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The development of glycine-like immunoreactivity in the dorsal cochlear nucleus
ELSEVIER
Hearing Research 89 (1995) 172-180
The development of glycine-like immunoreactivity in the dorsal cochlear nucleus Garrett H. Riggs a Edward J. Walsh b, Laura Schweitzer a,* Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40292, USA b Boys Town National Research Hospital, Omaha, NE, USA Received 29 November 1994; revised 5 June 1995; accepted 11 June 1995
Abstract
Both anatomical and physiological lines of evidence suggest that inhibitory influences are active early in the development of the dorsal cochlear nucleus (DCN). Data from physiological/pharmacological studies suggest that in the adult these inhibitory influences may be mediated at least in part by the neurotransmitter glycine. Using a polyclonal antibody to examine the development of glycine in the DCN, we have observed that glycine-like immunoreactive cell bodies and punctate label are present in the DCN by the day of birth in the hamster and in the kitten at least 10 days prior to birth. In contrast to the development of immunoreactivity for glutamic acid decarboxylase (GAD) (Schweitzer et al., 1993), glycine-like immunoreactivity shows a homogeneous distribution throughout the DCN from the day of birth through adulthood. In addition, glycine immunoreactivity is present earlier than GAD-immunoreactivity and is present well before these brain structures become responsive to air-borne sounds. Thus, glycine is present in the very young animal and may mediate inhibitory effects that occur early in development. Keywords: Inhibition; Glycine; Hearing; Cochlear nucleus; Development; Glutamic acid decarboxylase
1. Introduction
Two of the most ubiquitous amino acid neurotransmitters thought to act as inhibitory neurotransmitters in the adult brainstem - - gamma-aminobutyric acid (GABA) and glycine - - are present in the adult dorsal cochlear nucleus (DCN). Recently, we reported that glutamic acid decarboxylase (GAD) the enzyme for GABA, has a rather delayed and prolonged spatiotemporal pattern of development in the DCN of both kittens and hamsters (Schweitzer et al., 1993). GAD-immunoreactivity is present in the kitten DCN at birth, but not until postnatal day (PND) 3 in the hamster, arising first near the dorsal acoustic stria, next appearing in a band in the fusiform cell layer, and only later filling in to assume its adult distribution throughout the nucleus, but most dense in the molecular layer. Changes in spontaneous and evoked activity of posterior cochlear nucleus (CN) neurons following the iontophoresis of glycine and GABA in very young kittens suggests that the effects of both GABA and glycine are expressed along
* Corresponding author: Tel.: (502) 852-7288; Fax: (502) 852-6228; INTERNET: LFSCHW0 [email protected]. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 1 3 9 - 5
similar time lines (E.J. Walsh, unpublished observation). Previous studies have shown that long-latency IPSPs are present in the CN of the mouse on the 4th PND, and that in the adult mouse, CN disynaptic IPSPs are probably mediated by glycine (Wu and Oertel, 1986; Hirsch and Oertel, 1988). These physiological data suggest that glycine might be present in the neonatal CN where it could subserve inhibition. Based on these results one might predict that the expression of glycine is similar to that of GABA. The present study was designed to test this prediction and compare the time and pattern of development of glycine immunoreactivity with our previous GAD-related data. Glycine, the smallest of the amino acids, probably acts as an inhibitory neurotransmitter in the brainstem auditory nuclei in general (e.g., Caspary et al., 1979; Martin et al., 1982; Sanes et al., 1987; Wenthold et al., 1987; Peyret et al., 1987; Aoki et al., 1987; Code and Rubel, 1989; Winter et al., 1989; Benson and Potashner, 1990) and the CN in particular (e.g., Altschuler et al., 1986a, b; Wu and Oertel, 1986; Wenthold, 1987; Wenthold et al., 1987, 1988). Various studies describe the distribution of tritiated glycine uptake and strychnine-labeled glycine receptors (e.g., Schwartz, 1981; Frostholm and Rotter, 1985, 1986; Sanes
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et al., 1987; Glendenning and Baker, 1988) or the glycine molecule itself (e.g., Godfrey et al., 1977, 1978; Wenthold et al., 1987; Saint Marie et al., 1991; Kolston et al., 1992) in the adult CN. The D C N appears to have one of the highest concentrations of glycine among any of the subdivisions of the CN, and like GABA, glycine in the DCN may be most abundant in the two most superficial laminae (i.e., the molecular layer and fusiform cell layer; Godfrey et al., 1977, 1978). We have chosen to investigate the development of glycine-like immunoreactivity using a well-characterized and widely used polyclonal antibody to the glycine molecule (Wenthold et al., 1987). These data were compared to our data on GAD distribution during development and a significant spatial and temporal disparity was found between the detection of GAD and glycine using these immunohistochemical techniques.
2. Materials and methods 2.1. Animals
The models chosen are the golden Syrian hamster ( Mesocricetus auratus) and the domestic cat ( Felis domesticus). These species were chosen because (1) the kitten is
a commonly used model for auditory development and, in specific, data regarding GABA and glycine efficacy during development of the CN is available (Walsh et al., 1990; and unpublished observations); (2) the DCN of the golden Syrian hamster is a laminated structure that matures postnatally and in which afferent ingrowth and cell differentiation have been well characterized (e.g., Schweitzer and Cant, 1984, 1985); and (3) this study was designed to compare with previous studies in these species on GAD distribution during development (Schweitzer et al., 1993). Hamsters were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and a breeding colony was maintained in the University of Louisville Research Resources Center. Infant hamsters were weaned at postnatal day (PND) 21 and housed with a maximum of 3 same-sex littermates. A total of 47 hamsters was used for the present study. Hamsters at ages PND 0 (n = 9), PND 5 (n = 9), PND 10 (n = 5), PND 14 (n = 3), PND 30 (n = 2), PND 40 (n = 2), and adult ( > PND 60, n = 17) were injected intraperitoneally with an overdose of sodium pentobarbital (100 m g / k g , i.p.) and perfused transcardially with 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, brains were removed from the skull and postfixed in the perfusate for approximately 2 h. After postfixing, brains were transferred to a solution of 30% sucrose in 1.25% buffered glutaraldehyde overnight. DCN from 7 kittens were also studied: 1 from a PND 3 kitten; 2 born prematurely at 59 days gestation (i.e., 8 days premature); and 4 kittens born prematurely at 57 days gestation (i.e., 10 days premature). In a subset of these
kittens, the DCN on one side was studied electrophysiologically while the kitten was deeply anesthetized with sodium pentobarbital (40 m g / k g , i.p.). After collection of electrophysiologic data, kittens were perfused transcardially with the fixative as described above. After perfusion, kitten brains were transferred to a solution of 30% sucrose in 1.25% buffered glutaraldehyde, shipped to Louisville, KY, overnight and processed identically to hamster brains. 2.2. Cytochemistry
Frozen sections were cut into 0.05 M Tris-buffered saline (TBS, pH 7.6, 2.5% NaC1). Sections of hamster DCN were either 100 /xM thick (PND 0 and some PND 5 cases) or 50 /zM thick (some PND 5 cases and all ages > PND 5); sections of kitten DCN were all 50 /xM thick. Sections were rinsed in fresh TBS then incubated in a solution of T B S / 3 % H202/methanol (5:4:1) for 10 min to quench endogenous peroxidases. Sections were rinsed again with fresh TBS (from this step on TBS was pH 7.6, 0.9% NaC1) and then incubated for 1 h in 0.3% Triton X-100. After rinsing with fresh TBS, sections were blocked with 20% normal goat serum for 1 h, then rinsed again in TBS. The primary antibody, generously supplied by Dr. Robert Wenthold, was reconstituted to a final working dilution of 1:200 or 1:400 in TBS and applied to the sections for 24 h. (See Wenthold et al. (1987) for the biochemical characterization of this polyclonal antibody and Wickesberg et al. (1994) for a discussion of the reliability of the antibody's ability to label neurons that use glycine as a neurotransmitter.) Control sections (every 4th section) were incubated in TBS alone with no primary antibody (omission control).
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Age (days) Fig. 1. Cross-sectionalarea of cells labeled with the antibody to glycine on PND 0, 5, 10, 30, 40 and adult ( > 60). Means are represented by the bars and SEM are represented by the vertical lines. Labeled cells are larger at the older ages (ANOVA: F = 67.95; df = 5,95; P < 0.01) with growth extending beyond PND 40. Asterisks indicate a significantchange (Fisher's PLSD post-hoctest: P < 0.05) from the previous age.
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Fig. 3. A histogram of the number of cells with form factors falling into 0.05 tzm bins within an individual section of an adult hamster DCN. Every labeled cell was drawn and the form factor calculated. Form factors can range from 0 for flat cells to 1 for perfectly round cells. The number under each bar represents the greatest form factor within that bin. Many more rounded cells are seen than flattened cells.
Fig. 2. Transverse sections through adult hamster DCN reacted with DAB to visualize glycine-like immunoreactivity. A: immunoreactive cell bodies (arrows) are distributed throughout the nucleus. Arrowheads indicate immunoreactive puncta. B: labeled cells with elongated cell somata located deep in the DCN adjacent to the dorsal acoustic stria. C: a labeled cell with a rounded cell soma. This cell is located superficial in the fusiform cell layer and is likely a cartwheel cell. Scale bars: A = 40 /zm; B,C = 10 /xm.
On the following day, all sections were rinsed with TBS and a Vectastain ABC Elite kit (catalog # P K 6101, Vector Laboratories, Burlingame, CA) was used for the remaining
cytochemical processes. Reconstitution of all ABC reagents was carried out in TBS. Sections were incubated with biotinylated secondary antibody (goat anti-rabbit IgG, 1:100) for 1 h. In order to minimize non-specific binding, the solution of secondary antibody also contained 5% normal goat serum (final concentration). After 1 h in secondary antibody, sections were rinsed in TBS and then incubated in the A B C - H R P complex for 1 h. Sections were rinsed again in TBS, incubated for 30 min in 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma catalog #D9015 and then reacted with 0.1% H 2 0 z. The DAB
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Fig. 4. Transverse section through hamster DCN at PND 0. A: low-power photomicrograph showing immunoreactive cell bodies (arrows) in the DCN. B : higher magnification of panel A showing two immunoreactive somata. Arrows in panels A and B occupy the same positions. C: under oil immersion, some punctate label is intensely immunoreactive (arrowheads) while other punctate labeling shows intermediate degrees of immunoreactivity. Scale bars: A = 100 /xm; B = 20 /xm; C = 10 /.tm.
G.H. Riggs et al./ Hearing Research 89 (1995) 172-180
reaction was carried out in 0.1 M phosphate buffer (pH 7.4). After the DAB reaction, sections were mounted onto gelatin-coated slides out of phosphate buffer, dehydrated and coverslipped.
2.3. Quantitative histological methods To determine if the cells that are labeled change in size with age, the cross-sectional areas of 16 immunoreactive cell somata from at least 2 cases each on PND 0, 5, 10, 30, 40 and adult ( > PND 60) were measured. In addition, in 1 adult, the cross-sectional profiles of every immunoreactive cell was drawn in one section midway along the rostral to caudal extent of the DCN. The form factor ( F F = (47ra/p2), where a is somatic cross-sectional area and p is the cell perimeter) for each cell was calculated and a histogram of the form factors was plotted. Form factor is an index of circularity, ranging from an FF of 1 for perfectly round cells to an FF of 0 for flat cells.
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somewhat more in the deep layer. Several immunoreactive cells can even be found in the cell-sparse molecular layer in each section. Elongated cells with form factors less than 0.7 are less common than rounded cells (Fig. 3) and are present throughout the nucleus although they are frequently found near or within the dorsal acoustic stria (Fig. 2B). Rounded cells (Fig. 2C) are found scattered throughout the nucleus although there is commonly a row of these cells superficial in the fusiform cell layer. Similarly, punctate label is present throughout the DCN. Like immunoreactive somata, its distribution does not conform to a restricted laminar pattern, although in some cases punctate labeling does appear to be slightly heavier in the regions of the fusiform cell and molecular layers.
2.4. Animal welfare The care and use of the animals reported in this study were approved by the University of Louisville and Boys Town National Research Hospital Institutional Animal Care and Use Committees.
A
3. Results
Immunoreactive labeling was present at all ages studied in both species. In addition to a fairly generalized distribution of punctate label, cell somata were distinctly labeled. The punctate label probably represents both transected axons and axon terminals. Elongated labeled processes are also seen, especially in the dorsal acoustic stria where immunoreactive axons are cut lengthwise. All control sections showed minimal, non-specific background staining.
B
3.1. Glycine-like immunoreactivity in the adult hamster DCN In the mature mammalian DCN, 3 laminae may be distinguished by a variety of methods; from superficial to deep, these are the molecular layer, the fusiform cell layer, and the deep layer. In contrast, neither the punctate glycine-like label nor the labeled cell somata obeyed any consistent laminar distribution. In the adult, labeled cell somata are generally small (Fig. 1) in comparison with projection neurons in the nucleus which exceed 200 /xm 2 in cross-sectional area (Schweitzer et al., 1987). The labeled neurons are present throughout the nucleus. The cells do not conform to a laminar distribution (Fig. 2A). From section to section there is variability with respect to the location of the cells; in some sections there are somewhat more cells in the fusiform cell layer and in others there are
C Fig. 5. Composite schematic camera lucida drawings showing the distribution of immunoreactive cell somata at PND 0 (A), PND 5 (B) and PND 10 (C). Observe that in no case do the somata conform to a laminar distribution. All drawings were made at the same magnification, medial is to the left, dorsal is to the top. Scale bar = 40 /zm.
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Fig. 6. Transverse sections through hamster DCN at PND 5. A: immunoreactive cell (arrow) and process (large arrowhead). Note an adjacent process that is labeled (asterisk). B: low-power photomicrograph showing immunoreactive cells, processes and puncta (small arrowheads). Scale bars: A = 25 /xm; B = 30 /xm.
3.2. Glycine-like immunoreactivity in the neonatal hamster DCN On the day of birth (PND 0) immunoreactive cell bodies are present in the DCN (Fig. 4A, B). These cell
bodies appear round (Fig. 2) and smaller (Fig. 1) than at the older ages; other details of cellular morphology (dendrites, axons) are not usually clear at this age. The number of immunoreactive cell bodies present at this age is substantially less than that observed in the adult DCN (Fig. 2)
Fig. 7. Transverse sections through kitten DCN. A: 57 days of gestation (pre-natal day 10). Note immunoreactive axons in the dorsal acoustic stria (arrows). Faint immunoreactive cell bodies are indicated at arrowheads. The very dark profiles are reactive blood cells. B: 57 days gestation (pre-natal day 10). Control section from which the primary antibody was omitted. Arrows indicate an edge artifact. C: 59 days of gestation (pre-natal day 8). Immunoreactive cell bodies are clearly present (arrowheads). D: PND 3. Many immunoreactive cell bodies are present (arrowheads). Axons in the dorsal acoustic stria (to the left of the asterisk) retain the immunoreactivity first evidenced 8 days before birth. An axon in the dorsal acoustic stria projecting into the DCN is indicated by the asterisk. E is a higher magnification view of part of the section shown in D. The same location in D and E is indicated by an asterisk. Scale bars: A,B,D = 40 /.tm; C = 27/zm; E = 13 /xm.
G.H. Riggs et aL/ HearingResearch 89 (1995) 172-180 and large expanses of the DCN in the neonate lack immunoreactive cell bodies (Fig. 5A). Punctate label is present throughout the nucleus at this age. This label probably represents labeled axons, axon terminals and transected axons (Fig. 4C). Some of this punctate label is intensely immunoreactive, while other is less so, although punctate labeling can always be clearly distinguished from background. On PND 5, immunoreactive cell somata are still scattered throughout the nucleus (Fig. 5B); punctate label is present as well. In a few cases, labeled puncta appear to be slightly more concentrated in the superficial DCN (fusiform cell and molecular layers) than in the deeper DCN, but this observation is not consistent across cases or across sections within a case. The number of strongly immunoreactive puncta is somewhat greater on PND 5 than on PND 0. On PND 5 it is sometimes possible to discern the processes of immunoreactive cells (Fig. 6). Cell bodies occasionally present elongated profiles. There is little substantive change in the pattern of immunoreactivity between PND 5, PND 10, and subsequent ages including the adult (Fig. 2, Fig. 5). Although the number of immunoreactive cell somata is higher in the older animals, the density of labeled cell bodies appears somewhat lower on PND 10 (Fig. 5C) likely because the volume of the DCN more than doubles between PND 5 and 10 (Schweitzer and Cecil, 1992). The number of immunoreactive cells and intensely immunoreactive punctate label appears greater in the mature DCN than on PND 10. 3.3. Glycine-like immunoreactivity in the prenatal kitten
DCN In the kitten, immunoreactive axons are present in the dorsal acoustic stria and deep DCN 10 days prior to birth; some hints of lightly immunoreactive cell bodies are present at this time (Fig. 7A,B). Eight days prior to birth, immunoreactive axons are still seen, and immunoreactive cell bodies are clearly defined (Fig. 7C). On PND 3, axons remain darkly labeled, and large numbers of immunoreactive cell bodies are seen throughout the nucleus (Fig. 7D,E).
4. Discussion The present study is one of the first to detail the development of glycine-like immunoreactive labeling in the mammal. Using the Wenthold antibody, a well-known and well-characterized polyclonal antibody to the glycine molecule, we have observed immunoreactive cell bodies and punctate labeling throughout the DCN as early as the day of birth in the hamster and prenatally in the cat. In general, a gradual increase in punctate label and the number of immunoreactive cells is found across ages studied,
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with mature levels reached by about PND 10 in the hamster, 6 days prior to the onset of function (as defined by the ability to evoke brainstem responses to clicks at 54 d B pSPL, Schweitzer, 1987). Similarly, glycine-like immunoreactivity is seen before the onset of function in the cat. Although a complete developmental profile is not available for the cat, the presence of significant glycine-like immunoreactivity in the cochlear nucleus of the prenatal cat suggests that inhibitory circuits, presumably mediated through glycine, may be organizing relatively long before the first responses to high level airborne sounds are observed developmentally (Walsh and McGee, 1987). This finding is consistent with the view promulgated by Walsh et al. (1990) that inhibitory amino acid receptors and associated ionophores are in place and are operational among the majority of cochlear nucleus neurons in perinatal cats.
4.1. Glycine-like immunoreactivity in the adult DCN In the adult DCN of the hamster, the labeled cells can be classified as small cells (with somata about 196/xm 2 in cross-sectional area) in comparison to the cross-sectional areas of the large projection neurons (about 225 /zm 2 and 270 /.Lm2 for fusiform and giant cells, respectively; Schweitzer et al., 1987). Yet these cells are not as small as the smallest neurons in the nucleus - - the granule cells ( = 30 ~ m 2, L. Schweitzer, unpublished observations). The labeled cells are a variety of shapes but the majority of cells are rounded. In particular, rounded cells are often found in a discontinuous row in the superficial fusiform cell layer. The shape and distribution of these labeled cells suggests that they may be cartwheel neurons, small, rounded interneurons found in this location. Our results from the hamster support the observation summarized in Wickesberg et al. (1994) that there is not a clear, restricted, laminar distribution for glycine-like immunoreactive cell somata or punctate label in the adult. It should be noted that only a few immunoreactive cell somata were found in the molecular layer in each animal. However, it is the case that relatively few cell somata of any kind are contained in the molecular layer, a layer that is commonly referred to as a 'somata-sparse' layer. Thus, the scant number of labeled cell bodies here does not indicate a true distribution which is restricted by layer. Certainly, the distribution of labeled puncta in the molecular layer is comparable to that in the deeper layers.
4.2. Comparison of GAD and glycine development Although colocalization of glycine-and GABA-immunoreactivity appears to be quite common in the adult DCN (Wenthold et al., 1987; Wenthold and Hunter, 1990; Kolston et al., 1992), the present data on development of glycine-like immunoreactivity in the DCN do not bear any obvious relationship to the development of GAD-immuno-
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reactivity. Schweitzer et al. (1993) have shown that the development of GAD begins with a distribution of immunoreactivity in the neuropil of the deep DCN on PND 3, followed by an increase in immunoreactivity in the fusiform cell layer, which in turn is followed by an increase in GAD-immunoreactivity in the molecular layer. No GADimmunoreactive cell bodies are seen until PND 14. At that time, small- to medium-sized cells immunoreactive for GAD are observed in the superficial part of the fusiform cell layer (likely cartwheel neurons) as well as throughout the deep layer. We observe no laminar-related or deep-to-superficial progression in the development of glycine-like immunoreactivity. Rather, punctate label and glycine-like immunoreactive somata appear to be present in a homogeneous distribution throughout the DCN even on PND 0. Thus the pattern of development of GAD- and glycine-immunoreactivity in the DCN of the hamster are very different. Furthermore, the age at which we could successfully label immunoreactive cell somata using the two methods are very different. GAD-immunoreactivity appears late, especially the labeling of somata, which only shortly precedes the age when function was first observed. Glycinepositive somata were found much earlier in both kitten and hamster. This could relate to a true difference in expression of the two inhibitory amino acids, or could relate to the differences in our ability to detect the molecules given the limitations of the methods. Regardless of the reason, the ability to label glycine-like immunoreactivity in somata at such an immature age - - in embryonic kitten and perinatal hamsters - - was an unexpected finding. 4.3. Glycine-like immunoreactivity in the developing D C N
Very early in development glycine-immunoreactivity was found in the DCN of both hamster and kitten although at the early ages the cells labeled in the hamster were considerably smaller than at older ages. This is likely due to cell growth rather than the labeling of different populations at different ages. Measurements done on other cell populations in the DCN suggest that other cells grow during this time and like the labeled cells here, continue to grow even after PND 40 (Schweitzer, 1991). The early presence of inhibitory neurotransmitters in the DCN found in this study, correlates well with physiological data that suggest both excitatory and inhibitory inputs are present early in the development of the nucleus. Long-latency IPSPs are present at the earliest age studied (PND 4) in the immature mouse (Wu and Oertel, 1987; Hirsch and Oertel, 1988). It has also been shown that disynaptic IPSPs in the adult mouse are probably mediated by glycine. Wu and Oertel (1986) observed that the response characteristics of neurons in the DCN were consistent with the action of inhibitory interneurons present within the DCN proper. Furthermore, they used intracellular recordings from slice preparations of the VCN to
show that exogenously applied GABA and glycine could mediate IPSPs. However, only low levels (1 /xM) of strychnine, which binds specifically to glycine receptors, were needed to block all IPSP activity. By comparison, blockers of GABA-mediated inhibition (bicuculline and picrotoxin) were inconsistent in blocking IPSPs even when present at concentrations as high as 100 /xM (see also Hirsch and Oertel, 1988). The logical deduction based on these physiologic data is that glycine could potentially mediate inhibition at an early age in the DCN. Our data demonstrate the presence of glycine-like immunoreactivity in the DCN of the hamster by a gestational age at least equivalent to that reported by Wu and Oertel (1987) and Hirsch and Oertel (1988). Our anatomical data correlate well with data from slice recordings in the mouse and lend support to the hypothesis that glycine, present at an early age, could mediate the inhibition that has been demonstrated physiologically. A recent report by Gleich and Vater (1994) has shown only diffuse glycine-like immunoreactivity in the DCN of the gerbil on PND 1 and PND 2. In contrast to the present results, no immunoreactive cell bodies were reported at that time. By PND 9, some cell somata showed weak glycine-like immunoreactivity. In the young adult (3 months old), the neuropil of the molecular layer and fusiform cell layer showed substantial glycine-like immunoreactivity. Immunoreactive cell bodies were present, were of variable size, and were more prominent in the deep layer and fusiform cell layer. In addition to a neurotransmission function for glycine, the non-transmission roles, if any, of glycine early in the development of auditory function are unclear. Both GABA and glycine appear to produce depolarizing, activating actions in certain embryonic tissue-cultured spinal cord neurons of chicks, with typical inhibitory actions developing slightly later (Obata et al., 1978). Cherubini et al. (1991) reported similar findings among hippocampal neurons, concluding that GABA-mediated depolarizing actions are the dominant form of excitation during development and postulated that the role played by such activity is trophic. Hyson (1991) reported on the depolarizing actions of GABA and Kandler and Friauf (1993) on the depolarizing actions of glycine, on auditory brainstem neurons of vertebrates early in development. In addition, Sanes and Hafidi (1993) found that blockade of glycine receptors produced an increase in dendritic arborizations within the mature superior olive in vitro. These results raise the possibility that 'inhibitory' amino acid actions may be neuromaturational within the auditory system early in its development. Consistent with this changing role, Friauf et al. (1994) have shown that at least two isoforms of glycine receptor exist during development. One is present neonatally in the auditory brainstem, while a second 'adult' isoform becomes predominant on PND 9 (i.e., shortly before the onset of hearing on PND 12). In summary, we have demonstrated that glycine-like
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immunoreactivity is present in a relatively homogeneous distribution throughout the DCN from the day of birth through adulthood in the hamster. Immunoreactivity was found prenatally in the kitten as well. Glycine was detected earlier than GAD, the synthetic enzyme for the other major inhibitory amino acid in the CN. These data add to previously published reports suggesting that glycine may mediate inhibition early in the development of the DCN. It remains to be determined whether it also plays a trophic role in the early auditory system. Acknowledgements The authors gratefully acknowledge Tina Cecil for her expert technical assistance, This work was supported by NIH Grants DC00233 (L.S.) and DC01007 (E.J.W.). References Altschuler, R.A., Betz, H., Parakkal, M.H., Reeks, K.A., and Wenthold, R.J. (1986a) Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res. 369, 316-320. Altschuler, R.A., Hoffman, D.W., and Wenthold, R.J. (1986b) Neurotransmitters of the cochlea and cochlear nucleus: Immunocytochemical evidence. Am. J. Otolaryngol. 7, 100-106. Aoki, C., Milner, T.A., Sheu, K.F., Blass, J.P., and Pickel, V.M. (1987) Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate neurotransmission. J. Neurosci. 7, 22142231. Benson, C.G. and Potashner, S.J. (1990) Retrograde transport of [3H]glycine from the cochlear nucleus to the superior olive in the guinea pig. J. Comp. Neurol. 296, 415-426. Caspary, D.M., D.C. Havey and C.L. Faingold (1979) Effects of microiontophoretically applied glycine and GABA on neuronal response patterns in the cochlear nuclei, Brain Res., 172 179-185. Cherubini, E., Gaiarsa, J.L. and Ben-Ari, Y. (1991) GABA: An excitatory transmitter in early postnatal life. Trends Neurosci. 14, 515-519. Code, R.A. and Rubel, E.W. (1989) Glycine-immunoreactivity in the auditory brain stem of the chick. Hear. Res. 40, 167-172. Friauf, E., Hammerschmidt, B., Kirsch, J., and Betz, H. (1994) Development of glycine receptor distribution in the rat auditory brainstem: Transition from the 'neonatal' to the 'adult' isoform. Assoc. Res. Otolaryng. Abstr., 10. Frostholm, A. and Rotter, A. (1985) Glycine receptor distribution in mouse CNS: Autoradiographic localization of [3H]strychnine binding sites. Brain Res. Bull. 15, 473-486. Frostholm, A. and Rotter, A. (1986) Autoradiographic localization of receptors in the cochlear nucleus of the mouse. Brain Res. Bull. 16, 189-203. Gleich, O. and Vater, M. (1994) Preliminary data on the postnatal development of GABA- and glycine-like immunoreactivity in the gerbil cochlear nucleus. Assoc. Res. Otolaryng. Abstr., 10. Glendenning, K.K. and Baker, B.N. (1988) Neuroanatomical distribution of receptors for three potential inhibitory neurotransmitters in the brainstem auditory nuclei of the cat. J. Comp. Neurol. 275, 288-308. Godfrey, D.A., Carter, J.A., Berger, S.J., Lowry, D.H., and Matschinsky, F.M. (1977) Quantitative histochemical mapping of candidate neurotransmitter amino acids in the cat cochlear nucleus. 1. Histochem. Cytochem. 25, 417-431.
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Godfrey, D.A., J.A. Carter, O.H. Lowry and F.M. Matschinsky (1978) Distribution of gamma-aminobutyfic acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat. J. Histochem. Cytochem. 26, 118-126. Hirffch, J.A. and Oertel, D. (1988) Synaptic connections in the dorsal cochlear nucleus of mice, in vitro. J. Physiol. 396, 549-562. Hyson, R.L. (1991) A depolarizing inhibitory response to GABA in the brain stem auditory system of the chick. Assoc. Res. Otolaryngol. Abstr. 14, 141. Kandler, K. and Friauf, E. (1993) Pharmacology of early synaptic transmission in the superior olivary complex of the rat. Soc. Neurosci. Abstr. 19, 889. Kolston, J., Osen, K.K., Hackney, C.M., Ottersen, O.P., and StormMathisen, J. (1992) An atlas of glycine- and GABA-Iike immunoreactivity and colocalizatiou in the cochlear nuclear complex of the guinea pig. Anat. Embryol. 186, 443-465. Martin, M.R., Dickson, J.W., and Pex, J. (1982) Bicuculline, strychnine and depressant amino acid responses in the anteroventral cochlear nucleus of the cat. J. Neuropharmacol. 21,201-207. Obata, K. Oida, M. and Tanaka, H. (1978) Excitatory and inhibitory actions of GABA and glycine on embryonic chick spinal neurons in culture. Brain Res. 144, 179-184. Peyret, D., Campistron, G., Geffard, M., and Aran, J.-M. (1987) Glycine immunoreactivity in the brainstem auditory and vestibular nuclei of the guinea pig. Acta Otolaryngol. (Stockh.) 104, 71-76. Saint Marie, R.L., Benson, C.G., Ostapoff, E.-M., and Morest, D.K. (1991) Glycine immunoreactive projections from the dorsal to the anteroventral cochlear nucleus. Hear. Res. 51, 11-28. Sanes, D.H., Geary, W.A., Wooten, G.F., and Rubel, E.W. (1987) Quantitative distribution of the glycine receptor in the auditory brain stem of the gerbil. J. Neurosci. 7, 3793-3802. Sanes, D.H. and Hafidi, A. (1993) Glycine receptor blockade influences dendritic arborizations in organotypic culture. Soc. Neurosci. Abstr. 19, 616. Schwartz, I.R. (1981) The differential distribution of label following uptake of 3H-labelled amino acids in the dorsal cochlear nucleus of the cat. Exp. Neurol. 73, 601-617. Schweitzer, L. (1987) Development of brainstem auditory evoked responses in the hamster. Hear. Res. 25, 249-255. Schweitzer, L. (1991) Morphometric analysis of developing neuronal geometry in the dorsal cochlear nucleus of the hamster, Dev. Brain Res. 59, 39-47. Schweitzer, L., Bell, J.M. and Slotkin, T.A. (1987) Impaired morphological development of the dorsal cochlear nucleus in hamsters treated postnatally with alpha-difluoromethylomithine. Neuroscience 23, 1123-1132. Schweitzer, L. and Cant, N.B. (1984) Development of the cochlear innervation of the dorsal cochlear nucleus of the hamster. J. Comp. Neurol. 225, 228-243. Schweitzer, L. and Cant, N.B. (1985) Differentiation of the giant and fusiform cells in the dorsal cochlear nucleus of the hamster. Dev. Brain Res. 20, 69-82. Schweitzer, L. and Cecil, T. (1992) Morphology of HRP-labeUed cochlear nerve axons in the dorsal cochlear nucleus of the developing hamster. Hear. Res. 60, 34-44. Schweitzer, L., Cecil, T., and Walsh, E.J. (1993) Development of GADimmunoreactivity in the dorsal cochlear nucleus of the hamster and cat: light and electron microscopic observations. Hear. Res. 65, 240-251. Walsh, E.J. and McGee, J. (1987) Postnatal development of auditory nerve and cochlear nucleus neuronal responses in kitten. Hear. Res. 28, 97-116. Walsh, E.J., McGee, J., and Fitzakerley, J.L. (1990) GABA actions within the caudal cochlear nucleus of developing kittens. J. Neurophysiol. 64, 961-977. Wenthold, R.J. (1987) Evidence for a glycinergic pathway connecting the
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two cochlear nuclei: an immunocytochemical and retrograde transport study. Brain Res. 415, 183-187. Wenthold, R.J. and Hunter, C. (1990) Immunocytochemistry of glycine and glycine receptors in the central auditory system. In: O.P. Ottersen and J. Storm-Mathisen (Eds.), Glycine Neurotransmission, Wiley, Chichester, NY, pp. 391-416. Wenthold, R.J., Huie, D., Altschuler, R.A., and Reeks, K.A. (1987) Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex. Neuroscience 22, 897-912. Wenthold, R.J., Parakkal, M.H., Oberdorfer, M.D., and Altschuler, R.A. (1988) Glycine receptor immunoreactivity in the ventral cochlear nucleus of the guinea pig. J. Comp. Neurol. 276, 423-435.
Wickesberg, R.E., Whitlon, D. and Oertel, D. (1994) In vitro modulation of somatic glycine-like immunoreactivity in presumed glycinergic neurons. J. Comp. Neurol. 339, 311-327. Winter, I.M., Robertson, D., and Stewart Cole, K. (1989) Descending projections from auditory brainstem nuclei to the cochlea and cochlear nucleus of the guinea pig. J. Comp. Neurol. 280, 143-147. Wu, S.H. and Oertel, D. (1986) Inhibitory circuitry in the ventral cochlear nucleus is probably mediated by glycine. J. Neurosci. 6, 2691-2706. Wu, S.H. and Oertel, D. (1987) Maturation of synapses and electrical properties of cells in the cochlear nuclei. Hear. Res. 30, 99-110.
Hearing Research 89 (1995) 172-180
The development of glycine-like immunoreactivity in the dorsal cochlear nucleus Garrett H. Riggs a Edward J. Walsh b, Laura Schweitzer a,* Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40292, USA b Boys Town National Research Hospital, Omaha, NE, USA Received 29 November 1994; revised 5 June 1995; accepted 11 June 1995
Abstract
Both anatomical and physiological lines of evidence suggest that inhibitory influences are active early in the development of the dorsal cochlear nucleus (DCN). Data from physiological/pharmacological studies suggest that in the adult these inhibitory influences may be mediated at least in part by the neurotransmitter glycine. Using a polyclonal antibody to examine the development of glycine in the DCN, we have observed that glycine-like immunoreactive cell bodies and punctate label are present in the DCN by the day of birth in the hamster and in the kitten at least 10 days prior to birth. In contrast to the development of immunoreactivity for glutamic acid decarboxylase (GAD) (Schweitzer et al., 1993), glycine-like immunoreactivity shows a homogeneous distribution throughout the DCN from the day of birth through adulthood. In addition, glycine immunoreactivity is present earlier than GAD-immunoreactivity and is present well before these brain structures become responsive to air-borne sounds. Thus, glycine is present in the very young animal and may mediate inhibitory effects that occur early in development. Keywords: Inhibition; Glycine; Hearing; Cochlear nucleus; Development; Glutamic acid decarboxylase
1. Introduction
Two of the most ubiquitous amino acid neurotransmitters thought to act as inhibitory neurotransmitters in the adult brainstem - - gamma-aminobutyric acid (GABA) and glycine - - are present in the adult dorsal cochlear nucleus (DCN). Recently, we reported that glutamic acid decarboxylase (GAD) the enzyme for GABA, has a rather delayed and prolonged spatiotemporal pattern of development in the DCN of both kittens and hamsters (Schweitzer et al., 1993). GAD-immunoreactivity is present in the kitten DCN at birth, but not until postnatal day (PND) 3 in the hamster, arising first near the dorsal acoustic stria, next appearing in a band in the fusiform cell layer, and only later filling in to assume its adult distribution throughout the nucleus, but most dense in the molecular layer. Changes in spontaneous and evoked activity of posterior cochlear nucleus (CN) neurons following the iontophoresis of glycine and GABA in very young kittens suggests that the effects of both GABA and glycine are expressed along
* Corresponding author: Tel.: (502) 852-7288; Fax: (502) 852-6228; INTERNET: LFSCHW0 [email protected]. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 1 3 9 - 5
similar time lines (E.J. Walsh, unpublished observation). Previous studies have shown that long-latency IPSPs are present in the CN of the mouse on the 4th PND, and that in the adult mouse, CN disynaptic IPSPs are probably mediated by glycine (Wu and Oertel, 1986; Hirsch and Oertel, 1988). These physiological data suggest that glycine might be present in the neonatal CN where it could subserve inhibition. Based on these results one might predict that the expression of glycine is similar to that of GABA. The present study was designed to test this prediction and compare the time and pattern of development of glycine immunoreactivity with our previous GAD-related data. Glycine, the smallest of the amino acids, probably acts as an inhibitory neurotransmitter in the brainstem auditory nuclei in general (e.g., Caspary et al., 1979; Martin et al., 1982; Sanes et al., 1987; Wenthold et al., 1987; Peyret et al., 1987; Aoki et al., 1987; Code and Rubel, 1989; Winter et al., 1989; Benson and Potashner, 1990) and the CN in particular (e.g., Altschuler et al., 1986a, b; Wu and Oertel, 1986; Wenthold, 1987; Wenthold et al., 1987, 1988). Various studies describe the distribution of tritiated glycine uptake and strychnine-labeled glycine receptors (e.g., Schwartz, 1981; Frostholm and Rotter, 1985, 1986; Sanes
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et al., 1987; Glendenning and Baker, 1988) or the glycine molecule itself (e.g., Godfrey et al., 1977, 1978; Wenthold et al., 1987; Saint Marie et al., 1991; Kolston et al., 1992) in the adult CN. The D C N appears to have one of the highest concentrations of glycine among any of the subdivisions of the CN, and like GABA, glycine in the DCN may be most abundant in the two most superficial laminae (i.e., the molecular layer and fusiform cell layer; Godfrey et al., 1977, 1978). We have chosen to investigate the development of glycine-like immunoreactivity using a well-characterized and widely used polyclonal antibody to the glycine molecule (Wenthold et al., 1987). These data were compared to our data on GAD distribution during development and a significant spatial and temporal disparity was found between the detection of GAD and glycine using these immunohistochemical techniques.
2. Materials and methods 2.1. Animals
The models chosen are the golden Syrian hamster ( Mesocricetus auratus) and the domestic cat ( Felis domesticus). These species were chosen because (1) the kitten is
a commonly used model for auditory development and, in specific, data regarding GABA and glycine efficacy during development of the CN is available (Walsh et al., 1990; and unpublished observations); (2) the DCN of the golden Syrian hamster is a laminated structure that matures postnatally and in which afferent ingrowth and cell differentiation have been well characterized (e.g., Schweitzer and Cant, 1984, 1985); and (3) this study was designed to compare with previous studies in these species on GAD distribution during development (Schweitzer et al., 1993). Hamsters were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and a breeding colony was maintained in the University of Louisville Research Resources Center. Infant hamsters were weaned at postnatal day (PND) 21 and housed with a maximum of 3 same-sex littermates. A total of 47 hamsters was used for the present study. Hamsters at ages PND 0 (n = 9), PND 5 (n = 9), PND 10 (n = 5), PND 14 (n = 3), PND 30 (n = 2), PND 40 (n = 2), and adult ( > PND 60, n = 17) were injected intraperitoneally with an overdose of sodium pentobarbital (100 m g / k g , i.p.) and perfused transcardially with 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, brains were removed from the skull and postfixed in the perfusate for approximately 2 h. After postfixing, brains were transferred to a solution of 30% sucrose in 1.25% buffered glutaraldehyde overnight. DCN from 7 kittens were also studied: 1 from a PND 3 kitten; 2 born prematurely at 59 days gestation (i.e., 8 days premature); and 4 kittens born prematurely at 57 days gestation (i.e., 10 days premature). In a subset of these
kittens, the DCN on one side was studied electrophysiologically while the kitten was deeply anesthetized with sodium pentobarbital (40 m g / k g , i.p.). After collection of electrophysiologic data, kittens were perfused transcardially with the fixative as described above. After perfusion, kitten brains were transferred to a solution of 30% sucrose in 1.25% buffered glutaraldehyde, shipped to Louisville, KY, overnight and processed identically to hamster brains. 2.2. Cytochemistry
Frozen sections were cut into 0.05 M Tris-buffered saline (TBS, pH 7.6, 2.5% NaC1). Sections of hamster DCN were either 100 /xM thick (PND 0 and some PND 5 cases) or 50 /zM thick (some PND 5 cases and all ages > PND 5); sections of kitten DCN were all 50 /xM thick. Sections were rinsed in fresh TBS then incubated in a solution of T B S / 3 % H202/methanol (5:4:1) for 10 min to quench endogenous peroxidases. Sections were rinsed again with fresh TBS (from this step on TBS was pH 7.6, 0.9% NaC1) and then incubated for 1 h in 0.3% Triton X-100. After rinsing with fresh TBS, sections were blocked with 20% normal goat serum for 1 h, then rinsed again in TBS. The primary antibody, generously supplied by Dr. Robert Wenthold, was reconstituted to a final working dilution of 1:200 or 1:400 in TBS and applied to the sections for 24 h. (See Wenthold et al. (1987) for the biochemical characterization of this polyclonal antibody and Wickesberg et al. (1994) for a discussion of the reliability of the antibody's ability to label neurons that use glycine as a neurotransmitter.) Control sections (every 4th section) were incubated in TBS alone with no primary antibody (omission control).
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Age (days) Fig. 1. Cross-sectionalarea of cells labeled with the antibody to glycine on PND 0, 5, 10, 30, 40 and adult ( > 60). Means are represented by the bars and SEM are represented by the vertical lines. Labeled cells are larger at the older ages (ANOVA: F = 67.95; df = 5,95; P < 0.01) with growth extending beyond PND 40. Asterisks indicate a significantchange (Fisher's PLSD post-hoctest: P < 0.05) from the previous age.
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Form Factors 40
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Fig. 3. A histogram of the number of cells with form factors falling into 0.05 tzm bins within an individual section of an adult hamster DCN. Every labeled cell was drawn and the form factor calculated. Form factors can range from 0 for flat cells to 1 for perfectly round cells. The number under each bar represents the greatest form factor within that bin. Many more rounded cells are seen than flattened cells.
Fig. 2. Transverse sections through adult hamster DCN reacted with DAB to visualize glycine-like immunoreactivity. A: immunoreactive cell bodies (arrows) are distributed throughout the nucleus. Arrowheads indicate immunoreactive puncta. B: labeled cells with elongated cell somata located deep in the DCN adjacent to the dorsal acoustic stria. C: a labeled cell with a rounded cell soma. This cell is located superficial in the fusiform cell layer and is likely a cartwheel cell. Scale bars: A = 40 /zm; B,C = 10 /xm.
On the following day, all sections were rinsed with TBS and a Vectastain ABC Elite kit (catalog # P K 6101, Vector Laboratories, Burlingame, CA) was used for the remaining
cytochemical processes. Reconstitution of all ABC reagents was carried out in TBS. Sections were incubated with biotinylated secondary antibody (goat anti-rabbit IgG, 1:100) for 1 h. In order to minimize non-specific binding, the solution of secondary antibody also contained 5% normal goat serum (final concentration). After 1 h in secondary antibody, sections were rinsed in TBS and then incubated in the A B C - H R P complex for 1 h. Sections were rinsed again in TBS, incubated for 30 min in 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma catalog #D9015 and then reacted with 0.1% H 2 0 z. The DAB
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Fig. 4. Transverse section through hamster DCN at PND 0. A: low-power photomicrograph showing immunoreactive cell bodies (arrows) in the DCN. B : higher magnification of panel A showing two immunoreactive somata. Arrows in panels A and B occupy the same positions. C: under oil immersion, some punctate label is intensely immunoreactive (arrowheads) while other punctate labeling shows intermediate degrees of immunoreactivity. Scale bars: A = 100 /xm; B = 20 /xm; C = 10 /.tm.
G.H. Riggs et al./ Hearing Research 89 (1995) 172-180
reaction was carried out in 0.1 M phosphate buffer (pH 7.4). After the DAB reaction, sections were mounted onto gelatin-coated slides out of phosphate buffer, dehydrated and coverslipped.
2.3. Quantitative histological methods To determine if the cells that are labeled change in size with age, the cross-sectional areas of 16 immunoreactive cell somata from at least 2 cases each on PND 0, 5, 10, 30, 40 and adult ( > PND 60) were measured. In addition, in 1 adult, the cross-sectional profiles of every immunoreactive cell was drawn in one section midway along the rostral to caudal extent of the DCN. The form factor ( F F = (47ra/p2), where a is somatic cross-sectional area and p is the cell perimeter) for each cell was calculated and a histogram of the form factors was plotted. Form factor is an index of circularity, ranging from an FF of 1 for perfectly round cells to an FF of 0 for flat cells.
175
somewhat more in the deep layer. Several immunoreactive cells can even be found in the cell-sparse molecular layer in each section. Elongated cells with form factors less than 0.7 are less common than rounded cells (Fig. 3) and are present throughout the nucleus although they are frequently found near or within the dorsal acoustic stria (Fig. 2B). Rounded cells (Fig. 2C) are found scattered throughout the nucleus although there is commonly a row of these cells superficial in the fusiform cell layer. Similarly, punctate label is present throughout the DCN. Like immunoreactive somata, its distribution does not conform to a restricted laminar pattern, although in some cases punctate labeling does appear to be slightly heavier in the regions of the fusiform cell and molecular layers.
2.4. Animal welfare The care and use of the animals reported in this study were approved by the University of Louisville and Boys Town National Research Hospital Institutional Animal Care and Use Committees.
A
3. Results
Immunoreactive labeling was present at all ages studied in both species. In addition to a fairly generalized distribution of punctate label, cell somata were distinctly labeled. The punctate label probably represents both transected axons and axon terminals. Elongated labeled processes are also seen, especially in the dorsal acoustic stria where immunoreactive axons are cut lengthwise. All control sections showed minimal, non-specific background staining.
B
3.1. Glycine-like immunoreactivity in the adult hamster DCN In the mature mammalian DCN, 3 laminae may be distinguished by a variety of methods; from superficial to deep, these are the molecular layer, the fusiform cell layer, and the deep layer. In contrast, neither the punctate glycine-like label nor the labeled cell somata obeyed any consistent laminar distribution. In the adult, labeled cell somata are generally small (Fig. 1) in comparison with projection neurons in the nucleus which exceed 200 /xm 2 in cross-sectional area (Schweitzer et al., 1987). The labeled neurons are present throughout the nucleus. The cells do not conform to a laminar distribution (Fig. 2A). From section to section there is variability with respect to the location of the cells; in some sections there are somewhat more cells in the fusiform cell layer and in others there are
C Fig. 5. Composite schematic camera lucida drawings showing the distribution of immunoreactive cell somata at PND 0 (A), PND 5 (B) and PND 10 (C). Observe that in no case do the somata conform to a laminar distribution. All drawings were made at the same magnification, medial is to the left, dorsal is to the top. Scale bar = 40 /zm.
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Fig. 6. Transverse sections through hamster DCN at PND 5. A: immunoreactive cell (arrow) and process (large arrowhead). Note an adjacent process that is labeled (asterisk). B: low-power photomicrograph showing immunoreactive cells, processes and puncta (small arrowheads). Scale bars: A = 25 /xm; B = 30 /xm.
3.2. Glycine-like immunoreactivity in the neonatal hamster DCN On the day of birth (PND 0) immunoreactive cell bodies are present in the DCN (Fig. 4A, B). These cell
bodies appear round (Fig. 2) and smaller (Fig. 1) than at the older ages; other details of cellular morphology (dendrites, axons) are not usually clear at this age. The number of immunoreactive cell bodies present at this age is substantially less than that observed in the adult DCN (Fig. 2)
Fig. 7. Transverse sections through kitten DCN. A: 57 days of gestation (pre-natal day 10). Note immunoreactive axons in the dorsal acoustic stria (arrows). Faint immunoreactive cell bodies are indicated at arrowheads. The very dark profiles are reactive blood cells. B: 57 days gestation (pre-natal day 10). Control section from which the primary antibody was omitted. Arrows indicate an edge artifact. C: 59 days of gestation (pre-natal day 8). Immunoreactive cell bodies are clearly present (arrowheads). D: PND 3. Many immunoreactive cell bodies are present (arrowheads). Axons in the dorsal acoustic stria (to the left of the asterisk) retain the immunoreactivity first evidenced 8 days before birth. An axon in the dorsal acoustic stria projecting into the DCN is indicated by the asterisk. E is a higher magnification view of part of the section shown in D. The same location in D and E is indicated by an asterisk. Scale bars: A,B,D = 40 /.tm; C = 27/zm; E = 13 /xm.
G.H. Riggs et aL/ HearingResearch 89 (1995) 172-180 and large expanses of the DCN in the neonate lack immunoreactive cell bodies (Fig. 5A). Punctate label is present throughout the nucleus at this age. This label probably represents labeled axons, axon terminals and transected axons (Fig. 4C). Some of this punctate label is intensely immunoreactive, while other is less so, although punctate labeling can always be clearly distinguished from background. On PND 5, immunoreactive cell somata are still scattered throughout the nucleus (Fig. 5B); punctate label is present as well. In a few cases, labeled puncta appear to be slightly more concentrated in the superficial DCN (fusiform cell and molecular layers) than in the deeper DCN, but this observation is not consistent across cases or across sections within a case. The number of strongly immunoreactive puncta is somewhat greater on PND 5 than on PND 0. On PND 5 it is sometimes possible to discern the processes of immunoreactive cells (Fig. 6). Cell bodies occasionally present elongated profiles. There is little substantive change in the pattern of immunoreactivity between PND 5, PND 10, and subsequent ages including the adult (Fig. 2, Fig. 5). Although the number of immunoreactive cell somata is higher in the older animals, the density of labeled cell bodies appears somewhat lower on PND 10 (Fig. 5C) likely because the volume of the DCN more than doubles between PND 5 and 10 (Schweitzer and Cecil, 1992). The number of immunoreactive cells and intensely immunoreactive punctate label appears greater in the mature DCN than on PND 10. 3.3. Glycine-like immunoreactivity in the prenatal kitten
DCN In the kitten, immunoreactive axons are present in the dorsal acoustic stria and deep DCN 10 days prior to birth; some hints of lightly immunoreactive cell bodies are present at this time (Fig. 7A,B). Eight days prior to birth, immunoreactive axons are still seen, and immunoreactive cell bodies are clearly defined (Fig. 7C). On PND 3, axons remain darkly labeled, and large numbers of immunoreactive cell bodies are seen throughout the nucleus (Fig. 7D,E).
4. Discussion The present study is one of the first to detail the development of glycine-like immunoreactive labeling in the mammal. Using the Wenthold antibody, a well-known and well-characterized polyclonal antibody to the glycine molecule, we have observed immunoreactive cell bodies and punctate labeling throughout the DCN as early as the day of birth in the hamster and prenatally in the cat. In general, a gradual increase in punctate label and the number of immunoreactive cells is found across ages studied,
177
with mature levels reached by about PND 10 in the hamster, 6 days prior to the onset of function (as defined by the ability to evoke brainstem responses to clicks at 54 d B pSPL, Schweitzer, 1987). Similarly, glycine-like immunoreactivity is seen before the onset of function in the cat. Although a complete developmental profile is not available for the cat, the presence of significant glycine-like immunoreactivity in the cochlear nucleus of the prenatal cat suggests that inhibitory circuits, presumably mediated through glycine, may be organizing relatively long before the first responses to high level airborne sounds are observed developmentally (Walsh and McGee, 1987). This finding is consistent with the view promulgated by Walsh et al. (1990) that inhibitory amino acid receptors and associated ionophores are in place and are operational among the majority of cochlear nucleus neurons in perinatal cats.
4.1. Glycine-like immunoreactivity in the adult DCN In the adult DCN of the hamster, the labeled cells can be classified as small cells (with somata about 196/xm 2 in cross-sectional area) in comparison to the cross-sectional areas of the large projection neurons (about 225 /zm 2 and 270 /.Lm2 for fusiform and giant cells, respectively; Schweitzer et al., 1987). Yet these cells are not as small as the smallest neurons in the nucleus - - the granule cells ( = 30 ~ m 2, L. Schweitzer, unpublished observations). The labeled cells are a variety of shapes but the majority of cells are rounded. In particular, rounded cells are often found in a discontinuous row in the superficial fusiform cell layer. The shape and distribution of these labeled cells suggests that they may be cartwheel neurons, small, rounded interneurons found in this location. Our results from the hamster support the observation summarized in Wickesberg et al. (1994) that there is not a clear, restricted, laminar distribution for glycine-like immunoreactive cell somata or punctate label in the adult. It should be noted that only a few immunoreactive cell somata were found in the molecular layer in each animal. However, it is the case that relatively few cell somata of any kind are contained in the molecular layer, a layer that is commonly referred to as a 'somata-sparse' layer. Thus, the scant number of labeled cell bodies here does not indicate a true distribution which is restricted by layer. Certainly, the distribution of labeled puncta in the molecular layer is comparable to that in the deeper layers.
4.2. Comparison of GAD and glycine development Although colocalization of glycine-and GABA-immunoreactivity appears to be quite common in the adult DCN (Wenthold et al., 1987; Wenthold and Hunter, 1990; Kolston et al., 1992), the present data on development of glycine-like immunoreactivity in the DCN do not bear any obvious relationship to the development of GAD-immuno-
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reactivity. Schweitzer et al. (1993) have shown that the development of GAD begins with a distribution of immunoreactivity in the neuropil of the deep DCN on PND 3, followed by an increase in immunoreactivity in the fusiform cell layer, which in turn is followed by an increase in GAD-immunoreactivity in the molecular layer. No GADimmunoreactive cell bodies are seen until PND 14. At that time, small- to medium-sized cells immunoreactive for GAD are observed in the superficial part of the fusiform cell layer (likely cartwheel neurons) as well as throughout the deep layer. We observe no laminar-related or deep-to-superficial progression in the development of glycine-like immunoreactivity. Rather, punctate label and glycine-like immunoreactive somata appear to be present in a homogeneous distribution throughout the DCN even on PND 0. Thus the pattern of development of GAD- and glycine-immunoreactivity in the DCN of the hamster are very different. Furthermore, the age at which we could successfully label immunoreactive cell somata using the two methods are very different. GAD-immunoreactivity appears late, especially the labeling of somata, which only shortly precedes the age when function was first observed. Glycinepositive somata were found much earlier in both kitten and hamster. This could relate to a true difference in expression of the two inhibitory amino acids, or could relate to the differences in our ability to detect the molecules given the limitations of the methods. Regardless of the reason, the ability to label glycine-like immunoreactivity in somata at such an immature age - - in embryonic kitten and perinatal hamsters - - was an unexpected finding. 4.3. Glycine-like immunoreactivity in the developing D C N
Very early in development glycine-immunoreactivity was found in the DCN of both hamster and kitten although at the early ages the cells labeled in the hamster were considerably smaller than at older ages. This is likely due to cell growth rather than the labeling of different populations at different ages. Measurements done on other cell populations in the DCN suggest that other cells grow during this time and like the labeled cells here, continue to grow even after PND 40 (Schweitzer, 1991). The early presence of inhibitory neurotransmitters in the DCN found in this study, correlates well with physiological data that suggest both excitatory and inhibitory inputs are present early in the development of the nucleus. Long-latency IPSPs are present at the earliest age studied (PND 4) in the immature mouse (Wu and Oertel, 1987; Hirsch and Oertel, 1988). It has also been shown that disynaptic IPSPs in the adult mouse are probably mediated by glycine. Wu and Oertel (1986) observed that the response characteristics of neurons in the DCN were consistent with the action of inhibitory interneurons present within the DCN proper. Furthermore, they used intracellular recordings from slice preparations of the VCN to
show that exogenously applied GABA and glycine could mediate IPSPs. However, only low levels (1 /xM) of strychnine, which binds specifically to glycine receptors, were needed to block all IPSP activity. By comparison, blockers of GABA-mediated inhibition (bicuculline and picrotoxin) were inconsistent in blocking IPSPs even when present at concentrations as high as 100 /xM (see also Hirsch and Oertel, 1988). The logical deduction based on these physiologic data is that glycine could potentially mediate inhibition at an early age in the DCN. Our data demonstrate the presence of glycine-like immunoreactivity in the DCN of the hamster by a gestational age at least equivalent to that reported by Wu and Oertel (1987) and Hirsch and Oertel (1988). Our anatomical data correlate well with data from slice recordings in the mouse and lend support to the hypothesis that glycine, present at an early age, could mediate the inhibition that has been demonstrated physiologically. A recent report by Gleich and Vater (1994) has shown only diffuse glycine-like immunoreactivity in the DCN of the gerbil on PND 1 and PND 2. In contrast to the present results, no immunoreactive cell bodies were reported at that time. By PND 9, some cell somata showed weak glycine-like immunoreactivity. In the young adult (3 months old), the neuropil of the molecular layer and fusiform cell layer showed substantial glycine-like immunoreactivity. Immunoreactive cell bodies were present, were of variable size, and were more prominent in the deep layer and fusiform cell layer. In addition to a neurotransmission function for glycine, the non-transmission roles, if any, of glycine early in the development of auditory function are unclear. Both GABA and glycine appear to produce depolarizing, activating actions in certain embryonic tissue-cultured spinal cord neurons of chicks, with typical inhibitory actions developing slightly later (Obata et al., 1978). Cherubini et al. (1991) reported similar findings among hippocampal neurons, concluding that GABA-mediated depolarizing actions are the dominant form of excitation during development and postulated that the role played by such activity is trophic. Hyson (1991) reported on the depolarizing actions of GABA and Kandler and Friauf (1993) on the depolarizing actions of glycine, on auditory brainstem neurons of vertebrates early in development. In addition, Sanes and Hafidi (1993) found that blockade of glycine receptors produced an increase in dendritic arborizations within the mature superior olive in vitro. These results raise the possibility that 'inhibitory' amino acid actions may be neuromaturational within the auditory system early in its development. Consistent with this changing role, Friauf et al. (1994) have shown that at least two isoforms of glycine receptor exist during development. One is present neonatally in the auditory brainstem, while a second 'adult' isoform becomes predominant on PND 9 (i.e., shortly before the onset of hearing on PND 12). In summary, we have demonstrated that glycine-like
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immunoreactivity is present in a relatively homogeneous distribution throughout the DCN from the day of birth through adulthood in the hamster. Immunoreactivity was found prenatally in the kitten as well. Glycine was detected earlier than GAD, the synthetic enzyme for the other major inhibitory amino acid in the CN. These data add to previously published reports suggesting that glycine may mediate inhibition early in the development of the DCN. It remains to be determined whether it also plays a trophic role in the early auditory system. Acknowledgements The authors gratefully acknowledge Tina Cecil for her expert technical assistance, This work was supported by NIH Grants DC00233 (L.S.) and DC01007 (E.J.W.). References Altschuler, R.A., Betz, H., Parakkal, M.H., Reeks, K.A., and Wenthold, R.J. (1986a) Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res. 369, 316-320. Altschuler, R.A., Hoffman, D.W., and Wenthold, R.J. (1986b) Neurotransmitters of the cochlea and cochlear nucleus: Immunocytochemical evidence. Am. J. Otolaryngol. 7, 100-106. Aoki, C., Milner, T.A., Sheu, K.F., Blass, J.P., and Pickel, V.M. (1987) Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate neurotransmission. J. Neurosci. 7, 22142231. Benson, C.G. and Potashner, S.J. (1990) Retrograde transport of [3H]glycine from the cochlear nucleus to the superior olive in the guinea pig. J. Comp. Neurol. 296, 415-426. Caspary, D.M., D.C. Havey and C.L. Faingold (1979) Effects of microiontophoretically applied glycine and GABA on neuronal response patterns in the cochlear nuclei, Brain Res., 172 179-185. Cherubini, E., Gaiarsa, J.L. and Ben-Ari, Y. (1991) GABA: An excitatory transmitter in early postnatal life. Trends Neurosci. 14, 515-519. Code, R.A. and Rubel, E.W. (1989) Glycine-immunoreactivity in the auditory brain stem of the chick. Hear. Res. 40, 167-172. Friauf, E., Hammerschmidt, B., Kirsch, J., and Betz, H. (1994) Development of glycine receptor distribution in the rat auditory brainstem: Transition from the 'neonatal' to the 'adult' isoform. Assoc. Res. Otolaryng. Abstr., 10. Frostholm, A. and Rotter, A. (1985) Glycine receptor distribution in mouse CNS: Autoradiographic localization of [3H]strychnine binding sites. Brain Res. Bull. 15, 473-486. Frostholm, A. and Rotter, A. (1986) Autoradiographic localization of receptors in the cochlear nucleus of the mouse. Brain Res. Bull. 16, 189-203. Gleich, O. and Vater, M. (1994) Preliminary data on the postnatal development of GABA- and glycine-like immunoreactivity in the gerbil cochlear nucleus. Assoc. Res. Otolaryng. Abstr., 10. Glendenning, K.K. and Baker, B.N. (1988) Neuroanatomical distribution of receptors for three potential inhibitory neurotransmitters in the brainstem auditory nuclei of the cat. J. Comp. Neurol. 275, 288-308. Godfrey, D.A., Carter, J.A., Berger, S.J., Lowry, D.H., and Matschinsky, F.M. (1977) Quantitative histochemical mapping of candidate neurotransmitter amino acids in the cat cochlear nucleus. 1. Histochem. Cytochem. 25, 417-431.
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