 middle and apical neurons (Fig. 9D) and having a significantly smaller sag magnitude for the base compared with the apex (Fig. 9E). This suggests that Ih current activity increased along the P14 cochlear contour from apex to base. While retaining heterogeneity, the significant differences in sag magnitude currents and voltages between the cAMP and control conditions eliminated the average differences between regions in the cAMP condition (Fig. 9D,E). As expected from adaptation comparisons, RMP levels were significantly depolarized in cAMP compared with the control condition (Fig. 9F). By comparison, the gradations and significant differences in Ih current parameters observed tonotopically were not observed across adaptation categories (Ih sag current for control SA = −752.07 ± 68.27 pA, RA = −740.76 ± 39.72 pA, UA = −771.08 ± 39.74 pA; cAMP SA = −978.82 ± 114.07 pA, RA = −1192.17 ± 57.41 pA, UA = −1204.67 ± 103.54 pA; p > 0.05). This indicates that averaged Ih activity is graded tonotopically, but not between adaptation groups.
SGNs supplemented with 8-Br-cAMP displayed selective responses by electrophysiological parameter and tonotopic location. A-C, APmax, action potential latency and duration, respectively, did not differ between region or condition. D, The current required to reach a constant negative potential from which comparisons between sag magnitude were made (−185 mV) was significantly different between regions in control, but not in the cAMP condition. E, Ih sag magnitude differed significantly between base and apex neurons in vehicle control and between vehicle control and 8-Br-cAMP conditions for each tonotopic region. F, RMP level was significantly elevated in the 8-Br-cAMP condition compared with vehicle control for each tonotopic region. G, H, APD and APR threshold current levels, respectively, differed only between base and apex neurons in the vehicle control condition. I, APRE threshold current was not significantly different between regions or conditions. J-L, APD, APR, and APRE threshold voltage levels, respectively, did not differ between regions or conditions. M, N, Linear fits to apex, middle, and base APR threshold voltages plotted as a function of input current showed slopes with a greater divergence between tonotopic regions in control than in the 8-Br-cAMP condition, respectively. The slopes (mV/pA) and R2 values are as follows: control: apex = 0.0413, 0.886, middle = 0.0319, 0.713, base = 0.0164, 0.561; cAMP: apex = 0.0328, 0.835, middle = 0.0243, 0.414, base = 0.0259, 0.767). O, Significant differences in Rin between tonotopic categories in the control condition are no longer present in the cAMP condition.
Also shown for adaptation categories (Fig. 7D,E), APD, and APR threshold current differed significantly according to cochlear location (Fig. 9G,H), suggesting the presence of dual neural responsivity gradients within the ganglion. This did not apply to APRE threshold current. The significant differences observed for APRE between adaptation categories (Fig. 7F) were not observed regionally (Fig. 9I), indicating that channel types in addition to or other than Ih correlate with this parameter. Overall, greater current levels were required to reach APD and APR threshold for basal neurons compared with apical ones (Fig. 9G,H). This was consistent with significant differences in regional Rin levels between control base and apex neurons (Fig. 9O; 83.18 ± 4.85 and 134.996 ± 11.58 mΩ, respectively; p < 0.01) but not between the base and apex neurons in the cAMP condition (90.55 ± 8.77 and 100.74 ± 10.47 mΩ, respectively), similar to findings across adaptation categories (Figs. 6D and 7L). What is dissimilar from the adaptation categorization, however, was that the gradations in APD and APR current threshold levels were not observed in cAMP (Fig. 9G,H), nor were there significant differences in voltage threshold at the −60 mV holding potential used herein (Fig. 9J–L). In combination, these observations are consistent with the idea that Ih currents play a role in the tonotopic regulation of APD and APR current thresholds. Consistent with this, cAMP has been shown to increase Ih activity in SGNs by shifting the voltage dependence of activation to depolarized levels (Mo and Davis, 1997b); moreover, the same pattern of RMP shifts, without altered voltage threshold, have been previously observed with an Ih pharmacological antagonist (Liu et al., 2014a). By contrast, LVA ion channel α-subunits not directly modulated by cAMP with the capacity to directly alter threshold and APmax simultaneously, such as KV1.1 and KV1.2, have the potential to regulate responsivity and sensitivity gradients associated with accommodation that potentially account for increased APRE resolution.
Despite the significant differences in averaged APD and APR threshold current levels, heterogeneity is, nevertheless, retained. Assessments in both control and cAMP conditions would be expected to show that this response range is shifted relative to voltage. Most clearly observed for the hyperpolarization-activated APR responses, the tonotopic substructure observed within the population (Fig. 9M) becomes essentially overlapping in the cAMP condition (Fig. 9N). Expectedly, Rin is no longer significantly different between adaptation categories in the cAMP condition (Fig. 9O). These results indicate that, separate from AP duration regulation by cAMP, which is associated with a single adaptation class, cAMP shifts threshold current for the full neuron population according to cochlear location. These different responses, in addition to the dual adaptation- and location-related responsivity gradations, indicate that the voltage-gated ion channels that regulate duration, RMP, adaptation, and voltage threshold have unique distributions within the ganglion to coordinate their distinct electrophysiological response properties.
To examine the significant differences observed tonotopically in greater detail, APD and APR current thresholds and Rin were separately evaluated in each location for UA, RA, and SA neurons in control and cAMP conditions (Fig. 10). This analysis sheds light on the dual adaptation and regional gradients postulated for these parameters. The first gradient between adaptation categories was significantly different for each electrophysiological parameter in the middle and apical regions for control conditions (Fig. 10A–C). By contrast, the second gradient extended across the tonotopic regions for all three parameters measured in control conditions, but only for the RA neurons (Fig. 10A–C). As noted previously, the tonotopic gradient displayed by RA neurons was eliminated in the cAMP condition, as was the significance in the middle for adaptation classes (Fig. 10D–F). Although the low percentage of SA recordings were limited in number when divided into multiple categories, a significant tonotopic difference for Rin emerged in this adaptation category for the cAMP condition (Fig. 10F), further distinguishing RA from SA neurons. What is common between each of the parameters, however, was that significant differences between adaptation classes were restricted to the apex and that the RA tonotopic gradient was abolished in the cAMP condition (Fig. 10D–F). Overall, these results show that the orthogonal gradients are separately established and differentially modulated, allowing different patterns of activity to emerge within the ganglion because of neuromodulation.
Dual gradients of APD and APR threshold current levels and Rin observed between adaptation classes and cochlear locations were altered by cAMP. A-C, For the control condition, significant tonotopic differences were restricted to RA neuron measurements, while significant differences between adaptation categories were consistently found in the middle and apex. D-F, In the cAMP condition, tonotopic differences displayed by RA neurons were eliminated and adaptation-specific differences were limited to the apex. Interestingly, a new tonotopic difference emerged for SA neurons, but only for Rin (F).
The dynamic compression, expansion, shifts, and realignment of intrinsic electrophysiological properties in response to hyperpolarization and increased cAMP reveal neuromodulation of electrophysiological heterogeneity as a hallmark of neural coding in the spiral ganglion. This mechanism depends upon both the types and distributions of endogenous voltage-gated ion channels within primary auditory afferents and their independent regulation by multiple neuromodulatory elements. While it was not possible in a single study to examine the full range of neuromodulators or to target every possible ion channel type within these neurons, our focused study, nevertheless, revealed something quite remarkable about the spiral ganglion. This small population of neurons has the capability of orchestrating countless responses considering how the vast array of neuromodulators present in the system can target myriad ion channel types differentially distributed within and among three different neuron classes.
The contribution of complex electrophysiological regulation to peripheral sensory coding is not a unique observation in the auditory system. In reptiles, for example, frequency-specific electrical “ringing” in cell membranes resulting from the interplay between calcium channels and calcium-activated K+ channels has been observed in turtle cochlea hair cells (Art and Fettiplace, 1987; Art et al., 1995). While tonotopic variations remain (Fettiplace, 2017; Coate et al., 2019), electrical tuning is either missing or minor in inner hair cells of the mammalian cochlea (Marcotti et al., 2003; Johnson, 2015). Thus, it appears that these types of intrinsic electrophysiological specializations in mammals are first observed at the next stage of auditory processing: the primary afferents of the spiral ganglion.
These neurons are replete with endogenous membrane specializations that have been observed using multiple approaches (