Breed differences in deafferentation-induced neuronal cell death and shrinkage in chick cochlear nucleus

Hearing Research 127 (1999) 62^76

Breed di¡erences in dea¡erentation-induced neuronal cell death and shrinkage in chick cochlear nucleus Joseph L. Edmonds Jr. a , Larry A. Hoover a , Dianne Durham b

a;b;

*

a Department of Otolaryngology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7380, USA Ralph L. Smith Mental Retardation Research Center, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7380, USA

Received 4 November 1996; received in revised form 12 September 1998; accepted 21 September 1998

Abstract Removal of functional presynaptic input can result in a variety of changes in postsynaptic neurons in the central nervous system, including altered metabolism, changes in neuronal cell size, and even death of the postsynaptic cell. Age-dependent neuronal cell death and shrinkage has been documented in second order auditory neurons in the chick brainstem (nucleus magnocellularis, NM) following cochlea removal (Born and Rubel, 1985. J. Comp. Neurol. 231, 435^445). Here we examined whether the extent of neuronal cell death and shrinkage is also breed-dependent. We performed unilateral cochlea removal on both hatchling and adult birds of either a broiler breed (Arbor Acres Cross) or egg layer breed (Hy-Line, H and N) and killed birds one week later. Changes in neuronal cell number and cross sectional area were determined from Nissl-stained sections. We observed 25% neuronal cell loss and a 15^20% decrease in neuronal cross sectional area after cochlea removal in either broiler or egg layer hatchling birds. In adult birds, however, neuronal cell loss is breed-dependent. Adult egg layer birds lose an average of 37% of NM neurons after cochlea removal, while adult broiler birds show no cell loss. In both breeds of adult birds, cochlea removal results in a 20% decrease in neuronal cross sectional area. These results suggest that analysis of differences between breeds as well as ages of birds will prove fruitful in determining how afferent input controls neuronal survival and metabolism. z 1999 Elsevier Science B.V. All rights reserved. Key words: Auditory; Cochlea removal; Avian; Nucleus magnocellularis

1. Introduction A¡erent input plays an important role in both the development and the maintenance of central nervous system (CNS) pathways (Shatz, 1990; Globus, 1975 ; Walker et al., 1975; Armstrong and Montminy, 1993 ; Bornstein, 1989). Although the consequences of a¡erent manipulation are well known, the mechanisms by which presynaptic input controls postsynaptic neurons are not as completely known. Although increasing evidence suggests that reorganization of CNS connections can occur in adult animals (Kaas, 1991; Recanzone et al., 1993 ; Rausell et al., 1992), in most systems a period of greatest susceptibility to insult occurs early in develop-

* Corresponding author. Tel.: +1 (913) 588-6731; Fax: +1 (913) 588-6708; E-mail: [email protected]

ment, when synaptic connections critical to neuronal survival are becoming established (Durham and Woolsey, 1984 ; Farbman et al., 1988; Brunjes and Borror, 1983 ; Moore, 1990; Hashisaki and Rubel, 1989). Neurons in the central auditory system provide an accessible location in which to study presynaptic control of neuronal survival and metabolism. In both mammalian and avian species, alteration of cochlear input via the eighth nerve can be accomplished easily, and the resulting neuronal changes are more pronounced in the developmentally immature. The most extensively studied example of these changes in the auditory system is second-order neurons in nucleus magnocellularis (NM) (Rubel et al., 1990). NM neurons, homologous to large spherical cells in the mammalian anteroventral cochlear nucleus, receive their only excitatory input from the ipsilateral cochlea via the eighth nerve (Boord, 1969 ; Parks and Rubel,

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1978). Removal of the cochlea results in a constellation of rapid metabolic changes in NM neurons, including decreases in glucose use (Lippe et al., 1980; Heil and Scheich, 1986 ; Born et al., 1991) and protein synthesis (Steward and Rubel, 1985; Garden et al., 1994; Garden et al., 1991) accompanied by increases in oxidative capacity (Durham and Rubel, 1985; Hyde and Durham, 1990 ; Durham et al., 1993; Hyde and Durham, 1994b) and glial components (Rubel and MacDonald, 1992 ; Lurie and Rubel, 1994; Canady and Rubel, 1992). Within several days, 25^40% of NM neurons will die and the remainder will shrink in size (Born and Rubel, 1985 ; Hyde and Durham, 1990). The response of NM neurons to cochlea removal is also age-dependent. When cochlea removal is performed in adult animals, little neuronal death and few other metabolic changes are observed (Born and Rubel, 1985; Steward and Rubel, 1985 ; Durham and Rubel, 1985; Hyde and Durham, 1990). Recent observations have suggested that the age difference in the response of NM neurons to dea¡erentation may be breed-dependent. In a study designed to examine short-term changes in protein synthesis after cochlea removal in adult birds, decreases in [3 H]leucine uptake and incorporation reminiscent of those seen in hatchlings were observed (Debel, Durham and Rubel, unpublished observations). These results di¡ered from previously published work in adults, in which no changes in [3 H]leucine incorporation were observed in birds treated similarly (Steward and Rubel, 1985). The two groups of adult birds were, however, of di¡erent breeds. Two types of birds are routinely available from commercial suppliers. Broiler birds (Arbor Acres Cross, Hubbards, etc.) have been developed by breeders to grow quickly in size and muscle mass and are sold for meat production. In contrast, egg layer birds (H and N, Hy-Line, White Leghorn, Cornish) are bred to reach a smaller adult size and are used by commercial suppliers primarily to obtain eggs. Birds used in the published [3 H]leucine study, in which NM neurons in adults showed little response to cochlea removal, were of a broiler breed. The more recent unpublished results, in which rapid decreases in [3 H]leucine label were observed in adults, occurred in egg layer birds. The purpose of the experiments described here was to determine whether breed di¡erences do exist in the response of adult birds to cochlea removal. The rapid changes in protein synthesis described above have been correlated with neuronal cell death in NM neurons (Steward and Rubel, 1985; Rubel et al., 1991), and a number of investigators have studied mechanisms of cell death in NM (e.g. Rubel et al., 1990; Garden et al., 1994) Therefore, we used neuronal cell death and shrinkage as the dependent measures in these experiments. Preliminary results have been reported previously in abstract form (Edmonds et al., 1993).

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2. Materials and methods All hatchling birds (either sex) were received from commercial suppliers at two days of age and housed in communal brooders with free access to food and water until the time of surgery. Con Agra Hatchery (Batesville, AR) supplied broiler birds of the Peterson/ Arbor Acres cross strain ; Marty's Hatchery (Phoenix, AZ) supplied egg layer birds (H and N strain). Adult birds were all soon-to-be-retired breeding hens obtained at 66 weeks of age from commercial suppliers. Broiler adults were Arbor Acres hens used as breeders at the Con Agra Hatchery. Egg layer adults were either the Hy-Line breed (Cal-Maine Foods, Hutchinson, KS) or the H and N breed (H and N International, Redmond, WA). Adults were housed communally with free access to food and water until the time of surgery. A total of 20 adult and 19 hatchling birds provided data for this study. The care and use of animals reported here was approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee (IACUC). 2.1. Surgery and histology Hatchling birds underwent cochlea removal at 2 or 3 weeks of age, and adults underwent surgery at 67 weeks of age. Cochlea removal was performed as described previously (Durham and Rubel, 1985). Brie£y, animals ¢rst were anesthetized with Nembutal (18.85 mg/kg, i.p.) and Vetalar (80 mg/kg, i.m.) The area surrounding the external ear was £ooded with lidocaine and a small incision was made in the external ear. The tympanic membrane was then punctured, followed by removal of the middle ear bone (columella). The cochlea was removed through the oval window with ¢ne forceps and examined to insure completeness of the procedure. Any bleeding was controlled with thermal cautery. The external closure was accomplished by applying cyanoacrylate glue. After a survival period of 7 days, the animals were again deeply anesthetized with Nembutal and transcardially perfused with saline followed by 10% phosphate bu¡ered formalin (0.1 M, pH 7.4). The brains were stored at 4³C for an additional 7 days, soaking in fresh ¢x. The brains then were dehydrated through graded alcohols to xylene and embedded in para¤n. Coronal sections were cut at either 8 or 10 Wm and a one in four series of sections was mounted on slides, stained with thionin, dehydrated to xylene, and coverslipped with DPX. 2.2. Quantitative analyses 2.2.1. Neuronal counts Neuronal number was estimated by counting nucle-

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olar pro¢les in a one-in-four series of thionin-stained sections throughout the rostral to caudal extent of NM. NM neurons were examined under bright¢eld illumination with a 40U planapochromatic objective. Only those neurons with a visible nucleolus surrounded by a clear nuclear area and stained cytoplasm were counted (Born and Rubel, 1985). Total neuronal number was then estimated by multiplying the summed counts by four. Counts were done by two di¡erent investigators, and all slides to be counted were `blinded' by a third individual not involved in the experiment. Several brains were counted by both investigators and in each case the counts were within 4% of each other. In each brain, neurons were counted in both ipsilateral and contralateral NM. Systematic di¡erences in nucleolar size are a potential source of bias in the pro¢le counts we used to determine neuronal cell number (Abercrombie, 1946; Konigsmark, 1970 ; Coggeshall and Lekan, 1996). To insure that such di¡erences did not a¡ect our results, we made measurements of nucleolar diameter in 2 animals undergoing cochlea removal from each age and breed (total of 8 animals). In each of these animals, a section located at 50% of the rostral to caudal extent of NM was examined under bright¢eld illumination with a 100U oil immersion objective. The image was captured by a Dage-MTI CCD72 video camera mounted onto the microscope and sent to a Macintosh IIci computer equipped with a frame grabber board. The digitized images were evaluated using the NIH image program. We traced the diameters of nucleoli in 45^80 NM neurons on each side of the brain on a digitizing pad. In neurons with two nucleoli, the diameter was measured between their centers, as suggested by Clarke and Oppenheim (1995). All measurements were made by one investigator blind to the identity of the tissue. Mean nucleolar diameter then was compared among animals. 2.2.2. Neuronal cell area Neuronal cross sectional area was measured in one section from each bird at a point 50% of the rostral to caudal extent of NM. Neuronal cell area measurements were made in both ipsilateral and contralateral NM using a 60U oil immersion objective and the image analysis system described above. The outline of each neuron at its largest point was traced on a digitizing tablet and the cross sectional area determined by NIH Image. Beginning at the lateral edge of the nucleus, only those neurons with a visible nucleolus surrounded by a clear nucleus and stained cytoplasm were measured. An average of 50 neurons were measured on each side of the brain in each animal. As with the neuronal cell counts, cell size measurements were done by one of two investigators `blind' to the identity of the subject. Four brains were measured by both investigators and the area measurements were within 5% of each other.

2.2.3. Data analysis Each NM receives input only from the ipsilateral eighth nerve. Therefore, neurons in NM contralateral to cochlea removal can serve as a within-animal control for both neuronal number and cell area measurements. Additional controls were provided by age- and breedmatched unoperated animals. Di¡erences in absolute neuronal number among animals of all ages and treatments were evaluated ¢rst with a one way ANOVA for both ipsilateral and contralateral NM. Post-hoc pairwise comparisons were made with the Fisher PLSD (protected least squares di¡erence) test. In addition, for each animal a percent neuronal loss value was calculated by the formula [13(ipsilateral NM number/contralateral NM number)]U100. These derived ratio data were evaluated using a nonparametric overall analysis (Kruskal-Wallis) followed by post-hoc pairwise comparisons made with Mann-Whitney U-tests. It is di¤cult to evaluate absolute di¡erences in neuronal size between animals because the amount of tissue shrinkage that occurs during para¤n embedding can vary signi¢cantly from brain to brain. Therefore, we evaluated the e¡ects of cochlea removal on neuronal size by calculating the ipsilateral vs. contralateral percent change in cross sectional area. For each animal, average cross sectional area measurements for each side of the brain were used to determine the percent area change: [(13(average ipsilateral area/average contralateral area)]U100. These derived percent change values were compared among all treatment groups with a Kruskal-Wallis test followed by post-hoc pairwise Mann-Whitney U comparisons as described above for percent neuronal loss. 3. Results 3.1. Neuronal number The amount of neuronal cell loss in NM resulting from cochlea removal depends both on the breed of the animal and the age at the time of dea¡erentation. Fig. 1 shows photographs of NM neurons from broiler birds undergoing cochlea removal. In hatchling birds (C,D) neuronal cell loss is evident in ipsilateral compared to contralateral NM. However, in adult birds (A,B), no cell loss is apparent. These results are similar to those obtained previously in broiler birds (Born and Rubel, 1985). In Fig. 2, which shows NM neurons from egg layer birds, neuronal cell loss is apparent in ipsilateral NM in both hatchlings (C,D) and in adult birds (A,B,). Thus, only broiler birds display age-dependent di¡erences in the amount of neuronal cell loss after cochlea removal, at least at the ages we have examined. Table 1 gives the results of neuronal counts in NM for all eight groups of birds. Data are presented for

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Fig. 1. Photomicrographs of NM neurons from adult (A,B) and hatchling (C,D) broiler birds killed 1 week after unilateral cochlea removal. Ipsilateral neurons are shown in (A) and (C); contralateral neurons from the same animals are shown in (B) and (D). Neuronal cell loss is evident in ipsilateral NM of the hatchling but not the adult bird. Scale bar in (D) is 250 Wm.

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Fig. 2. Photomicrographs of NM neurons from an adult (A,B) and a hatchling (C,D) egg layer bird killed 1 week after unilateral cochlea removal. Ipsilateral neurons are shown in (A) and (C); contralateral neurons from the same animals are shown in (B) and (D). At both ages cell loss is evident in ipsilateral NM. Scale bar in (D) is 250 Wm.

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Table 1 Neuronal cell loss and shrinkage after cochlea removal Treatment Adult birds Broiler (3, 4) control Broiler (3, 3) dea¡erented Egg layer (6, 6) control Egg layer (5, 7) dea¡erented Hatchling birds Broiler (3, 3) control Broiler (4, 7) dea¡erented Egg layer (4, 3) control Egg layer (4, 4) dea¡erented

Ipsilateral cell number

Contralateral cell number

% Cell number decrease

% Cell area decrease

4821 (402) 4365 (839) 3885 (307) 2654 (421)

5073 (560) 4440 (796) 3910 (332) 4213 (290)

4.33 (2.33) 1.33 (6.96) 0.33 (1.05) 37.0 (8.33)

0.0 (2.49) 20.0 (6.08) 32.17 (2.1) 28.8 (2.29)

3152 (71) 2752 (170) 3653 (107) 2688 (278)

3112 (232) 3677 (295) 3779 (184) 3752 (332)

32.33 (5.21) 24.8 (3.45) 3.25 (3.09) 28.5 (1.55)

2.04 (3.12) 16.0 (4.34) 1.07 (5.20) 20.5 (5.16)

Values in parentheses in the lefthand column indicate the number of animals included in the analyses (neuronal cell number, neuronal cell size). Values in parentheses for all other columns indicate S.E.M.

four di¡erent variables, including absolute cell number on each side of the brain, percent cell loss (dea¡erented compared to control in each animal), and percent cell size di¡erence (dea¡erented compared to control in each animal). A between-group one way ANOVA showed no e¡ect of Group for absolute neuron number on the left (unoperated) side of the brain [F(7,24) = 1.87; P = 0.12]. For the dea¡erented side of the brain, we did observe with ANOVA a main e¡ect of Group [F(7,24) = 4.52 ; P = 0.0025)]. In post-hoc tests, only adult egg layer birds showed reliable di¡erences in Table 2 Nucleolar size measurements Group Adult birds Broiler dea¡erented

Egg layer dea¡erented

Hatchling birds Broiler dea¡erented

Egg layer dea¡erented

Ipsilateral nucleolar size

Contralateral nucleolar size

2.59 (0.56) 2.52 (0.53) 2.60 (0.51) 2.88 (0.64)

2.49 (0.53) 2.54 (0.42) 2.64 (0.70) 3.03 (0.60)

2.72 (0.56) 2.53 (0.41) 2.64 (0.68) 2.53 (0.46)

2.79 (0.56) 2.76 (0.56) 2.68 (0.52) 2.49 (0.58)

Mean nucleolar diameter in Wm measured from a section located at 50% of the rostral to caudal extent of NM. Each value is the mean (S.D.) from 45^70 NM neurons in one animal.

absolute number comparing operated animals to unoperated controls (Fisher PLSD, P 6 0.05). Both hatchling groups showed di¡erences in cell number that approached but did not meet signi¢cance, suggesting that we did not have su¤cient numbers of animals to demonstrate di¡erences based on absolute cell number. To determine whether systematic di¡erences in nucleolar size might contribute to the di¡erences in neuronal cell number we observed, we evaluated nucleolar diameter in dea¡erented animals of each breed and age. Our measurements, summarized in Table 2, show that deafferentation does not change nucleolar size in any group

Fig. 3. Graph showing average percent neuronal cell loss for both breeds and ages of birds. Note similar loss for either age of egg layer bird. For broilers, only hatchlings demonstrate neuronal cell loss. All but broiler adults show reliably di¡erent cell loss compared to age- and breed-matched unoperated control birds (Fisher PLSD, P 6 0.05).

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Fig. 4. Photomicrographs of NM neurons from an adult (A,B) and a hatchling (C,D) broiler bird killed one week after unilateral cochlea removal. Ipsilateral neurons are shown in (A) and (C); contralateral neurons from the same animals are shown in (B) and (D). For both ages decreases in cross sectional area are evident in ipsilateral NM. Scale bar in (D) is 50 Wm.

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Fig. 5. Photomicrographs of NM neurons from an adult (A,B) and a hatchling (C,D) egg layer bird killed one week after unilateral cochlea removal. Ipsilateral neurons are shown in (A) and (C); contralateral neurons from the same animals are shown in (B) and (D). For both ages decreases in cross sectional area evident in ipsilateral NM. Apparent age-related di¡erences in absolute cross sectional area, (A) vs. (C), are likely to be an artifact of tissue processing. Scale bar in D is 50 Wm.

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(ipsilateral vs. contralateral measurements), nor are there di¡erences in nucleolar size as a function of age or breed. Thus, we employed no correction factors in our estimates of neuronal cell number from pro¢le counts. We also evaluated neuronal cell death among di¡erent groups by calculating a percent cell loss in each animal, comparing neuronal number on the unoperated to that on the operated side. We evaluated these ratio data using nonparametric statistics (Kruskal-Wallis tests for overall di¡erences; Mann-Whitney U pairwise post-hoc tests). We observed an overall e¡ect of Group using a between-group analysis of variance (H = 22.92 ; P = 0.0018). For both hatchling groups, operated animals showed reliably more cell loss than unoperated controls (egg layers, P = 0.02; broilers, P = 0.03). The amount of cell loss was not reliably di¡erent between the two hatchling breeds (P = 0.39). For adults, only egg layer birds showed reliable neuronal cell loss (egg layers, P = 0.006; broilers, P = 0.51). For egg layer birds, the magnitude of cell loss in adults was not di¡erent from that seen in hatchlings (P = 0.14). A summary of all the neuronal cell number data is presented graphically in Fig. 3. Both groups of hatchling birds showed similar amounts of neuronal cell loss. In adults, however, only egg layer birds showed neuronal cell loss; in broiler adults, cochlea removal does not induce neuronal cell death. 3.2. Neuronal size Unlike neuronal cell death, the amount of neuronal shrinkage caused by cochlea removal does not depend on either age or breed. In all animals, cochlea removal causes an 16^29% decrease in cross sectional area. Fig. 4 shows photographs of NM neurons from broiler birds undergoing cochlea removal. At both ages, neuronal shrinkage is evident in ipsilateral compared to contralateral NM. These results are di¡erent from those obtained previously in broiler birds (Born and Rubel, 1985), in that we observed shrinkage in adult animals while the previous study did not. In Fig. 5, which shows NM neurons from egg layer birds, neuronal shrinkage is apparent in ipsilateral NM in both hatchlings (C,D) and in adult birds (A,B,). Thus, all birds display neuronal cell shrinkage after cochlea removal, at all ages examined. Neuronal cell shrinkage was evaluated using nonparametric statistics. A between-group Kruskal-Wallis analysis of variance on percent di¡erence in cross sectional area showed a reliable e¡ect of Group (H = 28.38; P = 0.0002). Post-hoc pairwise comparisons showed that for all groups, animals undergoing cochlea removal demonstrated reliable shrinkage when compared to unoperated control animals (Mann-Whitney U, P 6 0.05). The magnitude of neuronal shrinkage

Fig. 6. Graph showing average percent decrease in neuronal cross sectional area for both breeds and ages of birds. All groups of birds showed decreases in cross sectional area that were not reliably different in magnitude among any group (Fisher PLSD, P s 0.05). All groups show reliably di¡erent percent area loss compared to ageand breed-matched unoperated control birds (Fisher PLSD, P 6 .05).

did not di¡er between any groups examined (MannWhitney U, P s 0.05). In Fig. 6 the average percent neuronal shrinkage is shown for the four groups of birds. As con¢rmed by the statistical analysis, all groups demonstrated signi¢cant neuronal shrinkage. 4. Discussion Our primary results are, ¢rst, that neither the amount of neuronal cell death nor the degree of soma shrinkage that accompanies cochlea removal varies with breed in hatchling birds. However, dea¡erentation-induced neuronal cell death in adult birds depends on breed. Our results agree with published data (Born and Rubel, 1985) that NM neurons in adult broiler birds do not die after cochlea removal, while those in adult egg layer birds die in numbers equivalent to that seen in hatchlings. In addition, cochlea removal causes a 16^29% amount of neuronal soma shrinkage at all ages and in all breeds of birds. After consideration of some methodological issues, discussion of possible mechanisms underlying the manner in which NM neurons of di¡erent breeds respond to dea¡erentation will be separated into two broad areas ^ di¡erences in the presynaptic signals that might occur with cochlea removal, and di¡erences in the response properties of the postsynaptic neurons (Rubel et al., 1990). 4.1. Methodological considerations Our results in broiler birds di¡er somewhat from

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those reported previously by Born and Rubel (1985). They observed neuronal soma shrinkage only in hatchling birds, while we report neuronal soma shrinkage in adult birds indistinguishable from that seen in hatchlings. We can o¡er no obvious explanations for the discrepancy. They measured soma size at two di¡erent locations within NM (33% and 66% of the rostral to caudal extent of NM) while our measurements were made at only one (50%). However, additional measurements should make di¡erences more, not less, likely to be observed. Our results are more consistent with ¢ndings in mammalian auditory system. Cochlear ablation elicits cell shrinkage in gerbil (Pasic and Rubel, 1989 ; Pasic et al., 1994 ; Hashisaki and Rubel, 1989), mouse (Trune, 1982), rat (Coleman and O'Connor, 1979), ferret (Moore and Kowalchuk, 1988; Moore, 1990) and cat (Powell and Erulkar, 1962; Jean-Baptiste and Morest, 1975), even in the absence of neuronal cell death. Unlike Born and Rubel, however, we were unable to demonstrate reliable decreases in absolute neuronal number in ipsilateral NM following cochlea removal. This result likely re£ects relatively low animal numbers. Percent changes in number were robust. In other neuronal systems more dramatic responses to dea¡erentation in young animals can often be linked with the immaturity of neuronal connections at the time of the lesion (Durham and Woolsey, 1984; LeVay et al., 1980). As summarized in Born and Rubel (1985), this explanation is unlikely here, as the avian auditory system is well developed by several weeks of age (reviewed in Rubel and Parks, 1988). In addition, both the broiler and egg layer adults we used are considered by breeders to be at the end of their `useful' reproductive age, when egg production slows and few developmental changes are likely to occur in the CNS. However, the designation of these birds as adults is an operational one adopted by commercial poultry breeders for economic reasons. The normal `free range' lifespan of either of these commercial breeds is not known. If broiler birds have a shorter `free range' lifespan than egg layers, then comparisons between the two breeds at 66 weeks of age would not represent an age-matched evaluation. Determining the amount of neuronal cell death caused by cochlea removal in 2 or 3 year old egg layer birds would address this possibility directly. 4.2. Presynaptic signals Substantial evidence suggests that the presynaptic signal responsible for neuronal cell death following cochlea removal is interruption of eighth nerve activity. In both chick and mammal, reversible blockade of action potentials with tetrodotoxin (TTX) causes neuronal cell death (Born and Rubel, 1988; Rubel et al., 1991 ; Szewczyk and Durham, 1997), soma shrinkage (Sie and Rubel, 1992; Born and Rubel, 1988; Pasic

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and Rubel, 1989 ; Pasic and Rubel, 1991), oxidative upregulation (Robb et al., 1988; Rubel et al., 1991), glial responses (Canady and Rubel, 1992), and decreases in ribosomes and protein synthesis (Rubel et al., 1991 ; Sie and Rubel, 1992 ; Born and Rubel, 1988) indistinguishable from that seen with cochlear ablation. Di¡erences in the way adult and hatchling birds respond to cochlea removal could be explained by an unequal decrease in eighth nerve activity at the two ages. Born and colleagues investigated this question by measuring both electrophysiological activity and glucose uptake after cochlea removal at the two ages and found equivalent decreases in both parameters at both ages (Born et al., 1991). Unfortunately, the adult birds used in that study were egg layers, which we now know to undergo neuronal cell death. The interpretation of several other studies using adult egg layer birds to examine age-related di¡erences in the response to cochlea removal should be reevaluated (Hyde and Durham, 1990 ; Canady et al., 1994). Studies in the slice preparation have shown in young birds that presynaptic release of a trophic substance governs the decreases in protein synthesis that precede neuronal death in NM (Hyson and Rubel, 1989, 1995). The neurotransmitter at the eighth nerve synapse is glutamate, most likely acting at AMPA/kainate receptors (Sivaramakrishnan and Laurent, 1995 ; Zhou and Parks, 1992 ; Otis et al., 1995). It is possible that changes occur with age in the characteristics of glutamate release by eighth nerve terminals onto NM neurons. In hatchlings, approximately 70% of the surface of NM neurons are covered by eighth nerve excitatory synapses (Parks, 1981), but neither age- nor breed-related di¡erences in a¡erent morphology have been examined. The other major source of a¡erent innervation of NM neurons is GABAergic synapses (Code et al., 1989 ; von Bartheld et al., 1989 ; Code and Churchill, 1990), which arise from neurons in the superior olivary complex (Lachica et al., 1994). Unlike other GABAergic synapses, those in chick NM act to depolarize NM neurons (Hyson et al., 1995) and may play a role in a¡erent regulation of these cells. Again, although developmental changes have been reported in GABA terminals and receptors (Code et al., 1989; Code and Churchill, 1990), no attempt has been made to examine changes in GABA terminals in adult birds of di¡erent breeds. In contrast to the abrupt decrease in a¡erent activity caused by cochlea removal, a more graded decrease in a¡erent input might result from gradual loss of cochlear hair cells or ganglion cells that accompany aging. Such deterioration of cochlear hair cells or ganglion cells has been reported in several mammalian species (Keithley and Feldman, 1979, 1982 ; Henry, 1982 ; Willott et al., 1994, 1998; Johnsson and Hawkins, 1972) and in quail (Ryals and Westbrook, 1988). Removal of a cochlea

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with signi¢cant hair cell loss may well have a di¡erent e¡ect on cochlear nucleus neurons that removing an anatomically normal cochlea. Recent work in our laboratory suggests that in commercially raised adult birds, cochleae of both broiler and egg layer birds do show evidence of hair cell damage, particularly in the basal, high frequency region (D. Durham, D.L. Park, D.A. Girod, unpublished observations). In general, this preliminary scanning electron microscopic analysis of the sensory epithelium suggests that broiler adults are more likely to show cochlear damage and that the extent of damage is more severe than in egg layer adults. However, more detailed knowledge of the prevalence, time course and functional consequences of this adult cochlear damage will be necessary before we can discern its relevance to the age and breed-dependent NM response to cochlea removal. Elegant work by Willott and colleagues (Willott et al., 1994; Erway et al., 1993) has shown that several commercially available mouse strains (C57BL/6J, DBA/2J and BALB/c) all show signi¢cant loss of hair cells and ganglion cells in the basal cochlea with age. However, in only two of the three strains is any neuronal cell death seen in AVCN (Willott et al., 1998), and neuronal cell death in AVCN following cochlear damage in young animals is the exception rather than the rule (reviewed in Willott et al., 1994). In neither the present study nor previous work by Born and Rubel (1985) did we observe any age-related decline in NM number in unoperated animals. If anything, counts of absolute neuronal number in the present study suggest that in broiler birds NM neurons are more numerous in adults than in hatchling birds (Table 1, control animals). It is possible that new NM neurons are being added throughout life in broiler birds, as is known to occur in some parts of the nervous system (Alvarez-Buylla et al., 1994; Altman and Das, 1965 ; Calof et al., 1996). However, no evidence for mitotic activity among NM neurons has been observed in unoperated birds (Lurie and Rubel, 1994). 4.3. Postsynaptic responses to cochlea removal The second broad category of potential explanations for age or breed di¡erences in the response to cochlea removal are characteristics of the postsynaptic NM neuron. Both developmental and pathological cell death are areas of intense research and provide a framework in which to examine neuronal cell death in NM (Ellis et al., 1991 ; Clarke, 1990; Richter and Kass, 1991 ; Oppenheim, 1991 ; Pittman et al., 1994; Johnson and Deckwerth, 1993). Many aspects of development and normal metabolism are under hormonal control, including neuronal cell death and remodeling. Alterations in neuronal form and number occur in response to changing levels of ecdysone during metamorphosis (Truman and Reiss,

1995), thyroxin (Samuels et al., 1988; Dussault and Ruel, 1987; Sperry and Grobstein, 1985), and testosterone (Suga et al., 1987 ; Nottebohm et al., 1986; Nottebohm, 1980). The two breeds of birds we examined are very di¡erent with respect to ultimate body weight and developmental growth rate; it is not di¤cult to imagine that there may be di¡erences in the levels of circulating systemic hormones during development. Discussions with the breeders indicate that exogenous hormones are not administered as part of normal animal husbandry; however, genetically controlled endogenous hormonal di¡erences could also be playing a role. The e¡ects of hormones on neuronal form and function can be modi¢ed by dea¡erentation (Nottebohm, 1980; Bottjer et al., 1986), suggesting some interplay between the two control mechanisms. Genetic control of developmental or programmed cell death has received considerable attention (Pittman et al., 1994 ; Steller, 1995). Characterization of families of neurotrophic factors (Thoenen, 1991 ; Barde, 1989; Qin-Wei et al., 1994 ; Bonhoe¡er, 1996 ; Johnson et al., 1997), as well as the description of a set of genes and gene products that control apoptotic cell death (Yang and Korsmeyer, 1996; Ellis et al., 1991; Cohen, 1997), have allowed a more detailed evaluation of the cell death process. The distinction between this apoptotic cell death and `necrotic' cell death induced by damage to the nervous system has become more blurred in recent years as the same insult has been shown to result in both types of death (Choi, 1996; Pittman et al., 1994). A potential relationship between developmental and dea¡erentation-induced death in NM raises an intriguing possibility in NM. During embryogenesis, neurons that form NM are born at about 60 hours of incubation; between 11 and 13 days of incubation, about 18% of NM neurons in Red Cornish (broiler) birds undergo developmental programmed cell death (Rubel et al., 1976). In White Leghorn (egg layer) birds, however, no cell death is seen during development (Parks, 1979). It may be that NM neurons in egg layer birds retain the capacity for programmed cell death by virtue of not expressing such genes during embryonic development. Finally, increases in intracellular calcium have been implicated in many types of neuronal cell death (Cheung et al., 1986 ; Siesjo and Bengtsson, 1989; Farber, 1981 ; Trump and Berezesky, 1992 ; Coyle and Puttfarcken, 1993). Johnson and colleagues have suggested that levels of intracellular calcium are critical for normal development and that the `set point' for tolerable calcium levels changes during development (Johnson et al., 1992 ; Johnson and Deckwerth, 1993). Several lines of evidence suggest that an imbalance in calcium homeostasis is intimately involved in neuronal cell death in NM. In our laboratory we have described a rapid upregulation of oxidative metabolism in NM within

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hours of cochlea removal (Hyde and Durham, 1990 ; Durham et al., 1993), mediated in part by massive proliferation of mitochondria (Hyde and Durham, 1994b). Attenuation of this mitochondrial upregulation with chloramphenicol doubles neuronal cell death (Hyde and Durham, 1994a). Because mitochondria have been shown to bu¡er intracellular calcium levels in the cell (Werth and Thayer, 1994; White and Reynolds, 1995), we hypothesized that excess intracellular calcium is involved in NM neuronal cell death. Recent results in another laboratory have provided elegant direct evidence for this hypothesis. Glutamate release by presynaptic a¡erent terminals activate a metabotropic glutamate receptor on NM neurons that stimulates adenylate cyclase second messenger systems to bu¡er calcium (Lachica et al., 1995). Dea¡erentation increases calcium levels several fold in NM neurons (Zirpel et al., 1995). Taken together, these results suggest that the ability to bu¡er increases in intracellular calcium by NM neurons is important for neuronal survival after dea¡erentation in hatchlings. Direct calcium imaging with fura-2 will prove di¤cult in adult birds, as loading of the dye can only be accomplished in embryonic tissue. However, indirect measures of calcium bu¡ering capacity or second messenger systems in adult birds may prove fruitful. Preliminary evidence suggests that broiler birds do not demonstrate increases in mitochondrial capacity with dea¡erentation (Franklin and Durham, 1994); further analyses of mitochondrial capacity in adult birds are underway. It is interesting to note that in other systems, aging is associated with increases in mitochondrial DNA mutations and a decrease in mitochondrial enzyme activity (Wallace, 1994; Luft, 1994; Coyle and Puttfarcken, 1993). We might predict the opposite result if oxidative capacity helps adult NM neurons survive dea¡erentation. In summary, we have shown that two types of adult birds exist, those in which all NM neurons survive deafferentation and those in which 30% of cells die. The addition of this part of the avian model will allow analysis in which age can be held constant in evaluation of cell death mechanisms. We do not know whether the survival strategies used by adult neurons are similar to those used by the majority of neurons in hatchling birds that survive cochlea removal. In the auditory system especially the ability to restore electrical activity with cochlear implants or even with regeneration of cochlear hair cells (Tsue et al., 1994; Cotanche et al., 1994) makes understanding how a¡erent input regulates CNS neuronal metabolism of even greater importance. Continued analysis of the neuronal survival mechanisms at work in adult birds will help insure that a functional CNS awaits renewal of eighth nerve activity by either strategy.

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Acknowledgments The authors thank Brian McCullough, Ginny Morris and Judith Rose Debel for excellent technical support; Jo Anna Schroer for her assistance in procuring adult broiler birds; Irene Garrett for editorial assistance; Je¡ Radel and Deb Park for comments on the manuscript; Susan Jackson for help with statistics, and `anonymous' for pointing out di¡erences in developmental naturallyoccurring neuronal cell death in NM. Supported by NIDCD grants DC00520 and DC01589 (D2 ), funds from the Research Institute of the University of Kansas Medical Center (D2 ), and the Otolaryngology Department at the University of Kansas Medical Center.

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