Material and methods the top of the brainstem. The stimulus threshold for the

H . Straka et al. Brain Research 880 2000 70 –83 71 from other semicircular canal nerves. Since the dendrites the sylgard floor. The temperature of the bath was elec- of 28VN are long and select their appropriate semicircular tronically controlled and maintained at 1460.28C. canal input it is also conceivable that the cell bodies of 28 For electrical stimulation of individual semicircular canal nerves with an input from a particular semicircular canal nerve branches we used single constant current canal are clustered and form modular zones that represent pulses 0.2 ms; 8–12 mA across suction electrodes inner different vestibular endorgans in a manner similar to diameter 120–150 mm; see [34]. For stimulation of the retinotopic, somatotopic or tonotopic maps of other sen- VIIIth nerve we used single constant current pulses 0.2 sory systems. ms; 5–30 mA across a concentric bipolar electrode tip We analyzed these possibilities first with evoked field diameter 25 mm that was located about 2 mm more potentials recorded in vitro in the isolated frog brain. proximal than the electrodes stimulating the individual Separate electrical stimulation of individual semicircular semicircular canal nerves [34]. Constant current pulses canal nerves activated field potentials that consisted of two were produced by a stimulus isolation unit WPI A 360 at or more negative components. The spatial distribution of a repetition rate of 0.33 Hz. For extracellular recordings vestibular nerve evoked N and N field potentials, glass microelectrodes were fabricated with a horizontal 1 representing regional presynaptic N and postsynaptic puller P-87 Brown Flaming, beveled 308, 20 mm tip N response components [28], was mapped systematical- diameter and filled with 2 M sodium chloride 1–3 MV. 1 ly. We compared these results with the location of 28VN, Electrodes for intracellular recordings were filled with 2 M identified by their monosynaptic responses following elec- potassium acetate 90–120 MV or 2 M potassium chlo- trical stimulation of the horizontal, anterior vertical or ride 80–100 MV. Vertical displacements of the recording posterior vertical semicircular canal nerve branch. The electrodes were controlled with a nanostepper. Horizontal physiological response properties of these neurons had displacements were performed with a two axes micro- been described elsewhere [34]. manipulator. Preliminary parts of this study had been published in In order to standardize our results field potentials were abstract form [35]. recorded at the beginning of each experiment at the same standard recording site 0.4 mm caudal to the caudal end of the entry of the VIIIth nerve at a depth of 0.4 mm below

2. Material and methods the top of the brainstem. The stimulus threshold for the

evoked N component of the field potential was similar for 1 In vitro experiments were performed on the isolated each of the three semicircular canal nerves and ranged brains of grass frogs Rana temporaria and comply with usually between 2 and 3 mA. Stimulus intensities are given the Principles of Animal Care of the National Institutes of as multiples of these threshold values 3T and were Health, publication no. 86-23, revised 1985. Permission for restricted to intensities of maximally 43T. these experiments was granted by Regierung von Ober- In order to cast our electrophysiological results into a bayern 211-2531-31 95. Grass frogs were deeply anes- normalized geometry of the vestibular nuclei we used the thetized 0.1 3-aminobenzoic acid ethyl ester; MS-222 same reference frame for a three-dimensional coordinate and perfused transcardially with iced Ringer solution 75 system as in a parallel anatomical study [24]. Distances in mM NaCl; 25 mM NaHCO ; 2 mM CaCl ; 2 mM KCl; the rostro-caudal direction refer to the caudal end of the 3 2 0.5 mM MgCl ; 11 mM glucose; pH 7.4 [33]. The skull entry of the VIIIth nerve, in dorso-ventral direction to the 2 and the bony labyrinth were opened by a ventral approach top of the brainstem and in medio-lateral direction to the and the three semicircular canals on either side were medial surface of the brainstem at the particular rostro- sectioned. Then, the brain was removed with the labyrin- caudal level Fig. 1B–D. For the latter measurement the thine endorgans attached to the VIIIth nerve. The isolated midline of the brainstem was not practical because of the brain was submerged in iced Ringer and the dura, the variability in the shape of the alar plate after the choroid labyrinthine endorgans and the choroid plexus above the plexus had been removed. Measurements rostral to the IVth ventricle were removed. The forebrain was discon- caudal end of the entry of the VIIIth nerve and medial to nected and in part of the experiments also the cerebellum the top of the brainstem were denoted with a negative sign was removed. Brains were stored overnight at 68C in see Fig. 1B,D. The mean distance between the caudal end continuously oxygenated Ringer solution with a pH of of the cerebellum and the obex was 3.7460.07 mm n5 7.560.1 and were used up to 5 days after their isolation. 15. The distance between the left and right top of the For some experiments the brainstem was glued with brainstem at the level of the VIIIth nerve was 2.6860.07 cyanoacrylate glue to a plastic net with the ventral side mm n515 and the distance between the top of the down. This net was fixed with insect pins to the sylgard brainstem and the midline floor of the IVth ventricle at floor of a chamber volume 2.4 ml which was continuous- the level of the VIIIth nerve was 0.7860.04 mm n515. ly perfused with oxygenated Ringer solution at a rate of These measurements were used to define a standard size 1.3–2.1 ml min. In other experiments the brainstem was and to normalize the brains of different individuals. The sectioned along the midline and fixed with insect pins to normalized location of the vestibular nuclei of grass frogs 72 H Fig. 1. Schematic presentation of the vestibular nuclei in the brainstem and how the spatial distribution of semicircular canal nerve evoked field potentials was explored. A,C,E Frontal A, parasagittal C and oblique medio-lateral views of the vestibular nuclei as reconstructed from data published by Matesz [23] and Kuruvilla et al. [22]. Dashed lines in A indicate the orientation of the parasagittal 1 and oblique medio-lateral 2 planes shown in C and E, respectively. B,D,F Electrode tracks from surface to 0.8 mm in depth through the vestibular nuclei. Arrows indicate the laterality B or the rostro-caudal position D,F of electrode tracks. Zero laterality B refers to the top of the brainstem, zero rostro-caudal position D,F refers to the caudal end of the entry of the VIIIth nerve in the brainstem. Same calibrations for A and B and for C–F, respectively. CB, cerebellum; DN, dorsal nucleus; DVN, descending vestibular nucleus; LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; s.l., sulcus limitans; SVN, superior vestibular nucleus. in a frontal Fig. 1A,B, parasagittal Fig. 1C and in an In the latter case electrodes were advanced perpendicularly oblique medio-lateral plane Fig. 1E was used to specify to the surface of the medial wall of the brainstem dashed the search area for this mapping study. line 2 in Fig. 1A. The touch point was about 20.4 mm The spatial distribution of the evoked field potentials medial to the top of the dorsal brainstem. Typically, was mapped systematically with recording tracks from the records were taken every 0.1 mm in depth. However, in the surface of the brainstem to a depth of 0.8 mm in three oblique medio-lateral plane we recorded every 0.05 mm up different planes. Electrode tracks in the frontal plane were to a depth of 0.3 mm because of the relatively thin strip positioned rostral 20.4 mm or caudal 0.4 mm or 0.7 that represents the medial vestibular nucleus Fig. 1A,E. mm to the caudal end of the entry of the VIIIth nerve and Deeper measurement points were separated by 0.1 mm up consisted in each case of five depth tracks separated by to a depth of 0.8 mm see grid in Fig. 1F. 0.15 mm in laterality arrows in Fig. 1B. In the parasagit- Averages from 20 single sweeps of the evoked field tal plane, seven depth tracks explored the dorsal brainstem potentials at a given recording site were digitized CED at a laterality of 0 mm dashed line 1 in Fig. 1A. The 1401, Cambridge Electronic Design, stored on computer recording sites were located 20.7, 20.4 or 0 mm rostral and analyzed off-line SIGAVG, Cambridge Electronic and 0.4, 0.7, 1.0 or 1.5 mm caudal to the caudal end of the Design. From the averaged field potentials separate depth entry of the VIIIth nerve arrows in Fig. 1D. The oblique profiles were constructed for N and N potentials for each 1 medio-lateral plane was explored by another seven elec- recording track and for each stimulated nerve branch. trode tracks see arrows in Fig. 1F at the same rostro- Initially, we searched for the largest N and N field 1 caudal locations as used for parasagittal recording tracks. potential component in the rostro-caudal extension of the H . Straka et al. Brain Research 880 2000 70 –83 73 vestibular nuclear complex after stimulation of the VIIIth nerve or of one of its canal nerve branches. To minimize the variability of results we averaged the amplitudes recorded in a given depth track separately for each stimulated canal nerve, compared these averaged values between different parasagittal depth tracks and normalized the averaged values according to the largest values evoked by a given canal nerve branch in a particular track see Fig. 3. This procedure facilitated a comparison of the results following stimulation of different semicircular canal nerves or of the VIIIth nerve and a comparison of data from different experiments. From the analysis of data obtained in parasagittal depth tracks we obtained evidence for a differential spatial distribution of the largest evoked field potential amplitudes following stimulation of different semicircular canal nerves Fig. 3. In later experiments we estimated the maximal response amplitude of N or N in 1 each series of depth tracks for a given nerve branch and normalized all other responses evoked by the same nerve branch. In order to include depth information in our analysis field potentials are continuous functions and recording tracks were relatively close to each other in space isopotential surface plots were calculated through linear interpolation between maximal response amplitudes Stanford graphics, 3-D visions, Torrance, CA. Four groups of relative response amplitudes up to 25, 50, 75 or 100 were represented by different intensities of gray tones. Statistical analyses were performed with the aid of Fig. 2. Field potentials in the vestibular nuclear complex evoked by commercially available computer software INSTAT; stimulation of the VIIIth nerve or its semicircular canal nerve branches on Graphpad, San Diego, CA. Statistical differences in the ipsilateral side. A–D Pre- N and postsynaptic N field potential 1 latencies, amplitudes and areas were calculated according components evoked by stimulation of the VIIIth nerve A, the horizontal to the Wilcoxon signed rank test test for paired parame- canal HC nerve B, the anterior canal AC nerve C or the posterior ters. Graphical presentations were performed with the aid canal PC nerve branch D. Each of the responses in A–D were recorded at the same site. Dashed lines indicate baseline and arrow head of commercially available computer software Origin, the onset of stimulus. Calibration bars in B apply also for C and D. Each Microcal Software, Northampton, MA; Designer, Microg- record represents an average of 20 responses. rafx, Richardson, TX. differed between nerve branches and recording sites.

3. Results Horizontal canal HC nerve evoked field potentials were