Neurotoxicologyand Teratology, Vol. 13, pp. 189-193. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Glutamate Neurotoxicity in Rat Auditory System: Cochlear Nuclear Complex L A U R A S C H W E I T Z E R , * K A R L F. JENSEN'~ A N D R A E L Y N J A N S S E N t
*Department of Anatomical Sciences and Neurobiology University of Louisville School of Medicine, Louisville, K Y 40292 ~Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 R e c e i v e d 25 M a y 1990
SCHWEITZER, L., K. F. JENSEN AND R. JANSSEN. Glutamate neurotoxicity in rat auditory system: Cochlear nuclear complex. NEUROTOXICOL TERATOL 13(2) 189-193, 1991.--In other systems such as the hypothalamus and hippocampus, it has been shown that cells postsynaptic with respect to glutamatergic inputs degenerate when exposed to large doses of glutamate ("glutamate neurotoxicity"). We have shown that large doses of glutamate administered intraperitoneally are toxic to spiral ganglion cells in the inner ear of the rat. In the present study, we have investigated whether similar levels of glutamate cause alterations in the neurons of the cochlear nuclei. Specifically, we have studied the morphology and size of the cochlear nuclear complex and its subdivisions as well as the size and density of cochlear nucleus neurons following administration of glutamate. The morphological evidence indicates that glutamate caused severe anatomical alteration of the cochlear nuclei. The changes were most pronounced in the anteroventral cochlear nucleus, especially in the neurons that receive terminals of the end bulbs of Held from the cochlear nerve. This could be a direct effect of glutamate in the cochlear nuclei or secondary to degeneration of cochlear nerve fibers in the inner ear. Auditory
Cochlear nerve
Cochlear nucleus
Degeneration
Excitotoxicity
Glutamate
Neurotoxicity
were housed individually in breeding cages, given food and water ad lib until delivery. Pups from several litters were randomized and redistributed to the nursing mothers. Litter size was kept to 8 pups (4 male, 4 female) to maintain a standard nutritive stares. From postnatal days 2 through 9, pups were given daily intraperitoneal injections of L-glutamic acid (Glu) in amounts of 4 mg/g body weight (n= 9), or an equal volume (10 p,l/g b.wt.) of physiological saline (Sal, n = 9). This regime was selected based on the excitotoxic effects of Glu (17, 23, 30, 32). On PND 1719 the rats were implanted with surface electrodes for electrophysiological studies performed on PND 22 and reported in Janssen et al. (18). The offspring remained with their mothers until PND 21 when they were weaned and housed with littermates of the same sex.
WE have recently investigated the effects of early postnatal exposure to large doses of L-glutamic acid (Glu) on the electrophysiology and anatomy of the inner ear of the developing rat (18). The observed shifts in threshold of the brainstem auditory evoked response (BAER) and a selective loss of spiral ganglion ceils in the cochlea suggests that glutamate is neurotoxic or excitotoxic at the hair cell/cochlear nerve fiber synapse. In addition, brainstemrelated waves of the BAER such as P2 which is associated with cochlear nucleus (5, 20, 28) were reduced in amplitude by 20% and increased in latency by 6% following treatment. The present study details the toxic effects of glutamate on the anatomy of the cochlear nuclei of these animals. There is considerable evidence suggesting that excitatory amino acids may serve as neurotransmitters at afferent synapses of the cochlear nuclei (1, 10, 13, 14, 25). Glutamic acid, one of these, is present and may serve as a neurotransmitter there (1-3, 8, 1315, 19, 21, 24, 26, 31, 35, 40-42). However, it has not been established that glutamate is actually a neurotransmitter used by the cochlear nerve. High doses of glutamate are toxic to neurons that receive glutamate as a neurotransmitter (33,36). If glutamate is a neurotransmitter used by cochlear nerve afferents, excess glutamate might cause degeneration of postsynaptic targets of the cochlear nerve. The present study describes the pattern of morphological alteration we have found in the cochlear nuclei following early exposure to excess L-glutamic acid.
Nissl Preparations and Quantitative Histology To determine whether cells in the cochlear nuclei atrophy or die following Glu treatment, cell types that are known to receive cochlear input were studied with Nissl stains. On postnatal day 21, 22, or 23, 9 treated pups and 9 pups in the control group were deeply anesthetized with sodium pentobarbital and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brainstems were removed and frozen sections 25 p,m thick were cut in the transverse plane through the entire cochlear nuclear complex and were stained with cresyl violet. Three measures were obtained using the Nissl-stained sections. First, the volumes of the cochlear nuclear complex and of its anteroventral, posteroventral, and dorsal subdivisions, each minus the area taken up by the nerve root, were calculated. The border
METHOD
Experimental Subjects Timed pregnancy Long-Evans hooded rats (Charles River) 189
190
SCHWEITZER, JENSEN AND JANSSEN
between the anteroventral and posteroventral subdivisions is defined as the zone of bifurcation of the incoming cochlear fibers. In transverse sections this is difficult to define. For the purpose of precision and consistency the anteroventral subdivision was defined in this study as being contained in sections rostral to the sections containing a severed cochlear nerve bundle. To aid in visualization of changes in their size and shape as a result of glutamate treatment, three-dimensional reconstructions of the cochlear nuclei from several Glu- and Sal-treated animals were made using a computerized program (PC3D, Jandel Scientific). Second, the somatic areas of five types of cells of the cochlear nuclei were measured. The circumference of five cell bodies of each type from specific locations in each animal were drawn at a 400-fold magnification. The cell types measured were 1) fusiform cells and 2) giant cells from midway along the rostral to caudal boundaries of the dorsal cochlear nucleus (where lamination of the nucleus is the most definite), 3) octopus cells from the caudal-most posteroventral cochlear nucleus, 4) the large cells adjacent to the cochlear nerve root in the caudal anteroventral cochlear nucleus and 5) the spherical bushy ceils in the rostral pole of the anteroventral cochlear nucleus. Only cells with visible nucleoli were chosen for the analysis. The cross-sectional area of each cell body was determined with a computerized planimeter. Finally, an index of cell density in the anteroventral cochlear nucleus was obtained. This was done in the rostral pole of the anteroventral cochlear nucleus where cell size and density is the most uniform. Counts were made of the number of spherical cells with visible nucleoli within a square 100 Ixm on a side. The square was placed 100 ~m from the rostral tip of the anteroventral cochlear nucleus. Counts from one section were taken from each cochlear nucleus to avoid duplicate cell counts. ,N
Statistical Analyses Somatic areas were considered separately for each cell type, as were the density indices and measured volumes for each subdivision of the cochlear nuclear complex. To assess treatment effects each measure was subjected to an analysis of variance. When significant F ratios were obtained, Scheffe's post hoc analyses with an alpha level of 0.05 were used to determine significant differences between individual means.
FIG. 1. Dorsal cochlear nuclei (DCN) of Glu-treated and control rats (PND 7) stained with the Nadler et al. (29) stain to show degeneration. (A) Degenerating fibers (arrows) that are medial to the ventral region of the dorsal cochlear nucleus. Scale = 100 ~m. (B) Higher-power photomicrograph of the same section showing the degenerating axons. The arrows mark the same location as in (A). Scale = 25 p.m. (C) Saline-injected rat, arrowheads indicate the same location as in (A) but no degeneration product is apparent. Same magnification as in (A). d: dorsal, m: medial.
Stains for Neuronal Degeneration To determine whether the cochlear nerve or cells in the cochlear nuclei undergo degenerative changes following treatment, a modified Gallyas silver stain for neuronal degeneration (29) was used. Pups from a litter of dosed (Glu) and a litter of control (Sal) animals, treated dally from postnatal day 2, were overdosed with barbiturates on postnatal days 5, 7, 9, or 22, and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Frozen sections cut in the transverse plane at a thickness of 25 p.m through the entire cochlear nuclear complex were stained and examined. The stain was differentiated to reveal either degenerating cell bodies or nerve fibers and their terminals (11,12), RESULTS
Degeneration in the Cochlear Nuclear Complex A consistent but limited pattern of degeneration was seen on postnatal days 7 and 9. Degenerating axons were restricted in each brain to an area medial to the ventral region of the dorsal cochlear nucleus (Fig. 1). Degenerating axons were observed in every dosed rat processed at these ages and was never seen in the
control rats. The degeneration product appeared the same on day 7 (on the sixth day of glutamate treatment) and on day 9 (twelve hours after the eighth and final day of glutamate treatment). No degenerating ceils nor degenerating axons were observed in either the five-day-old rats or the 22-day-old rats.
Cochlear Nuclei Volumes The volume of the cochlear nuclear complex in rats treated with L-glutamic acid was reduced by 35% (Glu-treated: 0.73 ---0.04 mm3; Sal-treated: 0.48+-0.02 mm 3) on postnatal day 22 compared to their controls, F(1,17)=34.56, p
0892-0362/91 $3.00 + .00
Glutamate Neurotoxicity in Rat Auditory System: Cochlear Nuclear Complex L A U R A S C H W E I T Z E R , * K A R L F. JENSEN'~ A N D R A E L Y N J A N S S E N t
*Department of Anatomical Sciences and Neurobiology University of Louisville School of Medicine, Louisville, K Y 40292 ~Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 R e c e i v e d 25 M a y 1990
SCHWEITZER, L., K. F. JENSEN AND R. JANSSEN. Glutamate neurotoxicity in rat auditory system: Cochlear nuclear complex. NEUROTOXICOL TERATOL 13(2) 189-193, 1991.--In other systems such as the hypothalamus and hippocampus, it has been shown that cells postsynaptic with respect to glutamatergic inputs degenerate when exposed to large doses of glutamate ("glutamate neurotoxicity"). We have shown that large doses of glutamate administered intraperitoneally are toxic to spiral ganglion cells in the inner ear of the rat. In the present study, we have investigated whether similar levels of glutamate cause alterations in the neurons of the cochlear nuclei. Specifically, we have studied the morphology and size of the cochlear nuclear complex and its subdivisions as well as the size and density of cochlear nucleus neurons following administration of glutamate. The morphological evidence indicates that glutamate caused severe anatomical alteration of the cochlear nuclei. The changes were most pronounced in the anteroventral cochlear nucleus, especially in the neurons that receive terminals of the end bulbs of Held from the cochlear nerve. This could be a direct effect of glutamate in the cochlear nuclei or secondary to degeneration of cochlear nerve fibers in the inner ear. Auditory
Cochlear nerve
Cochlear nucleus
Degeneration
Excitotoxicity
Glutamate
Neurotoxicity
were housed individually in breeding cages, given food and water ad lib until delivery. Pups from several litters were randomized and redistributed to the nursing mothers. Litter size was kept to 8 pups (4 male, 4 female) to maintain a standard nutritive stares. From postnatal days 2 through 9, pups were given daily intraperitoneal injections of L-glutamic acid (Glu) in amounts of 4 mg/g body weight (n= 9), or an equal volume (10 p,l/g b.wt.) of physiological saline (Sal, n = 9). This regime was selected based on the excitotoxic effects of Glu (17, 23, 30, 32). On PND 1719 the rats were implanted with surface electrodes for electrophysiological studies performed on PND 22 and reported in Janssen et al. (18). The offspring remained with their mothers until PND 21 when they were weaned and housed with littermates of the same sex.
WE have recently investigated the effects of early postnatal exposure to large doses of L-glutamic acid (Glu) on the electrophysiology and anatomy of the inner ear of the developing rat (18). The observed shifts in threshold of the brainstem auditory evoked response (BAER) and a selective loss of spiral ganglion ceils in the cochlea suggests that glutamate is neurotoxic or excitotoxic at the hair cell/cochlear nerve fiber synapse. In addition, brainstemrelated waves of the BAER such as P2 which is associated with cochlear nucleus (5, 20, 28) were reduced in amplitude by 20% and increased in latency by 6% following treatment. The present study details the toxic effects of glutamate on the anatomy of the cochlear nuclei of these animals. There is considerable evidence suggesting that excitatory amino acids may serve as neurotransmitters at afferent synapses of the cochlear nuclei (1, 10, 13, 14, 25). Glutamic acid, one of these, is present and may serve as a neurotransmitter there (1-3, 8, 1315, 19, 21, 24, 26, 31, 35, 40-42). However, it has not been established that glutamate is actually a neurotransmitter used by the cochlear nerve. High doses of glutamate are toxic to neurons that receive glutamate as a neurotransmitter (33,36). If glutamate is a neurotransmitter used by cochlear nerve afferents, excess glutamate might cause degeneration of postsynaptic targets of the cochlear nerve. The present study describes the pattern of morphological alteration we have found in the cochlear nuclei following early exposure to excess L-glutamic acid.
Nissl Preparations and Quantitative Histology To determine whether cells in the cochlear nuclei atrophy or die following Glu treatment, cell types that are known to receive cochlear input were studied with Nissl stains. On postnatal day 21, 22, or 23, 9 treated pups and 9 pups in the control group were deeply anesthetized with sodium pentobarbital and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brainstems were removed and frozen sections 25 p,m thick were cut in the transverse plane through the entire cochlear nuclear complex and were stained with cresyl violet. Three measures were obtained using the Nissl-stained sections. First, the volumes of the cochlear nuclear complex and of its anteroventral, posteroventral, and dorsal subdivisions, each minus the area taken up by the nerve root, were calculated. The border
METHOD
Experimental Subjects Timed pregnancy Long-Evans hooded rats (Charles River) 189
190
SCHWEITZER, JENSEN AND JANSSEN
between the anteroventral and posteroventral subdivisions is defined as the zone of bifurcation of the incoming cochlear fibers. In transverse sections this is difficult to define. For the purpose of precision and consistency the anteroventral subdivision was defined in this study as being contained in sections rostral to the sections containing a severed cochlear nerve bundle. To aid in visualization of changes in their size and shape as a result of glutamate treatment, three-dimensional reconstructions of the cochlear nuclei from several Glu- and Sal-treated animals were made using a computerized program (PC3D, Jandel Scientific). Second, the somatic areas of five types of cells of the cochlear nuclei were measured. The circumference of five cell bodies of each type from specific locations in each animal were drawn at a 400-fold magnification. The cell types measured were 1) fusiform cells and 2) giant cells from midway along the rostral to caudal boundaries of the dorsal cochlear nucleus (where lamination of the nucleus is the most definite), 3) octopus cells from the caudal-most posteroventral cochlear nucleus, 4) the large cells adjacent to the cochlear nerve root in the caudal anteroventral cochlear nucleus and 5) the spherical bushy ceils in the rostral pole of the anteroventral cochlear nucleus. Only cells with visible nucleoli were chosen for the analysis. The cross-sectional area of each cell body was determined with a computerized planimeter. Finally, an index of cell density in the anteroventral cochlear nucleus was obtained. This was done in the rostral pole of the anteroventral cochlear nucleus where cell size and density is the most uniform. Counts were made of the number of spherical cells with visible nucleoli within a square 100 Ixm on a side. The square was placed 100 ~m from the rostral tip of the anteroventral cochlear nucleus. Counts from one section were taken from each cochlear nucleus to avoid duplicate cell counts. ,N
Statistical Analyses Somatic areas were considered separately for each cell type, as were the density indices and measured volumes for each subdivision of the cochlear nuclear complex. To assess treatment effects each measure was subjected to an analysis of variance. When significant F ratios were obtained, Scheffe's post hoc analyses with an alpha level of 0.05 were used to determine significant differences between individual means.
FIG. 1. Dorsal cochlear nuclei (DCN) of Glu-treated and control rats (PND 7) stained with the Nadler et al. (29) stain to show degeneration. (A) Degenerating fibers (arrows) that are medial to the ventral region of the dorsal cochlear nucleus. Scale = 100 ~m. (B) Higher-power photomicrograph of the same section showing the degenerating axons. The arrows mark the same location as in (A). Scale = 25 p.m. (C) Saline-injected rat, arrowheads indicate the same location as in (A) but no degeneration product is apparent. Same magnification as in (A). d: dorsal, m: medial.
Stains for Neuronal Degeneration To determine whether the cochlear nerve or cells in the cochlear nuclei undergo degenerative changes following treatment, a modified Gallyas silver stain for neuronal degeneration (29) was used. Pups from a litter of dosed (Glu) and a litter of control (Sal) animals, treated dally from postnatal day 2, were overdosed with barbiturates on postnatal days 5, 7, 9, or 22, and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Frozen sections cut in the transverse plane at a thickness of 25 p.m through the entire cochlear nuclear complex were stained and examined. The stain was differentiated to reveal either degenerating cell bodies or nerve fibers and their terminals (11,12), RESULTS
Degeneration in the Cochlear Nuclear Complex A consistent but limited pattern of degeneration was seen on postnatal days 7 and 9. Degenerating axons were restricted in each brain to an area medial to the ventral region of the dorsal cochlear nucleus (Fig. 1). Degenerating axons were observed in every dosed rat processed at these ages and was never seen in the
control rats. The degeneration product appeared the same on day 7 (on the sixth day of glutamate treatment) and on day 9 (twelve hours after the eighth and final day of glutamate treatment). No degenerating ceils nor degenerating axons were observed in either the five-day-old rats or the 22-day-old rats.
Cochlear Nuclei Volumes The volume of the cochlear nuclear complex in rats treated with L-glutamic acid was reduced by 35% (Glu-treated: 0.73 ---0.04 mm3; Sal-treated: 0.48+-0.02 mm 3) on postnatal day 22 compared to their controls, F(1,17)=34.56, p
