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1Department of Physiology, Ludwig-Maximilians-Universitöt München, 80336 Munich, Germany; and 2Laboratoire de Neurobiologie des Réseaux Sensorimoteurs, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7060, Université Paris 5, 75270 Paris Cedex 06, France
Submitted 3 February 2004; accepted in final form 22 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Johnston et al. 1994; Serafin et al. 1991a,1991b; Uno et al. 2003). In particular, MVN neurons of rats (Gallagher et al. 1985; Johnston et al. 1994) and guinea pigs (Serafin et al. 1991a,1991b) have been segregated in type A and type B cells that differ from each other in a number of interrelated membrane and discharge properties (see Vidal et al. 1999). Type A vestibular neurons have a more regular resting discharge and more tonic response dynamics and might form a specific channel for lower-frequency, low- and medium-amplitude linear signals. In contrast, type B vestibular neurons have a more irregular resting discharge and more phasic response dynamics that is well-suited for the transmission of higher-frequency, high-amplitude nonlinear signals (Beraneck et al. 2003a; Ris et al. 2001). These two types of MVN neurons, however, might in fact represent extreme endpoints of a continuum as suggested by du Lac and Lisberger (1995) for avian MVN neurons rather than two distinct populations. The separation of vestibular neurons into populations with different response dynamics is also mirrored at the level of their presynaptic input (see Goldberg 2000 for review). Hence, it was suggested that the various profiles of membrane properties found in vestibular networks could be the substrate for their partition in frequency and amplitude-tuned channels. However, the dynamic processing of vestibular information throughout the vestibulo-ocular and -spinal pathways remains elusive because of the complex organization of these motor responses in mammals. Frogs offer an attractive model to investigate this problem because the isolated whole brain preparation allows stable intracellular recordings together with selective activation of the nerve branches innnervating the different labyrinthine end organs.
In frog, vestibular hair cells as well as vestibular nerve afferent fibers segregate with respect to a number of interrelated morphological and physiological properties (Baird 1994a,1994b; Blanks and Precht 1976; Flock and Orman 1983; Honrubia et al. 1981, 1989; Lewis and Li 1975; Lewis et al. 1982; Myers and Lewis 1990). In particular, utricular hair cells subdivide according to their frequency sensitivity and response dynamics as well as active and passive membrane properties (Baird 1994a,1994b; Baird and Lewis 1986; Lewis and Li 1975). These distinct physiological properties correlate with different hair bundle morphologies and different locations in the utricular macula (Baird 1994a,1994b; Baird and Lewis 1986; Lewis et al. 1982). Similar morphological differences exist among the hair cells in the semicircular canal cristae (Baird and Lewis 1986; Gioglio et al. 1995; Guth et al. 1994; Lewis and Li 1975; Myers and Lewis 1990) and are associated with distinct micromechanics of the hair bundles (Flock and Orman 1983). Assuming that these morphological variations reflect the same differences in physiological properties as in the utricular macula, the segregation of vestibular hair cells according to their frequency sensitivity and response dynamics is likely to be a general property of all labyrinthine end organs.
A similar segregation is found in vestibular nerve afferent fibers that contact these hair cells and is correlated with a number of interrelated properties as fiber diameter, origin in the sensory epithelium, discharge regularity, and response dynamics (Baird and Schuff 1994; Honrubia et al. 1981, 1989). In particular, thick afferent nerve fibers tend to have a more irregular resting activity, respond with a more phasic discharge to step-like accelerations, larger gains and smaller phase shifts. In contrast, thin afferent nerve fibers tend to have a more regular resting activity, a more tonic discharge to step-like accelerations, lower gains, and larger phase shifts (Honrubia et al. 1981, 1989). The putative separation into different channels on the sensory side is mirrored on the motor side of the vestibulo-ocular network. Abducens motoneurons in frogs have been shown to fall into several subgroups that differ in their response dynamics (Dieringer and Precht 1986). Two groups of large- and medium-sized, phasic abducens motoneurons are complemented by a group of small, particularly tonic motoneurons with a very long time constant for evoked responses (Dieringer and Precht 1986).
Altogether then, frog vestibular hair cells, as vestibular nerve afferent fibers and abducens motoneurons, form subpopulations of neurons with heterogeneous membrane properties. This confers to these cells distinct dynamic properties, which suggests that as in rodents, the vestibulo-ocular pathways of frogs may be organized in parallel frequency-tuned channels. However, a crucial step to validate that hypothesis is the description of the membrane properties of 2°VN, which have not been investigated so far in these animals. In addition, the frog model offers the unique opportunity to link the membrane properties of 2°VN with their anatomical location and their afferent and efferent connectivity. In particular, the spatial convergence pattern of semicircular canal and macular signals on 2°VN has been well described (Straka et al. 1997, 2002). Almost half of the 2°VN receive convergent afferent nerve input from one macula and one canal organ in a spatially specific manner (Straka et al. 2002). Utricular (UT) signals converge mainly with horizontal canal (HC) signals, whereas lagenar (LA) signals converge only with anterior vertical (AC) or posterior vertical canal (PC) signals. The remaining half of 2°VN receives afferent nerve signals from only one canal or one macula organ.
The passive membrane properties and discharge dynamics of frog 2°VN were determined by intracellular injection of current steps and ramp-like currents. Vestibular neurons were recorded in the isolated frog brain and identified as 2°VN by the presence of monosynaptic EPSPs after stimulation of the utricular nerve, the lagenar nerve, or one of the nerve branches innervating an individual semicircular canal. Part of the identified 2°VN were classified as projection neurons by the presence of antidromic spikes evoked by stimulation of the upper spinal cord and/or of the oculomotor nuclei in the midbrain. Differences in spike shape, spike discharge and passive membrane properties allowed a subdivision of frog 2°VN into two distinct functional types of neurons. Possible correlation of labyrinthine nerve inputs, projection patterns and spatial location with the two different functional types of 2°VN were investigated. Preliminary results were published in abstract form (Straka et al. 2003b).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Straka and Dieringer 1993), the animals were deeply anesthetized with 0.1% 3-aminobenzoic acid ethyl ester (MS-222) and perfused transcardially with iced Ringer solution [which contained (in mM) 75 NaCl, 25 NaHCO3, 2 CaCl2, 2 KCl, 0.5 mM MgCl2, and 11 glucose; pH 7.4). Thereafter, the skull and the bony labyrinth were opened by a ventral approach. After dissecting the three semicircular canals on each side, the brain was removed with all labyrinthine end organs attached to the VIIIth nerve. Subsequently, the brain was submerged in iced Ringer, and the dura, the labyrinthine end organs, and the choroid plexus covering the IVth ventricle were removed. In all experiments, the cerebellum remained attached to the brain stem while the forebrain was removed. Brains were used 
4 days after their isolation and were stored overnight at 6°C in continuously oxygenated Ringer solution with a pH of 7.5 ± 0.1. For the experiments, the brain stem was fixed with insect pins to the silicone elastomer (Sylgard) floor of a chamber (volume: 2.4 ml), which was continuously perfused with oxygenated Ringer solution at a rate of 1.3–2.1 ml/min. The temperature was electronically controlled and maintained at 14 ± 0.1°C.
The recorded neurons were identified as second-order vestibular neurons by their monosynaptic response to electrical stimulation of at least one of the individual labyrinthine nerve branches. For this purpose, all three semicircular canal nerves were stimulated separately. In some experiments, either the utricular or the lagenar nerve was stimulated in addition to the canal nerves. Single constant current pulses (0.2 ms; 1–15 µA) applied across suction electrodes (diameter: 120–150 µm) were used for electrical stimulation. The use of suction electrodes facilitated the isolation of an individual nerve branch and allowed separate stimulation of each of the canal and otolith nerves (see Straka et al. 1997, 2002, 2003a). For the antidromic identification of 2°VN with ascending axons, a concentric bipolar electrode (tip diameter: 25 µm; Science Products GmbH) was placed in the midline between the two oculomotor nuclei in the midbrain. For the antidromic identification of 2°VN with descending axons, a pair of Teflon-coated silver wires with chlorided tips (diameter: 250 µm) was inserted into the ventrolateral spinal cord on the left and right side at the C1/C2 level. Single constant current pulses (0.2 ms; 30–150 µA) were used for all stimuli. Thus depending on the origin of the antidromic spike, 2°VN were classified either as vestibulo-ocular or vestibulo-spinal neurons. Pulses were produced by a stimulus isolation unit (WPI A 360) at a rate of 0.5 Hz. Glass microelectrodes used for extra- and intracellular recordings were made with a horizontal puller (P-87 Brown/Flaming). Electrodes for extracellular field potential recordings were beveled (30°, 20 µm tip diameter) and filled with a 2 M solution of sodium chloride (
1 M
). Electrodes for intracellular recordings were filled with a 3 M solution of potassium chloride which gave a final resistance of 70–90 M.
At the beginning of each experiment, field potentials evoked by separate stimulation of the labyrinthine nerve branches were recorded at a reference recording site to optimize the positions of the stimulus electrodes and to determine the stimulus threshold for each branch (see Straka et al. 1997, 2002). This reference recording site was located 0.4 mm caudal to the VIIIth nerve root at a depth of 0.4 mm below the top of the brain stem. The stimulus threshold for the postsynaptic N1 field potential component (Precht et al. 1974) was similar for each of the canal and macular nerve branches and ranged from 1.5 to 3.1 µA. Stimulus intensities were indicated as multiples of these threshold values (xT). Intracellular single-cell recordings were not started unless the semicircular canal or otolith nerve-evoked N1 component exceeded 0.15 mV. The position of the stimulation electrode in the midbrain and spinal cord was optimized by maximizing the amplitude of the short-latency, antidromic field potentials recorded at the standard recording site in the vestibular nuclei. Maximum amplitudes usually ranged from 0.3 to 0.5 mV. The stimulus threshold intensity (T) for these antidromic field potentials was 10–20 µA and was similar for the spinal and midbrain stimulation electrodes. During intracellular recordings the stimulation intensity to evoke an antidromic spike was limited to 5xT. Action potentials were considered antidromic if there was an all or nothing response in the absence of an underlying prepotential at threshold stimulus intensity (see Straka et al. 2002). Vestibular neurons were recorded between 0.4 mm rostral and 0.7 mm caudal to the entry of the VIIIth nerve at a depth between 0.05 and 0.8 mm below the dorsal surface of the brain stem. This recording area included all four vestibular subnuclei (lateral, superior, descending, and medial) except the most medial parts of the medial vestibular nucleus. The position of the recorded vestibular neurons was mapped rostrocaudally with respect to the entry of the VIIIth nerve in the brain stem and dorsoventrally with respect to the dorsal surface of the hindbrain as in earlier studies (Straka et al. 2000, 2003a). As reported in earlier studies, most of the vestibular neurons recorded in the isolated frog brain had no spontaneous discharge at their resting membrane potential (Straka and Dieringer 1996, 2000; Straka et al. 1997, 2002, 2003a). Only neurons with a membrane potential more negtive than –55 mV were included in this study.
Membrane properties of second-order vestibular neurons identified by their monosynaptic input from a particular canal and/or macular nerve branch were determined by intracellular current injection. Time constants, input resistance, current-voltage relationships as well as firing properties in response to long, positive current steps (1 s) and to ramp-like currents were used to classify the recorded neurons as phasic or tonic 2°VN. Spike parameters were analyzed by determining firing threshold, spike amplitude, width, rise time, and fall time of the evoked spikes. Firing threshold was determined as the point at the beginning of each spike where the voltage trace reached 10 mV/ms. The time constant and input resistance were obtained from responses to small negative current pulses of 200-ms duration. The membrane hyperpolarization was fitted with a double-exponential Eq. 1 that allowed a determination of the first (
0) and second (1) time constant
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Synaptic transmission was blocked in some 2°VN (n = 10) by increasing the concentration of Mg2+ (4 mM) and decreasing the concentration of Ca2+ (0.4 mM) in the Ringer solution. With these concentrations, the monosynaptic EPSPs evoked in vestibular neurons by stimulation of individual vestibular nerve branches were completely blocked within 12–15 min. Single sweeps of the responses were digitized (CED 1401, Cambridge Electronic Design), stored on computer and analyzed off-line (SIGNAL, Cambridge Electronic Design). Synaptic potentials were analyzed from averages of 20–30 single sweeps after electronic subtraction of the extracellular field potential recorded in the vicinity. Statistical differences in parameters were calculated according to the Mann-Whitney U test (unpaired parameters) and the Wilcoxon signed-rank test (paired parameters; Prism, Graphpad Software). All averaged results were expressed as means ± SD. Graphical presentations were made with the aid of commercially available computer software (Origin, Microcal Software; Corel Draw, Corel).
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RESULTS |
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Intracellular recordings were obtained from 249 vestibular neurons in 33 isolated in vitro frog brain preparations with 0.5 mM Mg2+ present in the bath solution except otherwise stated. Recordings were obtained from all vestibular nuclei. Only those neurons that were identified as second-order vestibular neurons (2°VN) by the presence of a monosynaptic EPSP (HC in Fig. 1A1; PC in B1) after separate electrical stimulation of the utricular nerve, the lagenar nerve or of one of the three ipsilateral semicircular canal nerves were taken into account for further analysis. As in an earlier study (Straka et al. 2002), intracellular EPSPs triggered by semicircular canal or utricular (UT) nerve stimulation were considered as monosynaptic if their latency ranged between 3.4 ms (average onset of the postsynaptic N1 field potential) and 5.2 ms (earliest possible onset of disynaptic EPSPs after adding another synaptic delay of 1.8 ms). These values were slightly shorter for lagenar (LA) nerve-evoked responses (between 2.9 and 4.7 ms) due to the shorter distance between the stimulation electrode and the recording site (Straka et al. 2002). Differences in the diameter of the fibers supplying the different equilibrium organs can be ruled out as an explanation for the difference in onset latencies. In fact, Dunn (1978) has shown in the frog that the number of the thickest fibers (>10 µm) as well as the average diameter of fibers is similar for all vestibular nerve branches. The chemically mediated monosynaptic EPSP increased in amplitude with stimulus intensity, whereas the onset latency remained constant (Fig. 1A2; c in B2). Corresponding to the N1 field potential components, the latency of monosynaptic EPSPs was similar for responses evoked by horizontal (HC), anterior vertical (AC), posterior vertical canal (PC), or UT nerve stimulation (mean: 3.8 ± 0.5 ms; n = 269), whereas the EPSPs evoked by lagenar nerve stimulation (mean: 3.4 ± 0.6 ms; n = 38) had significantly shorter latencies (P 0.001).
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; Straka and Dieringer 1996; Straka et al. 1997). The latency of this early depolarization (mean: 1.7 ± 0.3 ms; n = 37) was independent of the stimulus intensity (Fig. 1B2) and reflected an electrically mediated EPSP component. The threshold of this electrical EPSP was as low as the threshold of the chemical EPSP in a given neuron. In contrast with the latter one, however, the electrical EPSP increased only slightly with stronger stimulus intensity and saturated at amplitudes ranging from 1.0 to 2.5 mV (Fig. 1B2). The low threshold and the relatively early saturation suggest a preferred activation of these electrical EPSPs by thick vestibular nerve afferent fibers, a finding compatible with earlier results (Straka and Dieringer 1996; Straka et al. 1997).
According to the presence of monosynaptic EPSPs from a particular canal or macular nerve branch, recorded neurons were categorized as particular 2° canal, 2°UT, or 2°LA neurons or as particular 2° macula + canal or 2° canal + canal neurons if monosynaptic EPSPs were evoked from more than one vestibular nerve branch (Table 1). In 2° canal neurons that received a monosynaptic EPSP from only one canal nerve branch, the remaining two semicircular canal nerves, respectively, evoked either EPSPs with a di- or polysynaptic onset (Fig. 1, A, 3 and 4, and B5) or no response (B4). The presence of many 2°AC, 2°HC, or 2°PC neurons and few 2° macula neurons or convergent 2° canal + macula neurons (Table 1) was due to the fact that the UT or LA nerve branches were stimulated in addition to the three semicircular canal nerve branches only in some experiments. Therefore 2° macula neurons were less numerous and thus the convergence pattern of canal and macular signals observed in this study differs from that of a detailed, systematic earlier study (Straka et al. 2002). However, independent of the relatively small number of convergent neurons, UT nerve afferent signals converged predominantly with HC nerve signals, whereas LA nerve afferent signals converged mainly with AC and to a minor extent with PC nerve afferent signals as reported earlier (Straka et al. 2002).
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Classification of frog vestibular neurons according to their discharge patterns
Because of the lack of a spontaneous discharge in vestibular neurons recorded in the isolated in vitro frog whole brain (see Straka et al. 2002), the classification of vestibular neurons was performed using long (1s duration) positive current steps that evoked spikes in identified frog 2°VN (Fig. 1, A6 and B7). This contrasts with the situation in guinea pigs or rats (Gallagher et al. 1985; Johnston et al. 1994; Serafin et al. 1991a), where the different types of vestibular neurons were identified from spikes during spontaneous activity. Using positive current steps, the evoked spike discharge of frog 2°VN fell into two categories that differed in their discharge behavior (Fig. 1, A6 and B7). Above spike threshold, the majority of 2°VN (n = 194; 78%) exhibited a phasic discharge pattern that was characterized by a high-frequency burst of up to five spikes and the absence of a subsequent continuous discharge (Fig. 1A6, inset). This phasic discharge pattern did not change after inhibition of the chemical synaptic transmission by perfusion of a high-Mg2+/low-Ca2+ Ringer solution. Under this condition, the afferent EPSPs in these neurons were completely blocked. Hence, a recurrent inhibition as the responsible mechanism for the absence of a tonic discharge in these neurons can be ruled out. In a second, smaller group of 2°VN (n = 55; 22%), positive current steps well above spike threshold evoked a continuous, tonic discharge (Fig. 1B7). According to these two clearly different discharge patterns, frog 2°VN were subdivided and classified as "phasic" or "tonic" vestibular neurons.
This categorization of 2°VN into phasic or tonic vestibular neurons was not correlated with an origin of the monosynaptic labyrinthine response from a particular canal and/or macular nerve branch (Table 1). In fact, similar percentages of 2°VN with monosynaptic inputs from particular labyrinthine nerve branches were encountered among phasic as well as among tonic vestibular neurons (Table 1). This included nonconvergent 2° canal and 2° macula neurons as well as convergent 2° canal + macula neurons and 2°VN with multiple canal and macular nerve inputs.
A possible differential contribution of thicker and thinner vestibular nerve afferent fibers to monosynaptic canal or macular inputs in phasic and tonic vestibular neurons was studied by determining the threshold and saturation of the monosynaptic EPSP amplitude with increasing stimulus intensity. This test is based on the fact that thick vestibular nerve afferent fibers are recruited at lower stimulus intensities, whereas thinner afferent fibers have higher thresholds for their activation (Straka and Dieringer 2000; see Goldberg 2000 for a discussion of this issue). The threshold of monosynaptic EPSPs with respect to the stimulus intensity was evaluated in a subset of recorded 2°VN (n = 29). It was found to be close to the threshold of the monosynaptic N1 field potential component, and was very similar in phasic (1.2 ± 0.2xT; n = 20) and in tonic vestibular neurons (1.3 ± 0.2xT; n = 9). This low threshold indicated that monosynaptic EPSPs in phasic as well as in tonic vestibular neurons were mediated at least in part by thick vestibular nerve afferent fibers. Such a contribution was further supported by the presence of short-latency electrical EPSP components preceding the monosynaptic chemical EPSP in both types of vestibular neurons. An electrical coupling between vestibular nerve afferent fibers and 2°VN occurred in about similar proportions in phasic (28 of 194 neurons; 14%) and tonic (9 of 55 neurons; 16%) vestibular neurons. Thus both types of vestibular neurons received afferent inputs from thick vestibular nerve afferent fibers.
The monosynaptic EPSPs increased in amplitude with stimulus intensity (Fig. 1, A1 and B1) at a similar rate for phasic and tonic 2°VN. Amplitudes started to saturate at current intensities that ranged between 5xT and 7.5xT for different neurons. At even higher stimulus intensities action potentials were triggered that were superimposed on the EPSPs (Fig. 1B3).
The projection of phasic and tonic 2°VN to oculomotor and spinal targets was determined in a subset of 2°VN (n = 79). According to the origin of the antidromic spike(s), phasic 2°VN (n = 62) were subdivided into vestibulo-ocular (n = 15), vestibulo-spinal (n = 40), and vestibulo-oculo-spinal neurons (n = 7). Correspondingly, tonic 2°VN (n = 17) were subdivided into vestibulo-ocular (n = 7) and vestibulo-spinal neurons (n = 10). Although, no predominance for a particular projection of one or the other type was observed, tonic neurons (41%) tended to have a relatively larger proportion of ascending projections than phasic neurons (32%).
The location of phasic and tonic 2°VN in the vestibular nuclei was determined by systematically mapping the rostrocaudal and dorsoventral position of each recorded 2°VN as in earlier studies (Straka et al. 2000, 2003a). Plotting the stereotactic position of phasic and tonic 2°VN indicated that both types were intermingled, and no particular vestibular area with a predominance or an absence of one or the other type was observed (Fig. 2E). Because the recordings covered all vestibular nuclei, the two vestibular cell types were not restricted to a particular nucleus but were present throughout the whole frog vestibular nuclear complex. The smaller numbers of both types of neurons around the entry of the VIIIth nerve (zero in Fig. 2E) is due to the presence of the large bundle of afferent axons entering the brain stem and the general paucity of vestibular neurons at that level (see Straka et al. 2003a).
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The response of phasic 2°VN to positive current steps consisted of a transient discharge that increased from one spike just above threshold to a short, high-frequency burst of 5 spikes (2.7 ± 0.8; n = 194; Figs. 1A6 and 2A). The first spike after injection of a positive current step that reached spike threshold occurred in all phasic neurons within the first 4 ms after the onset of the current step. After the spike or the brief burst of spikes, neurons stopped firing until the end of the current step, indicating a rapid adaptation (Fig. 2C, 
, 
, 
). The frequency of the discharge within the burst was the highest for the first interspike interval and decreased by almost 50%, respectively, for the following interval(s) (Fig. 2C, , , ). The spike rate for the first interspike interval increased with intensity of the current step (see Fig. 2A, inset, and C and D, , , ) to reach a maximal rate of almost 300 spikes/s (mean: 176.5 ± 42.5 spikes/s; n = 56; Fig. 3C).
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, 
, 
, 
, 
, 
, and D, , and 3C). With larger current steps, a brief, rapidly adapting burst that usually was limited to the first two spikes preceded the constant tonic discharge (Fig. 2C, , , ). The spike rate of the first interspike interval increased with current intensity and saturated at maximum rates that varied between 90 and 190 spikes/s in different neurons (mean: 144.3 ± 30.1 spikes/s; n = 17; Figs. 2D, , and 3C). However, this value was significantly lower (P 0.01; Mann Whitney test) than the discharge rate of the first two spikes within the burst evoked in phasic 2°VN. The increase in frequency of the constant discharge with current intensity in tonic 2°VN was fairly linear over a wide range of injected current (Fig. 3A). However, the rate of the increase varied between different neurons as indicated by the different slopes of the curves in Fig. 3A. Part of this variation is most likely due to differences in passive impedance between individual tonic 2°VN. Taking these differences into account and to obtain the rate of increase with respect to voltage, the spike rate within the linear part was fitted with a linear regression and plotted with respect to the calculated voltage (Fig. 3B). A calculation of the voltage rather than obtaining it from the evoked depolarization was necessary because determination of the actual membrane potential in the presence of action potentials, in particular at higher discharge rates is very difficult. This approach is justified because the I-V curve of tonic 2°VN was linear. After this procedure, an increase of the discharge of tonic 2°VN at an average rate of 3.4 ± 1.9 spikes·s–1·mV–1 after reaching spike threshold (Fig. 3B, - - -) was obtained. No relation between threshold and slope of the discharge was found.
Among a number of parameters, type A and type B vestibular neurons in guinea pig can be differentiated in the in vitro whole brain by their spontaneous discharge regularity (Babalian et al. 1997). Although, the discharge regularity could not be obtained at rest in 2°VN recorded in the in vitro frog brain, it was possible to determine the regularity of the spike discharge in tonic (but not in phasic) 2°VN during persistent firing evoked by positive current steps. The discharge regularity, indicated by the coefficient of variation (CV), depended on the firing rate as shown in Fig. 3D. The CV decreased with increasing spike rate, reached 0.34 at 10 Hz and was constant at firing rates >40 Hz with a value of 0.15 (Fig. 3D).
Dynamics of the spike discharge during ramp-like current injection
To reveal the discharge dynamics of phasic and tonic 2°VN, slow-rising (2 s) ramp-like currents were injected (Fig. 4). In all phasic 2°VN, these ramp-like currents, however, failed to evoke a spike discharge (Fig. 4A1). Instead, the membrane potential slowly depolarized during the current ramps and exceeded spike threshold without triggering a spike in any of the phasic 2°VN. However, action potentials could be triggered in the same neurons when faster rising current ramps were applied (Fig. 4A2, 2). Because current ramps with a slightly slower (critical) slope failed to trigger a spike (Fig. 4A2, 1), a threshold rate of membrane depolarization that was necessary to evoke an action potential could be defined for each phasic 2°VN. This threshold rate was determined for each phasic 2°VN by applying a series of current ramps that changed the slope of the membrane potential around the critical slope in intervals of 0.2–0.3 mV/ms. Accordingly, the rate of the change of the membrane potential must exceed on the average 1.4 ± 0.7 mV/ms (n = 33; Fig. 4A3), and the membrane potential must reach spike threshold to trigger a spike. Thus the failure of evoking a discharge with slow but not with fast ramp-like currents is in accordance with the particularly fast adapting responses to positive current steps.
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Spike parameters of phasic and tonic second-order vestibular neurons
Second-order vestibular neurons in the in vitro frog brain were not spontaneously discharging at rest in this as in earlier studies (Straka and Dieringer 1996; Straka et al. 1997, 2002). To characterize the shapes of the action potentials in phasic and tonic 2°VN and to determine various spike parameters, action potentials were evoked by positive current steps. Comparison of the action potentials in phasic and tonic 2°VN revealed that both types of neurons not only differed in their particular characteristic spike discharge pattern but also in the shape of their evoked action potentials. Phasic 2°VN were characterized by action potentials with a small monophasic afterhyperpolarization (e.g., 5A1, arrow) followed by a small after-depolarization (Fig. 5A1, 1 in A3). During current steps that evoked bursts in phasic 2°VN, the second spike followed the first spike without changing the time course and size of the monophasic afterhyperpolarization (Fig. 5A2, 2–5 in Fig. 5A3). At current intensities that were just above threshold for a second spike, this spike was evoked on top of the small afterdepolarization (2 in Fig. 5A3). At even higher current intensities, this second spike was evoked at a progressively shorter delay with respect to the first spike (2–5 in Fig. 5A3).
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0.05; Mann Whitney U test) compared with the corresponding single afterhyperpolarizaton in phasic 2°VN, although the distributions overlapped to a large extent (Figs. 5, A1 and B1, compare single arrows, and 7B; Table 2A). Apart from these differences in the shape of the action potentials, and in particular the size of the afterhyperpolarization between phasic and tonic 2°VN, other parameters as spike threshold (see arrow in Fig. 5, A1 and B1) spike amplitude, width, and rise and fall times were not significantly different (see Table 2A).
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The differences in the shape of the action potentials and in the discharge properties of phasic and tonic 2°VN were correlated with their passive membrane properties. These passive membrane properties differed even though the average resting membrane potential of the two types of 2°VN (mean: –69.4 ± 8.9 mV; n = 249) were essentially identical (see Table 2B). To determine the input resistance and time constants of phasic and tonic 2°VN small negative current pulses were applied (Fig. 6, A, 1 and 2, and B, 1 and 2). The passive input resistance and the first two time constants (0 and 1) were determined by fitting responses to hyperpolarizing voltage steps with a double-exponential equation (see Eq. 1 in METHODS).
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) that decreased to lower values during the subsequent plateau phase (Fig. 6A3, ) was present in phasic but not in tonic 2°VN (see B3, and 
). This distinct initial transient and the subsequent rectification are compatible with the burst-like discharge of these neurons and the absence of a continuous firing evoked by positive current steps. In contrast, such a sharp initial depolarizing transient was absent in tonic 2°VN and both the initial (Fig. 6B3, ) as well as the plateau component (B3, ) exhibited a similar current voltage relation during the depolarizing current steps (Fig. 6B4). Because the sharp, initial depolarizing transient was restricted to phasic 2°VN, this parameter was quantified by calculating the ratio of the amplitudes of the initial transient response/plateau response of phasic 2°VN to depolarizing steps at current intensities just subthreshold to spike generation. The presence of an initial transient in phasic 2°VN in response to positive current steps is also indicated by the significantly different slopes (P 0.0001; Wilcoxon signed-rank test) of the I-V curve for the initial transient and the plateau component during positive current steps (Fig. 7E). Because tonic 2°VN did not exhibit such a transient peak, the ratio between the largest response obtained within a window of 100 ms after the onset of the current pulse and the final plateau response was calculated for these neurons. After this procedure, the ratio was found to be well >1 (mean: 1.87 ± 0.46) in phasic 2°VN (Fig. 7F, 
). In contrast, this ratio was centered around 1 (mean: 0.97 ± 0.09) in tonic 2°VN (Fig. 7F, ) because the early and the late component in these neurons had always similar amplitudes. The two values were significantly different (P 0.0001; Mann-Whitney U test). The absence of overlap of these values for phasic and tonic 2°VN implies that this parameter can serve as a decisive property to separate phasic and tonic 2°VN using subthreshold depolarizing current pulses. Thus frog 2°VN can be classified as phasic and tonic 2°VN even in the absence of action potentials. The responses of individual phasic and tonic 2°VN to current pulses were used to calculate the average I-V curves of the two types of neurons (Fig. 7E). The average slope of the I-V curve for tonic 2°VN (mean: 25.1 ± 14.8 mV/nA; n = 22) was significantly steeper (P 0.001; Mann-Whitney U test) than for phasic 2°VN (mean: 13.0 ± 7.7 mV/nA; n = 44; Fig. 7E), which corroborated the significant difference in input resistance between the two types of neurons using small hyperpolarizing current pulses. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Second-order vestibular neurons with different response dynamics in the frog
In comparison to mammals, the absence of a spontaneous resting discharge in 2°VN recorded in the in vitro frog whole brain (Straka and Dieringer 1996, 2000; Straka et al. 1997, 2002) appears largely due to the very hyperpolarized resting membrane potential of these neurons (about –69 mV). This is likely to result from the loss of excitatory inputs from vestibular nerve afferent fibers, which discharge at low spontaneous rates of 1–10 spikes/s in vivo in frog (Blanks and Precht 1976). Thus the average in vivo resting firing rate of 2°VN, which also ranges from 1 to 10 spikes/s (Dieringer and Precht 1977), might largely depend on the spontaneous activity of vestibular nerve afferent fibers. As in the frog, guinea pig 2°VN recorded in slices or in the in vitro whole brain have significantly lower resting activities (10–15 spikes/s) (Babalian et al. 1997) than in vivo (40 spikes/s) (Ris and Godaux 1998; Ris et al. 1995).
However, the absence of a spontaneous resting activity in 2°VN in the in vitro experiments in frog might be explained, at least in part, by the lower temperature (14°C) at which the experiments on the isolated frog brain were preformed in comparison to most in vivo recordings at room temperature (about 20–22°C) (see Blanks and Precht 1976). This is based on the fact that the spontaneous activity is reduced at lower temperatures. In fact, it has been shown for frog auditory nerve fibers recorded in vivo that the spontaneous activity is reduced by a factor of 1.6 (Q10) for a reduction in temperature by 10°C (van Dijk et al. 1990). According to this result, a resting activity of 10 Hz in 2°VN should decrease to 7.5 Hz when the temperature is lowered from 21 to 14°C. This is only minor compared with the complete absence of a resting activity in the isolated in vitro frog brain. Hence, the lower recording temperature cannot solely explain the absence of the spontaneous activity of 2°VN in the in vitro frog brain. Because frogs are poikilothermic vertebrates and are functional at a range of temperatures, a comparison of neuronal resting activities between frogs and mammalian species must be always accompanied by the temperature at which the recordings were obtained.
The absence of a resting discharge in frog 2°VN in vitro prohibited a classification of these neurons according to their spontaneous firing pattern. The 2°VN were therefore segregated into two distinct subtypes, phasic and tonic neurons, using injection of long positive current steps to evoke spikes. Because no neurons with intermediate response patterns were encountered, phasic and tonic 2°VN in frog represent two separate populations rather than a continuum. This classification of frog 2°VN does not seem to be restricted to a particular vestibular nucleus because the 249 2°VN were recorded throughout all vestibular nuclei without any noticeable difference in the topography of the two cell types.
The distinction between phasic and tonic neurons was corroborated by differences in other active and passive membrane properties. The transient response of phasic 2°VN was associated with the existence of a threshold for the rate of voltage change to evoke spikes during ramp-like currents (Fig. 4A2). When current steps did not reach the spike threshold, a sharp initial voltage transient was visible at the beginning of the membrane potential response (Fig. 6A3). In contrast, the continuous discharge of tonic 2°VN was associated with a linear increase of the spiking rate over a wide range as soon as the spike threshold was reached.
The rapidly adapting, transient responses of phasic 2°VN could be due either to particular membrane properties and/or to a synaptic circuitry involving a recurrent inhibition. In fact, a combination of both mechanisms has been shown to be responsible for the phasic responses to current steps displayed by the goldfish Mauthner cell (Nakayama and Oda 2003). In the isolated whole brain, a recurrent inhibition can be ruled out for phasic frog 2°VN because the phasic discharge pattern was not modified by a blockade of the chemical synaptic transmission. Thus it is likely that the phasic response properties of these neurons are mainly due to particular membrane properties. However, a recurrent inhibition might still contribute to shape the burst response of phasic 2°VN in vivo because an inhibitory feedback loop has been demonstrated in frog vestibular nuclei (Straka and Dieringer 1996, 2000; Straka et al. 1997). One of the important implications of the rapid and prominent adaptation capabilities of phasic neurons is that these 2°VN are silent in the absence of fast synaptic inputs even at membrane potentials above spike threshold. This is compatible with and might in fact explain the presence of spontaneously inactive frog 2°VN in vivo (Dieringer and Precht 1977).
Considerably more phasic than tonic 2°VN were recorded in the course of this study. This suggests an important role of phasic signals in the stabilization of gaze and posture in the frog. However, the larger number of phasic 2°VN might simply result from a sampling bias. Because the average input resistance of phasic 2°VN was significantly lower than that of tonic 2°VN, the latter neurons are likely to be smaller and thus more difficult to record. Both phasic and tonic 2°VN with a vestibular nerve branch-evoked electrical EPSP had a lower input resistance than the neurons where such an electrical component was absent. This suggests that in those 2°VN that are electrically coupled to presynaptic vestibular nerve afferent fibers, the input resistance is shunted by the presence of gap-junctions between the two cells.
Phasic and tonic 2°VN exhibited a similar convergence of labyrinthine nerve afferent input (Table 1), indicating that none of the two types was restricted to either canal or macular pathways. Rather both types of vestibular neurons are part of the network that mediates angular and linear head acceleration signals. However, a particular correlation of one or the other type of 2°VN with macular signals might have been underestimated by the fact that the lagenar and the utricular nerve branch were not stimulated in all experiments in addition to the canal nerve branches. Although tonic 2°VN tended to have more ascending projections and phasic 2° VN more descending projections, both types projected to spinal and/or to ocular motor target areas. Altogether, the presence of phasic and tonic 2°VN with similar afferent inputs and efferent targets suggests the presence of parallel pathways within the vestibulo-ocular and -spinal networks.
Parallel frequency-tuned channels underlying vestibulo-ocular responses in frogs
In the vestibular sensory organs of frogs, low- and high-frequency linear and angular head acceleration components are discriminated by different types of hair cells that differ in their response properties (Baird 1994a,1994b; Baird and Lewis 1986; Flock and Orman 1983; Lewis and Li 1975; Lewis et al. 1982; Myers and Lewis 1990). For the utricular hair cells, it has been shown that nonadapting hair cells display largely passive voltage responses, whereas others are endowed with rapidly adapting responses mediated by fast ionic currents (Baird 1994a,1994b). These different hair cells would be best suited to code either tonic (nonadapting cells) or phasic (adapting cells) components of head movements (Baird 1994a,1994b). Assuming that similar morphological differences among canal as among macular hair cells, respectively, (Baird and Lewis 1986; Gioglio et al. 1995; Guth et al. 1994; Lewis and Li 1975; Myers and Lewis 1990) are paralleled by similar differences in physiological properties, a general functional segregation of hair cells in all labyrinthine end organs is likely.
A separation into pathways that differ in their signal content and response dynamics is also found at the level of vestibular nerve afferent fibers (see Goldberg 2000). However, the segregation of afferent fibers might be less clear than for hair cells. In fact, afferent fibers might rather form a broad spectrum of parallel pathways between two extremes. Nonetheless, thick utricular afferent fibers tend to innervate hair cells characterized by fast dynamics, whereas thin afferent fibers innervate hair cells that are nonadapting and have a low-frequency sensitivity (Baird and Schuff 1994). This suggests that the response dynamics of afferent fibers might be determined by the rate of adaptation of the innervated hair cells. It is also compatible with the idea that these parallel pathways originating in multiple types of hair cells are prolonged at the level of the vestibular nerve fibers (Honrubia et al. 1981 1989).
A similar distinction into separate pathways that differ in response dynamics is also found on the motor side of the frog vestibulo-ocular reflex (Dieringer and Precht 1986; Dieringer and Rowlerson 1984). Abducens motoneurons can be subdivided into different types that differ in
, the average maximal rate, respectively. D: spike discharge regularity of the continuous discharge in tonic 2°VN indicated by the coefficient of variation (CV) as a function of the discharge rate. The CV decreased with spike rate and was constant >40 Hz at values of 
