Differential intrinsic response dynamics determine synaptic signal processing in frog vestibular neurons

Abstract

Central vestibular neurons process head movement-related sensory signals over a wide dynamic range. In the isolated frog whole brain, second-order vestibular neurons were identified by monosynaptic responses after electrical stimulation of individual semicircular canal nerve branches. Neurons were classified as tonic or phasic vestibular neurons based on their different discharge patterns in response to positive current steps. With increasing frequency of sinusoidally modulated current injections, up to 100 Hz, there was a concomitant decrease in the impedance of tonic vestibular neurons. Subthreshold responses as well as spike discharge showed classical low-pass filter-like characteristics with corner frequencies ranging from 5 to 20 Hz. In contrast, the impedance of phasic vestibular neurons was relatively constant over a wider range of frequencies or showed a resonance at approximately 40 Hz. Above spike threshold, single spikes of phasic neurons were synchronized with the sinusoidal stimulation between approximately 20 and 50 Hz, thus showing characteristic bandpass filter-like properties. Both the subthreshold resonance and bandpass filter-like discharge pattern depend on the activation of an I(D) potassium conductance. External current or synaptic stimulation that produced impedance increases (i.e., depolarization in tonic or hyperpolarization in phasic neurons) had opposite and complementary effects on the responses of the two types of neurons. Thus, membrane depolarization by current steps or repetitive synaptic excitation amplified synaptic inputs in tonic vestibular neurons and reduced them in phasic neurons. These differential, opposite membrane response properties render the two neuronal types particularly suitable for either integration (tonic neurons) or signal detection (phasic neurons), respectively, and dampens variations of the resting membrane potential in the latter.

Figure 1.

Discharge pattern of phasic and tonic 2°VNs. A, B, A short burst of two spikes (A) and a continuous discharge (B) evoked by injection of positive current steps of 1.1 nA (trace below tonic cell) differentiate between phasic and tonic 2°VNs; the inset in A shows the short initial burst (gray area) of the phasic 2°VNs at an extended timescale. C, The evoked burst discharge in all phasic 2°VNs (76%) was confined to the first 100 ms (black bar); the continuous discharge of all tonic 2°VNs (24%) began immediately after stimulus onset and lasted throughout the entire length of current step (gray bar). Insets in C show monosynaptic EPSPs evoked by stimulation of the horizontal (HC) and the anterior (AC) vertical canal nerve at two stimulus intensities, given as multiples of the N₁ field potential component (xT) in the phasic and tonic 2°VNs illustrated in A and B. Horizontal dashed lines in A and B and insets in C indicate resting membrane potentials at −65 and −69 mV, respectively. Arrowheads in the insets in A and C indicate stimulus onset. Records in A and B are single sweeps and in C averages of 24 responses. The calibration in A applies also to B. V, Voltage; C, current.

Figure 2.

Response dynamics of tonic 2°VNs. A, Response evoked by injection of a small-amplitude, sinusoidally modulated current that increased logarithmically in frequency up to 100 Hz (ZAP). B, Responses of a tonic 2°VN to injection of multiple cycles of sinusoidally modulated currents at discrete frequencies of 1 Hz (B₁), 10 Hz (B₂), and 50 Hz (B₃). Horizontal dashed lines indicate the resting membrane potential at −74 mV (A) and −67 mV (B₁–B₃). Calibration in B₃ applies to B₁ and B₂. C, Bode plots of impedance and phase of the responses of tonic 2°VNs tested with discrete frequencies (n = 5; C₁) and Bode plots obtained from tonic 2°VNs tested with ZAP stimuli (n = 12; C₂). The impedance of each neuron was normalized to the value obtained at a stimulation frequency of 0.4 Hz in C₁ and of 1 Hz in C₂; thick lines in C₁ and C₂ are the average of the respective population of neurons, and the dashed lines are the corner frequency (Cf). V, Voltage; F, frequency; C, current.

Figure 3.

Response dynamics of phasic 2°VNs. A₁–A₃, Typical examples of impedance profiles of three different phasic 2°VNs calculated from responses to injection of small-amplitude ZAP currents. Based on their shape, responses were classified as nonresonant (A₁), simple resonant (A₂), or trough resonant (A₃). Simple-resonant responses exhibit a single resonance peak (*), and trough-resonant responses exhibit a trough-like depression (black diamond) before the resonance peak (**). Horizontal dashed lines indicate resting membrane potential (−67 mV in A₁; −66 mV in A₂; −63 mV in A₃). Calibration in A₃ applies to A₁ and A₂. B, Bode plots comparing impedance and phase profiles of all phasic (filled circles) and tonic (filled stars) 2°VNs; phasic neurons were further separated as nonresonant (gray circles), simple resonant (gray circles), and trough resonant (gray triangles).V, Voltage; F, frequency; C, current.

Figure 4.

Membrane potential and temperature dependence of the impedance profile of phasic 2°VNs. A, Responses of a phasic 2°VN evoked by injection of small-amplitude ZAP currents at two membrane potentials at −63 mV (black trace) and −70 mV (gray trace). At the more depolarized membrane potential, the response exhibited a trough-like depression (black diamond) before the resonance peak (**) that is facilitated and shifted to higher frequencies compared with the resonance peak at the more hyperpolarized membrane potential. B, Bode plots of impedance and phase of the responses at the two membrane potentials (white and black circles) shown in A. C, Responses of a phasic 2°VN evoked by injection of small-amplitude, ZAP currents at two recording temperatures (14°C, black trace; 16°C, gray trace). Note the increased impedance of the response at higher stimulus frequencies and the pronounced resonance peak (*) at 16°C. D, Bode plots summarizing the impedance and phase of the responses of phasic neurons (n = 8) recorded at both 14°C (black circles) and 16°C (white circles). Vertical dashed lines indicate resonance peak frequencies. V, Voltage; F, frequency; C, current.

Figure 5.

Response asymmetry in tonic and phasic 2°VNs. A, The ratio of the depolarizing (black arrows in A₁, A₂)/hyperpolarizing half wave (red arrow in A₁, A₂) of the responses evoked by sinusoidally modulated currents is >1 for tonic and <1 for phasic 2°VNs (A₃). Horizontal dashed lines in A₁ and A₂ indicate resting membrane potential at −64 mV (A₁) and −66 mV (A₂); arrows and numbers in A₃ indicate mean ± SD of the ratios for tonic and phasic 2°VNs. Calibration in A₂ applies to A₁. B, Response of a phasic 2°VN evoked by ZAP currents. The response shows a marked asymmetry and a particular distortion in the falling phase of the depolarizing half wave at higher stimulus intensities (pink and black traces; see inset for extended timescale); the response peak (white arrowhead and vertical dashed line) occurs before the maximal depolarizing current is reached (vertical dotted line), and the membrane potential starts to hyperpolarize. C, D, Overlay of responses evoked by injection of ZAP currents at resting membrane potential (red traces) and during constant depolarization (black traces) of a tonic (C) and a phasic 2°VN (D). The impedance during depolarization of the membrane potential (V_m) increases in tonic but decreases in phasic 2°VNs at low- and mid-range frequencies (black triangles in C₂, D₂). Dashed lines in C₁ and D₁ indicate membrane potential before the onset of the stimulus. V, Voltage; F, frequency; C, current.

Figure 6.

Discharge dynamics of tonic 2°VNs. A, Example of the discharge evoked by injection of large-amplitude ZAP currents. B, Responses of a tonic 2°VN to injection of sinusoidally modulated currents at discrete frequencies of 1 Hz (B₁), 4 Hz (B₂), and 60 Hz (B₃). C, Transition from multiple to single spike discharge (8.7 Hz in C₁) and upper limit of single spike synchronization (16.9 Hz in C₂) of the neuron shown in A (see arrows in A). Responses in A–C are single sweeps, and horizontal dashed lines indicate resting membrane potential at −72 mV (A, C) and −82 mV (B). Calibration in B₃ applies to B₁ and B₂. D, Distribution of the upper limits of multiple spike modulation (black squares and solid line; F_mod) and of single spike synchronization (white circles and dashed line; F_max) among the tested tonic 2°VNs (n = 10). Note that <50% of the tonic 2°VNs fire multiple spikes above ∼6 Hz (dashed vertical line) and single spikes above ∼20 Hz (dotted vertical line) on each depolarizing half cycle. V, Voltage; F, frequency; C, current.

Figure 7.

Discharge dynamics of phasic 2°VNs. A, Discharge evoked by injection of ZAP currents. With increasing current intensity (A₁–A₃), responses increased in amplitude and triggered spikes (A₁, A₂) at a stimulus frequency of ∼20 Hz. B, Responses of a phasic 2°VN to injection of multiple cycles of sinusoidally modulated currents at constant frequencies of 4 Hz (B₁), 30 Hz (B₂), and 60 Hz (B₃). C, Responses of a phasic 2°VN evoked by injection of sinusoidally modulated currents below (gray) and above (black) spike threshold; spikes occurred at the resonance peak of the subthreshold response (gray trace). The inset in C shows superimposed subthreshold responses (gray) and the four spikes (black, amplitude truncated) riding on top of the depolarizing half cycles at the resonance peak (37 Hz) at an extended timescale. Responses in A–C are single sweeps, and horizontal dashed lines indicate resting membrane potential at −78 mV (A), −70 mV (B), and −67 mV (C). Calibration in A₁ and B₃ applies to A₂, A₃ and B₁, B₂, respectively. D, Frequency dependence of single (white circles) and multiple (white squares) spike modulation in tonic (n = 10) and phasic (filled triangles; n = 29) 2°VNs. The frequency dependence of the discharge probability for multiple spike discharge (white squares) in tonic 2°VNs was calculated by determining the proportion of neurons that fired two or more spikes per sine wave, respectively, at each one of the 16 tested frequencies between 1 and 100 Hz. Accordingly, the discharge probability for single spike synchronization in tonic (white circles) and phasic (filled triangles) 2°VNs was calculated as the proportion of neurons at each particular frequency point that fired one spike per sine wave. V, Voltage; F, frequency; C, current.

Figure 8.

Effect of 4-AP on impedance and resonance of phasic 2°VNs. A, Overlay of responses evoked by injection of ZAP currents (A₁) before (control, black) and after (red) application of 10 μm 4-AP. Bode plots illustrate the impedance increase of the phasic 2°VN shown in A₁ (A₂) and of the population of all tested phasic 2°VNs (A₃); in A₃, the relative impedance is the average of the values normalized to the impedance at 1 Hz of each neuron before drug application. B, Sinusoidally modulated responses of a phasic 2°VN before (control, black) and after (red) application of 10 μm 4-AP; the distortion in the falling phase of the depolarizing half wave (*) was abolished by 4-AP. C, Spike discharge of the 2°VN shown in A evoked by sinusoidally modulated current injection before (control, black *) and after (red *) application of 10 μm 4-AP; the control range of spike discharge (black arrow) is mainly extended toward lower frequencies (red arrow) by 4-AP. Horizontal dashed lines indicate resting membrane potentials at −65 mV (A₁, C) and −63 mV (B). D, Transverse section through the vestibular nuclei at the level indicated in the top left corner of D₁ treated with an antibody for the Kv1.1 potassium channel. Immunopositive neurons are located in the dorsal (auditory) nucleus (DN), the descending (DVN) and lateral (LVN) vestibular nuclei, but not the medial vestibular nucleus (MVN); higher magnification of labeled neurons in the lateral vestibular nucleus of this section (D₂) and in the descending vestibular nucleus of the adjacent rostral section (D₃). Outlines of vestibular nuclei are based on Kuruvilla et al. (1985). Scale bars: D₁, 100 μm; D₂, D₃, 20 μm. Arrow in D₁ indicates the sulcus limitans. V, Voltage; F, frequency; C, current.

Figure 9.

Differential synaptic processing of semicircular canal nerve afferent inputs in phasic and tonic 2°VNs. A, B, Monosynaptic EPSPs in a tonic (A) and a phasic (B) 2°VN evoked by electrical pulse stimulation of the anterior vertical canal (AC) nerve (white arrowheads). Different membrane potentials were obtained by current injection (A₁, A₂, B₁, B₂), or by preceding EPSPs in a stimulus train (A₃, A₄, B₃, B₄). A₁, B₁, EPSPs in a tonic (A₁) and a phasic (B₁) 2°VN during depolarizing (3) and hyperpolarizing (1) current steps (current traces plotted below) and in the absence of membrane polarization (2). A₃, B₃, Sequence of three EPSPs (1–3) in the same tonic (A₃) and phasic (B₃) 2°VNs evoked by a pulse train to the anterior vertical canal nerve at 50 Hz; EPSPs (1–3) start from successively more depolarized membrane potentials (dashed lines). Higher-magnification overlays (A₂, A₄, B₂, B₄) illustrate the increase of EPSP amplitudes in tonic (A₂, A₄) and the decrease of EPSP amplitudes in phasic 2°VNs (B₂, B₄) with depolarization. Horizontal dashed lines indicate membrane potentials at rest (−67 mV in A; −65 mV in B) and during polarization. C, Correlation between EPSP amplitude and membrane potential altered by depolarizing and hyperpolarizing current steps (C₁), respectively, or by preceding EPSPs (C₂) in tonic (white circles and squares with dashed lines) and phasic (black circles and squares with solid lines) 2°VNs; the slope of the linear regressions in C₁ and C₂ is significantly different from zero in all cases (p ≤ 0.001, except for white squares in C₂, for which p ≤ 0.05). In C₃, the inverse correlation between EPSP amplitude and membrane polarization (white and black circles from C₁; white and black squares from C₂) in tonic and phasic 2°VNs is corroborated by respective changes in input resistance during depolarization and hyperpolarizing current steps (white and black triangles). Values in C₃ were obtained by normalization using control values at resting membrane potential (dashed lines). V, Voltage; C, current.

Figure 10.

Filter properties of tonic and phasic 2°VNs affect their synaptic response dynamics. A, B, Schematic filter characteristics of tonic and phasic 2°VN discharge at resting membrane potential (A) shift to higher frequencies (arrow) during depolarization and/or increased temperature (B). C, D, Typical responses of a tonic (C) and a phasic (D) 2°VN to stimulation of the anterior vertical canal (AC) nerve with a train of electrical pulses that were sinusoidally modulated in frequency between 0 and 70 Hz (bottom trace). The discharge in the tonic 2°VN essentially mirrors the subthreshold compound response and has a relatively low rate (∼15 Hz); the discharge in the phasic 2°VN consists of three spikes at the response peak at a rate (∼40 Hz) that matches the resonance bandwidth of these neurons.