Bushy cells in the ventral cochlear nucleus convey firing of auditory nerve fibers to neurons in the superior olivary complex that compare the timing and intensity of sounds at the two ears and enable animals to localize sound sources in the horizontal plane. Three voltage-sensitive conductances allow bushy cells to convey acoustic information with submillisecond temporal precision. All bushy cells have a low-voltage-activated, α-dendrotoxin (α-DTX)-sensitive K+ conductance (gKL) that was activated by depolarization past −70 mV, was half-activated at −39.0 ± 1.7 (SE) mV, and inactivated ∼60% over 5 s. Maximal gKL varied between 40 and 150 nS (mean: 80.8 ± 16.7 nS). An α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, K+ conductance (gKH) was activated at voltages positive to −40 mV, was half-activated at −18.1 ± 3.8 mV, and inactivated by 90% over 5 s. Maximal gKH varied between 35 and 80 nS (mean: 58.2 ± 6.5 nS). A ZD7288-sensitive, mixed cation conductance (gh) was activated by hyperpolarization greater than −60 mV and half-activated at −83.1 ± 1.1 mV. Maximum gh ranged between 14.5 and 56.6 nS (mean: 30.0 ± 5.5 nS). 8-Br-cAMP shifted the voltage sensitivity of gh positively. Changes in temperature stably altered the steady-state magnitude of Ih. Both gKL and gKH contribute to repolarizing action potentials and to sharpening synaptic potentials. Those cells with the largest gh and the largest gKL fired least at the onset of a depolarization, required the fastest depolarizations to fire, and tended to be located nearest the nerve root.
Bushy cells receive information about the timing and fine structure of sounds from the temporal firing patterns in auditory nerve fibers and convey it to the superior olivary complex. Neurons in the MSO use phase-locking by large spherical bushy cells in the encoding of low-frequency sounds to compute the interaural phase and thus the relative time of arrival, of sounds at the two ears (Joris et al. 1998; Yin 2002). Neurons in the LSO compare the timing and frequency of excitation from ipsilateral small spherical bushy (Cant and Casseday 1986) and T (or planar) stellate cells (Doucet and Ryugo 2003) with inhibition from the medial nucleus of the trapezoid body (MNTB) that reflects the timing and frequency of firing of contralateral globular bushy cells in responses to high-frequency sounds to compute the relative intensities of sounds at the two ears (Tollin and Yin 2005; Yin 2002).
The three subtypes of bushy cells have been described in mammals that differ subtly in size and histological staining as well as in projection patterns: large spherical, small spherical, and globular bushy cells (Brawer et al. 1974; Cant and Casseday 1986; Cant and Morest 1979a,b; Osen 1969; Tolbert and Morest 1982a,b; Tolbert et al. 1982). Large spherical bushy cells in the rostral anterior VCN encode mainly low-frequency sounds and project to the lateral tuft in the ipsilateral and the medial tuft of dendrites of neurons in the contralateral MSO (Smith et al. 1993). Mice have little low-frequency hearing (Ehret 1974), a small and inconspicuous MSO and also few large spherical bushy cells (Willard and Ryugo 1983). In mice most bushy cells are of the globular and small spherical subtypes. Globular bushy cells are generally located near the root of the auditory nerve (Liberman 1991, 1993; Spirou et al. 1990; Tolbert and Morest 1982a,b; Tolbert et al. 1982), encode sounds of higher frequencies, and project to the MNTB through large axons that end in very large terminals, the calyces of Held (Brownell 1975; Liberman 1991; Sento and Ryugo 1989; Smith et al. 1991; Tolbert et al. 1982). Small spherical bushy cells are least well understood. Many project to the ipsilateral LSO, but it is unclear whether small spherical bushy cells or T stellate cells are the predominant source of ipsilateral excitation (Cant and Casseday 1986; Doucet and Ryugo 2003). Several lines of evidence indicate that globular and small spherical bushy cells are distinct. First, individually labeled globular bushy cells do not innervate the LSO (Smith et al. 1991). Second, monosynaptic, ipsilateral excitation of the LSO is matched in timing with disynaptic, contralateral inhibition through the MNTB and is therefore likely to be mediated through more slowly conducting axons (Joris and Yin 1995). In cats, the axons of globular bushy cells have exceptionally large diameters, small spherical bushy cells presumably have axons of intermediate diameter, and axons of T stellate cells have small diameters (Brownell 1975; Joris 1996; Tolbert et al. 1982).
Early recordings showed that bushy cells fire only one or two action potentials at the onset, whereas stellate cells fire tonically in response to a suprathreshold depolarizing current pulse (Fujino and Oertel 2001; Oertel 1983; Schwarz and Puil 1997; Wu and Oertel 1984). It has been reported that although all bushy cells fire transiently when they are depolarized, some seem to fire more than just one or two action potentials and require smaller currents to reach threshold (Francis and Manis 2000; McGinley and Oertel 2006; Wang and Manis 2006). Is it possible that early recordings were from only one of the three subtypes of bushy cells?
The anatomical and biophysical specializations of bushy cells play an integral role in their function. Bushy cells are innervated by a small number of auditory nerve fibers through large terminals, the end bulbs of Held (Brawer and Morest 1975; Liberman 1991; Sento and Ryugo 1989). Activation of end bulb synapses produces large and rapid synaptic currents that produce rapidly rising and falling excitatory postsynaptic potentials (EPSPs) in bushy cells (Oertel 1983; Oleskevich and Walmsley 2002; Oleskevich et al. 2000; Rhode et al. 1983; Smith and Rhode 1987; Zhang and Trussell 1994). Bushy cells can encode and convey information about the fine structure of sounds because their EPSPs are brief and sharply timed (Joris et al. 1998; Kopp-Scheinpflug et al. 2002; Rhode et al. 1983; Smith et al. 1987, 1993; Winter and Palmer 1990). The brevity and sharp timing of synaptic responses are made possible by the low input resistance and concomitant short membrane time constants, τm, of bushy cells (Oertel 1983; Wang and Manis 2006; Wu and Oertel 1984). Temporally precise signals are brought to bushy cells by primary auditory neurons that have a short τm (Mo and Davis 1997a). The temporal precision is preserved in pathways to the MSO and to the LSO through the MNTB.
The earliest recordings from bushy cells indicated that the voltage-sensitive conductances that gave them short τm's in the physiological voltage range were critical for their ability to encode timing (Oertel 1983; Wu and Oertel 1984). Later work suggested that a low-voltage-activated potassium conductance, gKL, coexists with a high-voltage-activated potassium conductance, gKH, to provide the short τm's (Manis and Marx 1991). Indeed gKL is present in many neurons at early stages of the auditory pathway (Bal and Oertel 2001; Dodson et al. 2002; Forsythe and Barnes-Davies 1993; Golding et al. 1995; Kuba et al. 2002, 2005; Mo et al. 2002; Rathouz and Trussell 1998; Reyes et al. 1994; Rothman and Manis 2003c; Scott et al. 2005; Wu 1999; Wu and Kelly 1995; Zhao and Wu 2001). Most of these neurons also have a hyperpolarization-activated, mixed cation conductance, gh, the partial activation of which at rest also contributes to the short τm (Chen 1997; Cuttle et al. 2001; Leao et al. 2005, 2006; Mo and Davis 1997a). We show here that gKL, gKH, and gh are prominent in bushy cells. The maximal values of gKL, and gh vary over a factor of about four and are correlated to transience in firing.
Preparation of slices
Recordings were made from coronal slices of the most caudal region of the cochlear nuclear complex from mice (ICR strain) between 18 and 21 days old. They were cut in normal physiological saline that contained (in mM) 130 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, and 10 glucose, saturated with 95% O2-5% CO2, pH 7.3–7.4, at between 24 and 27°C. The osmolality, measured with a 3D3 Osmometer (Advanced Instruments, Norwood, MA), was 306 mOsm/kg. All chemicals were from Sigma, unless stated otherwise. Slices, 200 μm thick, were cut with a vibrating microtome (Leica VT 1000S). Good recordings were most common in slices that were cut approximately in a coronal plane. The cut was tipped by ∼30° from the coronal so that the dorsal part of the slice was more anterior than the ventral part of the slice. After cutting, slices were transferred to the recording chamber (∼0.6 ml) and superfused continually at 5–6 ml/min. The temperature was measured in the recording chamber, between the inflow of the chamber and the tissue, with a Thermalert thermometer (Physitemp) the input of which comes from a small thermistor (IT-23, Physitemp, diameter: 0.1 mm). The output of the Thermalert thermometer was fed into a custom-made, feedback-controlled heater that heated the saline in glass tubing (1.5 mm) just before it reached the chamber to maintain the temperature at 33°C. An adjustable delay in the controller for the heater prevented oscillations. Slices were mounted on the stage of a compound microscope (Zeiss Axioskop) and viewed through a ×63 water-immersion objective. Recordings were generally made within 2 h after slices were cut.
Patch-clamp recordings were made with pipettes of borosilicate glass the resistances of which ranged between 4 and 6 MΩ. They were filled with a solution consisting of (in mM) 108 potassium gluconate, 9 HEPES, 9 EGTA, 4.5 MgCl2, 14 phosphocreatinine (Tris salt), 4 ATP (Na salt), and 0.3 GTP (tris salt) that had a final osmolarity 297 mOsm/kg. The pH was adjusted to 7.4 with KOH. Recordings were made with an Axopatch 200A amplifier (Axon Instruments). Records were digitized at 50 kHz and low-pass filtered at 10 kHz. All reported results were from recordings in which 80∼90% of the series resistance could be compensated on-line with 10 μs lag; no corrections were made for errors in voltage that resulted from uncompensated series resistance. The series resistance was 11.8 ± 0.7 MΩ (n = 60). With a cell capacitance 26.0 ± 2.6 pF, the time constant of the imposed voltage step was therefore ≥35 μs; recordings with changes in series resistance exceeding 2 MΩ were excluded from analysis. The output was digitized through a Digidata 1320A (Axon Instruments) and fed into a computer. Stimulation and recording was controlled by pClamp 8 software (Axon Instruments). The control solution contained (in mM) 138 NaCl; 4.2 KCl, 2.4 CaCl2, 1.3 MgCl2, 10 HEPES, and 10 glucose, pH 7.4, 306 mOsm/kg, and saturated with 100% O2. In voltage-clamp experiments, the voltage-sensitive sodium current was blocked by 1 μM tetrodotoxin (TTX), the voltage-sensitive calcium current was blocked by 0.25 mM CdCl2, glutamatergic and glycinergic synaptic currents were blocked with 40 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX) (Tocris Cookson, UK) and 1 μM strychnine respectively. In some experiments, 50 μM ZD7288, 10 mM TEA, or 50 nM α-DTX (Bal and Oertel 2000), were added to the control solution to isolate the IKL, IKH, and Ih. All reported voltages were compensated for a −12-mV junction potential.
The measurements of conductance from individual cells was fitted by a Boltzmann equation, g/gmax = 1 –1/[1+exp(V –V1/2)/κ]. Statistical analyses were made with Origin software (version 7.5); the results are given as means ± SE with n being the number of cells in which the measurement was made.
In some experiments, the physiological identification of bushy cells was verified anatomically. In these experiments, 0.1% biocytin was included in the pipette solution, and slices were fixed with 4% paraformaldehyde immediately after the recording. When the pipette was removed from the cell before fixation, even when fixation was initiated within seconds, cells were almost always damaged, leaving only clusters of labeled beads or beaded processes. To avoid such damage, fixative was introduced into the recording chamber for 5–10 min before the pipette was removed from the cell. Although the cell body and some processes were still sometimes lost, either torn away by the pipette or in the histological processing, the morphology of dendrites and the long stretches of straight axons show that these cells were well fixed even when some parts were lost. Slices were stored in 4% paraformaldehyde at 4°C. Before processing they were embedded in a block of gelatin and albumin that was cross-linked with glutaraldehyde and sectioned at 60 μm in the plane of the slice with a vibratome. Slices were incubated with avidin conjugated to horseradish peroxidase (Vector ABC kit, Vector Laboratories, Burlingame, CA), and cells were visualized after processing for horseradish peroxidase with cobalt and nickel intensification (Zhang and Oertel 1993). The sections were mounted on subbed slides, and counterstained with cresyl violet. Labeled cells were reconstructed with a camera lucida using a ×100 objective and digitized. To measure the distances of labeled cells from the nerve root, the outlines of the coronal sections of the VCN, the locations of the labeled cell, the granule cell areas and the nerve root were reconstructed using a ×10 objective and collapsed in the rostrocaudal dimension. The distance between the cell body and the nearest part of the root of the auditory nerve were measured in the coronal plane. We did not compensate for distance in the rostrocaudal dimension evident when labeled cells were not in the same sections as the nerve root because such compensation was approximate and affected the results only subtly.
Bushy cells have characteristic responses to current pulses
This study was based on 129 current- and voltage-clamp recordings. Of these, 69 were recorded with biocytin-filled pipettes and 24 were recovered and identified anatomically. Figure 1 shows reconstructions of eight of the labeled bushy cells. They are shown together with their responses to 0.4-nA depolarizing and 0.6-nA hyperpolarizing current pulses. Bushy cells characteristically have one or two primary dendrites that branch profusely in a bush of short dendrites. Dendrites in every cell were uneven in thickness; thickenings in dendrites of bushy cells presumably hold clumps of mitochondria (Cant and Morest 1979b). The axons of bushy cells were labeled for varying distances; in three cases, they could be followed into the trapezoid body (Fig. 1C). Axons were generally narrow where they emanated from the cell body and then widened. Responses to injected current follow a consistent pattern. Depolarizing current pulses evoked one or two action potentials that were followed by a small, flat depolarization in some bushy cells as was described previously (Fig. 1, D, F, and G) (Francis and Manis 2000; Leao et al. 2005, 2006; Oertel 1983; Wang and Manis 2006; Wu and Oertel 1984), but in others, depolarization evoked more action potentials and oscillations in voltage at the onset of a current pulse and even an occasional action potential in the middle of a pulse (Fig. 1, A–C, E, and H), a pattern that has not previously been observed in anatomically identified bushy cells. In every cell, the first action potential reached a more depolarized peak, to between −20 and 0 mV, than those that followed. Hyperpolarizing current pulses produced relatively larger voltage changes that sagged back toward rest. The shapes of the sags were variable. In some cells, sags were deep and relatively rapid (Fig. 1, A, D, F, and G), whereas in others, the sags were slower and less deep (Fig. 1, B, C, E, and H). The pattern is consistent with that described with sharp-electrode recordings earlier although some of the bushy cells fired more action potentials at the onset of a depolarization than was described before (Francis and Manis 2000; Oertel 1983; Wu and Oertel 1984). In no case was a cell with this pattern of responses found not to be a bushy cell when it was labeled anatomically. These findings confirm that transient firing is an identifying characteristic of bushy cells but also show that depolarizing current pulses can evoke more than just one or two action potentials.
It is possible that the subtle differences in responses to current reflect differing subtypes of bushy cells because bushy cells near the root of the auditory nerve tended to respond to depolarizing current pulses with few action potentials followed by a flat depolarization and to hyperpolarization with deep sags whereas those recorded somewhat more dorsally were more likely to fire more action potentials and have slower, shallower sags. To document differences in location, the distance from the auditory nerve in the coronal plane was plotted as a function of the maximum number of action potentials (Fig. 2). The plot shows that while differing types of bushy cells are intermingled, there was a correlation between firing patterns and their location. The r2 value of the linear regression was 0.22, showing that the trend is statistically significant (P < 0.05) even in the absence of the cell with the largest number of action potentials, linear regression showed a significant correlation with r2 = 0.18 and P < 0.05.
Firing in response to depolarizing current has served as an identifying characteristic of bushy cells and was therefore explored further. Most bushy cells fired only a single action potential at the onset of a depolarization, independent of the strength of the current (Figs. 1, D, F, and G, and 3, A and B, ○). In others, however, increasing current evoked first more, reached a maximum, and then fewer action potentials (Fig. 3B, ▵). On average, bushy cells fired maximally when they were depolarized with 0.6 nA (Fig. 3B, ▪).
The second identifying characteristic of bushy cells is the rectification in the voltage/current relationship in the depolarizing voltage range. A plot of the voltage changes at the end of 100-ms current pulses as a function of the strength of the injected current reveals rectification in the depolarizing voltage range in every cell (Fig. 3C). The slope of the V-I relationship at rest, a measure of the input resistance, was on average 67.2 ± 17.1 MΩ (n = 36). The sigmoid shape reflects the presence of noninactivating, voltage-sensitive conductances in both depolarizing and hyperpolarizing voltage ranges.
Tonic firing in bushy cells is prevented by an α-DTX-sensitive conductance. Recordings in Fig. 4 (left) show how three bushy cells responded to depolarizing current with transient, firing. One of these cells fired occasionally after the initial transient (Fig. 4Ab). The application of 50 nM α-DTX depolarized every bushy cell tested by between 2 and 4 mV and enabled it to fire tonically (Fig. 4B) independently of whether the cell fired only once (Fig. 4, a) or multiple times (Fig. 4, c). (The traces shown in Fig. 4B were recorded after bushy cells were brought back to their original resting potentials with hyperpolarizing current.) Even in the presence of α-DTX, the first action potential was larger than later ones. Large depolarizations resulted in a depolarization block in some bushy cells (Fig. 4Bb). Action potentials were broadened slightly (duration at threshold in responses to 0.1 nA in control conditions 1.7 ± 0.4 ms, α-DTX 2.4 ± 0.7 ms, n = 10), but their peaks did not change when α-DTX was applied (responses to 0.3 nA under control conditions 32.4 ± 2.7 mV, with α-DTX 33.1 ± 1.9 mV, n = 11). In the presence of α-DTX, prominent IPSPs were sometimes observed (Fig. 4B, a and b), suggesting that inhibitory interneurons have an α-DTX-sensitive K+ conductance. In contrast, the application of 10 mM TEA resulted in the broadening (duration at threshold in responses to 0.1 nA in control conditions 1.1 ± 0.1 ms, TEA 7.4 ± 2.9 ms, n = 5) and heightening of action potentials (responses to 0.3 nA under control conditions 36.9 ± 4.9 mV, with TEA 41.9 ± 8.6 mV, n = 6, Fig. 4, d and e).
Hyperpolarizing current causes a large hyperpolarization that sags back toward rest in bushy cells. The shape of the sag showed considerable variability in that the voltage sagged more quickly and more deeply in some cells than others (Figs. 1 and 5A). The sag toward rest could be reasonably well fit with a single-exponential function; the depth of the sag was quantified as the ratio between steady-state and peak voltage changes. Figure 5B shows that there was a positive correlation between the rate of the sag and its depth in responses to 0.8-nA hyperpolarizing current pulses in 40 bushy cells; in some cells the sag was deep (small b/a) and rapid (small τh), whereas in others, it was slower and less deep. The time course, and therefore also the depth of the sag, was correlated with the maximum number of action potentials elicited by depolarizing current pulses (Fig. 5C). Cells with the slowest and shallowest sags fired the most action potentials. Fifty micromolars ZD7288 blocked the sag but did not affect the firing of bushy cells (Fig. 5, D and E, n = 4).
Measurement of voltage-sensitive conductances
To examine the voltage-dependent conductances that give bushy cells their characteristic properties, conductances through different populations of ion channels were separated from one another and studied under voltage clamp. In voltage-clamp experiments, 1 μM TTX and 0.25 mM Cd2+ were added to the extracellular bathing solution to block Na+ and Ca2+ inward currents. In these experiments, spontaneous glutamatergic and glycinergic synaptic currents were also routinely blocked with 40 μM DNQX and 1 μM strychnine. As the voltage range of activation of gKL overlaps those of gKH and gh, one of each of the pairs of conductances had to be blocked to study the other. Fifty micromolars ZD7288 was used to block gh and 50 nM α-DTX to block gKL.
Low-voltage-activated K+ conductance (gKL)
The characteristics of gKL are illustrated in Fig. 6. Measurements of this conductance were made in the presence of 1 μM TTX, 0.25 mM Cd2+, 50 μM ZD7288, 40 μM DNQX, and 1 μM strychnine to block gNa, gCa, gh, and glutamatergic and glycinergic synaptic currents, respectively. From a holding potential of −80 mV, voltage pulses more depolarizing than −70 mV evoked outward currents whose rise was too rapid to be resolved from the capacitative transient in every bushy cell tested (Fig. 6A). Plots of peak outward currents as a function of voltage show that currents were activated at about −70 mV (Fig. 6B). As these currents are activated at relatively hyperpolarized potentials, they were dubbed low-voltage-activated currents (IKL). Although the voltage sensitivity of IKL was consistent between bushy cells, the magnitude of the current varied over a fourfold range (Fig. 6B, n = 14).
The reversal potential of IKL was determined from the reversal of tail currents. A voltage pulse to −50 mV was used to activate gKL. Tail currents were then measured with subsequent voltage pulses to between −60 and −110 mV. IKL reversed at −80.0 ± 3.1 mV (n = 6), near the theoretical equilibrium potential for K+, −85 mV.
At potentials more depolarized than −40 mV, not only IKL but also IKH were activated so that the two currents had to be separated pharmacologically as shown in Fig. 6C–E, to be studied. A family of voltage steps from −80 to +20 mV evoked a substantial outward current. The application of 50 nM α-DTX reduced the evoked outward current. Subtracting currents evoked in the presence of α-DTX from those under control conditions separated the α-DTX-insensitive current (Fig. 6D) from the α-DTX-sensitive current (Fig. 6E). The current that was blocked by α-DTX activated near −70 mV whereas that which remained after the application of α-DTX activated at voltages more depolarized than about −40 mV, indicating that α-DTX blocked essentially all of IKL and could be used to separate IKL from IKH (Fig. 6F). The clustering of currents in responses to large depolarizations (Fig. 6E) and the curvature in the I-V plot (Fig. 6F) indicate that the voltage was not perfectly clamped in the presence of large outward currents. The measured currents were converted to conductance with Ohm's law, gKL = IKL/(Vm –Erev) (Fig. 6G). The maximal gKL ranged between 40 and 140 nS among bushy cells and had a mean of 80.8 ± 16.7 nS (n = 11). Imperfections in clamping could make these underestimates of the maximal conductance. The sigmoidal, mean gKL was fit by a Boltzmann function with half-activation at −37.6 ± 2.1 mV and a slope factor of 10.2 ± 1.0 mV (n = 11).
Pharmacological blockers give an indication of what subunits form gKL. α-DTX blocks potassium channels of the Kv1 family that contain Kv1.1, Kv1.2, Kv1.3, and Kv1.6 α subunits (Dolly and Parcej 1996; Grissmer et al. 1994; Harvey 1997; Owen et al. 1997; Tytgat et al. 1995). DTX-K (40 nM), a blocker that is specific for channels that have Kv1.1 α subunits (Owen et al. 1997; Robertson et al. 1996; Wang et al. 1999a,b), blocked 72.3 ± 6.4% (n = 4) of the peak IKL. 100 nM Tityustoxin Kα, a blocker that is selective for Kv1.2 α subunits (Hopkins 1998; Werkman et al. 1993), blocked 57.8 ± 6.8% (n = 4) of the peak IKL at −45 mV. The currents blocked by DTX-K and tityustoxin Kα are together >100%, indicating that part of the current is sensitive to both blockers and that these toxins do not define mutually exclusive currents. IKL is thus mediated through heteromeric voltage-sensitive channels of the Kv1 family, some of which contain Kv1.1 α subunits and some of which contain Kv1.2 subunits. These results also show that α-DTX sensitivity can be used to separate IKL from other currents, including IKH.
High-voltage-activated K+ conductance (gKH)
The high-voltage-activated current, IKH, was defined by its insensitivity to α-DTX and its sensitivity to 10 mM TEA. In the presence of 50 nM α-DTX, voltage steps from a holding potential of −80 mV evoked outward currents that inactivated slowly (Fig. 7A). These currents were largely blocked by 10 mM TEA (Fig. 7B). The difference between these families of currents revealed the TEA sensitive, α-DTX-insensitive current, IKH (Fig. 7C). The clustering of currents in responses to large depolarizations indicates that the currents were not perfectly clamped at the highest voltages. The gKH in a group of bushy cells is shown in Fig. 7D. It becomes activated when bushy cells are depolarized more than about −45 mV. The maximum conductance, gKHmax, varied by a factor of about two and had a mean 58.2 ± 6.5 nS (n = 12). Fitting the average g-V relationship with a Boltzmann function showed that gKH was half activated at −18.4 ± 1.9 mV and had a slope factor of 8.7 ± 0.9 mV (n = 12).
The reversal potential for gKH was measured from tail currents in the presence of α-DTX. IKH reversed at −77.0 mV ±3.4 mV (n = 4). The difference between the reversal potentials of IKH and IKL was not statistically significant (Student's t-test, P = 0.24).
Several groups have reported the presence of A-type K+ conductances, conductances that are strongly inactivated near the resting potential, in cochlear nuclear cells (Rathouz and Trussell 1998; Rothman and Manis 2003a). In recordings from bushy cells, the magnitude of peak outward currents was not significantly affected by varying the holding potential between −70 and −90 mV (n = 3). We have observed A-type K+ conductances in other cells but never in a bushy cell.
Hyperpolarization-activated conductance (gh)
Hyperpolarizing voltage steps evoked a slowly activating and slowly deactivating inward current in bushy cells. To isolate Ih, recordings were made in the presence of 10 mM TEA, 50 nM α-DTX, 0.25 mM Cd2+, 1 μM TTX, 40 μM DNQX, and 1 μM strychnine. Control experiments showed that Ih was not significantly affected by the cocktail of drugs (data not shown). Figure 8A shows a family of inward currents that was evoked by hyperpolarizing voltage pulses. Immediately after the capacitative transient, there was an “instantaneous” change in current, which reflected current flowing through the resting conductance of the cell just before the voltage step. The inward current then increased slowly to a steady state, reflecting the activation of gh. After the capacitative transient at the end of the voltage step, the current declined back to the original holding level in a current tail as Ih deactivated. The bradicardiac agent, ZD7288, has been shown to block Ih selectively in many types of neurons including in neurons of the ventral cochlear nucleus (Bal and Oertel 2000; Maccaferri and McBain 1996). In bushy cells that were bathed in a cocktail of blockers, addition of 50 μM ZD7288 reduced the instantaneous current by 76.0 ± 2.7% (n = 8), prevented much of the slow activation of the inward current and substantially reduced the tail current. These results show that ZD7288 blocked most of the current, although some time- and voltage-dependent unblocking is evident as has also been observed in other types of neurons (Harris and Constanti 1995; Shin et al. 2001). Ih is inward even around rest, consistent with its being a mixed cation current. Plots of the I-V curve of steady-state currents in bushy cells are shown in Fig. 8B. Their magnitudes differed over a fivefold range among bushy cells. The reversal potential for Ih was measured as the point where chord conductances intersect (Bal and Oertel 2000). The mean reversal potential of Ih was −40.8 ± 2.3 mV (n = 5). The voltage sensitivity of gh was measured from tail currents after a family of voltage steps that encompassed the voltage range over which gh is affected by voltage. The activation of gh at the steady state by the family of voltage steps was reflected in the amplitude of tail currents on return to −77 mV after steps to voltages between −35 to −125 mV (Fig. 8C, ↓). The tail current when the voltage was stepped from a variable voltage to −77 mV reflects a change in activation of gh. This tail current reflects a deactivation when the voltage is stepped from more hyperpolarizing potentials to −77 mV and activation of Ih when the voltage is stepped from more depolarizing potentials to −77 mV (Fig. 8C). The tail currents saturated at large hyperpolarizing test potentials, as gh approached its maximum value and at depolarized potentials as gh approached the minimum. The relative amplitude of tail currents, measured immediately after the relaxation of the capacitative transient, is plotted in Fig. 8D. This plot shows the voltage sensitivity of gh in 10 bushy cells. The values of gh were derived from the absolute values of tail currents gh(V) = (I − Imin)/(Vm –Erev) and plotted as a function of the voltage of the preceding step (Vm). The maximum gh ranged from 14.5 to 56.6 nS and had an average of 30.0 ± 5.5 nS (n = 10) in bushy cells (Fig. 8D). A Boltzmann fit to the averaged activation curve gave V1/2 = −83.1 ± 1.1 mV and a slope factor k = 9.9 ± 1.1 mV (n = 10).
There was considerable variation between bushy cells in the rates at which Ih activated and deactivated, suggesting that the subunit composition of the ion channels may differ among bushy cells. Rates of activation depended on voltage (Fig. 8A); activation rates were higher in responses to large than to small hyperpolarizations. Variation in the rate of activation of Ih among bushy cells is illustrated by the superposition of normalized current traces in responses to −112 mV (Fig. 8E). The activation of Ih required fitting with double exponentials, τf and τs. In most bushy cells rates varied between a, 40% τf 78 ms and 60% τs 410 ms, and b, 60% τf 56 ms and 40% τs 277 ms, but in one cell, c, Ih activated more quickly (Fig. 8E).
One of the features that make gh biologically interesting is that its voltage-dependence is modulated by neurotransmitters. In cardiac and some neuronal cell types, including in T stellate cells of the VCN, Ih is modulated through a cAMP-dependent pathway (Banks et al. 1993; Leao et al. 2006; McCormick and Pape 1990a,b; Robinson and Siegelbaum 2003; Rodrigues and Oertel 2006), but in others, including octopus cells of the VCN the activation curves of which seem always to lie at the depolarizing extreme of the range of modulation, little modulation by cAMP was observed (Bal and Oertel 2000). Figure 9 (A–C) shows an example of the action of 500 μM 8-Br-cAMP, a membrane-permeable analogue of cAMP, on a bushy cell in which the activation curve shifted by ∼6 mV. On average 500 μM 8-Br-cAMP caused a depolarizing shift in the half-activation voltage of Ih of 6.8 ± 1.8 mV (n = 4).
Temperature also affects Ih differently in different cells. In octopus cells, a reduction in temperature causes not only changes in kinetics, as expected, but also changes in the amplitude of Ih that adapt to the original level over a few minutes (Cao and Oertel 2005). In T stellate cells, a reduction in temperature reduces the rates of activation and deactivation and the amplitude, but in contrast with octopus cells, changes in amplitude are stable (Rodrigues and Oertel 2006). Figure 9 (D–F) shows that in bushy cells, too, the reduction in rate and amplitude was stable over time and that the voltage sensitivity was not altered by lowering the temperature from 33 to 26°C. In bushy cells, V1/2 was −87.4 ± 2.3 mV at 33°C and −86.9 ± 1.8 mV at 26°C (n = 4).
Rate of depolarization (dV/dt) threshold
In every bushy cell tested, firing depended on the rate at which it was depolarized. Rapid depolarizations caused bushy cells to fire, whereas slow depolarizations did not. Each bushy cell has a threshold rate of depolarization that is independent of the magnitude of current when the current is suprathreshold (McGinley and Oertel 2006). Threshold rates of depolarization were measured by depolarizing bushy cells with currents that rose in ramps of varying amplitude. Measurements from three cells are illustrated in Fig. 10. For each ramp, the subthreshold rising phase of the voltage response was fit with a straight line the slope of which defined the dV/dt for that ramp (McGinley and Oertel 2006). Plots of the peak voltage reached during each ramp against the dV/dt revealed a step increase that was the threshold for firing in rate of depolarization (dV/dtthresh; Fig. 10, A–C, panels on the right). In bushy cells, the dV/dtthresh ranged from 1.4 to 4.2 mV/ms and had an average of 2.8 ± 0.5 mV/ms (n = 18).
Do biophysical properties define subclasses of bushy cells?
Expecting that mice might have globular and small spherical bushy cells and finding that their firing is correlated with position in the nucleus led us to question whether the biophysical properties of bushy cells fall into separate groups. An obvious difference between responses that were recorded with sharp electrodes and in the present group was in the firing of action potentials. Could bushy cells that fire more than two action potentials represent a population of more fragile bushy cells that was not represented in early sharp-electrode recordings? Figure 11 shows the relationship between the maximum number of action potentials fired by bushy cells and measurements of biophysical properties. The plots show that the bushy cells that fired fewest action potentials tended to have the largest maximal gKL, the largest maximal gh, the largest dV/dt threshold, the lowest input resistances, and the shortest τm. Maximum gKH was not strongly correlated with repetitive firing. Most of the relationships do not show distinct breaks, but dV/dt thresholds do fall into two groups with bushy cells that fire only one or two action potentials having high rate thresholds and bushy cells that fire more than two action potentials having lower rate thresholds. Cluster analysis revealed that each of the relationships in Fig. 11 falls best into two groups as indicated by the ovals. In all panels except B, the populations delineated by the ovals were statistically significantly different from one another (P < 0.05, Student's t-test). Whether bushy cells fall into two distinct groups is unclear, however. On the one hand, there were only 3/86 measurements inconsistent with the conclusion that bushy cells fall into two groups, one that fired one or two action potentials and another more than two action potentials when depolarized with square current pulses. On the other hand, the finding that the groupings differed could indicate that the population of bushy cells forms a continuum.
The present study confirms what had been reported incidentally (McGinley and Oertel 2006; Wang and Manis 2006): the criteria that were based on early recordings with sharp electrodes (Oertel 1983; Wu and Oertel 1984) were too narrow and excluded bushy cells that fire more action potentials at the onset of a depolarizing pulse. All bushy cells fired transiently when depolarized with current pulses; some bushy cells fired only once but others fired up to six action potentials. The gKL that reduces repetitive firing was present in every anatomically identified bushy cell tested. Being partly activated at rest, gKL and gh affected the τm near rest that determines the shape of synaptic potentials; τm varied between 0.6 and 2 ms in the population of bushy cells we studied. The presence of gKL distinguishes bushy cells from the T stellate cells in which gKL is weak or absent (Ferragamo and Oertel 2002; Rodrigues and Oertel, unpublished results).
The present results are consistent with bushy cells forming two separate populations of cells, perhaps globular and small spherical bushy cells, but they are not definitive. The dV/dt thresholds of bushy cells clearly fell into two groups. Cluster analysis showed that other features, too, fell largely, but not entirely, into two groups, those that fired maximally one or two action potentials and those that fired maximally three or more action potentials when they were depolarized with current pulses. There was a trend for bushy cells near the nerve root to fire fewer action potentials when depolarized with current pulses, have shorter τm, and have higher dV/dt thresholds than those that lie more dorsal to the nerve root. The finding that there are significant differences between bushy cells is consistent with responses to tones in vivo. Responses to tones also differ among bushy cells. The “primary-like-with-notch” responses to tones of globular bushy cells show a sharp onset transient, whereas “primary-like” responses have a broader onset transient and their firing is more tonic but the differences are sometimes subtle (Paolini et al. 2001; Rhode and Smith 1986; Smith et al. 1991; Winter and Palmer 1990). A high rate threshold enhances the sharpness of the onset transient in cells that sum many small inputs (Ferragamo and Oertel 2002; McGinley and Oertel 2006). If globular and small, spherical bushy cells differ biophysically, the present results suggest that globular bushy cells, more than small spherical bushy cells, are functionally specialized to detect the coincidence of multiple converging inputs. Incidental, but not systematic, estimates have been made of the number of converging auditory nerve fibers onto bushy cells in mice (Oertel 1985). In cats 6–69, but most often 15–23, auditory nerve fibers converge on a globular bushy cell (Liberman 1993; Spirou et al. 2005). Also in cats it has been shown that large spherical bushy cells receive input through about three fibers (Brawer and Morest 1975). Corresponding measurements have, however, not been made in small spherical bushy cells.
Voltage-sensitive conductances in bushy cells follow a pattern that has been observed in many auditory neurons that are known to receive and convey information in the timing of firing. The combination of gKL and gKH was first documented in dissociated cells that might have been bushy cells (Manis and Marx 1991; Pal et al. 2005; Rothman and Manis 2003a–c) and then found in other cells including avian homologues of bushy cells (Rathouz and Trussell 1998; Reyes et al. 1994), primary auditory neurons (Mo and Davis 1997a; Mo et al. 2002), octopus cells of the mammalian VCN (Bal and Oertel 2001; Cao and Oertel 2005; Golding et al. 1995, 1999), identified bushy cells (Leao et al. 2004), principal cells of the MNTB (Brew and Forsythe 1995; Brew et al. 2003; Dodson et al. 2002; Forsythe and Barnes-Davies 1993; Kopp-Scheinpflug et al. 2003), ventral nucleus of the lateral lemniscus (Wu and Kelly 1995), MSO (Scott et al. 2005) and its avian homologue, nucleus laminaris (Kuba et al. 2002, 2005; Reyes et al. 1996), and LSO (Barnes-Davies et al. 2004). Synaptic terminals also have this combination of conductances (Dodson et al. 2003; Ishikawa et al. 2003). Most of these cells also contain gh: primary auditory neurons (Chen 1997; Mo and Davis 1997b), octopus cells (Bal and Oertel 2000; Cao and Oertel 2005; Koch et al. 2004), bushy cells (Leao et al. 2005, 2006), MNTB neurons (Banks et al. 1993; Leao et al. 2005, 2006), ventral nucleus of the lateral lemniscus (Zhao and Wu 2001), MSO (Scott et al. 2005) and its avian homologue, nucleus laminaris (Reyes et al. 1996), and LSO (Barnes-Davies et al. 2004; Leao et al. 2006). Ih opposes IKL at the resting potential.
Although the overall pattern is consistent, the magnitudes and some of the functional characteristics of the conductances in auditory neurons are surprisingly variable. For example, we have shown that on average gKLmax is 80 nS in bushy cells but in octopus cells, the mean gKLmax is 515 nS (Bal and Oertel 2001), and in young MNTB cells, it is reported to be 0.5 nS (Leao et al. 2004). In dissociated cells no Ih seems to have been detected (Manis and Marx 1991; Rothman and Manis 2003a). At reduced temperatures, the rates of activation and inactivation as well as the absolute magnitudes of gKL, gKH, and gh are reduced differentially (Cao and Oertel 2005). In octopus cells, but not in bushy or T stellate cells, the magnitude of ghmax adapts to a constant value when the temperature is altered (Cao and Oertel 2005; Leao et al. 2006; Rodrigues and Oertel 2006). Activity also has been reported to affect the expression of potassium channels (Lu et al. 2004). As neurons mature, the magnitude of gh and gKL grows several-fold in rodents between 1 and 3 wk after birth (Cuttle et al. 2001; Scott et al. 2005). In bushy cells, the magnitudes of both ghmax and gKLmax are correlated with the number of action potentials evoked by depolarizing current pulses. As blocking gh does not affect firing, the correlation of ghmax with firing could result from a requirement that Ih balances IKL at rest to maintain a constant resting potential as it does in octopus cells (Oertel et al. 2000). Maturity also probably affects voltage sensitivity; for example in 2-wk-old mice, V1/2 of gh is reported to be −100 mV (Leao et al. 2005), whereas in 3-wk-old mice, we find it to be −83 mV. The voltage sensitivity varies within some of these cell populations (Mo and Davis 1997b). Measurements can also be affected by experimental parameters such as series resistance compensation (Rothman and Manis 2003a). Comparisons between populations of neurons are therefore most reliable within a study.
The presence of gKL enhances the ability of auditory neurons to encode timing with temporal precision. First, this conductance gives neurons a low input resistance and a rapid τm that makes synaptic potentials brief and sharply timed (Kuba et al. 2005; Manis and Marx 1991; Mo and Davis 1997a; Oertel 1983; Reyes et al. 1996). The low input resistance leads to a requirement for large synaptic currents that are delivered either through large calyceal endings of individual fibers or through smaller endings of large numbers of fibers. Second, gKL prevents repetitive firing (Barnes-Davies et al. 2004; Brew et al. 2003; Leao et al. 2004; Mo et al. 2002). Third, gKL contributes to the repolarization not only of action potentials but also of synaptic potentials, sharpening their peaks (Kuba et al. 2005; Oertel et al. 2000; Scott et al. 2005). Fourth, it makes neurons sensitive to the rate at which they are depolarized (Ferragamo and Oertel 2002; McGinley and Oertel 2006). The larger the rate of depolarization that is required for firing, the more closely coincident subthreshold inputs need to be to contribute to firing (McGinley and Oertel 2006).
gKL is mediated through K+ channels of the Kv1 (also termed shaker or KCNA) family. In bushy cells, gKL is blocked by α-DTX (present study; Leao et al. 2004), which, like DTX I (Lu et al. 2004; Rothman and Manis 2003a), blocks channels that contain Kv1.1, Kv1.2, Kv1.3, and Kv1.6 subunits (Dolly and Parcej 1996; Grissmer et al. 1994; Harvey 1997; Owen et al. 1997; Tytgat et al. 1995). Sensitivity of only 72% of IKL to DTX-K, a toxin that blocks channels with a Kv1.1 subunit (Owen et al. 1997; Robertson et al. 1996; Wang et al. 1999a,b), and of only 58% of IKL to tityustoxin Kα, a toxin that blocks channels with a Kv1.2 subunit (Hopkins 1998; Werkman et al. 1993), indicates that gKL comprises a population of heteromeric channels of which not all contain Kv1.1 and not all contain Kv1.2 subunits, much as in octopus cells (Bal and Oertel 2001). mRNA and protein for Kv1.1 and Kv1.2 subunits are prominent in the AVCN (Grigg et al. 2000; Pal et al. 2005). In MNTB neurons of young rats, 95% of IKL is sensitive to DTX-K, indicating that most channels contain Kv1.1 subunits (Dodson et al. 2003); IKL is, however, reduced but not eliminated in mice that lack Kv1.1 subunits (Brew et al. 2003). In the MSO, too, a larger proportion of IKL is sensitive to DTX-K than in bushy or octopus cells (Scott et al. 2005). gKL is graded with tonotopy in some auditory nuclei (Barnes-Davies et al. 2004; Kuba et al. 2005), but no tonotopic gradient was observed in the VCN in the magnitude of IKL (present study) nor in the levels of Kv1 mRNA (Grigg et al. 2000).
The depolarized activation range of gKH indicates that it is activated mainly by the peaks of action potentials and contributes to repolarizing them (Perney and Kaczmarek 1997; Rothman and Manis 2003c; Rudy et al. 1999; Rudy and McBain 2001; Wang et al. 1998); indeed blocking gKH with TEA broadened action potentials in bushy cells. gKH is likely mediated through channels that contain α subunits of the Kv3 (shaw or KCNC) family of potassium channels. mRNA for Kv3.1 and Kv3.3 is expressed in or near spherical and globular bushy cells (Grigg et al. 2000; Li et al. 2001; Perney and Kaczmarek 1997; Song et al. 2005; Weiser et al. 1994). In contrast with gKH in bushy cells, half-activated at −17 mV and nearly completely inactivated after 5 s, Kv3.1 channels in Xenopus oocytes are half-activated at about +15 mV and inactivate only slightly over seconds (McCormack et al. 1990; Rudy et al. 1999; Weiser et al. 1994). Kv3.1 subunits are variable, however. They are alternatively spliced, allowing channels to be differentially targeted and differentially modulated by phosphorylation (Macica et al. 2003; Ponce et al. 1997; Song et al. 2005). Currents through Kv3.3 channels in Xenopus oocytes resemble IKH in bushy cells more closely in that they inactivate strongly over seconds (Rudy and McBain 2001; Weiser et al. 1994). Furthermore, gKH in bushy cells is similar to that in octopus cells (Bal and Oertel 2001); in the octopus cell area the mRNA for Kv3.1 subunit is expressed only weakly, whereas the mRNA for the Kv3.3 subunit is strongly expressed (Bal and Oertel 2001; Grigg et al. 2000; Li et al. 2001; Perney and Kaczmarek 1997). No tonotopic gradient has been observed in IKH or in the expression of Kv3.1 or Kv3.3 mRNA in the VCN (Grigg et al. 2000). No A-type, high-voltage-activated current was detected in bushy cells in the present study, consistent with observations by Rothman and Manis (2003a) that type II cells do not have A-type K+ currents.
Hyperpolarization-activated currents have a less direct role in signaling in bushy cells. Being activated at the resting potential, gh contributes to setting the resting input conductance and Ih counteracts the resting IKL in setting resting potential. In all VCN cells, the reversal potential of Ih was near −40 mV, but its voltage sensitivity and maximum amplitude varied between cell types under comparable conditions. In bushy cells, V1/2 was relatively negative, −84 mV, so that a relatively small proportion of the conductance was activated at rest; it was even more negative, −88 mV, in T and D stellate cells, but much more positive, −65 mV, in octopus cells (Bal and Oertel 2000; Oertel and Fujino 2001; Rodrigues and Oertel 2006). The voltage sensitivity varies among primary auditory neurons (Mo and Davis 1997b). In bushy cells, ghmax averaged ∼30 nS, near the 20 nS average ghmax in T stellate and 30 nS in D stellate cells but smaller than the 150 nS in octopus cells. Other authors have reported more negative values of V1/2, −93 to −101 mV, and smaller ghmax, 2–6 nS in bushy cells of 12- to 14-day-old mice (Leao et al. 2005, 2006). In bushy cells, as in other auditory neurons, blocking Ih did not significantly affect the shape of action potentials (Bal and Oertel 2000; Leao et al. 2006).
Ih is mediated through HCN (hyperpolarization-activated, cyclic nucleotide-gated) channels. HCN1 and HCN2 subunits are clearly present in the anterior VCN (Koch et al. 2004). It is unclear how strongly HCN3 and HCN4 are expressed in the brain stem; some authors report that they are expressed at low levels (Moosmang et al. 2001) and others that they are expressed at substantial levels (Leao et al. 2006; Monteggia et al. 2000). The sensitivity of Ih to cyclic nucleotides is common (DiFrancesco and Tortora 1991; Ludwig et al. 1998; McCormick and Pape 1990a; Pape and McCormick 1989; Santoro et al. 1998; Tokimasa and Akasu 1990; van Ginneken and Giles 1991). In bushy, stellate, and MNTB cells, cAMP shifts the voltage sensitivity of Ih in the depolarizing direction, depolarizing cells and thus increasing their excitability, whereas in octopus cells, the voltage sensitivity of Ih is already maximally depolarized (Bal and Oertel 2000; Banks et al. 1993; Oertel and Fujino 2001; Rodrigues and Oertel 2006).
This work was supported by National Institute of Deafness and Other Communications Disorders Grant DC-00176.
Several people made substantial contributions to this work. M. J. McGinley, J. H. Wittig Jr., and A. Rodrigues initially showed that multiple spiking cells have α-DTX-sensitive conductances. P. Chang did the cluster analysis for Fig. 11. We thank J. Doucet and D. Ryugo for valuable discussions. M. J. McGinley and E. Lenhart read the manuscript critically and made valuable suggestions. We are also fortunate to have expert help from staff members in the department. Most especially we thank R. Kochhar, who keeps our computers running smoothly and L. Barnes and other members of the office staff for administrative support.
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