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Hyperpolarization-activated (Ih) conductances affect brainstem auditory neuron excitability
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Hearing Research 183 (2003) 126^136 www.elsevier.com/locate/heares
Hyperpolarization-activated (Ih ) conductances a¡ect brainstem auditory neuron excitability Aasef G. Shaikh, Paul G. Finlayson
Department of Otolaryngology, Wayne State University, 550 E Can¢eld Avenue, Rm. 327, Detroit, MI 48201, USA Received 20 February 2003; accepted 2 July 2003
Abstract Blockade of the hyperpolarization-activated cyclic-nucleotide-gated mixed-cationic conductance (Ih ) by ZD7288 markedly reduces excitability of neurons in the superior olivary complex (SOC), in vivo. Following pressure ejection application of 100 WM ZD7288, extracellular recorded single unit responses of 47/47 SOC neurons to monaural or binaural pure tone best frequency (BF) stimuli (30 dB above threshold) decreased by 49.7 : 19%, and background activity decreased by 56.3 : 18.1%. Pressure ejection of the vehicle did not affect excitability. The dose- and time-dependence of ZD7288 (10^100 WM) effects are consistent with specific blockade of Ih currents. SOC neuron responses to pressure-ejected glutamate were also decreased following application of 100 WM ZD7288 by 76.7 : 28.0%, which suggests a predominant direct effect of ZD7288 on auditory cell excitability. The considerable variability in the magnitude of ZD7288 effects between cells was only partially accounted for by greater effects on neurons with BFs greater than 16 kHz. Therefore, Ih channels significantly contribute to auditory brainstem neuron excitability, affecting their response level to acoustic stimuli. The variability in the ZD7288 reduction in excitability and its variation with the BF of units could be an indication of regulation and plasticity in neuronal encoding of sounds. @ 2003 Elsevier B.V. All rights reserved. Key words: Hyperpolarization-activated cyclic-nucleotide-sensitive non-selective cation channels; Auditory; Superior olivary complex; ZD7288; Excitability; Sound
1. Introduction Hyperpolarization-activated cyclic-nucleotide-gated mixed (or non-selective) cation channels carry prominent conductances (Ih ) a¡ecting membrane properties in most auditory neurons from spiral ganglion cells to neocortex (Manis, 1990; Banks et al., 1993; Hu, 1995; Smith, 1995; Tennigkeit et al., 1996; Fu et al., 1997;
* Corresponding author. Tel.: +1 (313) 577 8041; Fax: +1 (313) 577 8555. E-mail address: p¢[email protected] (P.G. Finlayson). Abbreviations: Ih , hyperpolarization-activated cyclic-nucleotidegated mixed-cationic conductance; cAMP, cyclic-AMP; SOC, superior olivary complex; LSO, lateral superior olive; PSTH, peristimulus time histograms; MNTB, medial nucleus of the trapezoid body; SPN, superior paraolivary nucleus; BF, best frequency; WGA-HRP, wheat-germ agglutinin horseradish peroxidase; aCSF, arti¢cial cerebrospinal £uid; HCN hyperpolarization-activated cyclic nucleotide-gated channel subunit
Mo and Davis, 1997; Schwarz and Puil, 1998; Golding et al., 1999; Adam et al., 1999, 2001; Bartlett and Smith, 1999; Bal and Oertel, 2000; Cuttle et al., 2001). In prior studies, the presence of Ih channels in auditory neurons was characterized in tissue slices from relatively young animals (less than 30 days old). The role(s) of Ih in adult animals and responses to acoustic stimuli have not been investigated. In the present study, we examine the role of Ih in determining auditory neuron excitability in the superior olivary complex (SOC) in adult anaesthetized animals. An important role of Ih channels is the regulation of membrane potential (McCormick and Pape, 1990; Banks et al., 1993; Adam et al., 2001; Cuttle et al., 2001), which can directly a¡ect neuronal excitability (Akasu et al., 1993; Maccaferri and McBain, 1996; Wang et al., 1997; Saitoh and Konishi, 2000; Seutin et al., 2001). This non-inactivating channel is activated by membrane hyperpolarization. The reversal potential
0378-5955 / 03 / $ ^ see front matter @ 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00224-7
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of Ih channels is between 333 and 348 mV in auditory brainstem neurons (Banks et al., 1993; Fu et al., 1997; Mo and Davis, 1997; Bal and Oertel, 2000; Cuttle et al., 2001), due to its mixed permeability for sodium and potassium. Therefore, opening of Ih channels (at rest and during hyperpolarization) produces a depolarizing in£uence, while decreased activation of Ih channels during depolarization (or blockade) hyperpolarizes cells (Robinson and Siegelbaum, 2003). As expected, blockade of Ih channels produces a hyperpolarization in membrane potential in lateral superior olive (LSO) neurons (Adam et al., 2001). Therefore, the membrane potential and excitability may be stabilized around a set point, dependent on the activation of Ih channels. The activation range for Ih is shifted towards more depolarized levels by increasing cyclic-AMP (cAMP) levels in central neurons (Bobker and Williams, 1989; Pape and McCormick, 1989; Tokimasa and Akasu, 1990; Pedarzani and Storm, 1993), and in the auditory system (Banks et al., 1993; Mo and Davis, 1997; Cuttle et al., 2001). The wide variation in Ih responses observed in spiral ganglion cells in culture may be attributed to intercellular di¡erences in cAMP levels (Mo and Davis, 1997), and may re£ect that Ih is also regulated physiologically. A role of Ih channels in determining the excitability of SOC neurons may have a signi¢cant impact on the encoding of essential acoustic information. LSO neurons are characterized by a linear relationship between their neuronal discharge activity and interaural intensity di¡erences (Boudreau and Tsuchitani, 1968; Goldberg and Brown, 1969; Tsuchitani and Boudreau, 1969; Caird and Klinke, 1983; Finlayson and Caspary, 1993; Finlayson, 1995). If the excitability of LSO neurons is modulated, their rate-code representation of sound source location can be shifted. Changes in excitability of other SOC neurons would a¡ect additional information for encoding sound source localization, and other features of sound. In this study and a preliminary report (Shaikh and Finlayson, 2002), we document that Ih conductances signi¢cantly a¡ect SOC neuron excitability.
2. Materials and methods 2.1. Animal preparation and surgery The Animal Investigation Committee at Wayne State University approved the care and use of animals reported in this study. Anesthesia of young adult male Long Evans rats (3^6 months in age) weighing 499^ 600 g was induced with a combination of ketamine (85 mg/kg) and xylazine (3 mg/kg), and followed 2^5 min later by an initial dose of 44 mg/kg sodium pento-
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barbital. Supplemental doses of anesthetics were administered as required every 2^3 h, indicated by the presence of toe pinch and eye blink re£ex, alternating between sodium pentobarbital (25 mg/kg) and ketamine (40 mg/kg). The animal’s rectal temperature was maintained at 37‡C by a thermostatically controlled DC heating pad. Following surgical exposure of the external auditory canals, the animal was placed in the stereotaxic plane (Paxinos and Watson, 1982) using ear bars. Following ¢xation of the skull with a custom-made head holder, ear bars were removed, and earphones sealed with the external auditory canals to produce closed auditory delivery paths. The foramen magnum was exposed and dilated. The dura was re£ected for access of electrodes to the brainstem through the cerebellum (for details of surgery, Finlayson and Adam, 1997). 2.2. Stimulus generation and delivery Binaural stimuli were digitally generated and delivered using the program A/Dvance (McKeller Designs) on a Macintosh Quadra 950 computer with National Instruments boards (MIO-161-9; DMA-2800). Outputs of the digital to analog converters were bandpass ¢ltered, ampli¢ed by an Amcron (Crown) ampli¢er, and attenuated under computer control (Medical Associates MA919 attenuators). Stimuli were transduced by impedance-matched headphones (Beyer-Dynamic 600 6, B4132.01, frequency range 0.05^35 kHz). Output of the stimulus system was calibrated o¡-line using a coupler with a 0.3 cc air space. Signals were measured with a 1/4-inch condenser microphone (Bruel and Kjaer) and a calibrated microphone preampli¢er (Bruel and Kjaer 2804). Calibration tables were used to determine intensities in dB SPL (re 0.0002 dyn/cm2 ) for tonal stimuli from 0.05 to 32 kHz. Brainstem auditory evoked potentials from scalp electrodes were recorded to monaural ipsilateral and contralateral clicks. 2.3. Extracellular single unit electrophysiology Double and triple-barrel piggyback electrodes were used for extracellular single unit recordings and simultaneous application of pharmacological agents. Piggyback electrodes were fabricated by gluing single or double-barrel pressure ejection pipet to the recording electrode, with the tip of the ejection pipet 150 Wm behind the recording electrode tip. Recording electrodes were ¢lled with 2% wheat-germ agglutinin horseradish peroxidase (WGA-HRP) dissolved in 2 M NaCl. Tip resistance of recording electrodes was 12^20 M6. The pressure ejection electrodes were ¢lled with the appropriate pharmacological agents. Electrodes were advanced at an angle 15‡ to the right of the mid-sagital
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axis and 49.5‡ up from the horizontal axis, to reach the left SOC. Signals were ampli¢ed using an A-M Systems (Seattle, WA, USA) 1800 ampli¢er and Kikisui (Japan) oscilloscope. Recordings were bandpass ¢ltered (between 1 or 10 Hz and 5 kHz). Spikes were identi¢ed on an oscilloscope and converted to pulses using a World Precision Instruments 121 window discriminator, and timing of spikes recorded with 10 Ws resolution.
(Tocris) was dissolved in arti¢cial cerebrospinal £uid (aCSF; NaCl 7.93 g/l, KCl 0.22 g/l, CaCl2 2H2 O 0.24 g/l, MgSO4 0.14 g/l, glucose 0.6 g/l, and HEPES 5.96 g/l). For aCSF controls and ZD7288 application, six pulses were given over a 1 min period. Recording sites or tracks were marked by iontophoretic application of WGA-HRP (5 WA, 500 ms pulses at 1 Hz). The recording locations were later con¢rmed histologically.
2.4. Classi¢cation of units
2.5. ZD7288 e¡ects on responses to excitatory amino acid agonists
Search stimuli were binaural clicks with the contralateral stimuli delayed by 20 ms relative to ipsilateral stimuli to discretely observe evoked potentials to either ear. As electrodes advanced into the SOC, robust evoked potentials to clicks to each ear were observed. Auditory neurons were initially characterized using 50 ms pure tone stimuli with 5 ms rise/fall ramps. Best frequencies (BF, the frequency of stimulation producing the greatest spike count) were determined from right and left monaural iso-intensity curves at 50 dB SPL. Iso-intensity curves of monaural ipsilateral and monaural contralateral stimulation were compared to con¢rm tonotopic alignment for binaurally excited neurons. Rate intensity functions to ipsilateral, contralateral and binaural stimuli at the unit’s BF were collected. Neurons were classi¢ed by their binaural responses patterns : ‘EE’ neurons excited from both sides, ‘EO’ only excited by ipsilateral stimuli, or ‘EI’ neurons excited by ipsilateral sounds but inhibited by contralateral stimuli. Excitability of units was monitored by sequentially recording responses to monaural or binaural BF stimuli delivered 30 dB above threshold. Peri-stimulus time histograms (PSTH), and total discharge rate to epochs of 50 stimuli were plotted. Agents were applied by pressure ejection, after baseline responses to at least 250 stimulus presentations were established, and e¡ects on responses monitored. A rate^intensity function was recorded prior to this paradigm, and in 19 cells, after a stable e¡ect of ZD7288 was established. Agents were applied with 30 PSI, 250 ms pressure pulses. ZD7288
Glutamate (20 mM) or AMPA (10 WM) (Sigma Alderich, St. Louis, MO, USA) dissolved in aCSF were applied by pressure ejection to directly excite recorded SOC neurons. The e¡ects of ZD7288 on tone-evoked and glutamate-evoked neuronal activity were compared to determine the relative role of ZD7288 blockade of Ih channels on the recorded neuron’s excitability. 2.6. Calibration of pressure ejection of drugs Volume of drugs ejected was determined by measuring the £uid level change in the pressure ejection pipet, and by examining histological marks of pressure-ejected WGA-HRP. During each ejection approximately 11.5 nl is ejected, and a total ejection volume of 70 nl with six pulses over a 1 min period. This volume ¢lls a sphere with a radius of over 280 Wm. Consistent with these estimates, pressure ejection of WGA-HRP into the brain produced spots with diameters of 500^800 Wm. Therefore, the expected volume of drugs applied initially delivers the agent over an area, which includes the somata and likely a large portion of the dendritic ¢eld of the recorded neuron. The concentration of drugs a¡ecting the recorded neuron somata and proximal dendrites is likely similar to concentrations of drugs in pressure ejection electrodes. As agents are diluted by di¡usion into surrounding tissue and circulation, the tissue concentration of agents will decrease over time. In addition, drug concentration at distal dendrites is expected to be lower than proximal locations.
Table 1 Tonotopic dependence of ZD7288 e¡ects BF of SOC neurons (kHz)
n
Percent decrease in activity following 100 WM ZD7288 Tone-evoked
1^2 2^4 4^8 8^16 16^32 a
2 8 7 21 9
Background activity
Mean : S.D. (%)
CV (%)
Mean : S.D. (%)
CV (%)
54 : 12 57 : 14 44 : 22%a 39 : 15%a 67 : 13
22 24 50 39 20
46 : 14 60 : 17 56 : 21 50 : 16 69 : 18
29 29 38 32 26
Signi¢cantly di¡erent from the 16^32 kHz group.
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Fig. 1. Application of the selective Ih channel antagonist, ZD7288, decreases the excitability of a LSO EI neuron. PSTH (A^C) and average response levels to 50 stimuli per point (D) to ipsilateral BF tone stimuli (4.0 kHz, 30 dB above threshold) show a selective decrease in excitability following pressure ejection of 100 WM ZD7288. Arrows (D) show application of aCSF and ZD7288 with six, 30 PSI, 250 ms pulses every 10 s. There was no change in tone-evoked (¢lled circles) or background (open circles) neuronal activity following pressure ejection of aCSF (B,D), while application ZD7288 (100 WM) decreased tone-evoked neuronal activity (C,D) by 59.6% (population mean: 49.7 : 19%). There was a gradual decrease in excitability following application of ZD7288.
2.7. Histology At the end of each experiment, animals were overdosed with sodium pentobarbital and perfused through the heart with 0.9% saline followed by paraformaldehyde ¢xative and sucrose-paraformaldehyde. Frozen sections (50 Wm) were cut in the plane of the electrode track, mounted, and reacted for peroxidase TMB/GOD (tetramethylbenzidine/glucose oxidase) chromogen reaction. Sections were counterstained with thionine. Tracings of relevant sections and electrode depth were used to identify locations of the neurons studied.
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(MNTB), including neurons in the superior paraolivary nucleus (SPN) and periolivary neurons around the SPN (n = 34). The location within the SOC of 18 cells could not be identi¢ed. In the SPN region, neurons with ‘O¡’ responses were also encountered, but were not routinely examined, as our goal was to determine the e¡ect of ZD7288 on excitatory tone-evoked responses. ZD7288 consistently decreased background ¢ring activity and tone-evoked neuronal activity in all cells tested with brief (1 min) applications of as low as 20 WM ZD7288. Application (six, 250 ms, 20^30 PSI pressure ejections over 1 min.) of ZD7288 at 100 WM decreased tone-evoked neuronal activity on average by 49.7 : 19% (range 18.3^94.3%, coe⁄cient of variation = 38%; n = 47), while background activity was decreased by 56.3 : 18.1% (n = 47; Figs. 1 and 2). There was no decrease in neuronal activity following application of aCSF (Figs. 1 and 2). Therefore, the decrease in activity to ZD7288 application was not due to mechanical effects of the pressure pulse or a reaction to the vehicle. The gradual decrease in neuronal activity following application of ZD7288 peaked after 2^3 min. Raster plots of individual responses con¢rm a gradual decrease, with no abrupt changes in responses after each pressure ejection. The reduction in SOC neuron excitability by 100 WM ZD7288 application was slightly lower (by 22.1 : 11%, n = 4) after a 20^32 min recovery period. Further recovery of excitability following 100 WM ZD7288 application was not studied over longer periods, as decreasing anesthetic levels over longer periods of time will also increase excitability.
3. Results 3.1. Selective antagonism of Ih channels decreases SOC neuronal excitability The e¡ect of ZD7288, a selective Ih channel antagonist, was examined in 71 SOC neurons (46 EE, 8 EI, 10 EO, and 7 cells, where only ipsilateral stimuli were tested (E-)) in 22 animals. Neurons localized by histology were recorded in the lateral nucleus of the trapezoid body (n = 4), LSO (n = 5), periolivary neurons around the LSO (n = 10), and in the region between the LSO and medial nucleus of the trapezoid body
Fig. 2. Decreased excitability of an EE neuron following application of the selective Ih channel antagonist, ZD7288. Background (open circles) and ipsilateral tone-evoked responses (4.615 kHz, 30 dB above threshold) were not a¡ected by application of aCSF, but decreased rapidly following application of 100 WM ZD7288. The decrease in excitability was slightly less 14 min after ZD7288 application.
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3.2. Dose- and time-dependence of ZD7288 induced decrease in excitability The decrease in excitability produced by a brief application (six, 250 ms, 20^30 PSI pressure ejections over 1 min) of ZD7288 was dose dependent (Fig. 3). Toneevoked neuronal activity was decreased by 44.9 : 20.8% (range 17.5^77%, coe⁄cient of variation = 46.3%; n = 7) after application of 50 WM ZD7288, and by 17.1 : 12.7% (range 2.8^32.6%, coe⁄cient of variation = 74.4%; n = 5) following application of 20 WM ZD7288. The decrease in tone-evoked neuronal activity was 4.3 : 6.8% (range 38.3^14.5%, coe⁄cient of variation = 158%, n = 7) following application of 10 WM ZD7288. This dose-dependence of ZD7288 e¡ects is very similar to dose-dependent blockade of Ih channels in vitro following brief application of ZD7288 (Shin et al., 2001). However, extended periods (15 min or longer) are required for low doses ( 6 50 WM) of ZD7288 to produce a signi¢cant blockade (Harris and Constanti, 1995). Time-dependent blockade of Ih channels was examined by applying low doses (10 WM) of ZD7288 over a 10^15 min period (pressure ejections every 20 s). In 4/4 cells (2 EE, 1 EI and 1 EO cell), 10 WM ZD7288 appliFig. 4. Excitability decreased slowly when 10 WM ZD7288 was applied over 13 min (250 ms, 30 PSI applications every 20 s). The time-dependent e¡ect of ZD7288 is similar to the time-dependent blockade of Ih channels, in vitro. Excitability recovered over a 20 min period following cessation of ZD7288 application.
cation produced a gradual decrease in activity (Fig. 4). The average maximal decrease produced by 10 WM ZD7288 was 67.1 : 17.1%. After cessation of ZD7288 application, a gradual increase in activity over 15 min was observed, reaching levels near prior control levels. 3.3. E¡ects of ZD7288 on responses to excitatory amino acid agonists
Fig. 3. ZD7288 decreased the rate of neuronal ¢ring in SOC neurons in a dose-dependent manner. Percent decrease in SOC neuron tone-evoked responses for individual cells (diamonds), and average percent decrease in responses (open circles) are plotted as a function of concentration of ZD7288 in the ejection pipet. Data were ¢tted with a sigmoid curve (line). The e¡ects of ZD7288 exhibited considerable variability at each dose, but consistently decreased excitability. The average e¡ect of ZD7288 did not signi¢cantly increase between 50 and 100 WM.
The e¡ects of ZD7288 (100 WM) on neuronal activity, directly stimulated by pressure ejection application of agonists, were determined and the magnitude of ZD7288 e¡ects on agonist-evoked excitation and toneevoked responses were compared in the same neurons. Glutamate (50 mM) or AMPA (10 WM) were applied by brief 150 ms pressure pulses. Lower concentrations of either agent had no e¡ect or produced small changes in ¢ring rate. Brief exogenous application of glutamate or AMPA evoked a rapid brief ( 6 500 ms) increase in ¢ring rate, which peaked after less than 400 ms. ZD7288 consistently decreased responses to glutamate in all eight cells examined. In six cells, where toneevoked responses were also examined, ZD7288 decreased glutamate-evoked ¢ring activity by 75.7 :
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neurons tested, which is likely related to excitotoxic e¡ects of AMPA. However, repeated glutamate application did not decrease neuronal excitability, which was decreased only following application of ZD7288. 3.4. Factors a¡ecting ZD7288 reduction in activity The e¡ects of ZD7288 on threshold- and intensitydependence of tone-evoked responses were examined in 19 SOC neurons. ZD7288 (100 WM) decreased overall excitability of these neurons by 47.5 : 9.3% (n = 19). Thresholds of responses in rate^intensity function were not a¡ected by ZD7288 application. This may be due to the similar, but slightly greater (6.9%) average percent decrease in background activity compared to tone-evoked responses following 100 WM ZD7288 application (r = 0.83, n = 47; Fig. 6, paired t-test, P 6 0.01). Furthermore, there was no relationship between unit thresholds and the percent decrease in toneevoked responses by ZD7288. The decrease in neuronal ¢ring rate to ZD7288 also did not vary signi¢cantly as a function of intensity of BF tonal stimuli in 13/19 cells. ZD7288 produced a lesser decrease in responses for higher intensity BF stimuli (average 21% change/10 dB) in four cells, while two cells exhibited the opposite trend. The absolute decrease in background and toneevoked ¢ring rate was related to absolute activity levels before application of ZD7288. The ZD7288 induced
Fig. 5. ZD7288 decreases both glutamate- and tone-evoked neuronal activity. Responses to pressure ejection (150 ms, 30 PSI pulses) application of glutamate (50 mM) were decreased following application of ZD7288 (100 WM). (A) PSTH of glutamate-evoked neuronal ¢ring before and after application of ZD7288. The average decrease in glutamate-evoked responses following ZD7288 application was greater than the average decrease in tone-evoked responses (C), which was observed in the majority (5/6) of cells (B).
18.8%, while it decreased tone-evoked neuronal activity by 50.6 : 17.2% (Fig. 5). Although, ZD7288 usually reduced glutamate-evoked neuronal ¢ring more than tone-evoked neuronal ¢ring, this was not statistically signi¢cant (paired t-test, P = 0.11), likely due to the small sample size. ZD7288 also decreased AMPAevoked neuronal activity by 48.8 : 31.6% (n = 4) while decreasing tone-evoked ¢ring activity by 50.8 : 7.5% (n = 4). There was no statistically signi¢cant di¡erence between the change in tone-evoked and AMPA-evoked neuronal ¢ring (t-test, P = 0.45). However, all neuronal activity was lost soon after AMPA application in other
Fig. 6. The decrease in background and tone-evoked activity following 100 WM ZD7288 application was similar in individual cells. The average percentage decrease in background activity, however, was signi¢cantly greater than the average percent decrease in toneevoked responses, with most points lying above the line of equality (dark line).
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greater in neurons with BFs between 16 and 32 kHz than neurons with BFs between 4^8 kHz and 8^16 kHz (Fig. 8, Table 1 (see Section 2.5), one-way analysis of variance (ANOVA), P 6 0.01; Tukey^Kramer honest statistical di¡erence (HSD). The e¡ect of ZD7288 on background activity followed a similar trend, but was not signi¢cant (P = 0.08, power = 0.6). Although, the excitability of EO neurons was signi¢cantly less a¡ected (32.4 : 5.5% reduction, n = 9) by ZD7288 than EE (51.5 : 3.1%, n = 29) and EI (72.3 : 9.6%, n = 3) neurons, 89% of EO neurons had BFs between 4 and 16 kHz. After excluding EO neurons, the e¡ect of ZD7288 (100 WM) on tone-evoked responses was signi¢cantly greater in neurons with BFs between 16 and 32 kHz
Fig. 7. The absolute decrease in background and tone-evoked (BF tones, 30 dB above threshold) ¢ring activity (spikes/s) produced by ZD7288 application was related to the pre-drug level of background and tone-evoked activity. There was a higher correlation for the relationship for ZD7288 e¡ects and background activity than for tone-evoked activity. Lines in each plot represent linear ¢ts (solid line) with 95% con¢dence intervals (dashed lines). The top panel shows percent decrease in the ¢ring rate in individual neurons, while the bottom panel shows the averaged reduction in ¢ring rate and standard error.
decrease in background ¢ring rate was highly correlated with the pre-drug background activity (correlation, r = 0.9, n = 45, Fig. 7). The ZD7288 induced decrease in tone-evoked ¢ring rate was correlated with the predrug tone-evoked response level, but exhibited less dependence (correlation, r = 0.75, n = 45, Fig. 7). The close relationship of background activity and sensitivity to ZD7288 is consistent with Ih channels playing an important role in determining resting potential and hence background ¢ring levels. The relative e¡ect of ZD7288 (100 WM) in decreasing tone-evoked neuronal activity was also dependent on the BF of neurons. SOC neurons were sorted into octave groups based on their BF. The e¡ect of ZD7288 (100 WM) on tone-evoked responses was signi¢cantly
Fig. 8. The magnitude of the decrease in excitability following ZD7288 application was related to neuronal BF. The top panel shows percent decrease in the ¢ring rate in individual neurons, while the bottom panel shows the averaged reduction in the ¢ring rate and standard error. Neurons with BFs greater than 16 kHz exhibited a signi¢cantly (ANOVA, P 6 0.0001) greater decrease in toneevoked activity following ZD7288 (100 WM) application than neurons with BFs between 4^8 kHz, and 8^16 kHz (bottom panel). ZD7288 decreased the excitability of EO cells to a lesser extent, but most of these neurons had BFs between 4 and 16 kHz.
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than neurons with BFs between 8 and 16 kHz (one-way ANOVA, P 6 0.02; Tukey^Kramer HSD). Other analysis on frequency-dependence of ZD7288 e¡ects for cells grouped by location or aurality was unreliable (power of analysis 6 0.5).
4. Discussion ZD7288, a selective blocker of Ih conductances, consistently decreased neuronal excitability of SOC neurons, in vivo. Pressure ejection of ZD7288 produced a gradual decrease of activity over 2^3 min, while the vehicle (aCSF) did not alter neuronal ¢ring. Therefore, mechanical e¡ects due to pressure ejection of pharmacological agents, and the vehicle were not responsible for the ZD7288-induced decrease in ¢ring activity. ZD7288 produced dose- and time-dependent, consistent decreases in superior olivary neuron tone-evoked and glutamate-evoked responses, in vivo. Together with the absence of e¡ects in controls, this is consistent with the selective blockade of Ih channels by ZD7288 directly reducing the excitability of SOC neurons. ZD7288 selectively blocks Ih channels, in vitro, at concentrations up to 100 WM (Harris and Constanti, 1995). High concentrations (300 WM) of ZD7288 produce a non-speci¢c, immediate cessation of ¢ring activity in dopamine neurons, which was also associated with a reduction in action potential amplitudes (Seutin et al., 2001). Lower concentrations (up to 100 WM) of ZD7288 have no reported e¡ects on other ion channels or non-speci¢c e¡ects, and do not a¡ect spike amplitude (nigral neurons, Harris and Constanti, 1995 ; vestibular neurons in vitro, Chabbert et al., 2001). The amplitude of spike after hyperpolarizations was increased by 100 WM ZD7288 (Harris and Constanti, 1995). Maximal reductions in SOC neuron ¢ring were observed following pressure ejection from pipets containing 50 and 100 WM ZD7288, and partial blockade at 20 WM. The dose response pro¢le (Kd = 40.5 : 3.3 WM) for brief (4 s) application of ZD7288 onto inside-out patches from HEK cells expressing mHCN1 (Shin et al., 2001) is very similar to the dose-dependent decrease in ¢ring rate by short duration (1 min) application of ZD7288 observed in SOC neurons, in vivo. ZD7288 progressively blocks Ih conductances during bath application, in vitro, where low doses (0.5^10 WM) require 15 to 30 min for complete blockade (Harris and Constanti, 1995). Since ZD7288 is a lipophilic drug, which acts at an intracellular domain of the Ih channel, delays in blockade are attributable to di¡usion through the tissue and across the neuronal membrane (Harris and Constanti, 1995). Therefore, dose-response curves measured after 15 min of ZD7288 application (between 0.9 and 4.3 WM ZD7288 produce a half-maximal de-
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crease in Ih , Harris and Constanti, 1995) are shifted to the right compared to the dose-dependent e¡ects of ZD7288 applied over a brief period (1 min in our study, and 4 s, Shin et al., 2001). Application of ZD7288 (10 WM) over a period of 15 min veri¢ed that low concentrations of ZD7288 produce a time-dependent decrease in SOC neuron excitability. Therefore, the dose- and time-dependence of ZD7288 e¡ects on SOC neuron excitability are consistent with prior studies showing selective blockade of Ih channels. Reversal of ZD7288 (10 WM) e¡ects was unexpected based on many prior studies. Factors a¡ecting reversal of ZD7288 e¡ects include clearance rate of ZD7288, the completeness of Ih channel blockade throughout the neuron, and reversibility in the blockade. The e¡ect of ZD7288 on Ih channels composed of di¡erent hyperpolarization-activated cyclic nucleotide-gated channel subunits (HCN) is still poorly understood. Shin et al. (2001) demonstrated that ZD7288 blockade of Ih channels composed of mHCN1 subunits is reversible, which appears to depend on three amino acids in the S6 pore region of the channel. These amino acids vary between subunits (Ludwig et al., 1998). Subunits (HCN1 to HCN4) of the Ih family of channels are di¡erential expressed in auditory neurons from cochlear nuclei to cortex (Monteggia et al., 2000), with most auditory neurons in the brainstem likely expressing all isoforms. The relatively fast activation of Ih conductances in LSO neurons (Adam et al., 1999, 2001) and SPN neurons (personal observations) is characteristic of Ih channels with HCN1 subunits (Franz et al., 2000). We observed a partial reversibility of the decreased excitability of SOC neurons to high (50 and 100 WM) doses of ZD7288. The recovery of SOC neuron excitability after 10 WM ZD7288 application could be due to the reversible blockade of Ih channels with HCN1 subunits. ZD7288 also decreased neuronal responses evoked by application of glutamate or AMPA, which is consistent with a direct action of ZD7288 on the recorded units. Most neurons in the SOC, including LSO, MSO, MNTB and SPN neurons exhibit a sag in hyperpolarizing responses, which has been consistently shown to be due to an Ih conductance. Therefore, ZD7288 is expected to directly a¡ect all these neurons. The major e¡ect of glutamate application on recorded activity is expected to be direct excitation of recorded neurons by activation of ionotropic glutamate receptors. ZD7288 typically decreased the responses to glutamate more than responses to sound. Pressure ejection of ZD7288 and glutamate likely a¡ect similar areas of the recorded neuron, whereas excitatory inputs to SOC neurons will impinge on the full extent of their often large dendritic ¢elds. Therefore, pressure application of ZD7288 may be expected to have a greater e¡ect on responses to pressure-ejected agonists. It is possible that ZD7288
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also decreases activation of interneurons by glutamate, which could contribute to the greater e¡ect of ZD7288 on glutamate-evoked responses. An e¡ect of ZD7288 blockade of Ih channels in presynaptic terminals cannot be ruled out. However, modulation and blockade of presynaptic Ih channels did not a¡ect synaptic transmission at the calyceal endings in the MNTB (Cuttle et al., 2001). Since the major e¡ect of glutamate application is expected to be a direct excitation of the recorded neuron, the marked decrease in excitability to glutamate following ZD7288 application is indicative of a predominant e¡ect of ZD7288 and Ih channel blockade on the recorded SOC neurons. The marked decrease in excitability of SOC neurons following ZD7288 application suggests a signi¢cant contribution of active Ih channels to the resting membrane potential and excitability of these neurons, in vivo. Non-selective blockade of Ih , in vitro, by cesium leads to membrane hyperpolarization in thalamic, rat fetal medullary, cochlear nucleus, amygdala, dorsal root ganglion, and LSO neurons (McCormick and Pape, 1990; Womble and Moises, 1993; Wang et al., 1997; Wellner-Kienitz and Shams, 1998; Bal and Oertel, 2000; Adam et al., 2001). Therefore, blockade of Ih channels by ZD7288 is expected to hyperpolarize SOC neurons, and thereby reduce their excitability, in vivo. Ih channels are expected to be partially active at resting membrane potentials, as for example, the activation range of Ih channels in thalamic neurons is between 360 and 395 mV (McCormick and Pape, 1990). The decrease in tone-evoked activity, but especially background neuronal activity by ZD7288 is consistent with Ih channels being active at rest in SOC neurons. The magnitude of the decrease in excitability produced by ZD7288 is highly variable between neurons. E¡ects of ZD7288 are not intensity dependent in most neurons. Variability in ZD7288’s e¡ects could depend on di¡erences in drug delivery. However, di¡erences in the magnitude of ZD7288 e¡ects in speci¢c cell groups, for example, EO neurons and neurons with BFs greater than 16 kHz, may be due to di¡erences in Ih channel activation or composition. The high correlation between pre-drug background activity and the absolute decrease in activity following ZD7288 application is also suggestive of di¡erential activation of Ih channels at rest in di¡erent cells. The activation range of Ih channels is dependent on the subunit composition of channels, but is also regulated by intracellular cAMP levels. Coexpression of HCN1 and HCN2 subunits in oocytes form heteromultimers composed of di¡erent HCN subunits, and unique properties compared to summative properties of these subunit expressed in isolation (Chen et al., 2001). The possible formation of Ih channels composed of di¡erent HCN subunits in auditory neurons potentially will produce Ih channels with
unique properties, including their sensitivity to cAMP. The activation range of Ih channels in cultured spiral ganglion cells exhibit a wide variability of over 40 mV for half maximal voltages (Mo and Davis, 1997). Furthermore, the activation range of Ih channels in MNTB neurons was shifted by over 35 mV in the presence of 8-Br-cAMP or norepinephrine, resulting in an increased activation of Ih channels at rest and a 2^3 mV depolarization at rest (Banks et al., 1993). Regulation of Ih channel activation in SOC neurons could be through transmitter systems known to modulate cAMP synthesis, and found in the SOC, such as metabotropic glutamate receptors (Kotak and Sanes, 1995), and cholinergic (Henderson and Sherri¡, 1991; Yao and Godfrey, 1998) or noradrenergic (Wynne and Robertson, 1996) innervation. The variability in the ZD7288-induced decrease in excitability of SOC neurons is consistent with varied properties of Ih channels in neurons due to expression of Ih channel subunits or their regulation by cAMP. The marked decrease in SOC neuron excitability following application of ZD7288 shows a signi¢cant role of Ih in determining neuronal excitability. Since the ability of LSO neurons to localize sound sources is rate dependent, Ih channels may play an important role in the encoding of information for sound localization. Plasticity in LSO responses may be adaptive to compensate for asymmetric temporary or permanent losses in peripheral hearing. Modulation of Ih channels, for example by cAMP, may also regulate information £ow to higher centers. Thus, Ih conductances potentially have an important role in sound localization perception and may impact processing of sounds, such as speech.
Acknowledgements The authors thank Sharjeel Farooqui for expert technical assistance. This research was supported by the Deafness Research Foundation (P.G.F.). A.S. was supported by an NIH training grant (T32 DC00026-14).
References Adam, T., Schwarz, D., Finlayson, P.G., 1999. Firing Properties of chopper and delay neurons in the Lateral Superior Olive of the rat. Exp. Brain Res. 12, 489^502. Adam, T., Finlayson, P., Schwarz, D., 2001. Membrane properties of principal neurons of the lateral Superior Olive. J. Neurophysiol. 86, 922^934. Akasu, T., Shoji, S., Hasuo, H., 1993. Inward recti¢er and low-threshold calcium currents contribute to the spontaneous ¢ring mechanism in neurons of the rat suprachiasmatic nucleus. P£ug. Arch. 425, 109^116.
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A.G. Shaikh, P.G. Finlayson / Hearing Research 183 (2003) 126^136 Bal, R., Oertel, D., 2000. Hyperpolarization-activated, mixed-cation current (Ih ) in octopus cells of the mammalian cochlear nucleus. J. Neurophysiol. 84, 806^817. Banks, M., Pearce, R., Smith, P., 1993. Hyperpolarization-activated cation current (Ih ) in neurons of the medial nucleus of the trapezoid body: voltage-clamp analysis and enhancement by norepinephrine and cAMP suggesting a modulatory mechanism in the auditory brainstem. J. Neurophysiol. 71, 119^128. Bartlett, E.L., Smith, P.H., 1999. Anatomic, intrinsic, and synaptic properties of dorsal and ventral division neurons in rat medial geniculate body. J. Neurophysiol. 81, 1999^2016. Bobker, D.H., Williams, J.T., 1989. Serotonin augments the cation current Ih in central neuron’s receptor subtypes in mammalian neurons. Neuron 2, 1535^1540. Boudreau, J.C., Tsuchitani, C., 1968. Binaural interactions in the cat superior olive S segment. J. Neurophysiol. 31, 442^454. Caird, D., Klinke, R., 1983. Processing of binaural stimuli by cat superior olivary complex neurons. Exp. Brain Res. 52, 385^399. Chabbert, C., Chambard, J.M., Valmier, J., Sans, A., Desmadryl, G., 2001. Hyperpolarization-activated (Ih) current in mouse vestibular primary neurons. NeuroReport 12, 2701^2704. Chen, S., Wang, J., Siegelbaum, S., 2001. Properties of hyperpolarization-activated pacemaker current de¢ned by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide. J. Gen. Physiol. 117, 491^504. Cuttle, M.F., Rusznak, Z., Wong, A.Y., Owens, S., Forsythe, I.D., 2001. Modulation of a presynaptic hyperpolarization-activated cationic current (Ih ) at an excitatory synaptic terminal in the rat auditory brainstem. J. Physiol. 534, 733^744. Finlayson, P.G., 1995. Decreased inhibition to lateral superior olive neurons in young and aged Sprague-Dawley rats. Hear. Res. 87, 84^95. Finlayson, P.G., Adam, T.J., 1997. Excitatory and inhibitory response adaptation in the superior olivary complex to monaural and binaural acoustic ‘maskers’. Hear. Res. 103, 1^18. Finlayson, P.G., Caspary, D.M., 1993. Response properties in young and old Fischer-344 rat lateral superior olive neurons - a quantitative approach. Neurobiol. Aging 14, 127^139. Franz, O., Liss, B., Neu, A., Roeper, J., 2000. Single-cell mRNA expression of HCN1 correlates with a fast gating phenotype of hyperpolarization-activated cyclic nucleotide-gated ion channels (Ih ) in central neurons. J. Eur. Neurosci. 12, 2685^2693. Fu, X., Brezden, B., Wu, S., 1997. Hyperpolarization activated inward current in neurons of the rat dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78, 2235^2245. Goldberg, J.M., Brown, P.B., 1969. Response of binaural neurons of dog superior olivary complex to dichotic tonal stimuli: some physiological mechanisms of sound localization. J. Neurophysiol. 32, 613^636. Golding, N., Ferragamo, M., Oertel, D., 1999. Role of intrinsic conductances underlying responses to transients in octopus cells of the cochlear nucleus. J. Neurosci. 19, 2897^2905. Harris, N.C., Constanti, A., 1995. Mechanism of block by ZD7288 of the hyperpolarization-activated inward rectifying current in guinea pig substantia nigra neurons in vitro. J. Neurophysiol. 74, 2366^ 2378. Henderson, Z., Sherri¡, F.E., 1991. Distribution of choline acetyltransferase immunoreactive axons and terminals in the rat and ferret brainstem. J. Comp. Neurol. 314, 147^163. Hu, B., 1995. Cellular basis of temporal synaptic signalling: an in vitro electrophysiological study in rat auditory thalamus. J. Physiol. 483, 167^182. Kotak, V.C., Sanes, D.H., 1995. Synaptically evoked prolonged depolarizations in the developing auditory system. J. Neurophysiol. 74, 1611^1620.
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Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F., Biel, M., 1998. A family of hyperpolarization-activated mammalian cation channels. Nature 393, 587^591. Maccaferri, G., McBain, C.J., 1996. The hyperpolarization-activated current (Ih ) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J. Physiol. 497 (Pt 1), 119^130. Manis, P.B., 1990. Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro. J. Neurosci. 10, 2338^2351. McCormick, D.A., Pape, H.-C., 1990. Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones. J. Physiol. 431, 291^318. Mo, Z.-L., Davis, R., 1997. Heterogenous voltage dependence of inward recti¢er currents in spiral ganglion neurons. J. Neurophysiol. 78, 3019^3027. Monteggia, L.M., Eisch, A.J., Tang, M.D., Kaczmarek, L.K., Nestler, E.J., 2000. Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Brain Res. Mol. Brain Res. 81, 129^139. Pape, H., McCormick, D., 1989. Noradrenaline and serotonin selectively modulate thalamic burst ¢ring by enhancing a hyperpolarization-activated cation current. Nature 340, 715^718. Paxinos, G., Watson, C., 1982. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Pedarzani, P., Storm, J.F., 1993. PKA mediates the e¡ects of monoamine transmitters on the K+ current underlying the slow spike frequency adaptation in hippocampal neurons. Neuron 11, 1023^ 1035. Robinson, R.B., Siegelbaum, S.A., 2003. Hyperpolarization-activated cation currents: from molecules to physiological function. Annual Rev. Physiol. 65, 453^480. Saitoh, F., Konishi, S., 2000. Excitability induced by K-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells. J. Neurophysiol. 84, 2026^ 2034. Schwarz, D., Puil, E., 1998. Firing properties of spherical bushy cells in the anteroventral cochlear nucleus of the gerbil. Hear. Res. 114, 127^138. Seutin, V., Massotte, L., Renette, M.F., Dresse, A., 2001. Evidence for a modulatory role of Ih on the ¢ring of a subgroup of midbrain dopamine neurons. NeuroReport 12, 255^258. Shaikh, A., Finlayson, P., 2002. Pharmacological manipulation of superior olivary complex ionic conductance. Assoc. Res. Otolaryngol. Winter Meet. 25, 113. Shin, K.S., Rothberg, B.S., Yellen, G., 2001. Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate. J. Gen. Physiol. 117, 91^ 101. Smith, P.H., 1995. Structural and functional di¡erences distinguish principal from nonprincipal cells in the guinea pig MSO slice. J. Neurophysiol. 73, 1653^1667. Tennigkeit, F., Schwarz, D.W., Puil, E., 1996. Mechanisms for signal transformation in lemniscal auditory thalamus. J. Neurophysiol. 76, 3597^3608. Tokimasa, T., Akasu, T., 1990. Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones. J. Physiol. 420, 409^429. Tsuchitani, C., Boudreau, J.C., 1969. Stimulus level of dichotically presented tones and cat superior olive S-segment cell discharge. J. Acoust. Soc. Am. 46, 979^988. Wang, Z., Van Den Berg, R.J., Ypey, D.L., 1997. Hyperpolarizationactivated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture. J. Neurophysiol. 78, 177^ 186.
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Wellner-Kienitz, M.C., Shams, H., 1998. Hyperpolarization-activated inward currents contribute to spontaneous electrical activity and CO2/H+ sensitivity of cultivated neurons of fetal rat medulla. Neuroscience 87, 109^121. Womble, M.D., Moises, H.C., 1993. Hyperpolarization-activated currents in neurons of the rat basolateral amygdale. J. Neurophysiol. 70, 2056^2065.
Wynne, B., Robertson, D., 1996. Localization of dopamine-beta-hydroxylase-like immunoreactivity in the superior olivary complex of the rat. Audiol. Neurotol. 1, 54^64. Yao, W., Godfrey, D.A., 1998. Immunohistochemical evaluation of cholinergic neurons in the rat superior olivary complex. Microsc. Res. Tech. 41, 270^283.
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Hyperpolarization-activated (Ih ) conductances a¡ect brainstem auditory neuron excitability Aasef G. Shaikh, Paul G. Finlayson
Department of Otolaryngology, Wayne State University, 550 E Can¢eld Avenue, Rm. 327, Detroit, MI 48201, USA Received 20 February 2003; accepted 2 July 2003
Abstract Blockade of the hyperpolarization-activated cyclic-nucleotide-gated mixed-cationic conductance (Ih ) by ZD7288 markedly reduces excitability of neurons in the superior olivary complex (SOC), in vivo. Following pressure ejection application of 100 WM ZD7288, extracellular recorded single unit responses of 47/47 SOC neurons to monaural or binaural pure tone best frequency (BF) stimuli (30 dB above threshold) decreased by 49.7 : 19%, and background activity decreased by 56.3 : 18.1%. Pressure ejection of the vehicle did not affect excitability. The dose- and time-dependence of ZD7288 (10^100 WM) effects are consistent with specific blockade of Ih currents. SOC neuron responses to pressure-ejected glutamate were also decreased following application of 100 WM ZD7288 by 76.7 : 28.0%, which suggests a predominant direct effect of ZD7288 on auditory cell excitability. The considerable variability in the magnitude of ZD7288 effects between cells was only partially accounted for by greater effects on neurons with BFs greater than 16 kHz. Therefore, Ih channels significantly contribute to auditory brainstem neuron excitability, affecting their response level to acoustic stimuli. The variability in the ZD7288 reduction in excitability and its variation with the BF of units could be an indication of regulation and plasticity in neuronal encoding of sounds. @ 2003 Elsevier B.V. All rights reserved. Key words: Hyperpolarization-activated cyclic-nucleotide-sensitive non-selective cation channels; Auditory; Superior olivary complex; ZD7288; Excitability; Sound
1. Introduction Hyperpolarization-activated cyclic-nucleotide-gated mixed (or non-selective) cation channels carry prominent conductances (Ih ) a¡ecting membrane properties in most auditory neurons from spiral ganglion cells to neocortex (Manis, 1990; Banks et al., 1993; Hu, 1995; Smith, 1995; Tennigkeit et al., 1996; Fu et al., 1997;
* Corresponding author. Tel.: +1 (313) 577 8041; Fax: +1 (313) 577 8555. E-mail address: p¢[email protected] (P.G. Finlayson). Abbreviations: Ih , hyperpolarization-activated cyclic-nucleotidegated mixed-cationic conductance; cAMP, cyclic-AMP; SOC, superior olivary complex; LSO, lateral superior olive; PSTH, peristimulus time histograms; MNTB, medial nucleus of the trapezoid body; SPN, superior paraolivary nucleus; BF, best frequency; WGA-HRP, wheat-germ agglutinin horseradish peroxidase; aCSF, arti¢cial cerebrospinal £uid; HCN hyperpolarization-activated cyclic nucleotide-gated channel subunit
Mo and Davis, 1997; Schwarz and Puil, 1998; Golding et al., 1999; Adam et al., 1999, 2001; Bartlett and Smith, 1999; Bal and Oertel, 2000; Cuttle et al., 2001). In prior studies, the presence of Ih channels in auditory neurons was characterized in tissue slices from relatively young animals (less than 30 days old). The role(s) of Ih in adult animals and responses to acoustic stimuli have not been investigated. In the present study, we examine the role of Ih in determining auditory neuron excitability in the superior olivary complex (SOC) in adult anaesthetized animals. An important role of Ih channels is the regulation of membrane potential (McCormick and Pape, 1990; Banks et al., 1993; Adam et al., 2001; Cuttle et al., 2001), which can directly a¡ect neuronal excitability (Akasu et al., 1993; Maccaferri and McBain, 1996; Wang et al., 1997; Saitoh and Konishi, 2000; Seutin et al., 2001). This non-inactivating channel is activated by membrane hyperpolarization. The reversal potential
0378-5955 / 03 / $ ^ see front matter @ 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00224-7
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of Ih channels is between 333 and 348 mV in auditory brainstem neurons (Banks et al., 1993; Fu et al., 1997; Mo and Davis, 1997; Bal and Oertel, 2000; Cuttle et al., 2001), due to its mixed permeability for sodium and potassium. Therefore, opening of Ih channels (at rest and during hyperpolarization) produces a depolarizing in£uence, while decreased activation of Ih channels during depolarization (or blockade) hyperpolarizes cells (Robinson and Siegelbaum, 2003). As expected, blockade of Ih channels produces a hyperpolarization in membrane potential in lateral superior olive (LSO) neurons (Adam et al., 2001). Therefore, the membrane potential and excitability may be stabilized around a set point, dependent on the activation of Ih channels. The activation range for Ih is shifted towards more depolarized levels by increasing cyclic-AMP (cAMP) levels in central neurons (Bobker and Williams, 1989; Pape and McCormick, 1989; Tokimasa and Akasu, 1990; Pedarzani and Storm, 1993), and in the auditory system (Banks et al., 1993; Mo and Davis, 1997; Cuttle et al., 2001). The wide variation in Ih responses observed in spiral ganglion cells in culture may be attributed to intercellular di¡erences in cAMP levels (Mo and Davis, 1997), and may re£ect that Ih is also regulated physiologically. A role of Ih channels in determining the excitability of SOC neurons may have a signi¢cant impact on the encoding of essential acoustic information. LSO neurons are characterized by a linear relationship between their neuronal discharge activity and interaural intensity di¡erences (Boudreau and Tsuchitani, 1968; Goldberg and Brown, 1969; Tsuchitani and Boudreau, 1969; Caird and Klinke, 1983; Finlayson and Caspary, 1993; Finlayson, 1995). If the excitability of LSO neurons is modulated, their rate-code representation of sound source location can be shifted. Changes in excitability of other SOC neurons would a¡ect additional information for encoding sound source localization, and other features of sound. In this study and a preliminary report (Shaikh and Finlayson, 2002), we document that Ih conductances signi¢cantly a¡ect SOC neuron excitability.
2. Materials and methods 2.1. Animal preparation and surgery The Animal Investigation Committee at Wayne State University approved the care and use of animals reported in this study. Anesthesia of young adult male Long Evans rats (3^6 months in age) weighing 499^ 600 g was induced with a combination of ketamine (85 mg/kg) and xylazine (3 mg/kg), and followed 2^5 min later by an initial dose of 44 mg/kg sodium pento-
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barbital. Supplemental doses of anesthetics were administered as required every 2^3 h, indicated by the presence of toe pinch and eye blink re£ex, alternating between sodium pentobarbital (25 mg/kg) and ketamine (40 mg/kg). The animal’s rectal temperature was maintained at 37‡C by a thermostatically controlled DC heating pad. Following surgical exposure of the external auditory canals, the animal was placed in the stereotaxic plane (Paxinos and Watson, 1982) using ear bars. Following ¢xation of the skull with a custom-made head holder, ear bars were removed, and earphones sealed with the external auditory canals to produce closed auditory delivery paths. The foramen magnum was exposed and dilated. The dura was re£ected for access of electrodes to the brainstem through the cerebellum (for details of surgery, Finlayson and Adam, 1997). 2.2. Stimulus generation and delivery Binaural stimuli were digitally generated and delivered using the program A/Dvance (McKeller Designs) on a Macintosh Quadra 950 computer with National Instruments boards (MIO-161-9; DMA-2800). Outputs of the digital to analog converters were bandpass ¢ltered, ampli¢ed by an Amcron (Crown) ampli¢er, and attenuated under computer control (Medical Associates MA919 attenuators). Stimuli were transduced by impedance-matched headphones (Beyer-Dynamic 600 6, B4132.01, frequency range 0.05^35 kHz). Output of the stimulus system was calibrated o¡-line using a coupler with a 0.3 cc air space. Signals were measured with a 1/4-inch condenser microphone (Bruel and Kjaer) and a calibrated microphone preampli¢er (Bruel and Kjaer 2804). Calibration tables were used to determine intensities in dB SPL (re 0.0002 dyn/cm2 ) for tonal stimuli from 0.05 to 32 kHz. Brainstem auditory evoked potentials from scalp electrodes were recorded to monaural ipsilateral and contralateral clicks. 2.3. Extracellular single unit electrophysiology Double and triple-barrel piggyback electrodes were used for extracellular single unit recordings and simultaneous application of pharmacological agents. Piggyback electrodes were fabricated by gluing single or double-barrel pressure ejection pipet to the recording electrode, with the tip of the ejection pipet 150 Wm behind the recording electrode tip. Recording electrodes were ¢lled with 2% wheat-germ agglutinin horseradish peroxidase (WGA-HRP) dissolved in 2 M NaCl. Tip resistance of recording electrodes was 12^20 M6. The pressure ejection electrodes were ¢lled with the appropriate pharmacological agents. Electrodes were advanced at an angle 15‡ to the right of the mid-sagital
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axis and 49.5‡ up from the horizontal axis, to reach the left SOC. Signals were ampli¢ed using an A-M Systems (Seattle, WA, USA) 1800 ampli¢er and Kikisui (Japan) oscilloscope. Recordings were bandpass ¢ltered (between 1 or 10 Hz and 5 kHz). Spikes were identi¢ed on an oscilloscope and converted to pulses using a World Precision Instruments 121 window discriminator, and timing of spikes recorded with 10 Ws resolution.
(Tocris) was dissolved in arti¢cial cerebrospinal £uid (aCSF; NaCl 7.93 g/l, KCl 0.22 g/l, CaCl2 2H2 O 0.24 g/l, MgSO4 0.14 g/l, glucose 0.6 g/l, and HEPES 5.96 g/l). For aCSF controls and ZD7288 application, six pulses were given over a 1 min period. Recording sites or tracks were marked by iontophoretic application of WGA-HRP (5 WA, 500 ms pulses at 1 Hz). The recording locations were later con¢rmed histologically.
2.4. Classi¢cation of units
2.5. ZD7288 e¡ects on responses to excitatory amino acid agonists
Search stimuli were binaural clicks with the contralateral stimuli delayed by 20 ms relative to ipsilateral stimuli to discretely observe evoked potentials to either ear. As electrodes advanced into the SOC, robust evoked potentials to clicks to each ear were observed. Auditory neurons were initially characterized using 50 ms pure tone stimuli with 5 ms rise/fall ramps. Best frequencies (BF, the frequency of stimulation producing the greatest spike count) were determined from right and left monaural iso-intensity curves at 50 dB SPL. Iso-intensity curves of monaural ipsilateral and monaural contralateral stimulation were compared to con¢rm tonotopic alignment for binaurally excited neurons. Rate intensity functions to ipsilateral, contralateral and binaural stimuli at the unit’s BF were collected. Neurons were classi¢ed by their binaural responses patterns : ‘EE’ neurons excited from both sides, ‘EO’ only excited by ipsilateral stimuli, or ‘EI’ neurons excited by ipsilateral sounds but inhibited by contralateral stimuli. Excitability of units was monitored by sequentially recording responses to monaural or binaural BF stimuli delivered 30 dB above threshold. Peri-stimulus time histograms (PSTH), and total discharge rate to epochs of 50 stimuli were plotted. Agents were applied by pressure ejection, after baseline responses to at least 250 stimulus presentations were established, and e¡ects on responses monitored. A rate^intensity function was recorded prior to this paradigm, and in 19 cells, after a stable e¡ect of ZD7288 was established. Agents were applied with 30 PSI, 250 ms pressure pulses. ZD7288
Glutamate (20 mM) or AMPA (10 WM) (Sigma Alderich, St. Louis, MO, USA) dissolved in aCSF were applied by pressure ejection to directly excite recorded SOC neurons. The e¡ects of ZD7288 on tone-evoked and glutamate-evoked neuronal activity were compared to determine the relative role of ZD7288 blockade of Ih channels on the recorded neuron’s excitability. 2.6. Calibration of pressure ejection of drugs Volume of drugs ejected was determined by measuring the £uid level change in the pressure ejection pipet, and by examining histological marks of pressure-ejected WGA-HRP. During each ejection approximately 11.5 nl is ejected, and a total ejection volume of 70 nl with six pulses over a 1 min period. This volume ¢lls a sphere with a radius of over 280 Wm. Consistent with these estimates, pressure ejection of WGA-HRP into the brain produced spots with diameters of 500^800 Wm. Therefore, the expected volume of drugs applied initially delivers the agent over an area, which includes the somata and likely a large portion of the dendritic ¢eld of the recorded neuron. The concentration of drugs a¡ecting the recorded neuron somata and proximal dendrites is likely similar to concentrations of drugs in pressure ejection electrodes. As agents are diluted by di¡usion into surrounding tissue and circulation, the tissue concentration of agents will decrease over time. In addition, drug concentration at distal dendrites is expected to be lower than proximal locations.
Table 1 Tonotopic dependence of ZD7288 e¡ects BF of SOC neurons (kHz)
n
Percent decrease in activity following 100 WM ZD7288 Tone-evoked
1^2 2^4 4^8 8^16 16^32 a
2 8 7 21 9
Background activity
Mean : S.D. (%)
CV (%)
Mean : S.D. (%)
CV (%)
54 : 12 57 : 14 44 : 22%a 39 : 15%a 67 : 13
22 24 50 39 20
46 : 14 60 : 17 56 : 21 50 : 16 69 : 18
29 29 38 32 26
Signi¢cantly di¡erent from the 16^32 kHz group.
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Fig. 1. Application of the selective Ih channel antagonist, ZD7288, decreases the excitability of a LSO EI neuron. PSTH (A^C) and average response levels to 50 stimuli per point (D) to ipsilateral BF tone stimuli (4.0 kHz, 30 dB above threshold) show a selective decrease in excitability following pressure ejection of 100 WM ZD7288. Arrows (D) show application of aCSF and ZD7288 with six, 30 PSI, 250 ms pulses every 10 s. There was no change in tone-evoked (¢lled circles) or background (open circles) neuronal activity following pressure ejection of aCSF (B,D), while application ZD7288 (100 WM) decreased tone-evoked neuronal activity (C,D) by 59.6% (population mean: 49.7 : 19%). There was a gradual decrease in excitability following application of ZD7288.
2.7. Histology At the end of each experiment, animals were overdosed with sodium pentobarbital and perfused through the heart with 0.9% saline followed by paraformaldehyde ¢xative and sucrose-paraformaldehyde. Frozen sections (50 Wm) were cut in the plane of the electrode track, mounted, and reacted for peroxidase TMB/GOD (tetramethylbenzidine/glucose oxidase) chromogen reaction. Sections were counterstained with thionine. Tracings of relevant sections and electrode depth were used to identify locations of the neurons studied.
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(MNTB), including neurons in the superior paraolivary nucleus (SPN) and periolivary neurons around the SPN (n = 34). The location within the SOC of 18 cells could not be identi¢ed. In the SPN region, neurons with ‘O¡’ responses were also encountered, but were not routinely examined, as our goal was to determine the e¡ect of ZD7288 on excitatory tone-evoked responses. ZD7288 consistently decreased background ¢ring activity and tone-evoked neuronal activity in all cells tested with brief (1 min) applications of as low as 20 WM ZD7288. Application (six, 250 ms, 20^30 PSI pressure ejections over 1 min.) of ZD7288 at 100 WM decreased tone-evoked neuronal activity on average by 49.7 : 19% (range 18.3^94.3%, coe⁄cient of variation = 38%; n = 47), while background activity was decreased by 56.3 : 18.1% (n = 47; Figs. 1 and 2). There was no decrease in neuronal activity following application of aCSF (Figs. 1 and 2). Therefore, the decrease in activity to ZD7288 application was not due to mechanical effects of the pressure pulse or a reaction to the vehicle. The gradual decrease in neuronal activity following application of ZD7288 peaked after 2^3 min. Raster plots of individual responses con¢rm a gradual decrease, with no abrupt changes in responses after each pressure ejection. The reduction in SOC neuron excitability by 100 WM ZD7288 application was slightly lower (by 22.1 : 11%, n = 4) after a 20^32 min recovery period. Further recovery of excitability following 100 WM ZD7288 application was not studied over longer periods, as decreasing anesthetic levels over longer periods of time will also increase excitability.
3. Results 3.1. Selective antagonism of Ih channels decreases SOC neuronal excitability The e¡ect of ZD7288, a selective Ih channel antagonist, was examined in 71 SOC neurons (46 EE, 8 EI, 10 EO, and 7 cells, where only ipsilateral stimuli were tested (E-)) in 22 animals. Neurons localized by histology were recorded in the lateral nucleus of the trapezoid body (n = 4), LSO (n = 5), periolivary neurons around the LSO (n = 10), and in the region between the LSO and medial nucleus of the trapezoid body
Fig. 2. Decreased excitability of an EE neuron following application of the selective Ih channel antagonist, ZD7288. Background (open circles) and ipsilateral tone-evoked responses (4.615 kHz, 30 dB above threshold) were not a¡ected by application of aCSF, but decreased rapidly following application of 100 WM ZD7288. The decrease in excitability was slightly less 14 min after ZD7288 application.
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3.2. Dose- and time-dependence of ZD7288 induced decrease in excitability The decrease in excitability produced by a brief application (six, 250 ms, 20^30 PSI pressure ejections over 1 min) of ZD7288 was dose dependent (Fig. 3). Toneevoked neuronal activity was decreased by 44.9 : 20.8% (range 17.5^77%, coe⁄cient of variation = 46.3%; n = 7) after application of 50 WM ZD7288, and by 17.1 : 12.7% (range 2.8^32.6%, coe⁄cient of variation = 74.4%; n = 5) following application of 20 WM ZD7288. The decrease in tone-evoked neuronal activity was 4.3 : 6.8% (range 38.3^14.5%, coe⁄cient of variation = 158%, n = 7) following application of 10 WM ZD7288. This dose-dependence of ZD7288 e¡ects is very similar to dose-dependent blockade of Ih channels in vitro following brief application of ZD7288 (Shin et al., 2001). However, extended periods (15 min or longer) are required for low doses ( 6 50 WM) of ZD7288 to produce a signi¢cant blockade (Harris and Constanti, 1995). Time-dependent blockade of Ih channels was examined by applying low doses (10 WM) of ZD7288 over a 10^15 min period (pressure ejections every 20 s). In 4/4 cells (2 EE, 1 EI and 1 EO cell), 10 WM ZD7288 appliFig. 4. Excitability decreased slowly when 10 WM ZD7288 was applied over 13 min (250 ms, 30 PSI applications every 20 s). The time-dependent e¡ect of ZD7288 is similar to the time-dependent blockade of Ih channels, in vitro. Excitability recovered over a 20 min period following cessation of ZD7288 application.
cation produced a gradual decrease in activity (Fig. 4). The average maximal decrease produced by 10 WM ZD7288 was 67.1 : 17.1%. After cessation of ZD7288 application, a gradual increase in activity over 15 min was observed, reaching levels near prior control levels. 3.3. E¡ects of ZD7288 on responses to excitatory amino acid agonists
Fig. 3. ZD7288 decreased the rate of neuronal ¢ring in SOC neurons in a dose-dependent manner. Percent decrease in SOC neuron tone-evoked responses for individual cells (diamonds), and average percent decrease in responses (open circles) are plotted as a function of concentration of ZD7288 in the ejection pipet. Data were ¢tted with a sigmoid curve (line). The e¡ects of ZD7288 exhibited considerable variability at each dose, but consistently decreased excitability. The average e¡ect of ZD7288 did not signi¢cantly increase between 50 and 100 WM.
The e¡ects of ZD7288 (100 WM) on neuronal activity, directly stimulated by pressure ejection application of agonists, were determined and the magnitude of ZD7288 e¡ects on agonist-evoked excitation and toneevoked responses were compared in the same neurons. Glutamate (50 mM) or AMPA (10 WM) were applied by brief 150 ms pressure pulses. Lower concentrations of either agent had no e¡ect or produced small changes in ¢ring rate. Brief exogenous application of glutamate or AMPA evoked a rapid brief ( 6 500 ms) increase in ¢ring rate, which peaked after less than 400 ms. ZD7288 consistently decreased responses to glutamate in all eight cells examined. In six cells, where toneevoked responses were also examined, ZD7288 decreased glutamate-evoked ¢ring activity by 75.7 :
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neurons tested, which is likely related to excitotoxic e¡ects of AMPA. However, repeated glutamate application did not decrease neuronal excitability, which was decreased only following application of ZD7288. 3.4. Factors a¡ecting ZD7288 reduction in activity The e¡ects of ZD7288 on threshold- and intensitydependence of tone-evoked responses were examined in 19 SOC neurons. ZD7288 (100 WM) decreased overall excitability of these neurons by 47.5 : 9.3% (n = 19). Thresholds of responses in rate^intensity function were not a¡ected by ZD7288 application. This may be due to the similar, but slightly greater (6.9%) average percent decrease in background activity compared to tone-evoked responses following 100 WM ZD7288 application (r = 0.83, n = 47; Fig. 6, paired t-test, P 6 0.01). Furthermore, there was no relationship between unit thresholds and the percent decrease in toneevoked responses by ZD7288. The decrease in neuronal ¢ring rate to ZD7288 also did not vary signi¢cantly as a function of intensity of BF tonal stimuli in 13/19 cells. ZD7288 produced a lesser decrease in responses for higher intensity BF stimuli (average 21% change/10 dB) in four cells, while two cells exhibited the opposite trend. The absolute decrease in background and toneevoked ¢ring rate was related to absolute activity levels before application of ZD7288. The ZD7288 induced
Fig. 5. ZD7288 decreases both glutamate- and tone-evoked neuronal activity. Responses to pressure ejection (150 ms, 30 PSI pulses) application of glutamate (50 mM) were decreased following application of ZD7288 (100 WM). (A) PSTH of glutamate-evoked neuronal ¢ring before and after application of ZD7288. The average decrease in glutamate-evoked responses following ZD7288 application was greater than the average decrease in tone-evoked responses (C), which was observed in the majority (5/6) of cells (B).
18.8%, while it decreased tone-evoked neuronal activity by 50.6 : 17.2% (Fig. 5). Although, ZD7288 usually reduced glutamate-evoked neuronal ¢ring more than tone-evoked neuronal ¢ring, this was not statistically signi¢cant (paired t-test, P = 0.11), likely due to the small sample size. ZD7288 also decreased AMPAevoked neuronal activity by 48.8 : 31.6% (n = 4) while decreasing tone-evoked ¢ring activity by 50.8 : 7.5% (n = 4). There was no statistically signi¢cant di¡erence between the change in tone-evoked and AMPA-evoked neuronal ¢ring (t-test, P = 0.45). However, all neuronal activity was lost soon after AMPA application in other
Fig. 6. The decrease in background and tone-evoked activity following 100 WM ZD7288 application was similar in individual cells. The average percentage decrease in background activity, however, was signi¢cantly greater than the average percent decrease in toneevoked responses, with most points lying above the line of equality (dark line).
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greater in neurons with BFs between 16 and 32 kHz than neurons with BFs between 4^8 kHz and 8^16 kHz (Fig. 8, Table 1 (see Section 2.5), one-way analysis of variance (ANOVA), P 6 0.01; Tukey^Kramer honest statistical di¡erence (HSD). The e¡ect of ZD7288 on background activity followed a similar trend, but was not signi¢cant (P = 0.08, power = 0.6). Although, the excitability of EO neurons was signi¢cantly less a¡ected (32.4 : 5.5% reduction, n = 9) by ZD7288 than EE (51.5 : 3.1%, n = 29) and EI (72.3 : 9.6%, n = 3) neurons, 89% of EO neurons had BFs between 4 and 16 kHz. After excluding EO neurons, the e¡ect of ZD7288 (100 WM) on tone-evoked responses was signi¢cantly greater in neurons with BFs between 16 and 32 kHz
Fig. 7. The absolute decrease in background and tone-evoked (BF tones, 30 dB above threshold) ¢ring activity (spikes/s) produced by ZD7288 application was related to the pre-drug level of background and tone-evoked activity. There was a higher correlation for the relationship for ZD7288 e¡ects and background activity than for tone-evoked activity. Lines in each plot represent linear ¢ts (solid line) with 95% con¢dence intervals (dashed lines). The top panel shows percent decrease in the ¢ring rate in individual neurons, while the bottom panel shows the averaged reduction in ¢ring rate and standard error.
decrease in background ¢ring rate was highly correlated with the pre-drug background activity (correlation, r = 0.9, n = 45, Fig. 7). The ZD7288 induced decrease in tone-evoked ¢ring rate was correlated with the predrug tone-evoked response level, but exhibited less dependence (correlation, r = 0.75, n = 45, Fig. 7). The close relationship of background activity and sensitivity to ZD7288 is consistent with Ih channels playing an important role in determining resting potential and hence background ¢ring levels. The relative e¡ect of ZD7288 (100 WM) in decreasing tone-evoked neuronal activity was also dependent on the BF of neurons. SOC neurons were sorted into octave groups based on their BF. The e¡ect of ZD7288 (100 WM) on tone-evoked responses was signi¢cantly
Fig. 8. The magnitude of the decrease in excitability following ZD7288 application was related to neuronal BF. The top panel shows percent decrease in the ¢ring rate in individual neurons, while the bottom panel shows the averaged reduction in the ¢ring rate and standard error. Neurons with BFs greater than 16 kHz exhibited a signi¢cantly (ANOVA, P 6 0.0001) greater decrease in toneevoked activity following ZD7288 (100 WM) application than neurons with BFs between 4^8 kHz, and 8^16 kHz (bottom panel). ZD7288 decreased the excitability of EO cells to a lesser extent, but most of these neurons had BFs between 4 and 16 kHz.
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than neurons with BFs between 8 and 16 kHz (one-way ANOVA, P 6 0.02; Tukey^Kramer HSD). Other analysis on frequency-dependence of ZD7288 e¡ects for cells grouped by location or aurality was unreliable (power of analysis 6 0.5).
4. Discussion ZD7288, a selective blocker of Ih conductances, consistently decreased neuronal excitability of SOC neurons, in vivo. Pressure ejection of ZD7288 produced a gradual decrease of activity over 2^3 min, while the vehicle (aCSF) did not alter neuronal ¢ring. Therefore, mechanical e¡ects due to pressure ejection of pharmacological agents, and the vehicle were not responsible for the ZD7288-induced decrease in ¢ring activity. ZD7288 produced dose- and time-dependent, consistent decreases in superior olivary neuron tone-evoked and glutamate-evoked responses, in vivo. Together with the absence of e¡ects in controls, this is consistent with the selective blockade of Ih channels by ZD7288 directly reducing the excitability of SOC neurons. ZD7288 selectively blocks Ih channels, in vitro, at concentrations up to 100 WM (Harris and Constanti, 1995). High concentrations (300 WM) of ZD7288 produce a non-speci¢c, immediate cessation of ¢ring activity in dopamine neurons, which was also associated with a reduction in action potential amplitudes (Seutin et al., 2001). Lower concentrations (up to 100 WM) of ZD7288 have no reported e¡ects on other ion channels or non-speci¢c e¡ects, and do not a¡ect spike amplitude (nigral neurons, Harris and Constanti, 1995 ; vestibular neurons in vitro, Chabbert et al., 2001). The amplitude of spike after hyperpolarizations was increased by 100 WM ZD7288 (Harris and Constanti, 1995). Maximal reductions in SOC neuron ¢ring were observed following pressure ejection from pipets containing 50 and 100 WM ZD7288, and partial blockade at 20 WM. The dose response pro¢le (Kd = 40.5 : 3.3 WM) for brief (4 s) application of ZD7288 onto inside-out patches from HEK cells expressing mHCN1 (Shin et al., 2001) is very similar to the dose-dependent decrease in ¢ring rate by short duration (1 min) application of ZD7288 observed in SOC neurons, in vivo. ZD7288 progressively blocks Ih conductances during bath application, in vitro, where low doses (0.5^10 WM) require 15 to 30 min for complete blockade (Harris and Constanti, 1995). Since ZD7288 is a lipophilic drug, which acts at an intracellular domain of the Ih channel, delays in blockade are attributable to di¡usion through the tissue and across the neuronal membrane (Harris and Constanti, 1995). Therefore, dose-response curves measured after 15 min of ZD7288 application (between 0.9 and 4.3 WM ZD7288 produce a half-maximal de-
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crease in Ih , Harris and Constanti, 1995) are shifted to the right compared to the dose-dependent e¡ects of ZD7288 applied over a brief period (1 min in our study, and 4 s, Shin et al., 2001). Application of ZD7288 (10 WM) over a period of 15 min veri¢ed that low concentrations of ZD7288 produce a time-dependent decrease in SOC neuron excitability. Therefore, the dose- and time-dependence of ZD7288 e¡ects on SOC neuron excitability are consistent with prior studies showing selective blockade of Ih channels. Reversal of ZD7288 (10 WM) e¡ects was unexpected based on many prior studies. Factors a¡ecting reversal of ZD7288 e¡ects include clearance rate of ZD7288, the completeness of Ih channel blockade throughout the neuron, and reversibility in the blockade. The e¡ect of ZD7288 on Ih channels composed of di¡erent hyperpolarization-activated cyclic nucleotide-gated channel subunits (HCN) is still poorly understood. Shin et al. (2001) demonstrated that ZD7288 blockade of Ih channels composed of mHCN1 subunits is reversible, which appears to depend on three amino acids in the S6 pore region of the channel. These amino acids vary between subunits (Ludwig et al., 1998). Subunits (HCN1 to HCN4) of the Ih family of channels are di¡erential expressed in auditory neurons from cochlear nuclei to cortex (Monteggia et al., 2000), with most auditory neurons in the brainstem likely expressing all isoforms. The relatively fast activation of Ih conductances in LSO neurons (Adam et al., 1999, 2001) and SPN neurons (personal observations) is characteristic of Ih channels with HCN1 subunits (Franz et al., 2000). We observed a partial reversibility of the decreased excitability of SOC neurons to high (50 and 100 WM) doses of ZD7288. The recovery of SOC neuron excitability after 10 WM ZD7288 application could be due to the reversible blockade of Ih channels with HCN1 subunits. ZD7288 also decreased neuronal responses evoked by application of glutamate or AMPA, which is consistent with a direct action of ZD7288 on the recorded units. Most neurons in the SOC, including LSO, MSO, MNTB and SPN neurons exhibit a sag in hyperpolarizing responses, which has been consistently shown to be due to an Ih conductance. Therefore, ZD7288 is expected to directly a¡ect all these neurons. The major e¡ect of glutamate application on recorded activity is expected to be direct excitation of recorded neurons by activation of ionotropic glutamate receptors. ZD7288 typically decreased the responses to glutamate more than responses to sound. Pressure ejection of ZD7288 and glutamate likely a¡ect similar areas of the recorded neuron, whereas excitatory inputs to SOC neurons will impinge on the full extent of their often large dendritic ¢elds. Therefore, pressure application of ZD7288 may be expected to have a greater e¡ect on responses to pressure-ejected agonists. It is possible that ZD7288
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also decreases activation of interneurons by glutamate, which could contribute to the greater e¡ect of ZD7288 on glutamate-evoked responses. An e¡ect of ZD7288 blockade of Ih channels in presynaptic terminals cannot be ruled out. However, modulation and blockade of presynaptic Ih channels did not a¡ect synaptic transmission at the calyceal endings in the MNTB (Cuttle et al., 2001). Since the major e¡ect of glutamate application is expected to be a direct excitation of the recorded neuron, the marked decrease in excitability to glutamate following ZD7288 application is indicative of a predominant e¡ect of ZD7288 and Ih channel blockade on the recorded SOC neurons. The marked decrease in excitability of SOC neurons following ZD7288 application suggests a signi¢cant contribution of active Ih channels to the resting membrane potential and excitability of these neurons, in vivo. Non-selective blockade of Ih , in vitro, by cesium leads to membrane hyperpolarization in thalamic, rat fetal medullary, cochlear nucleus, amygdala, dorsal root ganglion, and LSO neurons (McCormick and Pape, 1990; Womble and Moises, 1993; Wang et al., 1997; Wellner-Kienitz and Shams, 1998; Bal and Oertel, 2000; Adam et al., 2001). Therefore, blockade of Ih channels by ZD7288 is expected to hyperpolarize SOC neurons, and thereby reduce their excitability, in vivo. Ih channels are expected to be partially active at resting membrane potentials, as for example, the activation range of Ih channels in thalamic neurons is between 360 and 395 mV (McCormick and Pape, 1990). The decrease in tone-evoked activity, but especially background neuronal activity by ZD7288 is consistent with Ih channels being active at rest in SOC neurons. The magnitude of the decrease in excitability produced by ZD7288 is highly variable between neurons. E¡ects of ZD7288 are not intensity dependent in most neurons. Variability in ZD7288’s e¡ects could depend on di¡erences in drug delivery. However, di¡erences in the magnitude of ZD7288 e¡ects in speci¢c cell groups, for example, EO neurons and neurons with BFs greater than 16 kHz, may be due to di¡erences in Ih channel activation or composition. The high correlation between pre-drug background activity and the absolute decrease in activity following ZD7288 application is also suggestive of di¡erential activation of Ih channels at rest in di¡erent cells. The activation range of Ih channels is dependent on the subunit composition of channels, but is also regulated by intracellular cAMP levels. Coexpression of HCN1 and HCN2 subunits in oocytes form heteromultimers composed of di¡erent HCN subunits, and unique properties compared to summative properties of these subunit expressed in isolation (Chen et al., 2001). The possible formation of Ih channels composed of di¡erent HCN subunits in auditory neurons potentially will produce Ih channels with
unique properties, including their sensitivity to cAMP. The activation range of Ih channels in cultured spiral ganglion cells exhibit a wide variability of over 40 mV for half maximal voltages (Mo and Davis, 1997). Furthermore, the activation range of Ih channels in MNTB neurons was shifted by over 35 mV in the presence of 8-Br-cAMP or norepinephrine, resulting in an increased activation of Ih channels at rest and a 2^3 mV depolarization at rest (Banks et al., 1993). Regulation of Ih channel activation in SOC neurons could be through transmitter systems known to modulate cAMP synthesis, and found in the SOC, such as metabotropic glutamate receptors (Kotak and Sanes, 1995), and cholinergic (Henderson and Sherri¡, 1991; Yao and Godfrey, 1998) or noradrenergic (Wynne and Robertson, 1996) innervation. The variability in the ZD7288-induced decrease in excitability of SOC neurons is consistent with varied properties of Ih channels in neurons due to expression of Ih channel subunits or their regulation by cAMP. The marked decrease in SOC neuron excitability following application of ZD7288 shows a signi¢cant role of Ih in determining neuronal excitability. Since the ability of LSO neurons to localize sound sources is rate dependent, Ih channels may play an important role in the encoding of information for sound localization. Plasticity in LSO responses may be adaptive to compensate for asymmetric temporary or permanent losses in peripheral hearing. Modulation of Ih channels, for example by cAMP, may also regulate information £ow to higher centers. Thus, Ih conductances potentially have an important role in sound localization perception and may impact processing of sounds, such as speech.
Acknowledgements The authors thank Sharjeel Farooqui for expert technical assistance. This research was supported by the Deafness Research Foundation (P.G.F.). A.S. was supported by an NIH training grant (T32 DC00026-14).
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