Convergence of excitatory and inhibitory hair cell transmitters shapes vestibular afferent responses

Abstract

The vestibular semicircular canals respond to angular acceleration that is integrated to angular velocity by the biofluid mechanics of the canals and is the primary origin of afferent responses encoding velocity. Surprisingly, some afferents actually report angular acceleration. Our data indicate that hair-cell/afferent synapses introduce a mathematical derivative in these afferents that partially cancels the biomechanical integration and results in discharge rates encoding angular acceleration. We examined the role of convergent synaptic inputs from hair cells to this mathematical differentiation. A significant reduction in the order of the differentiation was observed for low-frequency stimuli after gamma-aminobutyric acid type B receptor antagonist administration. Results demonstrate that gamma-aminobutyric acid participates in shaping the temporal dynamics of afferent responses.

Fig. 1.

Topography of GABAergic hair cells. (A) Low-magnification multiphoton laser scanning projected image showing the spatially restricted distribution of GABAergic hair cells (Alexa 488, green) in relation to the ubiquitous glutamatergic hair cells (Alexa 568, red) across an entire crista ampullaris of a double-labeled canal crista. Vertical dashed lines demarcate the central 60% of the crista. (B) Locations of dendritic fields of 38 physiologically characterized, labeled afferents. The relative center of each dendritic field for pure velocity-sensitive (V, red squares), mixed velocity/acceleration-sensitive (V/A, yellow circles), and acceleration-sensitive (A, green circles) afferents are plotted on a normalized crista (after ref. 13).

Fig. 2.

Multiphoton laser scanning images of physiologically characterized, biocytin-injected afferents. (A) Acceleration-sensitive afferent (red) contacts GABA-immunolabeled hair cells (green) in the central crista. (Inset) The location of the afferent dendritic field is shown in the low-magnification projected image. (B) Velocity-sensitive afferent (red) ramifying in the peripheral crista, far away from the GABAergic hair cells (green) of the central region. (Inset) Low-magnification contextual image is shown.

Fig. 3.

Time course of change in gain slope and average phase in whole nerve, suction electrode recordings averaged over seven experiments before and after systemic injection of CGP 55845 (n = 2 at 1 mg), CGP 35348 (n = 2at 0.9 mg; n = 1 at 0.7 mg), and CGP 46381 (n = 2 at 0.5 mg). Data for individual fish are plotted vs. time relative to the time of half-maximal drug effect (vertical dashed line denotes t₅₀ = 15 ± 4.3 min). Error bars are interanimal standard deviations. ** indicates statistical significance at the 95% confidence level, and * indicate the 90% level with respect to preinjection control condition. (A) Gain slope calculated as the change in gain divided by change in frequency (log-log scale) by using lock-in suction electrode data at 5 and 2 Hz. (B) Phase is the average recorded at 5 and 2 Hz.

Fig. 4.

Bode plots of log gain (A, spikes per s per μm of canal indentation) and phase (B, spikes per s with respect to peak amplitude of stimulation) versus log frequency of stimulation for a single mixed velocity/acceleration afferent before and after CGP 55845 injection at 250 μg. Different symbols correspond to measurements obtained 15, 30, and 67 min postinjection. Gain slopes are indicated for the linear fits of the data. Gain slope is plotted overlying that of the suction electrode experiments in A Inset. The two-transmitter convergence model (Methods) was used to predict how the unit in the control condition (thick black solid line) would be expected to respond after GABA_B receptor blockade (thin red curved lines). Note correspondence between the predicted response (red curved lines) and the postdrug data (dashed line curve fits).

Fig. 5.

Bode plots of log gain (A, spikes per s per μm of canal indentation) and phase (B, spikes per s re: peak amplitude of stimulation) versus log frequency of stimulation for a single high-α afferent before and after CGP 55845 injection at 400 μg. Different symbols correspond to measurements obtained 13 and 30 min postinjection. Gain slopes are indicated for the linear fits of the data. Gain slope is plotted overlying that of the suction electrode experiments in A Inset. The two-transmitter convergence model (Methods) was used to predict how the unit in the control condition (thick black solid line) would be expected to respond after GABA_B receptor blockade (thin red curved lines). Note correspondence between the predicted response (red curved lines) and the postdrug data (dashed line curve fits).

Fig. 6.

Afferent adaptation to step excitatory and inhibitory stimuli (step amplitude indicated in B) of the mixed velocity/acceleration afferent illustrated in Fig. 4. Afferent discharge adaptation for excitatory stimuli was fit by using a double-exponential curve (slow and fast time constants t_s, t_f) and a single exponential for inhibitory stimuli (slow time t_s). (Inset) Summary statistics of afferent adaptation changes associated with GABA_B receptor administration. The data were fit as double exponentials by using: spikes/s = is the fast time constant, τ₂ is the slow time constant, A₁ is the fast component amplitude, A₂ is the slow component amplitude, and A₁+A₂ is the total adaptation amplitude. Error bars denote SEM. There was no statistically significant change in τ₁ or τ₂, but significant differences were observed in the relative proportions of A₁ and A₂ (** denotes 0.05 level) such that total time required to recovery lengthened after CGP administration.