The Rotary Hearing Centre, Department of Surgery (Otolaryngology), University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada
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ABSTRACT |
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Adam, T. J., P. G. Finlayson, and D.W.F. Schwarz. Membrane Properties of Principal Neurons of the Lateral Superior Olive. J. Neurophysiol. 86: 922-934, 2001. In the lateral superior olive (LSO) the firing rate of principal neurons is a linear function of inter-aural sound intensity difference (IID). The linearity and regularity of the "chopper response" of these neurons have been interpreted as a result of an integration of excitatory ipsilateral and inhibitory contralateral inputs by passive soma-dendritic cable properties. To account for temporal properties of this output, we searched for active time- and voltage-dependent nonlinearities in whole cell recordings from a slice preparation of the rat LSO. We found nonlinear current-voltage relations that varied with the membrane holding potential. Repetitive regular firing, supported by voltage oscillations, was evoked by current pulses injected from holding potentials near rest, but the response was reduced to an onset spike of fixed short latency when the pulse was injected from de- or hyperpolarized holding potentials. The onset spike was triggered by a depolarizing transient potential that was supported by T-type Ca2+-, subthreshold Na+-, and hyperpolarization-activated (IH) conductances sensitive, respectively, to blockade with Ni2+, tetrodotoxin (TTX), and Cs+. In the hyperpolarized voltage range, the IH, was largely masked by an inwardly rectifying K+ conductance (IKIR) sensitive to blockade with 200 µM Ba2+. In the depolarized range, a variety of K+ conductances, including A-currents sensitive to blockade with 4-aminopyridine (4-AP) and additional tetraethylammonium (TEA)-sensitive currents, terminated the transient potential and firing of action potentials, supporting a strong spike-rate adaptation. The "chopper response," a hallmark of LSO principal neuron firing, may depend on the voltage- and time-dependent nonlinearities. These active membrane properties endow the LSO principal neurons with an adaptability that may maintain a stable code for sound direction under changing conditions, for example after partial cochlear hearing loss.
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INTRODUCTION |
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In the lateral superior
olivary nucleus (LSO), inter-aural sound intensity differences (IIDs)
within narrow frequency bands are linearly related to firing rate. This
simple relationship appears to encode sound direction when measured
over short poststimulus time periods in the tonotopically arrayed
principal output neurons (Boudreau and Tsuchitani 1968;
Caird and Klinke 1983
; Finlayson 1995
; Finlayson and Caspary 1993
;
Goldberg and Brown 1969
). The linear relationship arises
through an integration of excitatory and inhibitory synaptic inputs
from the ipsilateral and contralateral ears. The excitatory input
originates in glutamatergic spherical bushy cells of the ipsilateral
ventral cochlear nucleus (VCN) (Cant and Casseday 1986
;
Glendenning et al. 1991
). The inhibitory input is
relayed from globular bushy cells in the contralateral VCN via
glycinergic neurons in the medial nucleus of the trapezoid body
(Cant and Casseday 1986
; Glendenning et al.
1991
; Moore and Caspary 1983
;
Spangler et al. 1985
). The result of this integration is
a highly regular discharge pattern of repetitive firing known as the
"chopper response," where spikes are precisely timed in a train, as
reflected in a multi-modal peristimulus time histogram (Goldberg
and Brown 1969
; Romand 1978
;
Tsuchitani 1982
).
The regularity of the chopper response, also observed in VCN stellate
cells (Pfeiffer 1966), has been attributed to diffuse synaptic inputs distributed along extensive dendritic arbors of these
neurons (Rhode et al. 1983
; Young et al.
1988a
,b
). Cable properties of the fine, long dendrites impose
long time and space constants that transform brief synaptic current
inputs into smooth, steady membrane polarization patterns (Rall
1989
) and, hence, regular firing of action potentials. Indeed,
excitatory postsynaptic responses to acoustic stimuli display slow
depolarization levels (Finlayson and Caspary 1989
), as
expected from cable properties of the large dendritic arborizations of
LSO neurons (Helfert and Schwartz 1987a
,b
;
Scheibel and Scheibel 1974
). The proposed passive role
of the dendritic membrane in the integration of inputs should be
reflected in linear membrane input/output relations. In models of VCN
stellate neurons, it has been demonstrated that the chopper response
is, in principle, compatible with linear voltage-current (V-I) relationships (Arle and Kim 1991
;
Banks and Sachs 1991
; Hewitt and Meddis
1993
). Linear V-I curves have, in fact, been reported for neurons recorded, in vitro, in the general region of the
LSO (Wu and Kelly 1991
, 1993
).
Not all features of the principal LSO (pLSO) neuron are, however,
easily reconciled with integration by passive cable properties alone.
For example, the contralateral inhibitory input affects the membrane
potential at the spike trigger zone through glycinergic synapses
located at the soma and proximal dendrites (Glendenning et al.
1991; Spangler et al. 1985
), compatible with
short inhibitory postsynaptic potential (IPSP) rise times. In contrast,
long excitatory postsynaptic potential (EPSP) rise times would be
imposed by cable properties mediating the excitatory input directed,
largely, to the dendritic periphery (Cant and Casseday
1986
). LSO principal neurons must evaluate coincident
excitation and inhibition over short time periods to detect, among
other features, the movement of a sound source. We would expect,
therefore, membrane mechanisms that are able to balance temporal
aspects of the ipsilateral excitatory and contralateral inhibitory
synaptic inputs. Thus nonlinear membrane properties may be required to
account for the linear relation between IID and firing rate.
The short, invariant onset latency that is prerequisite for the
multi-modal chopper response is not expected to emerge from EPSPs
conducted passively from the dendritic periphery to the spike trigger
zone. Long time constants and membrane potential fluctuations would be
expected to impose long and variable latencies. In fact, in LSO
principal neurons the onset emerges from a brief depolarizing transient
potential, visible in subthreshold voltage responses to injected
current pulses (Adam et al. 1997). The transient potential can be isolated from the spike by
Na+-current blockade with tetrodotoxin (TTX) and
is followed, during maintained constant current, by a partial
repolarization with a time course that is similar to the adaptation of
spike rate in the chopper response. A parsimonious hypothesis would
involve several voltage- and activity-dependent ion conductances in
this response pattern. Responses to hyperpolarizing current pulse
injections mirror the depolarizing responses: a transient peak
hyperpolarization shortly after stimulus onset is followed by a voltage
sag, back toward the resting potential, in spite of the continued
hyperpolarizing current (Adam et al. 1997
). Thus LSO
neurons are equipped with special membrane properties that emphasize
and balance the onset of both excitatory and inhibitory inputs. As a
functional consequence of the onset emphasis, pLSO neurons are
sensitive to the inter-aural time difference of low-frequency and
amplitude-modulated sounds that cause a phase-locked firing pattern
(Finlayson and Caspary 1991
; Joris 1996
;
Joris and Yin 1995
). The output signal depends, in magnitude, on the coincidence of inputs that would cause the peak
de- and hyperpolarization in the subthreshold range.
The responses to injected current suggest active membrane behavior that involves a variety of voltage- and time-dependent conductances throughout the physiological voltage range of pLSO neurons. V-I relations may appear linear under such conditions, provided a balance exists between the magnitudes of conductances activated in the depolarized and hyperpolarized voltage ranges. This type of linearity should be limited to a narrow range of membrane potentials from which the de- and hyperpolarizing test currents are injected. Here, we report that the V-I relations of LSO principal neurons differ in linearity when tested from different membrane potentials, due to contributions by several voltage- and time-dependent conductances that have a prominent influence on the firing pattern.
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METHODS |
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Long Evans rats, 9-16 days old, were deeply anesthetized with halothane and decapitated. A transverse brain stem block containing both LSO nuclei was quickly dissected in ice-cold artificial cerebrospinal fluid (ACSF) in which NaCl was replaced with sucrose. Using a Vibratome, we cut transverse brain stem slices, 300-350 µm thick, in the same solution and then transferred them to regular ACSF at 36°C. After an incubation period of 30 min, the slices were gradually cooled to 22°C and maintained at that temperature for up to 6 h before recording. The slices were then suspended on a nylon mesh in a recording chamber with a bath volume of 0.7 ml and perfused continually with ACSF at a rate of 4-6 ml per minute by means of a pump. The bath temperature was either 34 or 22°C. The conductances reported here were identified at either temperature, but we did not investigate temperature dependencies of their amplitudes or kinetics. The standard ACSF contained (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4-H2O, 10 D-glucose, and 25 NaHCO3. After saturation with 95% O2-5% CO2 the pH was 7.3, and the osmolarity was 312 mOsm.
For whole cell recording we used patch pipettes pulled from
borosilicate glass using a two-stage vertical puller (Narashige PP-83).
The electrodes were filled with a solution containing (in mM) 115 K-gluconate, 20 KCl, 10 Na-N-2-hydroxyethylpiperazine-N-2-ethanesulfate (HEPES), 4 Mg-ATP, 0.3 Na-guanidine triphosphate (Na-GTP), 2 CaCl2, and 10 ethylene glycol-bis
(-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA) to
yield a calculated free Ca2+ concentration of
10
8 M (MaxChelator 1.2 software). Pipette resistance was between 5 and 8 M
. The electrode
tips were placed on the slice surface within the LSO, as identified
under a Zeiss dissection microscope, and advanced into the nucleus by
means of a Newport micromanipulator system (actuator model 850B;
controller PMC100). Cells were typically encountered between 50 and 150 µm from the tissue surface.
We used an Axoclamp 2B amplifier (Axon Instruments) in bridge mode to record membrane potentials and inject current, with optimal compensation for electrode resistance and capacitance. We offset the tip potential to zero in the bath fluid before recording and measured any deviation from zero after a session to extrapolate required corrections of the membrane potential, but we did not attempt to correct for further junction potentials. We low-pass filtered recorded data at 3 kHz, amplified them, and sampled at 20 kHz via National Instruments boards (MIO-16H; DMA-2800) in a MacIntosh Quadra 650 computer using A/Dvance software (McKellarDesigns).
Using electrophysiological criteria that distinguish the two types of
morphologically identified LSO neurons in the rat (Adam et al.
1997), we selected LSO principal output cells for this report.
Access for whole cell recording was obtained with seals of >1 G
.
The neurons had overshooting action potentials, and membrane potentials
were stable over recording sessions that generally exceeded 1 h.
We measured voltage responses to 200-ms current pulses, injected
through the recording electrode, from the resting membrane potential
(RMP) or a different holding potential maintained with injected DC.
TTX was purchased from Tocris. All other chemicals and drugs were purchased from Sigma.
The experiments were approved by the Animal Care Committee of the University of British Columbia and conducted in compliance with the Canadian Guidelines for Animal Care.
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RESULTS |
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Eighty-three neurons recorded in the LSO responded to constant
supra-threshold current pulses, injected from rest, with highly regular
trains of action potentials of short fixed onset latency. As shown in
an earlier intracellular study (Adam et al. 1997), this
behavior accounts for the multi-modal poststimulus time histogram firing patterns of principal LSO neurons, with spike rates that rise
with current intensity, mimicking "chopper responses" in the intact
animal. Using whole cell recording techniques we now recorded RMPs of
62 ± 5.3 mV (mean ± SD), input resistances of 109 ± 64.4 M
, and membrane time constants of 8.5 ± 4.5 ms, measured at the offset voltage responses of <5 mV. The resistances and time
constants were significantly greater than those recorded previously
with intracellular (sharp) electrodes (P < 0.01)
(Adam et al. 1997
). On depolarization beyond threshold,
LSO principal neurons fired overshooting action potentials with
amplitudes of 81.3 ± 15.3 mV and half-amplitude durations of
0.9 ± 0.6 ms.
Rectification in LSO principal neurons
Voltage responses in all principal LSO neurons displayed time- and voltage-dependent nonlinear relationships in responses to current pulses injected from rest or de- and hyperpolarized holding potentials. However, steady-state V-I relations measured just before pulse offset often showed only moderate rectification, particularly when determined at rest (Fig. 1, B and E) or a hyperpolarized holding potential (Fig. 1, C and F).
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A depolarizing transient potential, visible in isolation in
subthreshold depolarizing responses, triggered the first action potential (Fig. 1, A-C). While the transient potential
varied in amplitude it increased, in all 83 neurons, on depolarization from negative holding potentials, suggesting contributions of conductances that are activated or de-inactivated in the hyperpolarized range. A contribution of strongly voltage-dependent inward conductances is suggested by an inflection in the rising phase of the transient potential in 58 neurons (Fig. 1, B and C). In all
83 neurons the transient potential was followed by a sustained outward
rectification (Fig. 1), suggesting contributions to its decaying phase
by outward currents activated in the depolarized range. Negative
current pulses evoked hyperpolarizing peak potentials followed by a
voltage sag back toward the RMP, resulting, within 100-150 ms, in a
steady-state inward rectification that was most pronounced when
measured from relatively positive holding potentials (Fig. 1,
A and D; n = 83). This
hyperpolarization-dependent inward rectification activated at
approximately 60 mV, and approached a maximum at
70 to
80 mV.
Thus hyperpolarization from such holding potentials evoked little or no
sag. Inward currents activated in the hyperpolarized range likely
account for this behavior.
Voltage dependence of action potential firing
We examined the influence of prevailing membrane potential on the
firing pattern because synaptic inputs may occur, in vivo, during
preexisting inhibition, and the "resting" membrane potential may
vary, for example, as result of neuromodulation. In 25 neurons, we
adjusted the amplitude of a test inward current pulse, injected from
rest, to evoke a short train of action potentials. We then repeated the
same stimulus immediately after a 200-ms prepulse that polarized the
membrane to a value varied systematically between 50 and
110 mV
(Fig. 2, A and B).
The prepulse amplitude had only a small influence on the latency of the
onset spike, which, presumably, was stabilized by the same conductances
responsible for the transient potential. The latency (measured at the
point the rising phase crossed 0 mV) was slightly shorter at rest than following hyper- or depolarization, resulting in a U-shaped function of
the latency versus prepulse potential amplitude (Fig. 2C;
n = 25). However, over a prepulse voltage range of
110 to
50 mV, the onset latency changed by only 3.2 ± 0.9 ms,
or by 0.05 ms/mV. Thus the onset latency of a chopper response is not
very sensitive to the preexisting membrane potential.
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In contrast, repetitive firing varied dramatically with the prepulse
potential. In all 25 neurons tested with prepulses, we evoked
repetitive firing from only a restricted range of prepotentials between
approximately 50 and
70 mV (Fig. 2B). The highest spike counts were always evoked from rest, while fewer spikes were elicited in 16 neurons after prepolarization in either direction (Fig. 2E). In the remaining 9 of 25 neurons, repetitive firing
could only be evoked from rest. Correspondingly, in 16 neurons the
interval between the 1st 2 spikes was shortest when the test response
arose from prepotentials close to rest (
50 to
55 mV) and longer
from more negative or positive prepotentials (Fig. 2D).
Following prepolarization between approximately 70 and
50 mV, the
test response was characterized by voltage oscillations that occurred
after the onset action potential and triggered firing of further action
potentials in 21 of 25 neurons (Fig. 2, A and B).
These oscillations may actively support regularity of firing by
maintaining firing times within the modes of the multi-modal chopper
response, even after failure of individual action potentials (Adam et al. 1997
). The oscillations faded in test
responses evoked from negative prepotentials (less than
70 mV), being
replaced by an extended spike afterhyperpolarization (AHP; Fig.
2A). In 18 of 21 neurons the magnitude of this AHP grew with
the level of prehyperpolarization as expected from an outward
conductance dependent on Ca2+ influx that is
inactivated in the depolarized range. Thus prehyperpolarization tends
to restrict firing to the onset spike, as observed in vivo when the
excitatory ipsilateral response was preceded by inhibitory contralateral stimulation (Tsuchitani 1988a
,b
).
Following predepolarization, firing after the onset spike was also
suppressed, due, possibly, to a shunting action during the sustained
outward rectification (Fig. 2B).
In summary, active membrane properties appear to guarantee short and precise onset latencies over a wide range of preexisting membrane potentials and support the multi-modal chopper response available from a narrower range of possible resting potentials.
Subthreshold Na+ conductance
We characterized voltage-dependent conductances that account for the active nonlinear membrane behavior in LSO principal neurons. To study voltage-dependent nonlinearities independent of action potential firing, we applied the Na+-channel blocker, TTX (600 nM) in the ACSF. This application invariably hyperpolarized the RMP by a small amount (average: 3 mV), indicating that a persistent Na+ conductance normally contributes to the resting potential of pLSO neurons. When testing membrane behavior at rest, we compensated for this hyperpolarization by DC injection. Due to its depolarization dependency, this voltage-activated conductance appears to contribute to the short stable onset latency and the transient potential (see Conductances contributing to the transient potential below).
Hyperpolarization-activated conductances
Injection of negative current pulses typically caused a
hyperpolarizing peak, followed by a depolarizing sag of the membrane potential, despite a maintained current stimulus (Figs. 1-4). On hyperpolarization from rest, this voltage sag varied considerably in
magnitude between neurons (compare Figs. 1A and
4A). Fifty-eight of the 83 neurons exhibited only slight
sags, as in Fig. 4A. We hypothesized that a
hyperpolarization-activated current,
IH, is responsible for the sag but is
masked to varying extents by simultaneous activation of an inwardly
rectifying potassium current, IKIR, as
in neurons of the auditory thalamus (Ströhmann et al.
1994; Tennigkeit et al. 1996
).
In the presence of TTX, we first evoked the voltage sag on
hyperpolarization (Fig. 3A)
and then applied Ba2+ to the bath at a
concentration that selectively blocks the inward rectifier (0.2 mM)
(Nichols and Lopatin 1997). Barium application depolarized the membrane by 7.2 ± 2.2 mV (P < 0.01), increased the input resistance by 61.8 ± 29.9%
(P < 0.01), and raised the slope resistance in the
hyperpolarized range, measured at the peak, by 77 ± 23.6% (Fig.
3D; n = 7/7). Thus
Ba2+ application greatly increased
hyperpolarization amplitudes, mainly of the peak, but also at steady
state (Fig. 3, B and D), suggesting a strong
contribution of IKIR to the membrane
conductance in the hyperpolarized range. In all seven neurons the
depolarizing sag in hyperpolarizing responses was markedly enhanced in
the presence of Ba2+, presumably due to unmasking
of the gradually activating IH by IKIR blockade. In the voltage range of
a well-developed sag (negative to approximately
65 mV), the slope
resistance was not significantly increased by
Ba2+ (Fig. 3E; n = 7/7), illustrating a major role of the remaining IH in the membrane conductance.
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Application of the IH blocker, Cs+ (3 mM; n = 6), to the ACSF (in addition TTX and Ba2+) completely eliminated the sag (n = 6/6). Cs+ increased the membrane resistance, measured close to the RMP, by 170.1 ± 3.3% (P < 0.01) and the slope resistance in the hyperpolarized range by 482 ± 176% (P < 0.01; cf. Fig. 3E). These values were low estimates as strong hyperpolarizations were incomplete at the end of the 200-ms pulse due to greatly prolonged time constants (Fig. 3C). The Cs+-induced increase in resistance (Fig. 3E) suggests that IH normally dominates the conductance in the hyperpolarized range in pLSO neurons. Furthermore, the Cs+ application hyperpolarized the RMP by 5.0 ± 2.1 mV, indicating that IH contributes to the RMP. The slow deactivation of the IH after termination of hyperpolarizing pulses led to a transient depolarizing afterpotential (Fig. 3B, arrowhead) that could serve to emphasize the onset of a response to depolarization (e.g., an EPSP) from negative potentials or rest in a voltage-dependent manner. Thus the strong expression of IH, and its masking by IKIR, may be of great functional importance.
Conductances contributing to the transient potential
In many respects, the depolarizing afterpotential following a
hyperpolarizing pulse was similar to the transient potential. Both
increased with the magnitude of hyperpolarization of a pulse or the
prevailing holding potential (Figs. 1 and 3-5), and both were greatly
amplified after application of Ba2+ (Figs.
3B, 4B, and 5B). An increase in
amplitude under Ba2+ was expected as a
consequence of K+ channel blockade (raised
resistance); however, a greater permeability of
Ca2+ channels to Ba2+,
compared with Ca2+, could also contribute
(Huguenard 1996). We investigated a possible contribution of the transient Ca2+ current,
IT, to the depolarizing afterpotential
and transient potential. We evoked depolarizing afterpotentials,
following hyperpolarization from rest, in the presence of TTX (600 nM)
to block any contamination by action potential firing (Fig.
4A). After an amplification of the depolarizing afterpotential with Ba2+ (0.2 mM; Fig. 4B), we applied Cs+ (3 mM;
Fig. 4C) to remove the large contribution of
IH to the depolarizing afterpotential.
We then added the T-channel blocker, Ni2+ (50 µM; Fig. 4D) to the ACSF. The result was an almost
complete elimination of the depolarizing afterpotential, consistent
with a contribution by a transient Ca2+ current,
IT (n = 5).
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The transient potential displayed qualitatively similar behavior. We
depolarized the neurons (n = 7) from a holding
potential of approximately 80 mV (deinactivating T-type currents) and
evoked transient potentials in the presence of TTX (Fig.
5A). Again, the transient
potentials were greatly amplified after addition of 0.2 mM
Ba2+ to the bath (Fig. 5, B
and C; n = 7/7). An addition of
Cs+ (3 mM) then removed the
IH contribution to the transient
potential, which now occurred with longer and more variable latencies
(Fig. 5, D and G; n = 6/6).
Paradoxically, the net amplitudes were raised in the presence of
Cs+, presumably due to a great increase in
impedance caused by the IH blockade.
Subsequent co-application of Ni2+ eliminated the
transient potential and thus reduced the depolarizing peak potentials
(Fig. 5F), but for a small emphasis of the
depolarization onset (Fig. 5E; n = 5/5).
Therefore IT seems to contribute to the transient potential, although its short and relatively constant latency depends on activation of an
IH.
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We characterized the role of Ca2+ influx by comparing voltage responses, including the depolarizing afterpotential and transient potential, measured after a 10-fold reduction of the Ca2+ concentration and in standard ACSF (n = 8). The depolarizing afterpotential, evoked in the presence of 2 mM Ca2+ (and TTX), was reduced in amplitude and duration in medium containing only 200 µM Ca2+ (Fig. 6; n = 8/8). Thus a Ca2+ influx seems to contribute to the transient depolarization for a longer period of time than the decaying IH. In contrast, a blockade of the IH with Cs+ led to prolonged (up to 100 ms) and variable latencies of the Ca2+-dependent depolarizing afterpotentials (e.g., Fig. 4C; n = 6). Apparently, the IH is largely responsible for the constant short onset latencies of depolarizing responses from hyperpolarized potentials (e.g., Fig. 2).
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A Ni2+-sensitive depolarizing conductance,
activating between approximately 70 and
55 mV, appeared to make a
large contribution to the amplitude of the transient potential (Fig. 5,
B, E, and F). Despite the
limited selectivity of the Ni2+ blockade, this
relatively negative voltage range of activation suggested a strong
contribution of a transient (T-type) Ca2+
conductance. As a confirmation, we compared depolarizations evoked from
negative holding potentials (
75 mV) in standard TTX containing ACSF
with and without 50 µM of Ni2+
(n = 7). Nickel application strongly reduced the
amplitudes of the transient potential (peak depolarization in Fig.
7, A and C) but
also diminished the steady-state depolarization to a lesser degree
(Fig. 7D; n = 7/7). This effect is
consistent with a contribution of a transient
Ca2+ current but does not exclude participation
of other conductances, particularly at depolarized levels. In the
absence of TTX, Ni2+ application (50 µM) raised
the current intensities required to trigger an action potential from
hyperpolarized holding potentials (
75 mV in Fig. 7D;
n = 5/5). No significant increase in current was
required, however, to evoke action potentials from holding potentials
positive to
60 mV. Therefore a Ni2+-sensitive
Ca2+ current that is inactivated at
60 mV seems
to have a major role, in concert with
IH, in the latency stabilization of
onset spikes evoked from a hyperpolarized voltage range (cf. Fig. 2).
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A Na+ current contributes to the RMP of LSO
principal neurons, as an application of TTX (300-600 nM) caused a
hyperpolarization (3 ± 0.4 mV) in 14/14 neurons. We compensated
for the TTX-induced hyperpolarization with depolarizing current (e.g.,
to the original RMP of 58 mV in Fig.
8A) to measure voltage
responses to hyper- and depolarizing current pulses under comparable
conditions before and after TTX application. The V-I
relations of the hyperpolarizing responses were identical; however, the
slope of the V-I curves representing the peak depolarization
decreased by 41.3 ± 10.2% in the voltage range between the RMP
and firing threshold as a consequence of TTX application (Fig.
8C). We repeated the same type of measurements from
hyperpolarized holding potentials (e.g.,
73 mV in Fig. 8,
D-F) and obtained TTX-reduced subthreshold
depolarization amplitudes in a similar voltage range. In about one-half
of the neurons (n = 6/14), steady-state depolarizations
measured at the end of the 200-ms pulse were also reduced by TTX
application, documenting the blocked depolarizing effect of a
long-lasting Na+ conductance. Thus a non- or
slowly inactivating, subthreshold Na+ conductance
contributes to depolarization amplitudes above approximately
60 mV,
potentially accelerating the onset response. It remains to be
elucidated which TTX-sensitive Na+ channels
account for the Na+ conductance at different
voltage levels in principal LSO neurons.
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Depolarization-activated K+ conductances
In spite of contributions by a persistent
Na+ conductance, the decaying phase of the
transient potential may be explained, to a large extent, by the decline
of the transient IT,
IH, and, possibly, subthreshold
INa. We hypothesized a further
contribution of depolarization-activated K+
currents because of an increase in rate of the transient potential decay with depolarization amplitude (e.g., Fig. 1). We therefore compared voltage responses before and after K+
channel blockade. Application of 4-aminopyridine (4-AP), a blocker of
transient K+ currents (McCormick
1991), produced a concentration-dependent depolarization of the
resting potential in the presence of TTX (300-600 nM;
n = 7). For example, 1 mM of 4-AP changed the RMP from
62.7 ± 2.7 mV to
42.0 ± 5.7 mV, and 4 mM to
38.5 ± 4.9 mV. 4-AP-sensitive outward currents are therefore at least
partially activated at rest. However, 50 and 200 µM 4-AP did not
significantly alter the RMP. These lower concentrations led to an
increase in voltage amplitude during and immediately after the
transient potential, evoked from hyperpolarized holding potentials
(Fig. 9A), and to enlarged
depolarization amplitudes early during a pulse elicited from holding
potentials close to rest (Fig. 9B). V-I relations showed that the low 4-AP concentrations led to amplitude increases of
the initial portions of depolarizing responses above approximately
50
mV, as shown for the voltage sag (arrow in Fig. 9A)
immediately following the transient potential in Fig. 9C.
Steady-state responses measured at the end of the pulses were
unaffected by 50 µM 4-AP (Fig. 9D). Higher 4-AP
concentrations (1-4 mM) were required to elevate steady-state
depolarizations (not shown). These data are compatible with a selective
blockade of transient A-type K+ currents by low
4-AP concentrations (McFarlane and Cooper 1991
; Storm 1990
) and illustrate an additional contribution of
sustained K+ currents, sensitive to higher 4-AP
concentrations, to depolarizing responses and the RMP.
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Activation of an A-type K+ current would be expected to accelerate the rate adaptation in a neuron's discharge pattern during depolarization. To investigate this possibility we applied 50 µM of 4-AP without TTX (n = 6). At higher 4-AP concentrations we were unable to maintain stable recording conditions, due to excessive spontaneous firing. A single onset spike response evoked, from rest or hyperpolarized holding potentials, by a threshold current pulse amplitude under control conditions was transformed into sustained repetitive firing after application of 50 µM 4-AP (Fig. 10). Suprathreshold depolarization from rest that resulted in a short, strongly adapting spike burst in control ACSF also caused sustained firing with very little, if any, adaptation in the presence of 4-AP (Fig. 10). Spike amplitudes and durations were also magnified after application of 4-AP. Therefore A-type K+ currents expressed in pLSO neurons accelerate the decay of action potentials and contribute strongly to the spike rate adaptation that normally characterizes the chopper response.
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Transient outward currents cannot explain the sustained depression of
depolarization following the transient potential (e.g., Figs. 1 and
5B), although the sustained current sensitive to high 4-AP
concentrations could. We considered contributions of further depolarization-activated K+ currents and applied
the broad spectrum K+-channel blocker,
tetraethylammonium (TEA) in ACSF containing 600 nM TTX
(n = 6). At concentrations between 5 and 30 mM, TEA had
no significant effect on the RMP. However, voltage responses to
depolarizing pulses were strongly amplified in the presence of 5 mM
TEA, both early and late during the pulse (Fig.
11, A and B). At
higher concentrations (20 mM in Fig. 11C) a depolarizing ramp potential led, in addition, to a peak at a long and variable latency during small current pulses. At higher stimulus intensities the
ramp triggered an all-or-nothing spike at an inflection point of
approximately 35 mV. After a broad peak, this high-threshold spike
(HTS) terminated in a plateau depolarization that lasted throughout the
duration of the pulse and some times longer (Figs. 11C and
12C). V-I relations for depolarizations from rest
(
61 mV in Fig. 11) demonstrate a linear increase in response
amplitude at 5 mM but, at 20 mM, a sudden strong increase in slope at
approximately
35 mV, reflecting the HTS early, and the plateau late
in the response (Fig. 11, D and E). Thus TEA
blockade of a sustained K+ conductance that does
not normally contribute to the RMP unmasked a regenerative depolarizing
process capable of producing HTSs with plateaus.
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We assessed the influence of the TEA-sensitive conductance on the
firing pattern by recording depolarizing responses without TTX in the
ACSF (n = 6). As a precaution against excessive firing, we injected positive current pulses from a holding potential of 80
mV, evoking, above threshold, a single onset spike under control conditions (Fig. 12A, cf.
Fig. 2). Addition of 5 mM TEA to the ACSF transformed this pattern. At
suprathreshold stimulus intensities, repetitive firing of spikes, with
gradually increasing amplitudes and durations, occurred after the onset
action potential, which, at moderate current strengths, was followed by
a brief pause in firing (Fig. 12B). The action of A-type
currents, not blocked by TEA, probably prevented spike discharge during
the pause. After application of 20 mM TEA the onset spike led, without
repolarization, to a plateau potential (Fig. 12C), caused,
presumably, by blockade of repolarizing K+
currents (Storm 1987
). A brief, negative voltage sag
between onset spike and plateau may also have been caused by A-current activity.
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Mechanisms supporting the high-threshold spike
We never observed an HTS under control conditions, but they
commonly occurred after application of high TEA concentrations (e.g.,
20 mM in Figs. 11C, 12C, and 13B).
Since they are readily evoked in the presence of TTX, we assumed that a
Ca2+ current accounted for both HTSs and
plateaus. We confirmed this assumption as follows: In the presence of
TTX we first recorded voltage responses to depolarizing current pulses
under control conditions (Fig.
13A) and then applied 20 mM
TEA. After establishing current pulse amplitudes that reliably evoked
HTSs and plateaus (Fig. 13B), we applied
Cd2+ at a concentration (50 µM) that
selectively blocks high-threshold Ca2+ currents
in other neurons (Huguenard 1996). On repetition of the
depolarizing pulses, we found that the presence of
Cd2+ completely prevented firing of the HTSs and
plateaus (Fig. 13C). Low-threshold
Ca2+ spikes and transient potentials evoked from
hyperpolarized potentials were not affected by application of 50 µM
Cd2+ (not shown). We also compared
Ca2+ spikes in control ACSF (2 mM
Ca2+) and after reduction of the
Ca2+ concentration to 200 µM (n = 5). This reduction of the external Ca2+
concentration eliminated the neuron's ability to fire either HTS or
LTS and reduced the amplitude of the transient potential (cf. Fig. 6).
These data show that LSO principal neurons are equipped with at least
two pharmacologically distinct Ca2+ conductances,
a Ni2+-sensitive transient low-threshold
conductance that accounts for LTS and amplifies the transient
potential, and a high-threshold conductance able to support HTSs and
sustained plateaus.
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DISCUSSION |
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Principal neurons in the LSO are thought to extract information about sound direction by combining the excitatory input from the ipsilateral ear with the contralateral inhibitory input. A regular firing pattern and smooth depolarization recorded at the soma during acoustic stimulation have led to the assumption that both inputs are integrated by cable properties of the large dendritic tree. Given normal fluctuations in membrane potential, this passive integration model would have difficulty accounting for the temporal firing pattern of pLSO neurons. In particular, this model is incompatible with the narrow range of onset latencies that is prerequisite for the chopper response. Active membrane properties appeared necessary to explain the firing pattern.
Here, we present evidence for a rich complement of voltage- and time-dependent conductances, which are expected to strongly affect the integration of postsynaptic potentials at the spike trigger zone and shape the pattern of output firing. Apart from the mechanisms for action potential generation, LSO neurons exhibit many voltage-dependent conductances that activate on depolarization, including a subthreshold Na+, low- and high-threshold Ca2+ currents, and 4-AP- and TEA-sensitive K+ currents. In addition, two distinct conductances, IH and IKIR are activated during hyperpolarization.
Principal neurons of the LSO transform the firing patterns of
excitatory and inhibitory input fibers, which are similar to auditory
nerve ("primary-like") responses, into a new signal termed the
chopper response. This multi-modal firing pattern characterizes pLSO
neuron responses to tonal stimuli, in vivo, and to current injection in
tissue slices. Temporal and spatial integration of excitatory input at
the periphery of the extensive dendritic tree probably accounts for
much of the prerequisite regularity. However, the combination of inward
and outward conductances activated on depolarization contributes a
strong current shunt that will attenuate voltage fluctuations,
promoting regularity. Furthermore, active membrane properties, albeit
unidentified, must be responsible for the voltage oscillations that
appear, in response to injected current pulses, at times of failed
action potentials or after cessation of firing (Adam et al.
1997; Fig. 2, A and B). We believe that
these oscillations also support regularity. It is certainly not true
that the regularity simply reflects passive linear membrane behavior,
since voltage-activated conductances cause different degrees of
nonlinearities in input/output relations at different preexisting
membrane potentials (Fig. 1).
Both input and output firing patterns share an emphasis of stimulus onset. However, the onset emphasis is not simply imported from the excitatory synaptic input. Active membrane properties of the output neurons amplify the response onset as evident in the transient potential. IT, IH, INa, and A-type currents contribute to the transient potential. Their magnitudes vary with the preexisting membrane potential, due to different activation voltage ranges. During ipsilateral excitation, in vivo, subthreshold Na+ and transient Ca2+ currents will accelerate depolarization and shorten the onset latency, in spite of long dendritic time constants. These nonlinear properties temporally fix the onset of the regular firing pattern at a short latency, enabling the multi-modal chopper response. Thus the low-pass filter properties of the long dendrites are largely compensated by intrinsic voltage- and time-dependent membrane conductances. 4-AP- and TEA-sensitive K+ conductances repolarize and shunt the membrane after the transient potential. They serve, therefore, as intrinsic mechanisms for spike rate accommodation. Since onset emphasis and adaptation are actively maintained, they probably are of critical importance for the output code of the LSO.
A hyperpolarization of the membrane potential has been recorded in pLSO
neurons during inhibitory contralateral acoustic stimulation (Finlayson and Caspary 1989). The extent of this
glycinergic hyperpolarization depends on the equilibrium potential for
chloride (ECl) and the activation of
voltage-dependent conductances in the negative voltage range, e.g.,
IKIR and
IH. Reversal potentials of
glycine-evoked IPSPs have been measured at
73.0 ± 7.1 mV in the
LSO of rat pups, aged 10 days or more, with micropipettes containing 2 M K-acetate (Kandler and Friauf 1995
). As this technique
imposes a negative bias on the ECl,
the chief inhibitory effect of the glycinergic contralateral input may
be a powerful shunt conductance at the proximal soma-dendritic
membrane, between the dendritic periphery (receiving excitation) and
the spike trigger zone, rather than hyperpolarization. What then, is
the role of the hyperpolarization-activated currents?
The activation kinetics of the two hyperpolarization-activated
currents, IH and
IKIR, differ. The
IKIR can rapidly amplify a
hyperpolarization, whereas the IH has
a slowly depolarizing effect. The temporal course of these conductances
roughly mirrors the resistance change evoked, on depolarization, by the
fast depolarizing subthreshold INa and
IT and the slower hyperpolarizing
K+ currents. The overall increase in conductance
results in a decrease in membrane time constant, allowing the cell's
membrane potential to change more rapidly. On balance, these
nonlinearities may serve to transform coinciding excitatory and
inhibitory inputs into an output that provides a linear code for IID
within, approximately, the first 50 ms of the stimulus onset. The onset
emphasis during both hyperpolarization and depolarization is likely
instrumental in the neuronal sensitivity for phase in low-frequency
stimuli (Finlayson and Caspary 1991) and
amplitude-modulated sounds (Joris 1996
; Joris and
Yin 1995
) found in the LSO.
We demonstrated that a Na+ conductance and the
IH contribute to the resting membrane
potential. It is reasonable to assume that these conductances also are
subject to adjustment. For example, the voltage range of activation for
IH typically changes under the
influence of neuromodulators such as histamine, acteylcholine, norepinephrine, or glutamate (via metabotropic receptors) (Pape 1996; Santoro et al. 2000
). Afferents from the
A5 cell group, dorsolateral to the LSO, are thought to release
norepinephrine within the LSO, possibly at discrete dendritic sites
(Woods and Azeredo 1999
; Wynne and Robertson
1996
). Glutamate released by ipsilateral afferents also
activates metabotropic receptors (Kotak and Sanes 1995
).
Vesicular acetylcholine transporter (Yao and Godfrey
1998
) and choline acetyl transferase are found in puncta within
the LSO (Henderson and Sherriff 1991
). A cholinergic
innervation could be derived from lateral olivocochlear (LOC) neurons
(Brown 1993
). Here, acetylcholine has been co-localized
with other neuromodulators, such as calcitonin gene-related peptide
(CGRP), dynorphins, and GABA (Abou-Madi et al. 1987
;
Altschuler et al. 1984
, 1988
; Lu et al. 1987
; Saffieddine and Eybalin 1992
;
Schwarz et al. 1988
). The RMP can therefore be predicted
to change with neuromodulation, possibly affecting the neuron's
ability to maintain repetitive firing (Fig. 2). Therefore it will be
simplistic to model LSO signaling at one RMP value.
The output of the LSO has been assumed to encode sound direction as
firing rate, or as the number of spikes over a time segment. Accordingly, a decaying rate would signal movement of the sound source.
To avoid ambiguities between adaptation to a stationary sound stimulus
and a moving sound source, an evaluation of rate would have to occur
over short time segments. Thus the discharges during the transient
potential and its decay may be more important for sound localization
than the steady-state firing rate, which, by virtue of its regularity,
seems well suited to indicate stability. The LSO output seems to
emphasize change over stability: even strong depolarizing current
pulses can lead to complete adaptation in firing (Adam et al.
1997). Here, we show that continuous firing strongly depends on
the preexisting membrane potential, being maximal during current
injection from rest. Currents causing the same depolarization from de-
or hyperpolarized prepotentials maintain the onset, but not the
steady-state response (Fig. 2). This behavior probably explains why, in
vivo, LSO neurons respond with only the onset response to an excitatory
stimulus that is preceded by inhibition (Tsuchitani
1988a
,b
). LSO neurons secure, then, the dynamic onset response
under varying conditions, but not the steady-state firing rate. Thus
change in stimulus position may be more securely encoded than position
itself. In the behaving animal the emphasis of change is reflected in
scanning head and pinna movements during precise localization of a
stationary sound.
Does the onset response contain sufficient information to encode sound
localization? Two parameters in the onset discharge reflect the
amplitude of a depolarizing current, and thus possibly, sound source
location: 1) the latency of the first spike and
2) the initial interspike intervals. In responses to current
pulses, the onset spike latency varies systematically with stimulus
intensity (Adam et al. 1997) and with the preexisting
membrane potential, but over a narrow range (Fig. 2C).
However, in vivo a wider latency range of the subthreshold transient
potential peaks (e.g., Fig. 1) may expand the range of firing
latencies, and hence resolution. Separate transient potentials, evoked
from numerous excitatory afferents converging onto a pLSO neuron at
different dendritic locations, would summate to the extent of their
coincidence. For similar reasons, the resolution and range of the first
spike interval measure would be greatly enhanced by the effects of
convergent excitation, particularly if the subthreshold transient
potentials coincide only partially, causing an expansion of transient
potential peak duration or even several peaks. The ranges of both onset latency and initial interval would further be widened by the slower onset of naturally occurring EPSPs, compared with current pulses. Thus
by virtue of intrinsic properties responsible for the transient potential, principal LSO neurons may be endowed with a dynamic intensity (location) coding capability that has, during the response onset, a far greater resolution in the behaving animal than apparent in
vitro in the current pulse response.
In conclusion, the active membrane of pLSO neurons, endowed with the voltage- and time-dependent conductances we have identified (and possibly more), can better account for the firing behavior observed in vivo, including the chopper response, than linear models based on soma-dendritic cable properties. As an added benefit, the corresponding ion channels are, in principle, plastically adjustable, either by changes in expression or by neuromodulation. Without plasticity, the translation of IID into firing rate would produce errors in the code for sound incidence angle as asymmetric hearing losses occur over a lifetime.
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ACKNOWLEDGMENTS |
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This study was supported by the Canadian Institutes for Health Research, the Deafness Research Foundation, and the Rotary Hearing Foundation, Vancouver.
Present address of P. G. Finlayson: Dept. of Otolaryngology, Wayne State University, Lande Building, Rm. 327, 550 E. Canfield, Detroit, MI 48201.
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FOOTNOTES |
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Address for reprint requests: D.W.F. Schwarz, Dept. of Surgery/ENT, University of British Columbia, F153, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada (E-mail: dsch{at}interchange.ubc.ca).
Received 7 December 2000; accepted in final form 23 April 2001.
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REFERENCES |
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