THE JOURNAL OF COMPARATIVE NEUROLOGY 386:317–327 (1997) Use of Calcium-Binding Proteins to Map Inputs in Vestibular Nuclei of the Gerbil GOLDA ANNE KEVETTER* AND ROBERT B. LEONARD Departments of Otolaryngology, Anatomy and Neurosciences, and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555 ABSTRACT We wished to determine whether calbindin and/or calretinin are appropriate markers for vestibular afferents, a population of neurons in the vestibular nuclear complex, or cerebellar Purkinje inputs. To accomplish this goal, immunocytochemical staining was observed in gerbils after lesions of the vestibular nerve central to the ganglion, the cerebellum, or both. Eleven to fourteen days after recovery, the brain was processed for immunocytochemical identification of calretinin and calbindin. After lesion of the vestibular nerve, no calretinin staining was seen in any of the vestibular nuclei except for a population of intrinsic neurons, which showed no obvious change in number or staining pattern. Calbindin staining was reduced in all nuclei except the dorsal part of the lateral vestibular nuclei. The density of staining of each marker, measured in the magnocellular medial vestibular nucleus, was signifi- cantly reduced. After the cerebellar lesion, no differences in calretinin staining were noted. However, calbindin staining was greatly reduced in all nuclei. The density of staining, measured in the caudal medial vestibular nucleus, was significantly lower. After a combined lesion of the cerebellum and vestibular nerve, the distribution and density of calretinin staining resembled that after vestibular nerve section alone, whereas calbindin staining was no longer seen. This study demonstrates that calretinin and calbindin are effective markers for the identification of vestibular afferents. J. Comp. Neurol. 386:317–327, 1997. r 1997 Wiley-Liss, Inc. Indexing terms: calbindin; calretinin; plasticity; compensation; sensory motor Normal operations of the vestibular system result in We are investigating the staining patterns of various stabilization of head position and the visual field via marker proteins in an effort to address these issues compensatory movements of the extremities, neck, and directly. Thus far, calbindin and calretinin are two proteins eyes. For example, if a head is passively rotated to the that may prove practical and effective in the effort. Calreti- right, then vestibular reflexes result in deviation of the nin immunoreactivity in the vestibular end organs is eyes to the left and an increased tone in the extensor limited to calyx endings in the apex of the crista or to the muscles of the extremities on the right. The vestibulospi- striola (the line demarcating orientation of the kinocilium) nal and vestibuloocular responses (VSR and VOR, respec- of the macula (Desmadryl and Dechesne, 1992; Leonard tively) involve minimally a three-neuron arc. The first and Kevetter, 1995). A count of the number of stained neuron is the vestibular afferent, the second is a neuron in fibers and the inability to identify label in any bouton the vestibular nuclei, and the third is a motoneuron in the endings led these authors to suggest that calretinin immu- spinal cord or oculomotor complex (Wilson and Melvill noreactivity is limited to the population of ‘‘calyx-only’’ Jones, 1979). units in the mammalian crista (Desmadryl and Dechesne, There is a need to characterize morphologically the 1992). interactions between various vestibular inputs on vestibu- lospinal and vestibuloocular neurons. Physiological stud- ies have shown that the inputs to vestibulospinal and Grant sponsor: National Institute on Deafness and Other Communica- vestibuloocular neurons may differ (see, e.g., Goldberg et tion Disorders; Grant number: 5KO4 DC00052; Grant sponsor: Deafness Research Foundation; Grant sponsor: UTMB Small Grants Program. al., 1987; Highstein et al., 1987; Minor and Goldberg, *Correspondence to: Dr. Golda Anne Kevetter, ENT Research, 7.102 1991). This includes differences in the physiological proper- MRB, Rt. 1063, University of Texas Medical Branch, Galveston, TX ties of the vestibular afferents. Thus far, it has not been 77555-1063. E-mail: [email protected] possible to examine directly these issues at synaptic levels. Received 3 April 1996; Revised 8 April 1997; Accepted 29 April 1997 r 1997 WILEY-LISS, INC. 318 G.A. KEVETTER AND R.B. LEONARD Cerebellar Purkinje cells and their terminals also stain Nembutal (60 mg/kg, i.p.) and were perfused with 4% with some calcium-binding proteins (e.g., calbindin: Celio, paraformaldehyde and 1% picric acid in phosphate buffer. 1990; Schwab et al., 1992). Some Purkinje cells project The brain and vestibular ganglia were dissected and directly to neurons in the vestibular nuclear complex postfixed overnight (4°C) in 20–30% sucrose fixative. Then, (VNC), bypassing the cerebellar nuclei (see, e.g., Ito, 1984; 30–40 µm sections were cut with a freezing microtome, Tan et al., 1995; Umetani, 1992; Walberg and Dietrichs, collected in phosphate-buffered saline (PBS), blocked in 1988). Cerebellar input to the VNC is thought to convey, the appropriate normal serum, and processed for immuno- among others, inputs related to vision and eye movements histochemical staining of calbindin-D (1:400 mouse mono- (Chubb et al., 1984; Stone and Lisberger, 1990a,b). Eye- clonal antibody; Sigma, St. Louis, MO) or calretinin (1:2,000 related cerebellar inputs travel in pathways that originate rabbit polyclonal antibody; Chemicon, Temecula, CA). Sec- in portions of the inferior olive, the pontine nuclei, and, tions were further processed with the avidin-biotin- indirectly, the paramedian pontine reticular formation peroxidase procedures for mouse or rabbit. A black or and accessory optic system, and pretectal areas, such as brown diaminobenzidine reaction was obtained. the nucleus of the optic tract (Balaban and Henry, 1988; Sections were analyzed with light microscopy and the Bernard, 1987; Blanks, 1990; Cohen et al., 1992b; Gerrits density of staining was measured for the medial vestibular et al., 1984; Katayama and Nissimaru, 1988; Takeda and nucleus (MVN). Measurements were made in three por- Maekawa, 1989a,b; Walberg et al., 1987; Yamada and tions of the MVN: the magnocellular portion (mc), the Noda, 1987). The floccular-nodular lobe and adjacent areas parvocellular portion (pc) near the genu of the seventh also receive massive inputs from the vestibular nerve (e.g., nerve, and the caudal portion (c) by using the cytoarchitec- nodulus: Barmack et al., 1993) and vestibular nuclei tural distinctions of Gerrits (1990). We chose to concen- (Blanks, 1990; Epema et al., 1990). The brainstem- trate on this nucleus because it contains both VOR and cerebellar-oculomotor circuits have been implicated in VSR neurons (Kevetter and Coffey, 1991; Kevetter and certain types of neuronal plasticity. For example, cells in Hoffman, 1991). For a control, a section from the same the flocculus may be critical for the modification in gain of brain that was processed together with the immunohisto- the VOR due to changes in visual input (Barmack and chemically stained sections but was not exposed to the Peorossi, 1985; Broussard et al., 1992; Cohen et al., primary antibody was also measured to determine back- 1992a,b; Favilla et al., 1984; Lisberger et al., 1984; Nagao, ground staining. Image analysis was performed with 1992; Waespe and Cohen, 1983). Knowledge about the Sigma Scan analysis software (Jandel Scientific Software, interaction of visual and vestibular input in both the San Rafael, CA). For each brain, measurements of average cerebellar cortex and the vestibular nuclei is crucial for density of staining, as transmittance of light, were made of understanding sensory-motor integration in the VOR. To 184 µm2 in three sections each for the MVNmc, the test the hypothesis that calretinin is a useful marker to adjacent MVNpc, the MVNc, a nucleus with no staining study vestibular afferent input within the VNC and that (hypoglossal nucleus), and a nucleus with maximal stain- calbindin stains only Purkinje cell and primary vestibular ing (the inferior olive for calbindin and the dorsal cochlear afferents within the VNC, lesions were made of the nucleus for calretinin). In animals in which the vestibular vestibular ganglion and/or the cerebellum, and fibers were ganglion was lesioned on one side, measurements of the allowed to degenerate. nuclei on the intact side served as controls. The density of staining on each side was compared with a t-test of paired values. MATERIALS AND METHODS Successful lesion of the vestibular ganglion was verified Gerbils were anesthetized with 20 mg/kg Nembutal, i.p., in 2 days. First, the dissection was performed from the and 2.5 mg ketamine, i.m.; anesthesia was maintained lateral side of the brainstem, a procedure that normally with supplements of ketamine, if necessary. The animals leaves the ganglion attached to the brainstem. Visual were positioned in a stereotaxic frame. Surgery was per- observation confirmed that the ganglion was removed formed by using sterile instruments. successfully on one side. Second, the vestibular nerve root To generate a lesion in the cerebellum, the skull overly- was carefully analyzed for histochemical staining. Incom- ing the cerebellum was removed. Subsequently, the cerebel- plete lesions always leave some vestibular afferents that lar vermis was gently aspirated until the fourth ventricle stain positively with the antibodies. Three gerbils received