 |
INTRODUCTION |
The cochlea receives an efferent innervation from olivocochlear (OC) neurons located in the superior olivary complex. The mammalian OC neurons have been anatomically classified into two groups by their origins and targets: the lateral and medial OC (LOC and MOC, respectively) neurons. LOC neurons are distributed within or around the lateral superior olivary nucleus (LSO). They receive inputs from the ipsilateral posteroventral cochlear nucleus and project mainly to the dendrites of ipsilateral type I spiral ganglion neurons that innervate cochlear inner hair cells. MOC neurons are located in the medial, ventral, or rostral periolivary zone. They receive inputs from the bilateral posteroventral cochlear nucleus and the ipsilateral inferior colliculus and make a main projection to the contralateral cochlear outer hair cells. The majority of OC neurons is known to be cholinergic from histochemical investigations (for reviews see Spangler and Warr 1991
; Warr 1992
).
Electrophysiological studies have shown that the stimulation applied to MOC axons inhibited afferent nerve activities (Fex 1962
; Galambos 1956
; Wiederhold and Kiang 1970
). Acetylcholine, a putative transmitter of OC neurons, hyperpolarized outer hair cells through the activation of Ca2+-activated K+ current (Doi and Ohmori 1993
; Kakehata et al. 1993
). Furthermore, outer hair cells are known to change their size by the level of membrane potential; depolarization decreased the length and hyperpolarization lengthened it (Brownell et al. 1985
). On the basis of these findings, several functional roles of the inhibitory effect of MOC neurons have been proposed, such as protection from noise damage (Cody and Johnstone 1982a
; Rajan 1988
; Reiter and Liberman 1995
), improvement in detection of auditory signal in noise (Winslow and Sachs 1987
), and control of the mechanical state of the cochlea through the motility of outer hair cells (LePage 1989
). On the other hand, the effect of LOC neurons on afferent nerve activities is likely facilitatory, because the focal application of acetylcholine increased the firing frequency of the spiral ganglion neurons (Felix and Ehrenberger 1992
). This facilitatory effect may be due to the suppression of a K+ current by acetylcholine (Yamaguchi and Ohmori 1993
). However, functional roles of LOC neurons in auditory signal coding have not yet been well investigated. Although many functions are not clarified, OC neurons likely hold a pivotal position in the auditory brain stem, integrating the binaural afferent information from cochlear nuclei and the inputs from higher centers and sending outputs directly to the cochlea to regulate peripheral auditory signal processing.
To understand how OC neurons integrate the information and how they exert efferent control on the cochlea, it is necessary to elucidate their intrinsic membrane properties. Despite numerous anatomic studies and much research about their influences on the afferent system, electrophysiological properties of OC neurons are known only marginally. In MOC neurons, there are only some extra- and intra-axonal recordings in vivo (Brown 1989
; Cody and Johnstone 1982b
; Fex 1962
; Liberman 1988a
,b
; Liberman and Brown 1986
; Robertson 1984
; Robertson and Gummer 1985
) and single-channel recordings from the isolated nerve terminals (Wangemann and Takeuchi 1993
). To the best of our knowledge there have been no electrophysiological recordings from the identified LOC neurons. Because of their sparse distribution in the brain stem, recordings from the somata of OC neurons were difficult irrespective of in vivo or in vitro preparation.
We recorded from both groups of OC neurons of the rat with the use of a whole cell patch-clamp technique in slice preparations. OC neurons were retrogradely labeled with a fluorescent tracer applied into the cochlea 2 days before the experiment. Stained neurons were identified under a fluorescence microscope and were subjected to electrophysiological recordings. The main purpose of this study is to characterize firing properties and to clarify underlying ionic conductances in the identified LOC and MOC neurons. Among ionic currents, we have mainly analyzed transient outward currents that would determine discharge properties of both groups of neurons.
 |
METHODS |
Retrograde labeling of OC neurons
OC neurons were retrogradely labeled in Wister rats of postnatal day (P) 3-9. The animal was anesthetized with ether, and the left tympanic bulla was opened by a ventral approach. The gelatinous matter within the bulla was carefully removed, and the cochlear wall was exposed. A small hole was drilled on the second turn of the cochlea, and the perilymph was aspirated. A small piece of gelatin sponge (Spongel, Yamanouchi) of a few cubic millimeters was soaked with 3 µl of 10% dextran-Texas Red (3000 MW, Molecular Probe) and was inserted into the cochlea. Dextran-Texas Red was dissolved in distilled water. Then the hole was sealed with fibrin glue (Bolheal, Fujisawa) to prevent leakage of the dye. The wound was sutured, and the rat was returned to the mother. After 36-48 h, the animal was used for slice preparations; these periods were reported to be sufficient to label OC neurons retrogradely in neonatal rats after intracochlear injection of the dye (Robertson et al. 1989
).
Slice preparation
Slices were made from the animals whose cochleae were injected with the fluorescent dye (P5-P11). The animal was reanesthetized with ether and decapitated. The brain was removed and submerged for several minutes in an oxygenated ice-cold high-glucose saline composed of (in mM) 130 NaCl, 4.5 KCl, 2 CaCl2, 30 glucose, and 5 piperazine-N,N
-bis(2-ethanesulfonic acid) (PIPES), pH adjusted to 7.4 with NaOH. The brain stem was isolated and embedded in 3% agarose gel (low gelling temperature, Nacalai tesque) dissolved in the high-glucose saline. Coronal sections (130-150 µm) including the superior olivary complex were made by a tissue slicer (DTK-2000, Dosaka). The slices were then incubated for
1 h in oxygenated artificial cerebrospinal fluid (ACSF) at room temperature (22-24°C). The composition of the ACSF was as follows (in mM): 138 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), pH adjusted to 7.4 with NaOH. One slice was then transferred to the recording chamber on an upright microscope (BH2, Olympus), and was perfused with the oxygenated ACSF at 2-3 ml/min.
Identification of OC neurons
The slice was surveyed with the upright microscope equipped with an epifluorescence and a Nomarski optics (BH2-RFCA, Olympus). Neurons emitting the red fluorescence from Texas Red were found mainly in the ipsilateral LSO (Fig. 1A), and in the bilateral ventral nucleus of the trapezoid body (VNTB; Fig. 1B); these two nuclei could be identified, and could be distinguished from each other under the Nomarski optics (Paxinos and Watson 1986
). These locations of the labeled neurons were identical with the sites of LOC and MOC neurons, respectively, reported in the previous anatomic studies (Robertson et al. 1989
; Vetter and Mugnaini 1992
; White and Warr 1983
). The crossed OC bundle, which is an axon bundle of the contralateral projecting MOC neurons, was often labeled just below the fourth ventricle together with the cell bodies of MOC neurons (Fig. 1C). In some animals, labeled neurons were also found around the bilateral facial genua and in the ipsilateral facial nucleus. These neurons could be the vestibular efferent neurons and the facial motor neurons (White and Warr 1983
). The facial motor neurons were probably labeled through the stapedius muscle. These locations were apart from the LSO, and from the VNTB (Paxinos and Watson 1986
). Therefore these neurons were easily distinguished from the labeled OC neurons, and were excluded from electrophysiological studies.

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| FIG. 1.
Location and morphology of labeled olivocochlear (OC) neurons. A: Texas Red fluorescence from lateral OC (LOC) neurons distributed in the left lateral superior olivary nucleus (LSO). B: medial OC (MOC) neurons distributed in the left ventral nucleus of the trapezoid body (VNTB). C: crossed OC bundle running just below fourth ventricle. D: typical LOC neuron of fusiform shape. E: typical MOC neuron of multipolar shape. F1: Texas Red-labeled OC neuron viewed with epifluorescence optics. F2: same neuron viewed with Nomarski optics. A-E were taken from the fixed specimens; and F1 and F2 were taken during the electrophysiological experiments. Ages of rats: postnatal day (P) 7-8. Thickness of slice: 130-150 µm. M, medial; D, dorsal; IV, fourth ventricle.
|
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The labeled LOC neurons observed in the present study were fusiform or ovoid in shape (Fig. 1D). The MOC neurons were multipolar, triangular, or fusiform in shape (Fig. 1E). These morphological features were consistent with the previous anatomic studies (Vetter and Mugnaini 1992
; White and Warr 1983
).
After observation with the epifluorescence optics (Fig. 1F1), the neuron was identified with the Nomarski optics (Fig. 1F2) and the whole cell recording was made (Edwards et al. 1989
; Takahashi 1990
; Umemiya et al. 1993
).
After electrophysiological recordings, some slices were fixed with 7% Formalin in sodium phosphate buffer (pH 7.2) for 12 h. These slices were mounted on gelatin-coated slide glasses and were dried for 1 day. They were then dehydrated with ethanol, cleared with xylene, counterstained with cresyl violet, and coverslipped. These preparations were subjected to light microscopic observation, and anatomic features (locations and cell shapes) of the labeled neurons were confirmed.
Electrophysiological recording and data analysis
The whole cell recording configuration of the patch-clamp technique (Hamill et al. 1981
) was applied to the identified OC neurons. Patch pipettes were made from borosillicate glass capillaries (GC150TF-10, Clark) and had a resistance of 7-10 M
when filled with a potassium-gluconate-based internal solution containing (in mM) 135 potassium gluconate, 5 KCl, 5 NaCl, 1 MgCl2, 2 ATP, 0.1 guanosine 5
-triphosphate (GTP), 10 HEPES, and 0.1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), pH adjusted to 7.25 with KOH. The pipettes were coated with silicone resin (Sylgard 184, Dow Corning), and their tips were heat polished before use. Whole cell membrane currents and potentials were recorded by a patch-clamp amplifier (EPC-7, List). Both electrode capacitance and series resistance (up to 70%) were compensated electronically. The liquid junction potential was corrected (Hagiwara and Ohmori 1982
). All experiments were performed at room temperature (22-24°C).
Current and voltage output signals from the amplifier were filtered at 10 kHz through a four-pole low-pass filter with Bessel characteristics (UF-BL2, NF), sampled with a 12-bit A/D converter (ANALOG PRO 2, Canopus), and stored on a hard disk drive by a 32-bit personal computer (PC-9801FA, NEC). Off-line analyses were performed on a workstation (Micro VAX station 2000, DEC). Numerical data are presented as means ± SD, with n being the number of cells.
The resting membrane potential was measured just after the whole cell recording condition was achieved. Only the neurons that had a resting membrane potential more negative than
55 mV were used for further analyses. The input resistance and the input capacitance were estimated from the steady-state membrane current and the transient capacitative current in response to a small hyperpolarizing voltage step from a holding potential of
75 mV. There were no significant differences between the two groups of cells in the resting membrane potential (LOC:
63.6 ± 4.6 mV,mean ± SD, n = 55 cell; MOC:
66.7 ± 5.2 mV, n = 41 cells), input resistance (LOC: 445 ± 191 M
, n = 32 cells; MOC: 501 ± 303 M
, n = 28 cells), or input capacitance (LOC: 40.2 ± 19.6 pF, n = 30 cells; MOC: 35.2 ± 8.9 pF, n = 25 cells). This result for input capacitance seems inconsistent with the anatomic somatic size of OC neurons, because MOC is much larger than LOC (White and Warr 1983
). This may be because the input capacitance contains both somatic and dentritic components, and because the major components of the capacitance we have measured could be originated in dendrites. The transient capacitative currents we have examined in LOC and MOC neurons showed biexponential decay kinetics; the fast component should represent the somatic component, and the slow component the dendritic one (Rall 1977
). The average size of fast component was smaller in LOC cells (5.3 ± 2.6 pF, n = 30 cells) than in MOC cells (9.0 ± 3.4 pF, n = 25 cells).
Solutions
All experiments except for the recording of Ca2+ currents were performed with the use of the potassium-gluconate-based internal solution. All the current-clamp experiments and the measurement of the passive membrane properties were performed in the ACSF.
In the recording of outward currents, Na+ currents were blocked by 1 µM tetrodotoxin (TTX, Sankyo) added to the ACSF. Outward currents were recorded in LOC neurons in the ACSF with extracellular Ca2+ concentration ([Ca2+]o) reduced to 0.5 mM by replacement with Mg2+. In MOC neurons, outward currents were recorded in the normal ACSF (2 mM [Ca2+]o). Ca2+ currents were much smaller in MOC neurons than in LOC neurons, and their time course of decay was much slower than the decay of transient outward current (see Fig. 11). Therefore the influence of Ca2+ currents on the kinetics of outward currents was neglected in MOC neurons. Inorganic Ca2+ channel blockers (e.g., La3+, Cd2+, Co2+) were not used, because they likely shift the voltage dependence of transient outward currents (Klee et al. 1995
; Mayer and Sugiyama 1988
).

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| FIG. 11.
Ca2+ currents and its influence on outward currents. Outward currents were recorded in the normal ACSF (extracellular Ca2+ concentration [Ca2+]o = 2 mM), and were compared with the record in the low-[Ca2+]o (0.5 mM) ACSF in the same neuron (A and C). Ca2+ currents were elicited by the same pulse protocol as in A and C, and were recorded in the normal ACSF (B1 and D1) or in the 10 mM Ba2+ solution (B2 and D2) with the CsCl-e t h y l e n e g l y c o l - b i s ( - a m i n o e t h y l e t h e r ) N,N,N ,N -tetraacetic acid (EGTA)-based internal solution. Na+ currents were blocked by 1 µM TTX in all experiments. Calibrations in B2 are also for B1, and calibrations for D2 apply to D1 as well. B3 and D3: current-voltage relationships for the Ba2+ current (B2 and D2) plotted after leak subtraction. , peak current. , current at pulse end. B2: current was activated at potentials more positive than 20 mV, and was maximum at 10 mV. D2: low-voltage-activated fast decaying component was observed together with the slow decaying component. The low-threshold fast current was activated at potentials more positive than 50 mV, and the high-threshold slow current was activated at potentials positive to 20 mV. Note that the steady-state outward currents of both LOC and MOC neurons were slightly decreased when the Ca2+ concentration was reduced (A and C).
|
|
Ca2+ currents were recorded with a CsCl-EGTA-based internal solution composed of (in mM) 160 CsCl, 2 ATP, 0.1 GTP, 5 EGTA, and 10 HEPES, pH adjusted to 7.25 with CsOH. The external solution for the recording of Ca2+ currents was the normal ACSF (2 mM [Ca2+]o) or 10 mM Ba2+ external solution (composition, in mM; 130 NaCl, 2.5 KCl, 10 BaCl2, 0.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH); both were enriched with 1 µM TTX.
4-Aminopyridine (4-AP; 0.5, 1, or 2 mM) or tetraethylammonium chloride (TEA; 20 mM) was bath applied. TEA was added to the ACSF by replacing equimolar concentrations of NaCl.
 |
RESULTS |
Firing properties of OC neurons
Neither LOC nor MOC showed spontaneous firing at the resting membrane potential. Both neurons showed spike trains of tonic pattern in response to depolarizing current pulses with a slight frequency adaptation (Fig. 2, A and B). Figure 2C shows the spike frequency plotted against the intensity of injected current averaged from six LOC and eight MOC neurons. In both neurons, spike frequency increased almost linearly with an increase in the intensity of injected current pulses. There was no significant difference in firing frequency between LOC and MOC neurons at any intensity of injected current tested (P > 0.12). There was no marked difference in firing properties between LOC and MOC neurons when trains of action potentials were generated from the resting membrane potential.

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| FIG. 2.
A and B: firing properties of typical LOC (A) and MOC (B) neurons elicited at the resting membrane potential. Both neurons showed tonic firing properties. Top traces (superimposed): injected currents. m.p., membrane potential. C: firing frequency of OC neurons. Action potentials were generated at the resting membrane potential, and the mean numbers of action potentials were plotted against the intensity of injected currents (averaged from 6 LOC and 8 MOC neurons). Duration of current pulse: 200 ms.
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However, we observed a clear difference in firing properties between these two groups of neurons when trains of action potentials were generated from hyperpolarized membrane potentials (Fig. 3). When the LOC neuron generated a train of action potentials from the more hyperpolarized membrane potential, a more prolonged first interspike interval (ISI) was observed (Fig. 3A). In contrast, the MOC neuron generated a train of action potentials with almost constant ISIs, but the latency to the first spike was lengthened with hyperpolarization (Fig. 3B). These observations suggest a presence of outward current that could be inactivated at the resting membrane potential in both types of neurons.

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| FIG. 3.
A and B: modulation of firing properties by the negative shift of membrane potential. Firing properties were examined at 3 different membrane potentials in the same neuron. The most positive membrane potential among the 3 was the resting membrane potential (r.m.p.). The stimulus intensity was adjusted to generate equal number of spikes during the current injection for each LOC and MOC neuron (except for the record of LOC neuron at 90 mV). Pulse duration: 200 ms. A: LOC duration of the 1st interspike interval (ISI) was lengthened with the negative shift of membrane potential. B: MOC duration of the 1st spike latency became longer with membrane hyperpolarization. C and D: typical firing properties of the LOC and MOC neurons elicited from a hyperpolarized membrane potential ( 74 mV). Pulse duration: 400 ms. C: response of an LOC neuron (resting potential 66 mV) to the various depolarizing currents. D: response of an MOC neuron (resting potential 70 mV). Note small hyperpolarizing notch at the beginning of current injections (arrowheads) in both LOC and MOC neurons. The membrane was hyperpolarized by direct current. Dotted lines: 0 current levels. Duration of hyperpolarization before pulse command: >10 s.
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These differences in the firing property became even more obvious when the intensity of depolarizing current was increased systematically from a hyperpolarized membrane potential (Fig. 3, C and D). The LOC neuron generated action potentials with a long latency to the first spike, and showed a gradually depolarizing potential in response to a small injected current (<0.2 nA; Fig. 3C). When a larger current was injected (>0.3 nA), the first spike was generated immediately after the current pulse onset, and a long first ISI was observed. This long first ISI was followed by a train of spikes with almost regular ISIs. These firing properties were observed in 18 of 25 LOC neurons when recorded at the membrane potentials of
72 to
76 mV. The presence of a long first ISI seems to characterize LOC neurons. The other seven LOC neurons also showed the tonic firing pattern; however, they showed a long first spike latency (not the long 1st ISI) even when injected with 0.4-nA current; the neurons demonstrated a firing property similar to that of the MOC neurons, as described later.
The MOC neuron generated the first spike after a certain latency (Fig. 3D). The latency became shorter with the increase of the injected current. However, the MOC neuron did not generate the first spike at the current pulse onset in all the trials in which we investigated its nature from the hyperpolarized membrane potentials of
72 to
76 mV with limited current injections (<0.4 nA). These firing properties were observed in 17 of 22 MOC neurons. Another four neurons showed a long first ISI rather than the long first spike latency, and the remaining neuron showed neither the marked first spike latency nor the long first ISI at this potential range (
72 to
76 mV). A small hyperpolarizing notch (Fig. 3, C and D, arrowheads) was observed at the onset of current injections in both neurons. This notch may be produced by the activation of transient outward currents (Chandler et al. 1994
; Ducreux and Puizillout 1995
; Tell and Bradley 1994
).
The threshold of action potentials was measured by injecting depolarizing currents in both neurons. The mean threshold was
38.4 ± 4.9 mV in LOC neurons (n = 26 cells) and
46.9 ± 5.2 mV in MOC neurons (n = 25 cells), and was not affected by the level of membrane potential (Fig. 3, A and B).
The unique firing properties of LOC and MOC neurons in hyperpolarized conditions are quantitatively evaluated in Fig. 4. Figure 4, A and B, demonstrates the spike number plotted against the timing of occurrence of spike. These data are from the traces demonstrated in Fig. 3, C and D. The MOC neuron showed a stronger adaptation in firing frequency than the LOC neuron. Figure 4, C and D, demonstrates the duration of the first spike latency and the first ISI; both were plotted against the intensity of injected currents. These data are averaged from five LOC neurons (Fig. 4C) and five MOC neurons (Fig. 4D). In LOC neurons, the first spike was generated immediately after the pulse onset when the injected current was >0.3 nA (mean 1st spike latency was 11.9 ± 2.1 ms at 0.3 nA), whereas MOC neurons generated the first spike after a delay even with a strong (0.4 nA) current pulse (mean 1st spike latency was33.0 ± 15.1 ms at 0.4 nA). The mean first ISI in LOC neurons was ~2 times longer than the mean first spikelatency in MOC neurons when the injected current was>0.3 nA.

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| FIG. 4.
Quantitative evaluation of the firing properties of LOC and MOC neurons at hyperpolarized membrane potentials. A and B: number of spikes plotted against the timing of occurrence from the LOC and MOC neurons as demonstrated in Fig. 3, C and D. C and D: duration of the 1st spike latency ( ) and duration of the 1st ISI ( ) averaged from 5 LOC and 5 MOC neurons. Pulse duration: 400 ms. Membrane potential was hyperpolarized to the level from 72 to 76 mV in each neuron.
|
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Effects of 4-AP on firing properties of OC neurons
As shown in the previous section, the firing properties of LOC and MOC neurons were strongly affected by the level of membrane potential. This phenomenon suggests the existence of K+ conductances that are inactivated around the resting membrane potential. 4-AP-sensitive transient K+ current is one of the likely candidates for the underlying conductance. The presence of a hyperpolarizing notch at the onset of current pulses (Fig. 3, C and D, arrowheads) also supports this idea. We therefore investigated the effects of 4-AP on the firing properties of OC neurons.
When 2 mM 4-AP was bath applied, the first ISI became shortened in LOC neurons (from 87.3 ± 18.2 ms in control to 53.3 ± 19.0 ms with 4-AP, n = 3 cells), and action potentials were regularly generated during current injections (0.25-0.35 nA) from the hyperpolarized membrane potentials of
75 to
80 mV (Fig. 5A). The mean duration of action potentials was prolonged, particularly from the second action potential (181 ± 13% of control; mean duration after the 2nd spike was compared, n = 3 cells), and the level of spike afterhyperpolarization was reduced. In MOC neurons, the characteristic long latency to the first spike almost disappeared in the presence of 4-AP (0.5-1 mM; from34.0 ± 2.6 ms in control to 15.3 ± 5.8 ms with 4-AP, n = 3 cells) when currents (0.3-0.35 nA) were injected at the hyperpolarized membrane potentials of
80 to
90 mV (Fig. 5B). The hyperpolarizing notch at the current pulse onset disappeared or decreased in depth. The mean duration of action potentials was similarly lengthened (152 ± 10% of control; mean duration after the 2nd spike was compared, n = 3 cells), and the level of spike afterhyperpolarization was decreased.

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| FIG. 5.
Effects of 4-aminopyridine (4-AP) on the firing properties of LOC and MOC neurons. A: membrane potential was maintained at the hyperpolarized level of 75 mV, and depolarizing current (0.25 nA) was applied. Application of 2 mM 4-AP shortened the 1st ISI. B: application of 0.5 mM 4-AP shortened the 1st spike latency. Action potentials were elicited from the hyperpolarized membrane potential of 90 mV by injection of 0.35-nA current.
|
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4-AP influenced the firing features in both LOC and MOC neurons. Unique firing features of these neurons in the hyperpolarized condition disappeared, and they generated action potentials almost in a tonic pattern, similar to those elicited at the resting membrane potential (Fig. 2, A and B). Thus the 4-AP-sensitive, A-type K+ channel seems crucial in featuring the pattern of spike generation in these two neurons. Moreover, the difference in the firing properties between LOC and MOC neurons in the hyperpolarized condition might be due to the difference in the kind of A-type channel expressed in these neurons. Therefore we further investigated the K+ currents of LOC and MOC neurons in voltage-clamp experiments. At the beginning of each experiment we examined the firing properties of neurons, and then we recorded their K+ currents. We analyzed the currents in cells whose firing properties were typical for either LOC or MOC neurons as described above.
Outward currents in LOC neurons
When a series of step membrane depolarizations was applied to a typical LOC neuron after a prepulse to
110 mV, outward currents with a transient peak were observed at potentials more positive than
60 mV (Fig. 6A1). The outward current developed almost instantaneously, and decayed with fast and slow kinetics; apparently the current consisted of two components. When depolarizations were applied after a prepulse to
50 mV, the fast component disappeared (Fig. 6A2). Therefore the fast transient outward current was carried through a channel that was inactivated at depolarized membrane potentials. A part of the slowly decaying current was also suppressed by the prepulse to
50 mV, and the trace-to-trace subtraction of Fig. 6, A2 from A1, at corresponding voltages revealed a fast transient current followed by a slowly decaying component (Fig. 6A3). The decay kinetics of the whole cell currents was exponentially fit by the following function
|
(1)
|
where A1 and A2 are amplitudes,
1 and
2 are time constants for the decay, and C is the time-independent component. Figure 6, B1-B3, illustrates three traces with an exponential fitting to the decaying kinetics. The first two traces were recorded by step depolarization to 0 mV after a prepulse to
110 mV (Fig. 6B1) or
50 mV (Fig. 6B2), and the third trace was made by subtraction of trace B2 from trace B1 (Fig. 6B3). Trace B1 could be fit with a biexponential function with two time constants of 63 and 868 ms, and trace B2 with a monoexponential function with a time constant of 1,137 ms. The subtracted trace B3 was best fit by a biexponential function with almost the same combination of time constant and amplitude for the fast component as in the fitting to trace B1. From the comparison of amplitudes between traces B1 and B3, the entire fast transient current and 30% of the slow component were left after subtraction. So, the voltage ranges of inactivation of these two current components partly overlapped at
50 mV. These two components could not be separated by this trace subtraction procedure. In this paper, therefore, the amplitudes and the time constants of these fast and slow current components were estimated from biexponential fitting to the total outward current (Fig. 6B1).

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| FIG. 6.
Outward currents and their sensitivity to K+ channel blockers in the LOC neuron. Outward currents were recorded in a low-Ca2+ (0.5 mM)/high-Mg2+ (2.5 mM) extrac e l l u l a r m e d i u m c o n t a i n i n g t e t r o d o t o x i n(TTX, 1 mM). Insets: pulse protocols. A1: outward currents were activated by depolarization of 2,000 ms ranging from 80 to +20 mV in 10-mV steps, applied after a 500-ms prepulse to 110 mV. Holding potential: 70 mV. Transient outward currents with fast and slow decaying kinetics were observed. A2: outward currents were activated from a prepulse of 500 ms to 50 mV by depolarizing and hyperpolarizing pulses (from 80 to +20 mV) of 2,000 ms. Note that the fast component was completely inactivated, whereas the slow component still remained. A3: trace A2 subtracted from A1. In addition to the fast component, the slow component was left to some extent. B1-B3: exponential fitting to the traces in A1-A3 (at 0-mV test potential). B1 was best fit by a biexponential function (Eq. 1). B2 was best fit monoexponentially, and B3 was best fit biexponentially. The amplitude and the decay time constant ( ) of the fast component were not significantly changed before (B1) and after (B3) subtraction. C: effects of 4-AP on the fast (IA-LOC) and slow (IKD) components of outward currents in LOC neurons. Amplitudes of IA-LOC and IKD were calculated by biexponential fitting to the total outward current. Application of 2 mM 4-AP reduced the amplitude of IA-LOC by 78% (at +20 mV), but IKD was enhanced by 18%. The effect was partially reversed by washing. D:e f f e c t s o f t e t r a e t h y l a m m o n i u m c h l o r i d e(TEA) on IA-LOC and IKD. Application of 20 mM TEA reduced the amplitude of IKD by 28% and reduced the amplitude of IA-LOC by 14% (at +20 mV). The effect was reversed by washing.
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These two currents had distinct pharmacological properties. When 4-AP (2 mM) was bath applied, most of the fast component disappeared (78 ± 10%, n = 7 cells; compared at +20 mV), whereas the amplitude of the slow component was enhanced (Fig. 6C). A similar increase of the slow component was observed in all seven cells tested. This might reflect an improvement of space-clamp condition by the block of 4-AP-sensitive K+ conductance. When TEA (20 mM) was bath applied, the fast component was slightly suppressed by 12 ± 10% (n = 7 cells, at +20 mV), and the slow component was blocked by 40 ± 8% (n = 7 cells, at +20 mV; Fig. 6D). The blocking effect of TEA was not as selective or extensive as that of 4-AP. We concluded, however, that these outward currents in LOC neurons consisted of at least two distinct currents, because there were several differences between these two currents in the sensitivity to the blockers, in the voltage dependence of inactivation, and in the decay kinetics. In this paper, we tentatively call the fast component IA-LOC, and the slow component IKD (Fig. 6B1). We are certain that IA-LOC is a single component because of its high sensitivity to 4-AP and its clear voltage dependence of inactivation kinetics, but we are not certain about IKD.
Transient outward currents in LOC neurons
Two components of the transient outward current in the LOC neuron were separated by the exponential fitting, and their steady-state natures are analyzed in Fig. 7. A series of outward currents was generated after a prepulse to
110 mV in Fig. 7A, and the peak amplitudes of the currents (
) are plotted against the test pulse potentials in Fig. 7B. By biexponential fitting, amplitudes of two current components were estimated and are plotted in Fig. 7B (
for IA-LOC and
for IKD). IA-LOC was activated at potentials more positive than
70 mV, IKD at about
50 mV. When outward currents were generated at 0 mV after a series of 5-s prepulses ranging from
110 to 0 mV (Fig. 7C), the voltage dependence of steady-state inactivation could be estimated. IA-LOC and IKD were separated by the biexponential fitting as performed previously. The peak of the total outward current (
),IA-LOC (
), and IKD (
) were plotted against the prepulse potentials (Fig. 7D). IA-LOC was completely suppressed at membrane potentials more positive than
60 mV, whereas the amplitude of IKD decreased gradually with depolarization. This difference in the voltage dependency of inactivation confirms that IA-LOC and IKD are distinct currents.

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| FIG. 7.
Voltage dependence of activation and inactivation kinetics of IA-LOC and IKD. A: outward currents activated by the same pulse protocol as in Fig. 6A1. B: Current-voltage relationships of the peak current (Ipeak) ( ), IA-LOC ( ), and IKD ( ). C: steady-state inactivation of the outward currents. Durations of prepulse and test pulse were 5 and 2 s, respectively. Both fast and slow components were inactivated during the long prepulse. D: current-voltage relationships of the peak current ( ), IA-LOC ( ), and IKD ( ). Amplitudes of IA-LOC and IKD were calculated by exponential fitting as in B. E: activation and steady-state inactivation kinetics of IA-LOC. Mean conductances for activation ( ; n = 8 cells) and inactivation ( ; n = 6 cells) were plotted against membrane potentials after normalization and were fit by a Boltzmann-type equation (Eq. 2). Ordinate: ratio of conductance (G) relative to the maximum conductance (Gmax). F: voltage dependence of the activation and inactivation of IKD. Amplitudes of IKD were normalized to the current at 20 mV ( ; activation) and 110 mV ( ; inactivation), and mean values were plotted against the test pulse ( ; n = 6 cells) or prepulse ( ; n = 6 cells) potentials. Vertical dashed lines in E and F: mean resting potential of LOC neurons.
|
|
The voltage dependence of IA-LOC activation was estimated as a voltage dependence of the conductance; current amplitudes plotted in Fig. 7B (
) were divided by the driving force for the K+ current assuming a potassium equilibrium potential of
104 mV. The conductances were normalized to the maximum value, and the mean values from eight tested neurons were plotted against the test pulse potentials (Fig. 7E,
). The voltage-dependent curve was fit by the following Boltzmann-type equation
|
(2)
|
with V1/2 =
48.2 mV and k =
12.4 mV (n = 8 cells). The voltage dependence of IA-LOC inactivation was similarly estimated, and is also shown in Fig. 7E (
). The normalized current amplitudes averaged from six tested neurons were plotted against the prepulse potentials, and were fit by Eq. 2, with V1/2 =
71.9 mV and k = 7.6 mV (n = 6 cells).
Current amplitudes of IKD shown in Fig. 7B were normalized to the maximum current recorded at +20 mV, and mean values of tested neurons are plotted against the test pulse potentials in Fig. 7F (
; n = 6 cells). Amplitudes of IKD in Fig. 7D were normalized to the maximum current at
110 mV, and are similarly plotted against the prepulse potentials as average values (
; n = 6 cells). The inactivation of IKD was voltage dependent. The amplitude of IKD increased with supralinear voltage dependence, and this indicates a presence of voltage-dependent activation kinetics (Fig. 7F). We have not further analyzed these voltage-dependent kinetics of IKD.
The voltage dependence of the steady-state inactivation suggests an extensive change in the open probability of the K+ channel, especially the channel responsible for IA-LOC. The degree of inactivation of IA-LOC (Fig. 7E,
) changed between ~10% and 50% within a ±10-mV range of the mean resting membrane potential (
63.6 mV; Fig. 7E, vertical dashed line). The K+ channel responsible for IA-LOC was activated (Fig. 7E,
) by ~70% at the threshold(
38.4 ± 4.9 mV) of the action potential in LOC neurons. This may indicate a possibility of sensitive and effective modulation of the membrane excitability around the resting membrane potential as a consequence of the voltage-dependent change of the open probability of IA-LOC. These natures of the K+ channel could explain the voltage dependence of firing properties as shown in Fig. 3A. So far, we do not have any clear explanation of the contribution of IKD to the firing properties of LOC neurons.
Transient outward current in MOC neurons
Figure 8A shows a series of outward currents elicited by membrane depolarizations applied to a typical MOC neuron. When the membrane was depolarized from a holding potential of
90 mV, transient outward currents were observed as in the LOC neuron at potentials more positive than
60 mV. When outward currents were generated at 0 mV after a prepulse to from
110 to
55 mV, the peak amplitude of the outward current was decreased with the depolarization of prepulse (Fig. 8B). Figure 8C illustrates outward currents elicited at 0 mV after a prepulse to
110 mV (C1) or
55 mV (C2). The transient current was completely suppressed by the prepulse to
55 mV, but the amplitude of steady-state component was not changed. The transient component of the outward current was suppressed by 4-AP (Fig. 8D). The peak amplitude of the total outward current was decreased to 71% of the control (at +20 mV), whereas the steady-state component was not affected by 0.5 mM 4-AP. Therefore, from the voltage dependence (Fig. 8C) and the 4-AP sensitivity (Fig. 8D), the transient outward current in the MOC neuron was confirmed as distinct from the steady outward current. The 4-AP-sensitive transient current was satisfactorily fit by a monoexponential function, and we call this current IA-MOC in this paper (Fig. 8E). IA-MOC was suppressed by 4-AP (0.5-1 mM) by 85 ± 13% (n = 6 cells at +20 mV).

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| FIG. 8.
Transient outward current in the MOC neuron. Outward currents were recorded in artificial cerebrospinal fluid (ACSF) containing 1 µM TTX. Insets: pulse protocols. A: outward currents were activated by 400-ms step depolarizations applied from a holding potential of 90 mV to +20 mV in 10-mV steps. B: steady-state inactivation of the outward currents. Currents were elicited at 0 mV after a prepulse ranging from 110 to 55 mV in 5-mV steps. Duration of both prepulses and test pulses: 400 ms. Holding potential: 70 mV. C: separation of the transient and the steady outward currents. Traces from B. C1: outward current was elicited at 0 mV from the prepulse to 110 mV. C2: current was elicited at 0 mV from the prepulse to 55 mV. D: effect of 4-AP on outward currents in the MOC neuron. E: total outward current trace was best fit monoexponentially. This fast transient outward current is called IA-MOC in this paper. F: voltage-dependent activation and inactivation of IA-MOC. Amplitudes of IA-MOC were calculated by monoexponential fitting. Mean normalized conductances for activation (n = 6 cells) and inactivation (n = 5 cells) were plotted against the test pulse potentials ( ; activation) or prepulse potentials ( ; inactivation). They were fit by a Boltzmann-type equation (Eq. 2). Vertical dashed line: mean resting potential of MOC neurons.
|
|
The voltage-dependent activation of IA-MOC was estimated from the pulse protocol shown in Fig. 8A, as performed in the LOC neuron. The mean normalized conductances for six tested neurons are plotted against the test pulse potentials in Fig. 8F (
). The steady-state inactivation of IA-MOC was estimated from the current traces in Fig. 8B. Current amplitudes were normalized to the maximum measured after a prepulse to
110 mV. Mean values of five tested neurons are also plotted in Fig. 8F (
) as a function of prepulse potential. These two voltage-dependent curves were fit by Eq. 2, with V1/2 =
41.9 mV and k =
8.9 mV (n = 6 cells) for the activation and V1/2 =
75.0 mV and k = 6.8 mV (n = 5 cells) for the inactivation.
The steady-state inactivation of IA-MOC (Fig. 8F,
) greatly changed between ~0% and 50% within a ±10-mV range of the mean resting membrane potential (
66.7 mV; Fig. 8F, vertical dashed line). About 40% of IA-MOC could be activated (Fig. 8F,
) at the threshold of action potentials (
46.9 ± 5.2 mV). This suggests a possibility of sensitive modulation of the membrane excitability around the resting membrane potential in MOC neurons, as was suggested in LOC neurons. These voltage dependencies are consistent with the firing features of the MOC neuron when the membrane potential was changed (Fig. 3B).
Kinetics of transient outward currents
The decay time constants (
) of the transient outward currents were calculated for IA-LOC, IA-MOC, and IKD at various membrane potentials (
50 to +20 mV), and the mean values are plotted against the potentials in Fig. 9A. All three decay
values became smaller with the positive shift of the membrane potential. The decay kinetics of IA-MOC was the fastest, with
= 33.0 ± 3.5 ms (n = 6 cells) at 0 mV, whereas that of IA-LOC was 85.5 ± 19.0 ms (n = 6 cells), and that for IKD was 853 ± 195 ms (n = 6 cells). This tendency of relative speed of decay kinetics was the same in these three K+ currents throughout the membrane potentials investigated (
50 to +20 mV). These differences in the inactivation time constants of the outward current between LOC and MOC neurons can be one of the factors to determine the nature of their firing in the hyperpolarized condition (Figs. 3, C and D, and 4, C and D). The difference in decay
values of IA-LOC and IA-MOC may be responsible for the difference in the duration of the first spike latency or the first ISI between LOC and MOC neurons.

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| FIG. 9.
Inactivation and activation time courses of outward currents. A: voltage dependence of the decay of transient outward currents. All values were estimated by exponential fitting to the pulse protocols as demonstrated in Figs. 6A and 8A. Mean values were plotted against the test pulse potentials. Left ordinate: IA-LOC and IA-MOC. Right ordinate: IKD. B: activation phase of the total outward current in LOC and MOC neurons. Currents were elicited at 40 mV from a holding potential of 90 mV, and the peak amplitudes were normalized. In this measurement, the time to half was measured from the end of the capacitative transient to the time at half of the peak amplitude. This shortened the time to half by ~1 ms compared with that measured from the timing of voltage pulse onset. Time to half was 3.0 ms in the LOC neuron and 1.5 ms in the MOC neuron. C: time to half of total outward currents at various test potentials ( 50 to +20 mV) averaged from 5 LOC and 6 MOC neurons.
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|
There was a similar difference between LOC and MOC neurons in the activation time course of their outward currents. The activation kinetics of these outward currents was much faster than the decay kinetics, and we have made a rough estimate of the activation of these outward currents. Figure 9B illustrates the activation process of the total outward currents recorded in typical LOC and MOC neurons superimposed. These currents were elicited at
40 mV from a holding potential of
90 mV, and the peak amplitudes were normalized. The time between the onset of step depolarization (strictly, the end of capacitative transient) and the time at the half of peak amplitude (defined here as "time to half") was measured as the indication of speed of activation. The activation was faster in the MOC neuron than in the LOC neuron. Outward currents were evoked at various testpotentials (
50 to +20 mV; 5 LOC neurons, 6 MOC neurons) from a holding potential of
90 mV, and mean values of the time to half were plotted against the test potentials (Fig. 9C). The time to half was nearly 2 times larger in LOC neurons than in MOC neurons irrespective of the potential. These differences in the activation speed of outward currents between LOC and MOC neurons, especially at potentials around the action potential threshold(
38.4 ± 4.9 mV in LOC neurons,
46.9 ± 5.2 mV in MOC neurons), can be one of the factors to determine the timing of the first spike (Figs. 3, C and D, and 4, C and D). The slow activation of the total outward current in LOC neurons may not suppress the generation of the first spike at the current pulse onset, whereas the spike generation is likely delayed by the fast activation of the outward current in MOC neurons.
The time-dependent recovery kinetics from inactivation was faster in the MOC neuron than in the LOC neuron (Fig. 10). Outward current was generated in the LOC neuron by a long depolarization to 0 mV (3 s), and the current was inactivated (Fig. 10A). After a step back to the holding level of
90 mV (10-150 ms in duration), a depolarization to 0 mV (500 ms in duration) was applied. Amplitudes of IA-LOC were measured by subtraction of the current measured at 500 ms from the peak current for both pre- and test depolarization steps. Relative amplitudes (IA-LOC
test/IA-LOC
pre) were plotted against the duration of intervals (Fig. 10B), and the time course of recovery was fit by the following exponential function
|
(3)
|
The mean recovery time constant (
rec) was 32.4 ± 4.8 ms (n = 4 cells) for LOC neurons. In the MOC neuron, the duration of both prepulses and test pulses was 200 ms, because the kinetics was faster in IA-MOC (Fig. 10C). The interval between these two pulses was increased from 2 to 36 ms in 2-ms steps. Amplitudes of IA-MOC were measured by subtraction of the steady-state current from the peak current. Figure 10D illustrates the time course of recovery of IA-MOC. The mean
rec was 14.8 ± 2.8 ms (n = 4 cells) for MOC neurons.

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| FIG. 10.
Time-dependent recovery kinetics from inactivation of the A-current (IA) in the LOC and MOC neurons. The kinetics was examined by double-pulse protocols with increasing interval (insets). In both neurons, each pulse was elicited by step depolarization from a holding potential of 90 mV to 0 mV. A: durations of prepulse and test pulse were 3 s and 500 ms, respectively. The interval between 2 pulses was increased from 10 to 150 ms in 10-ms steps (every other trace is shown). B: relative amplitudes (IA-LOC test/IA-LOC pre) plotted against the duration of intervals, and fit by the function in Eq. 3. Recovery time constant ( rec): 33.5 ms. C: duration of both prepulse and test pulse: as 200 ms. The interval between 2 pulses was increased from 2 to 36 ms in 2-ms steps (every other trace is shown). D: relative amplitudes(IA-MOC test/IA-MOC pre) plotted against the duration of intervals, and fit by the function in Eq. 3. rec: 11.8 ms.
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Ca2+ currents in OC neurons
In this study we elicited outward currents in a medium with reduced [Ca2+]o (0.5 mM) in LOC neurons. This is because the time course of outward currents was seriously affected by the flow of Ca2+ current in a normal medium (2 mM [Ca2+]o). Figure 11A illustrates the outward currents recorded in the normal medium with 2 mM [Ca2+]o and in the medium with reduced (0.5 mM) [Ca2+]o in the same LOC neuron. When recorded in 2 mM [Ca2+]o, the decay time course of the fast component was accelerated, and the slow component was not clear especially at potentials more positive than
10 mV. Because Ca2+ currents affected the decay time course of outward current in a concentration-dependent manner, we felt it necessary to know the amount of Ca2+ current flowing through the membrane. Furthermore, we wanted to clarify the current's contribution, if any, to the firing properties of the neurons. In the following analyses, we measured the Ca2+ current both in the standard 2 mM [Ca2+]o medium and in 10 mM [Ba2+]o medium. Standard 2 mM [Ca2+]o was used to know the normal level of Ca2+ current, and 10 mM [Ba2+]o to know the channel kinetics more clearly. Figure 11B1 illustrates the Ca2+ currents in the LOC neuron recorded with the CsCl-EGTA-based intracellular medium in the normal ACSF (2 mM [Ca2+]o). Ca2+ currents were generated after a prepulse to
110 mV by a series of depolarizations with the same pulse protocol as used in Fig. 11A. The mean maximum current amplitude was 850 ± 438 pA (n = 4 cells). Figure 11, B2 and B3, shows the current through the Ca2+ channel enhanced by recording in the 10 mM [Ba2+]o medium, and its current-voltage relationship. Irrespective of the external medium (2 mM [Ca2+]o or 10 mM [Ba2+]o), the currents through the Ca2+ channel were activated at membrane potentials more positive than
20 mV, were maximum at
10 mV, and demonstrated biphasic (fast and slow) decay kinetics. The decay
of the fast component (e.g.,
= 53 ms in Fig. 11B1) was comparable with that of IA-LOC, and the inward flow of Ca2+ currents will substantially mask the time course of IA-LOC as shown in Fig. 11A. However, the mean maximum amplitude of Ca2+ currents was <100 pA when recorded in 0.5 mM [Ca2+]o (n = 4 cells) and <7% of the mean peak amplitude of the outward current measured at
10 mV(1.53 ± 0.31 nA; n = 13 cells). This presence of small Ca2+ currents was neglected in our analysis of outward currents recorded in 0.5 mM [Ca2+]o. The contribution of Ca2+ currents to the first spike generation could be minimum in LOC neurons, because the Ca2+ currents were activated at potentials (
20 mV or greater) more positive than the action potential threshold (
38.4 ± 4.9 mV).
In MOC neurons, Ca2+ currents were much smaller than in LOC neurons. When recorded in the normal (2 mM) [Ca2+]o medium (Fig. 11D1), the mean maximum amplitude (at
10 mV) was 246 ± 154 pA (n = 4 cells), and at
10 mV it was 17% of the mean peak amplitude of the outward current (1.44 ± 0.35 nA, n = 13 cells). The Ca2+ currents decayed with
values of >500 ms, and this decay kinetics was much slower than that of IA-MOC. The shape of outward currents was not remarkably changed between recordings in 2 mM and 0.5 mM [Ca2+]o medium (Fig. 11C). The decay phase of outward currents followed a single-exponential time course in both media, and the difference in current amplitudes of IA-MOC between these two solutions was <10% in four neurons tested. This small influence of the Ca2+ currents on IA-MOC may be due to the slow decay kinetics of the Ca2+ currents. Therefore we have neglected the presence of Ca2+ currents in the recording of outward currents of MOC neurons. When the current through the Ca2+ channel was enhanced by the 10 mM [Ba2+]o medium, a low-voltage-activated fast decaying component was observed in addition to the slow decaying component (Fig. 11, D2 and D3). Neither the influence of this fast decaying component on the outward currents nor its contribution to the firing property was clear in the present study.
There were quantitative and qualitative differences in Ca2+ currents between LOC and MOC neurons. Moreover, steady-state outward currents of both LOC and MOC neurons were slightly decreased (5-15%) when [Ca2+]o was reduced (Fig. 11, A and C). This might be due to a presence of Ca2+-activated K+ current as a component of the steady-state current. However, we did not investigate these matters further in this paper.
 |
DISCUSSION |
The principal finding of the present study is that two identified OC neurons demonstrated different electrophysiological properties, although they had some characteristics in common. There was no marked difference in the firing properties from the resting membrane potential between LOC and MOC neurons (Fig. 2). Notable difference emerged in the firing properties when action potentials were generated from a slightly hyperpolarized membrane potential (Fig. 3, C and D). Both neurons showed a silent period before the onset of repetitive firing. LOC neurons had tendency to generate the first spike immediately after the pulse onset, and the silent period emerged as a long first ISI. In contrast, MOC neurons showed a certain delay to the first spike. These silent periods were usually longer in LOC neurons than in MOC neurons (Fig. 4, C and D).
These differences in firing properties between LOC and MOC neurons should be a consequence of the differences in the kinetics of their transient outward currents. The difference in the duration of the silent period could be largely due to the difference in decay
values of the fast transient outward currents in LOC and MOC neurons (Fig. 9A) rather than to the difference in their voltage dependence of inactivation (V1/2 =
71.9 mV, k = 7.6 mV in IA-LOC vs.V1/2 =
75.0 mV, k = 6.8 mV in IA-MOC). The difference in first spike generation could be attributed to the difference in the speed of activation of the total outward currents (Fig. 9C), rather than to the contribution of Ca2+ currents in LOC neurons (Fig. 11, B1-B3). The Ca2+ currents were only activated at more positive membrane potentials (
20 mV or greater) than the action potential threshold (
38.4 ± 4.9 mV) in LOC neurons.
In this study we used rats in neonatal stages (P5-P11). At later postnatal days, the growth of fibers was so dense that we could hardly identify labeled neurons or apply the patch-clamp technique. OC neurons are reported to be almost the same as adult neurons already at P0 in number and pattern of distribution (Robertson et al. 1989
). The synapse of the LOC terminal onto the afferent dendrites innervating the inner hair cell is well developed already at P1 (Lenoir et al. 1980
), and the innervation of the MOC neurons to the outer hair cell is close to the mature pattern by P6 (Cole and Robertson 1992
). Therefore the OC neurons at the stage of our experiment (P5-P11) seem to be almost matured morphologically. However, it remains uncertain whether they have the same electrophysiological properties as the adult animals or not.
Properties of MOC neurons
Previous studies performed in vivo recorded the spontaneous and sound-evoked single-unit activities of MOC neurons in their axons (Brown 1989
; Cody and Johnstone 1982b
; Fex 1962
; Liberman 1988a
,b
; Liberman and Brown 1986
; Robertson 1984
; Robertson and Gummer 1985
). In these reports, 12-18% of MOC fibers showed spontaneous firing. Both spontaneous and sound-evoked discharges of MOC fibers were regular, whereas both were irregular in auditory afferent fibers. The spike frequency increased approximately in proportion to the intensity of sound stimulation. The latency to the discharge from the onset of sound stimulation in each MOC unit was 10-40 ms. The intrinsic membrane properties of MOC neurons revealed in our study are generally consistent with those in vivo observations, although we have never seen any spontaneous spike activities. This difference may indicate the possibility that those spontaneous firings are due to the spontaneous synaptic inputs, not to their intrinsic membrane properties.
IA-MOC in the present study demonstrated similar properties to the fast transient outward currents (IA or A-current) reported in other mammalian neurons (Huguenard et al. 1991
; Klee et al. 1995
; McFarlane and Cooper 1991
; Numann et al. 1987
; Wu and Barish 1992
). They inactivated rapidly(
= 10-30 ms), had V1/2 values of
80 to
60 mV, showed a fast recovery from inactivation (
rec = 10-60 ms), and were blocked by low concentrations (
1 mM) of 4-AP. The firing properties of other central neurons that possess IA are similar to those of the MOC neuron in that they have some onset delay, and the duration of delay is voltage dependent and 4-AP sensitive (Ducreux and Puizillout 1995
; Wang and McKinnon 1995
).
Properties of LOC neurons
The firing property observed in LOC neurons is characterized by a long first ISI, and this behavior is very rare among mammalian central neurons. There are several reports of neurons with this property; for example, neurons in vagal motor nucleus (Yarom et al. 1985
), in superior colliculus (Lopez-Barneo and Llinás 1988
), in dorsal cochlear nucleus (Manis 1990
), and in nucleus tractus solitarii (Bradley and Sweazey 1992
).
IA-LOC demonstrated different properties from the typical fast transient outward current in the other reports (Huguenard et al. 1991
; Klee et al. 1995
; McFarlane and Cooper 1991
; Numann et al. 1987
; Wu and Barish 1992
). IA-LOC decayed relatively slowly (
= 60-120 ms) and demonstrated a relatively low sensitivity to 4-AP; IA-LOC wasnot completely blocked by 4-AP even at the concentration of 2 mM.
Several studies in mammalian neurons have previously reported slow inactivating outward currents apparently similar to IKD in this study. The current called ID was sensitive to dendrotoxin and to low concentrations (
100 µM) of4-AP (Luthi et al. 1996
; Storm 1988
; Wu and Barish 1992
). Other currents were diversely named (e.g., IK, IKS, IAS), and the sensitivity to the blockers (e.g., 4-AP, TEA, dendrotoxin) was not uniform among them (Foehring and Surmeier 1993
; Huguenard and Prince 1991
; Klee et al. 1995
; Nisenbaum et al. 1994
). These currents usually had a slow inactivation kinetics with a time constant of several hundreds of milliseconds to several seconds, and showed a slow kinetics in the recovery from inactivation (
rec = 200 ms-20 s). These neurons, which possessed such slow inactivating currents, showed a gradually depolarizing potential and demonstrated a very long first spike latency (Huguenard and Prince 1991
; Luthi et al. 1996
; Nisenbaum et al. 1994
; Storm 1988
). In LOC neurons in this study, the first spike latency of 810 ms maximum was observed (not shown). These slow inactivating currents were considered to enable a neuron to integrate the subthreshold depolarizing synaptic inputs over a long period of time (Storm 1988
). So far, we are not able to demonstrate this integrative function in LOC neurons.
Comparison with other auditory neurons
There are numerous reports about the auditory brain stem neurons in slice preparations. The bushy cells in anteroventral cochlear nucleus generated a single action potential at the onset of a depolarizing pulse (Wu and Oertel 1984
). Similar phasic properties are reported in the medial superior olivary nucleus (Smith 1995
) and the medial nucleus of the trapezoid body (Banks and Smith 1992
; Wu and Kelly 1991
). The large and low-threshold sustained outward currents that limit the timing of action potential generation at the pulse onset seem to determine these phasic properties (Forsythe and Barnes-Davies 1993
; Manis and Marx 1991
). On the other hand, the stellate cells in anteroventral cochlear nucleus (Wu and Oertel 1984
) and posteroventral cochlear nucleus (Oertel et al. 1990
) showed tonic firing properties. The principal cells of LSO, which are located together with the LOC neurons in the same nucleus, also showed a tonic firing property (Wu and Kelly 1991
). These tonic neurons showed a regular discharge at the resting membrane potential, and this does not seem to be different from LOC and MOC neurons at resting potential. However, there are no reports in these tonic neurons about their outward currents, nor about their firing properties when the membrane potential was changed from the rest. Therefore it is not clear whether OC neurons have the same firing properties as these tonic neurons or not. The simple-spiking cells in dorsal cochlear nucleus (Manis 1990
) could be the only reported auditory brain stem neurons whose discharge properties are similar to those of OC neurons, as far as we know.
Possible functional significance
MOC neurons had basically a tonic discharge property, and produced a quasilinear increase in firing frequency in response to the increase of injected current at the resting membrane potential. This tonic property should be suitable for making a feedback suppression to the cochlea in proportion to the intensity of sound stimulus. The functional role of the onset delay manifested in the hyperpolarized condition is not clear, but it might serve as a gate to the short synaptic inputs, as suggested by Cassell and McLachlan (1986)
.
LOC neurons could show a variety of firing properties in a single neuron depending on the membrane potential and the stimulus intensity. Relatively small fluctuation of the membrane potential could result in a substantial change in the initial phase of firing. Preceding inhibitory synaptic inputs or, if any, metabotropic synaptic responses that hyperpolarize the membrane, can change the linear input-output relationship to a nonlinear one. This neuron may play a profound functional role in auditory signal coding in addition to antagonistic regulation (facilitation and inhibition) with the MOC neuron.