DCN cell may be able to distinguish
between climbing fiber and mossy fiber input to the Purkinje cells and modify its output accordingly.
RDs are by no means unique to DCN neurons. In fact they are known to be
critical in controlling the spiking patterns of several types of
neurons exhibiting rhythmic behavior. Thalamic relay neurons, for
example, fire a RD in response to IPSPs originating in the
perigeniculate nucleus, which then causes them to feed back inputs to
this nucleus and thus generate a pacemaker type circuit (Bal et
al. 1995). Indeed, inferior olivary neurons as well as Purkinje
cells, both of which are interconnected with the DCN, have been shown
to exhibit RDs, and fire rhythmic bursts of action potentials
(Llinás and Sugimori 1980a
,b
; Llinás
and Yarom 1981
). This circuit has been proposed to underlie
different types of coordinated motor behavior and tremor
(Llinás 1985a
,b
). In addition, there is a striking
similarity between the bursting capabilities of DCN cells and B-type
cells of the medial vestibular nuclei (MVN), which are the analogous
cell type in the vestibulo-cerebellum. Both express low-threshold
spikes (or RDs), in both cases a small population was seen to burst
spontaneously, and both have a high propensity to burst in the presence
of apamin (de Waele et al. 1993
; Serafin et al.
1991a
,b
). This rhythmic behavior in MVN cells has been proposed
to be important in controlling certain types of eye movements. It
remains to be determined whether intrinsic bursting plays an analogous
role in a DCN-dependent motor behavior.
A third role for the RD in the DCN may be important for
activity-dependent synaptic plasticity at this site. A computational model for eye-blink conditioning has been proposed where the RD acts as
a type of Hebbian-style coincidence detector, which can compare the
timing between excitatory and inhibitory inputs and induce changes in
the excitatory drive accordingly (Mauk and Donegan 1997). Thus some of the learned behavior can be stored in the inputs to the DCN instead of exclusively occurring in the cerebellar cortex. In addition, there is experimental evidence that shows that the
degree of RD-driven spiking determines the polarity of synaptic
plasticity at the Purkinje cell to DCN inhibitory synapse (Aizenman et al. 1998
). Thus it is possible that the
spiking patterns of the DCN may act as a framework within which
excitatory and inhibitory inputs arrive and are modified accordingly.
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ACKNOWLEDGMENTS |
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Thanks to P. Manis, A. Kirkwood, and C. Hansel for comments on an earlier draft of the manuscript and to E. Ertel (Hoffmann-La Roche, Basel) for mibefradil.
This work was supported by the Develbiss Fund and a predoctoral fellowship from the Howard Hughes Medical Institute to C. D. Aizenman.
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FOOTNOTES |
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Address for reprint requests: D. J. Linden, Dept. of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205.
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 25 March 1999; accepted in final form 26 May 1999.
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REFERENCES |
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