THE JOURNAL OF COMPARATIVE NEUROLOGY 3131-16 (1991)
Calbindin-Like Immunoreactivity in the Central Auditory System of the Mustached Bat, Pteronotus parnelli M.L. ZETTEL, C.E. CARR, AND W.E. O’NEILL Departments of Physiology (M.L.Z., W.E.O.) and Neurobiology and Anatomy (C.E.C.), University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT With the aid of a polyclonal antibody specific for Calbindin D-28k, we studied the distribution of this calcium-binding protein in the central auditory system of the mustached bat, Pteronotus parnelli. Components of the cochlear nucleus (CN) that were calbindin-positive (cabp(+)) included the root of the auditory nerve, multipolar and globular bushy cells in the anteroventral CN, multipolar and octopus cells in the posteroventral CN, and small and medium-size cells in the dorsal CN. Not stained were spherical bushy cells of the anteroventral CN and pyramidal/fusiform cells in the dorsal CN. In the superior olivary complex, labeled cells were found in the lateral and medial nuclei of the trapezoid body, the ventral and ventromedial periolivary nuclei, and the anterolateral periolivary nucleus. No cellular labeling was seen in the lateral superior olive. In the medial superior olive, only marginal cells were cabp( +). Labeled fibers could be seen surrounding the ghosts of unlabeled cells in both the latter nuclei. Most cells in the intermediate nucleus and the columnar division of the ventral nucleus of the lateral lemniscus were cabp(+). However, the dorsal nucleus was cabp(-). A group of cabp( +) cells was also seen in the paralemniscal zone. The inferior colliculus had a relatively low density of cabp( +) cells. Labeled cells were more common in the caudal half of the central nucleus, and in the external nucleus and dorsal cortex. In the auditory thalamus, nearly every cell in the medial geniculate body was cabp(+), but those in the suprageniculate nucleus and in the posterior group did not stain. Small cells in the intermediate layer and giant cells in the deep layers of the superior colliculus were densely cabp(+). In the pons, cabp(+) cells and neuropil could be seen in the medial and lateral pontine nuclei (pontine gray). In conclusion, calbindin-like immunoreactivity was found in most of the brainstem auditory system, as well as in regions associated with acoustic orientation or control of vocalization. However, except for a minority of cells of the medial superior olive, it is conspicuously absent from the nuclei receiving binaural input below the level of the inferior colliculus. Key words: Calbindin D-28k, immunocytochemistry, brainstem, auditory system
Calcium-binding proteins of the calmodulin super-gene family have been found in specific cellular populations in many regions of the nervous system. Although the number of different calcium-binding proteins and their functions are still unclear, some have been linked to circuitry with high activity levels and metabolic demands, such as the auditory system. The calmodulin family of calcium-binding proteins contains, among others, calmodulin, parvalbumin, calbindin, and calretinin. Calbindin D-28k has been proposed to function as a cytosolic buffer for calcium ions in neurons that have high rates of discharge (Baimbridge et al., ’82). Q
1991 WILEY-LISS, INC.
Calbindin-like immunoreactivity has been reported in neurons of several sensory systems which have the ability to preserve temporal information with great precision (Carr, ’86). These include the electrosensory system of certain gymnotid fish (Maler et al., ’84) and the “timepathway” of the auditory system of the barn owl (TakaAccepted July 26,1991. Address reprint requests to William E. O’Neill, Dept. of Physiology, Box 642, Univ. of Rochester Medical Center, Rochester, NY 14642-8642. C.E. Carr is now a t the Dept. of Zoology, University of Maryland, College Park, MD 20742.
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hashi et al., '87). Neurons in these systems preserve in their firing patterns the phase of the electrical or acoustic stimulus, respectively. Thus it has been hypothesized that calbindin might be an important biochemical component of neurons that preserve temporal information (Maler et al., '84; Carr, '86). Unfortunately, the antibodies directed against calbindin that were used in the above studies also recognize the closely related 29 kD protein, calretinin (Rogers, '87; Carr, in preparation). This finding has confounded interpretations of the role and distribution of calbindin. It is now certain that much of the calbindin-like immunoreactivity reported by Takahashi et al. ('87) in the barn owl is attributable to calretinin (Rogers, '87). Rogers used both RNA blots and in situ hybridization to show that the cochlear nuclei and the nucleus laminaris express calretinin RNA. The calbindin-like immunoreactivity in the nucleus laminaris of the barn owl similarly appears to be attributed to calretinin (Carr, in preparation). Recently, studies have been published describing specific calbindin-like immunoreactivity in the feline and rodent superior olivary complexes (Matsubara, '90; Webster et al., '90). We have used a polyclonal antibody for calbindin that does not recognize calretinin (Buchan and Baimbridge, '88) to describe the distribution of this protein in the entire auditory brainstem and diencephalon of the mustached bat, Pteronotus pamelli, including regions related to vocal motor control. The mustached bat auditory system has been extensively investigated, and much is already known about the pathways that process specific acoustic cues important for echolocation (Zook and Casseday, '82a,b; Ross et al., '88; Frisina et al., '89; O'Neill et al., '89; Ross and Pollak, '89). In some of these pathways preservation of temporal information is thought to be important, but probably not for the processing of binaural cues. Rather, they process temporal information related to the estimation of distance to a target. Although calbindin immunoreactivity is widely distributed throughout the auditory system of the mustached bat, we found that in the brainstem it is primarily associated with monaural path-
ways and monaural afferents to nuclei receiving bilateral input.
METHODS The primary antiserum used in this research was a polyclonal antibody against rabbit antimonkey cerebellar calbindin, generously provided by Dr. Kenneth Baimbridge of the University of British Columbia (Canada). It has been found to detect calbindin in the nervous tissue of all mammals tested, including monkey, baboon, and human. In both one- and two-dimensionalgel electrophoresis immunoblots, this antibody detects a single band at 28-kD, unlike those antibodies raised against chick gut vitamin D-dependent calbindin, which appear to detect 27 kD and 29 kD bands (Buchan and Baimbridge, '88). Immunoprecipitation of rat cerebellar soluble proteins with this antibody followed by PAGE also yields a single 28 kD band (Buchan and Baimbridge, '88). In this report, structures showing calbindin-like immunoreactivity t o this antibody will be referred to as cabp(+). Results reported in this study were obtained from eight mature Jamaican mustached bats (Pteronotus parnelli) with one additional bat used for cresyl violet-stained reference sections. All bats were given an overdose of sodium pentobarbital (Nembutal60 mg/kg), exsanguinated with 30 ml phosphate-buffered saline (PBS; pH 7.4) and perfused intracardially with 100 ml filtered 4% paraformaldehyde containing 0.1 M PBS. The brain was postfixed for a minimum of 4 hours and cryoprotected by sinking in successive 10% and 30% sucrose/PBS solutions. The brain was placed in a small plexiglass chamber and aligned such that the superior surfaces of the cerebral cortex and cerebellum were parallel to the long axis of the chamber (standard stereotaxic position; Schuller et al., '86). The brain was embedded in fixed egg yolk, frozen in dry ice, and 36- or 50-pm sections were cut on a sliding microtome. The sections were stored in PBS at 4°C for a maximum of 24 hours prior to the immunohistochemistry reactions.
Abbreviations ALD ALP0 AN AVa AVCN AVm AVP cabp CBL CFiFM CN CTX DC DCN DMPO DNLL DPD EE EI EO EX
FM HP ICC IC INLL LL LNTB
anterolateral division of ICc anterolateral periolivary nucleus (=NCAT) auditory nerve anterior division of AVCN anteroventral CN medial division of AVCN posterior division of AVCN calbindin cerebellum constant frequencyiFM cochlear nucleus cerebral cortex dorsal cortex of the IC dorsal CN dorsomedial periolivary nucleus dorsal nucleus of the lateral lemniscus dorsoposterior division of ICc binaural excitatory contra excitatoryiipsi inhibitory contralateral monaural excitatory external nucleus of the IC frequency modulated hippocampus central nucleus of the IC inferior colliculus intermediate nucleus of the lateral lemniscus lateral lemniscus lateral nucleus of the trapezoid body
LPN LSO LT MD MGB MNTB MPN MSO NCAT NL PG PLZ PN PO PVCN, PV PVl PVm PVO
sc
SG SOC TB VMPO VNLL VNLLd VNLLV WO
lateral pontine nucleus lateral superior olive lateral tegmental zone (=PLZ) medial division of the ICc medial geniculate body medial nucleus of the trapezoid body medial pontine nucleus medial superior olive nucleus of the central acoustic tract (=ALP01 nucleus laminaris periaquaductal grey perilemniscal zone (=LT) pontine nuclei posterior group of thalamic nuclei posteroventral CN lateral division of PVCN medial division of PVCN caudal division (octopus cell region) of PVCN superior colliculus suprageniculate nucleus of the thalamus superior olivary complex trapezoid body ventromedial periolivary nucleus ventral nucleus of the lateral lemniscus dorsal division of the VNLL ventral division of the VNLL ventral periolivary nucleus
CALBINDIN IN THE BAT AUDITORY SYSTEM
Immunohistochemistry
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of a homogeneous population of small (10 pm) spherical bushy cells (Zook and Casseday, ’82a). These cells are not Sections from entire brains, or in some cases alternate immunoreactive but are embedded in densely stained neusections, were incubated in diluted (1:700) primary antiseropil and circumscribed by immunoreactive terminals (Fig. rum for 24-48 hours at 4°C. The secondary was a protein A 1F). When viewed with Nomarski optics, these large end(obtained from R. Sloviter, Helen Hayes Hospital, West ings appear to be end bulbs of Held from AN fibers. Haverstraw, NY) used at the recommended dilution of The posterior division of the AVCN (AVp; Fig. 1A) is a 1:400 and incubation time of 1hour. Both the primary and heterogeneous mixture of cells. One highly immunoreactive secondary antisera were diluted in PBS containing bovine cell type is a large (15-20 pm) round-to-ovoid cell found in serum albumin (BSA) and 0.1 to 0.3%Triton X-100 (both Sigma). The sections were further processed according to highest concentrations ventrally, intercalated within the AN fibers (Fig. 1E). Cell size, location, and the the avidin-biotin method (Vector Laboratories) with horse- ascending presence of an eccentrically placed nucleus (difficult to radish peroxidase (Sigma type VI)/diaminobenzidine hydrochloride (Sigma) histochemistry. In all cases except one, the observe in photomicrographs) indicate that these are globustandard Vector Avidin-Biotin kit was used, with good lar (large bushy) cells. Immunoreactive multipolar cells of results being obtained with the suggested dilutions. The various sizes (7-15 pm) are found throughout AVp (Fig. other set of sections was processed with thevector Elite kit, lC,H). Immunoreactive terminals are present on both which resulted in more sensitive staining but with higher stained and unstained cells (Fig. 1C). The marginal division (AVm; Fig. 1A) is unique to background. Metal intensification (1,250 mg of Nickel (11) Sulfate added to 50 ml Imidazole/Na Acetate buffer contain- mustached bats and can easily be distinguished in the ing 20 mg DAB) was used in all except two cases to improve caudal two-thirds of AVCN (Zook and Casseday, ’82a). It is the contrast of the staining. Both intensified and noninten- composed almost entirely of large multipolar cells (15-20 sified sections yielded identical staining patterns, with km) distinctly separate from other regions of AVCN. These immunoreactive neurons characterized by blue (metal- are the largest multipolar cells found in AVCN and are intensified) or brown reaction product within the perikaryal densely packed. These cells were highly immunoreactive cytoplasm. Those sections not treated with immunore- and were found nested among dark fibers, presumably AN agents were counterstained with cresyl violet, and all afferents and/or efferents leaving AVCN (Fig. 1G). Due to sections were dehydrated and coverslipped. Cell types were the intensity of cellular and fiber staining, it was not identified according to previously published studies as well possible to determine if labeled terminals were present on as personal consultation with Drs. J.M. Zook and R.D. these cells. The PVCN (“PV” in Fig. 1A) may be distinguished from Frisina. Serial dilution controls were performed with primary other divisions of the cochlear nucleus by the presence of a antiserum diluted 1:250, 1500, 1:700, 1:1,000 and 15,000, very darkly stained neuropil, comprised of dense plexuses of with the 1:700 dilution giving the best results. Control sec- immunoreactive fibers cut in cross section in the transverse tions were treated with identical procedures to those above view. These fibers are part of the descending branch of the except for the elimination of the primary antiserum. Non- auditory nerve. Medium-size (15 pm), cabp(+) multipolar specific staining was not evident in these sections. Monkey cells (Fig. 11) are found in both lateral (PVl) and medial cerebellar 28 kD calbindin was unavailable for protein (PVm) divisions, and small immunoreactive multipolar reabsorbtion controls, but the antibody had been exten- cells (10 pm) are also found in the medial division. Many of these multipolar cells have a n elongate appearance and run sively tested by Dr. Baimbridge prior to our experiments. in rows parallel to the fiber tract separating medial and lateral divisions. RESULTS The caudal division of PVCN, the octopus cell region Cochlear nucleus (PVo; Fig. 2A), is prominently cabp(+). The ovoid octopus As in other mammals, the cochlear nucleus (CN; Figs. 1, cells (15-20 pm) are immunoreactive (Fig. 2D). The fiber 2) is divided into three regions, each innervated by a plexuses are very densely stained and are oriented in a parallel branch of the auditory nerve (AN).The anteroven- variety of directions. Three of the four immunoreactive cell types contained in tral cochlear nucleus (AVCN; “AV” in Fig. 1A) receives input from the ascending branch of the AN, whereas the the DCN (Fig. 2A) are found along its lateral edge. In the descending branch divides to innervate the posteroventral mustached bat, DCN does not have the distinct laminar cochlear nucleus (PVCN; “PV” in Figs. lA, 2A) and the organization found in some other mammals, so the positive dorsal cochlear nucleus (DCN; Fig. 2A). The AVCN and the identification of cell types by location is not always possible. FVCN are prominent in the mustached bat, whereas the We therefore measured the perikaryon areas of both cabp(+) DCN is not. The AN fibers are immunoreactive, although and cabp(-) cells, and compared them to the areas of their level of staining is masked to a large extent by the projection neurons retrogradely labeled by HRP injections surrounding unstained myelin (Fig. lD,E). Figure 1D shows into the inferior colliculus in a previous series of experibundles of stained fibers oriented approximately perpendic- ments (Frisina et al., ’89).Cabp(+) cells were significantly ular to the incoming AN fiber tract. The bundles are smaller than either the cabp(-) cell “ghosts” outlined by presumably bifurcating AN fibers coursing dorsally and immunoreactive fibers in the deep DCN (Fig. 2F), or cells ventrally to innervate the AVCN and PVCN, respectively. projecting to the IC taking up HRP. Most lateral are very The fibers are more clearly visible as they enter the CN small (5-7 pm) immunoreactive cells that correspond to small cells and granule cells (Fig 2E). Just medial to these (compare C and D in Fig. 1). The AVCN (Fig. lA,C,E-H) contains immunoreactive small cells are densely cabp(+), medium-size cells (10-15 fibers in addition to stained cells and terminals. The pm) with dendrites extending out in various directions (Fig. staining intensity and pattern of immunoreactivity vary 2C). Some of these cells can be seen to terminate on large from one region of AVCN to another, and its divisions are cabp(-) ghosts (15-20 pm) in the adjacent region of the readily distinguished. The anterior division (AVa) consists DCN. They resemble cartwheel cells in the rat described
Figure I
CALBINDIN IN THE BAT AUDITORY SYSTEM
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F
l5
Fig. 2. Cabp immunoreactivity patterns in caudal CN. A. Caudal CN at the level of the octopus cell region (PVo) of PVCN and the DCN. B. Camera lucida drawing of hemisection associated with photomicrographs and drawings D-F. C. Photomicrograph of cabp(+) multipolar cells in the superficial layers of DCN, taken from a different section than that shown in A. These may be cartwheel cells (see text). D. Cabp(+) octopus cells of caudal PVCN. E. Densely immunoreactive small and granule cells of DCN. F.Camera lucida drawing of densely stained terminals surrounding ghosts of cabp(-) cells in deep (polymorphic) layer of DCN.
Fig. 1. Cabp immunoreactivity patterns in rostral cochlear nucleus (CN). A. Rostra1 CN showing anterior (AVa), posterior (AVp), and medial (AVm) divisions of AVCN, stump of auditory nerve (AN),and PVCN (PV). B. Camera lucida drawing of section from which A, C, E-I were taken. Labeled areas C, E, F, G , H, I in section shown in A mark regions enlarged in like-laheled accompanying figures. These and all subsequent figures were made from 50-km thick sections and have a similar format. C. Large cabp(+) terminal (open arrow) ending on unstained cell in AVp. Solid arrows indicate bundle of cabp(+) ascending AN fibers. D. Enlargment of the stump of the AN taken from different section, which showed better the stained AN fibers entering the CN and presumably turning in bundles to innervate the AVCN and PVCN. E. Cabp(+) cells embedded in AN fibers in the interstitial nucleus. F. Camera lucida drawing of a cluster of three cahp(-) small spherical bushy cells of AVa with cabp(+) end bulb-like terminal. G. Large cabp(+) multipolar cells of AVm. H. Intensely stained globular bushy cell and smaller multipolar cells of the AVp embedded in eabp(+i fibers, bordering the stump of the AN. I. Medium-size cabp(+) multipolar cells of the PVl.
with the Golgi technique (Wouterloud and Mugnaini, '841, and their projection to deeper lying cabp(-) cells, as well as their calbindin immunoreactivity, support this suggestion (Mugnaini et al., '87). Unfortunately, previous studies of mustached bat CN have not differentiated cartwheel cells as a distinct class (Zook and Casseday, '82a), so our identification of these cells is tentative. The remaining DCN contains unstained ghosts (15-20 pm) surrounded by fine immunoreactive fibers and terminals (Fig. 2F). These ghosts are similar in size to HRPlabeled projection neurons and are assumed to be pyramidal/ fusiform cells of the DCN. A few darkly stained, very large ( 20 pm) multipolar cells are also seen along the medial border of DCN adjacent to the acoustic stria (Fig. 2A). It is unclear whether these are part of DCN or a caudal extension of AYm. Zook and Casseday ('82a) also noted large multipolar cells along the medial edge of DCN.
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M.L. ZETTEL ET AL.
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Superior olivary complex Medial superior olive (MSO). Only one cabp(+) cell type, the marginal cell, was found in MSO. These large ( 15 pm) multipolar, or asymmetric bipolar, neurons are most highly concentrated along the medial margin of this nucleus (Fig. 3A). Few stained cells are found within the central core of the nucleus, which is occupied mostly by unstained cells outlined by immunoreactive endings (Fig. 3D). Lateral superior olive (LSO). The LSO is easy to distinguish in the reacted sections due to the heavily stained fiber plexus surrounding the nucleus (Fig. 3A). The nucleus has no immunoreactive cells, but, as in the MSO, many ghosts surrounded by immunoreactive terminals are seen. These terminals may arise from immunopositive cells of the medial nucleus of the trapezoid body (MNTB; see below). Periolivary nuclei. Some of the periolivary nuclei are also cabp(+). Cabp(+) cells include the large multipolar neurons (15 pm) of the anterolateral periolivary nucleus (ALPO, or nucleus of the central acoustic tract-NCAT; Casseday et al., '89; Fig. 3A,C); the large multipolar and the medium elongate cells (12-15 pm) of ventromedial and ventral periolivary nuclei (VMPO and VPO; Fig. 3A,E); and, at the lateral edge of the LSO, darkly stained cells (15 pm) of the lateral nucleus of the trapezoid body (LNTB; Fig. 3A). Cells of the dorsomedial periolivary nucleus (DMPO) and the interstitial nucleus (together comprising the olivocochlear bundle; Bishop and Henson, '87) were cabp( -). Medial nucleus of the trapezoid body (MNTB). The MNTB is highly immunoreactive to cabp-antibody (Fig. 4). Densely packed, medium-size (12-15 pm) cabp( +) principal cells are seen among a network of large caliber, cabp(+) fibers (Fig. 4A). Large cabp(+) terminals are present on many of the cells (Fig. 4B). Unfortunately, the density of somatic staining obscured the detailed morphology of these large endings. The drawing illustrating the terminals in Figure 4B underestimates the degree to which these endings envelope the cell body. Because we cannot clearly determine the boundary between ending and cell body in this material, we are uncertain whether to identify these terminals as calyces of Held. Calycean endings are the only known large terminal specializations on MNTB cells. They arise from axons of globular cells originatingin the contralatera1 AVp. Globular cells are highly cabp( +) (Fig. lE), and this makes it likely that the heavily labeled fibers and endings found in the MNTB arise from them. Cabp(+) fibers of the trapezoid body (TB) can be seen as a latticework running through the floor of the brainstem (Fig. 4A). As was the case for the auditory nerve, the labeling of these fibers was interrupted and partially obscured by myelin. The perisomatic labeling around cabp(-) ghosts in the LSO may arise from cabp(+) terminals of MNTB cells (Zook and DiCaprio, '88). Additionally, at least some of the cabp(+) terminals found in the VNLLv (see below) may also originate from the MNTB (Kuwabara et al., '89).
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Lateral lemniscus The lateral lemniscus of the mustached bat contains three distinct nuclei, which are easily differentiated using this technique (Fig. 5).The ventral nucleus (VNLL) and the intermediate nucleus (INLL) contained immunopositive cells (Fig. 5A), whereas the dorsal nucleus (DNLL) did not (Fig. 5C).
Ventral nucleus. Based on the organization of its cells, the VNLL has been divided into two regions (Zook and Casseday, '82a), each of which stains differently with this antibody. The dorsal region (VNLLd) is composed of small (10 pm) multipolar cells not arranged in any particular anatomical orientation. Some labeled 4 s .andhrminals are found in this region (Fig. 5A). The ventral region (VNLLv) has a distinct columnar arrangement of small multipolar cells (Fig. 5A), which are uniform in appearance and tightly packed lengthwise along the darkly stained fibers of the lateral lemniscus. These cells are numerous and very immunoreactive (Fig. 5D). Many delicate immunoreactive fibers surround the somata of these cells. These may be collaterals of globular cell afferents to the MNTB (Figs. l E , 4B; Zook and Casseday, '85), or, as mentioned above, the axons of labeled MNTB cells themselves (Spangler et al., '85; Kuwabara et al., '89). Intermediate nucleus. The INLL contains a heterogeneous population of cells, some of which are cabp(+) (Fig. 5A). The dorsolateral tip of the nucleus contains many darkly stained, very small (5-7 pm) round-to-ovoid cells dispersed among immunoreactive fibers, which appear to terminate on these cells (Fig. 5B). Cabp( +) medium-size ovoid cells (10-12 pm) are distributed irregularly throughout the rest of the nucleus. There are also large (15 pm) elongate immunopositive principal cells whose dendrites tend to r u n in a mediolateral direction. Cabp(-) cells surrounded by immunoreactive terminals are visible here as well. Darkly stained fibers running both mediolaterally and dorsoventrally can be seen (Fig. 5B). Dorsal nucleus. The DNLL (Fig. 5C) is visible using this technique because it forms such an obvious void within the otherwise richly immunopositive lateral lemniscal complex. The cells of DNLL are cabp(-), and no endings are visible on them. Paralemniscal zone. Another region of darkly stained cabp( +) cells (10-15 pm) is found just medial to the DNLL. This area may be the same as that refered to as the lateral tegmental (LT), or paralemniscal zone (Fig. 5A; Covey et al., '87). In horseshoe bats, cells in LT have been shown to be responsive to auditory stimuli, and many also become active just before and during vocalization of sonar pulses (Metzner, '89). Electrical stimulation in the vicinity of LT elicits sonar vocalizations as well (Schuller and RadtkeSchuller, '90). In the mustached bat, cells in this area receive projections from the deep layers of the superior colliculus (Covey et al., '871, where we also found very large cabp( + ) cells (see Fig. 8D).
Inferior colliculus The inferior colliculus (IC; Fig. 6 ) of the mustached bat is comprised of the central nucleus (ICc), the external nucleus
Fig. 3. Cabp immunoreactivity in the superior olivary complex (SOCI. A. Low power view including LSO, MSO, LNTB, ALPO, W O , VMPO. Note cabp(+) cells in all except the LSO, where only perisomatic cabp(+) profiles can be seen. Labeled cells in MSO are found mostly along medial edge of the nucleus, orthogonal to isofrequency laminae in the core of the nucleus. B. Drawing of section. C. Cabp(+) cell of ALPO with characteristic thick dendrite oriented orthogonally to ascending lemniscal fibers, many of which are also cabp(+). D. Detail of ventral (high frequency) portion of MSO, showing one of scattered population of cabp(+) cells, surrounded by ghosts of cabp(-) cells encircled by immunoreactive terminals (arrows). Diffuse grey blobs are cabp(+) cells lying out of plane of focus. E. Intensely stained large cells and neuropil of the VMPO.
Figure 3
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M.L. ZETTEL ET AL.
Fig. 4. Floor of the brainstem showing the medial nucleus of the trapezoid body (MNTB) (A, C). In A, dense lattice work of cabp(+) trapezoid body fibers course between the intensely labeled cells of the
MNTB on each side. Note cabp(-) pyramidal tracts lying below MNTB. B. Camera lucida drawing of labeled axons terminating on cabp(+) MNTB cells. C. Drawing of section.
(EX), and the dorsal cortex (DC, after Morest and Oliver, '84; formerly pericentral and dorsomedial nuclei). The mustached bat ICc is subdivided into dorsoposterior (DPD), anterolateral (ALD), and medial (MD) divisions, based on cytoarchitecture and frequency representation (Zook and Casseday, '82a; Zook et al., '85; O'Neill et al.,'89). Zook et al. ('85) showed that the ICc contains various size multipolar cells. Immunoreactive cells (7-18 km) were found in each of the three divisions of ICc (Fig. 6A), but the lack of
consistent dendritic labeling made it difficult to make further distinctions among cell types. From serial cross sections o f the IC from a single bat, labeled cells were counted on one side only at intervals of 100 km. Out of a total of 1,305 cells that could be considered immunopositive, 1157 (89%)were labeled at a level barely above background, when compared with labeled cells in the brainstem of this preparation. Only 148 (11%) were moderately to heavily labeled (i.e., comparable to that
CALBINDIN IN THE BAT AUDITORY SYSTEM seen in cells of the VNLLv). Rostrally in the ALD, lightly and heavily labeled cells were evenly dispersed. Progressing caudally, however, the number of heavily labeled cells declined. They were more commonly found near the midline at the dorsal tip of MD, bordering the commissure of the IC, as well as scattered about the external nucleus. By contrast, lightly labeled cells were distributed throughout the IC. The highest densities of labeled cells were found in the caudal half of the IC, at the level of, and posterior to, the collicular commissure. Overall, however, the level of cellular labelling was not striking, especially when compared to the medial geniculate body (see Fig. 7). The ALD and DPD, which are roughly similar in volume (O'Neill et al., '89), contained nearly the same number of labeled cells (38%and 33%,respectively, of the total), whereas MD contained 22%. Cabp(+) cells are also found in EX and DC; in the latter, there is a greater concentration of labeled cells and a more darkly stained neuropil dorsomedially, within the fibers of the collicular commissure. The IC is notable (especially when compared with the medial geniculate body; cf. Fig. 7) for the extensive labeling of a fine neuropil (Fig. 6B-D). Concentrations of cabp(+) fibers could be seen demarcating the different divisions of the ICc, and separating ICc from DC and EX. In the very deep, lateral, and caudal part of the ALD, heavily labeled lateral lemniscal fibers envelope a few stained, and many unstained, cells (Fig. 6C). The staining of neuropil seemed most dense in the ALD and least dense in DPD, with MD somewhere between the two.
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trolling the larynx (Schweizer et al., '81; Rubsamen and Betz, '86; Rubsamen and Schweizer, '86). The pontine nuclei make connections to the cerebellar cortex, and electrical stimulation suggests they play a role in modifying the sonar pulse (Schuller and Radtke-Schuller, '90).
DISCUSSION
Functional markers and calcium-binding proteins. The differential distribution of calcium-binding proteins in the central auditory system raises the intriguing question that similarly labeled structures share a functional organization. Therefore, before considering the patterns of labeling in detail, we first address the problem of cross-reactivity. As discussed below, the calbindin antibody used in this study shows a clear association with the monaural nuclei of the auditory brainstem. This association does not, however, address the nature or the role of the antigen(s). The antibody we used has been reported to be monospecific for monkey cerebellar calbindin (Buchan and Baimbridge, '88). Recent studies have shown, however, that antisera against calretinin and calbindin cross-react (Rogers et al., '89; Resibois et al., '89). Resibois et al. ('89) used antibodies against calbindin and calretinin, absorbed with calbindin and calretinin, to differentiate between the two patterns of immunoreactivity in the auditory nuclei of the rat. Although reported only briefly, a comparison of their results with those of this study yields both similar and interesting observations. They found both calbindin and calretinin immunoreactivity in the auditory nerve. They also found Auditory thalamus both calbindin and calretinin immunoreactive neurons in Compared with the IC, the medial geniculate body (MGB) the ventral cochlear nucleus but only calbindin immunoreis extremely immunoreactive with a high density of cells activity in the MNTB and the nuclei of the lateral lemnis(6-15 km) staining in all three (ventral, dorsal, medial) cus. In the absence of the calretinin antibody and purified divisions (Fig. 7). It is clearly differentiated from the calbindin and calretinin, we must assume that the similaradjacent thalamic areas, which were largely cabp(-), includ- ity of the staining patterns in our study and that seen by ing both the suprageniculate nucleus and the posterior these authors supports the contention that the calbindingroup (SG and PO, respectively; Fig. 7B,C). Faintly labeled like immunoreactivity observed is truly attributable to fibers could be found in the suprageniculate, consistent calbindin, with the possible exception of some cells in the with the projection it receives from the cabp(+> NCAT cochlear nucleus. However, immunoreactivity in the audi(ALPO). The dense fibers characteristic of the dorsal tory nerve and cochlear nucleus may be attributable to the division ("D" in Fig. 7A-C) are cabp(+) (Fig. 7B,D). In the co-localization of both types of calcium binding proteins. ventral division ("V" in Fig. 7A,B), which is the principal Calbindin not localized to nuclei receiving bilateral (but not exclusive) target of collicular efferents, the neuro- input in the brainstem. Antibodies directed against calpi1 is lightly labeled. bindin label a wide variety of structures in the auditory system of the mustached bat. Our major finding is that Superior colliculus, pons below the level of the IC, calbindin immunoreactivity is The superior colliculus (SC)showed cabp immunoreactiv- conspicuously absent from cells in nuclei known to process ity throughout its depth (Fig. 8A). Especially prominent binaural information. For example, the principal cells of the were small, 6-8 pm diameter cabp( +) cells in the intermedi- LSO and DNLL were universally cabp(-), although in the ate layer (stratum griseum, Fig. 8B) and giant cells (20-25 former they were surrounded by cabp( +) endings. Whereas pm) scattered throughout the deep layers (stratum griseum/ previous studies using this same antibody or calbindinstr. album; Fig. 8D). The layers of SC below stratum specific antibodies from different sources have found that opticum are noted in mammals for the presence of multimo- all cells in the MSO are cabp(-) (cat, Matsubara, '90; rat, dal cells sensitive to the location of auditory, visual, and Celio, '90; Webster et al., '90; chinchilla, Kelley et al., tactile stimuli (Wickelgren, '71; Drager and Hubel, '71.. submitted), we found that some cells in the mustached bat Knudsen, '82; Meredith and Stein, '83; Middlebrooks and MSO were cabp( +). However, this population of immunoreKnudsen, '84; Shimozawa et al., '84; Wong, '84; Palmer and active cells was greatly outnumbered by cabp(-) cells and was confined mainly to the medial and lateral margins of King, '85; Wise and Irvine, '85; Jay and Sparks, '87). We also observed extensive labeling of cells in both the the long axis of the nucleus. By their position in the MSO, medial (MPN) and lateral (LPN) pontine nuclei (Fig. 8E). their size, and dendritic orientation, we have identified These cells (10-15 km) receive substantial input from the them as marginal cells. Recent evidence from electrophysiological experiments ICc (Schweizer, '81; Frisina et al., '89), SC (Covey et al., '87), and auditory cortex (Olsen, '86) and connect to the has revealed the responses of cells in the mustached bat nucleus ambiguus, which contains the motorneurons con- LSO and MSO to binaural acoustic stimulation (Covey et
CALBINDIN IN THE BAT AUDITORY SYSTEM al., '91). Confirming earlier anatomical studies (Zook and Casseday, '82b; Ross et al., '88; Frisina et al., '891, Covey and colleagues found that the tonotopic axes are oriented along the long axes of both nuclei, with low frequencies represented laterally in the LSO and dorsolaterally in the MSO and high frequencies at the respective opposite poles. Except for the difference in the tilt of the MSO in the brainstem and an enormous overrepresentation of the dominant 60 kHz component of the species echolocation call, this is the same pattern seen in the cat (Adams, '79; Brunso-Bechtold et al. '81; Henkel and Spangler, '83; Aitkin and Schuck, '85; Yin and Chan, '90). As in the cat, Covey et al. found that cells in the mustached bat LSO were binaural, with the ipsilateral input being excitatory, and the contralateral input inhibitory (IE response type). With regard to the MSO, previous anatomical studies (Zook and Casseday, '85) had shown that it receives bilateral inputs from the same region of the cochlear nucleus (namely, the AVa) that projects to the MSO in other mammals. This observation suggested that, like the LSO, the MSO also would contain binaural neurons, but these authors pointed out that the input was unusual when compared to other mammals in that it was dominated by the contralateral side. Although the data are scant regarding the binaural properties of the mammalian MSO, most cells have been reported to be excited by stimulation to either ear (i.e., EE). Contrary to expectation, however, Covey and colleagues found that 79% of the cells in the mustached bat MSO were not binaurally excited. Rather, they were excited by the contralateral ear, but neither excited nor inhibited by the ipsilateral ear; i.e., they were monaural (EO). Only 7% of MSO cells were EE; 11% were EI. In hindsight, this surprising result was not unanticipated, as Ross and Pollak ('89) had previously shown a strong projection from the MSO to a monaural region of the dorsoposterior division of ICc. Based on these data, we must assume that many, if not all, the cabp(-) cells found in the MSO are functionally monaural. In the LSO, the perikarya of the unstained cells are surrounded by densely labeled terminals, whereas there is no apparent staining around dendrites. This pattern is consistent with the origin of afTerents to these cells: the dendrites receive their input from cabp( -) spherical cells of the ipsilateral AVa, whereas the cell body receives its inputs from the cabp(+) ipsilateral MNTB (Cant '84; Zook and DiCaprio, '88; Kuwabara et al., '89). The perikarya of some cabp(-) MSO cells were also surrounded by cabp(+) profiles. It is unlikely that these terminals arise from spherical bushy cells of the AVa, which project to the MSO, as they were cabp( -). The recent studies in the mustached bat by
11
Kuwabara and Zook ('90) and previous studies in the cat (Spangler et al., '85) have instead shown that axon collaterals of MNTB cells project to MSO somata, as in the LSO, and this projection is the likely source of these profiles. Considering the recent results of Covey et al. ('91), one would predict that the cells showing such cabp(+) profiles would have binaural interactions of the IE type. However, it is problematic that no binaural interactions of this type were reported in their study. Although there is no direct physiological evidence in bats that the DNLL receives binaural inputs, its connections to the LSO and MSO are highly suggestive that this is likely the case. As for the LSO and most of the MSO, the DNLL is conspicuous by the complete lack of calbindin-like immunoreactivity in cell bodies. The lack of cabp(+) endings is consistent with the observation that DNLL receives nearly all its input from the LSO and MSO. To summarize then, below the level of the IC, cells within nuclei which either receive bilateral input from monaural sources (i.e., the LSO and MSO) or bilateral input from binaural sources (i.e,, the DNLL) are mostly devoid of calbindin-like immunoreactivity. By contrast, cabp immunoreactivity is apparently widely distributed in monaural nuclei. One might be tempted to hypothesize a link between calbindin and monaural cells in general, were it not for the fact that the majority of cells in the mustached bat MSO are reported to be monaural, and our results show that most (but not all) MSO cells are cabp(-). In this regard, the mustached bat MSO differs from that in other mammals so far studied in that at least some cells are cabp(+). If the mustached bat MSO can be considered atypical, it may be possible to formulate a general hypothesis for a functional relationship between calbindin and the physiology of auditory brainstem neurons. This hypothesis is that calbindin is associated with monaural, but not binaural, pathways in the mammalian auditory brainstem. A correlary of this hypothesis is that binaural pathways might contain a different species of calcium binding protein. Further study is clearly needed on this issue. Comparison to findings in burn owl. Contrary to the findings in mammals, previous studies in the barn owl have reported extensive calbindin-like immunoreactivity in binaural nuclei in the brainstem, including nucleus laminaris (NL), the terminal fields of NL in the anterior division of the ventral lateral lemniscus, the superior olive, and the central nucleus of the inferior colliculus (Takahashi et al., '87). Since NL is homologous to the mammalian MSO, the results of Takahashi et al. ('87) are an apparent contradiction to the studies in mammals, which do not show cabp reactivity in the LSO or MSO. However, the contrary results are explained by the fact that the antibody used in the owl studies cross-reacted with the similar 29 kD calcium binding protein, calretinin (Rogers, '87), whereas the more Fig. 5. Pattern of cabp staining in the lateral lemniscus (LL). A. recent studies in mammals (including ours) have used a Low power view showing many immunopositive cells in the INLL (B) calbindin antibody which does not cross-react with calretiand VNLLv (D), a few cells in the VNLLd, but none in the DNLL (0.nin, at least above the level of the cochlear nucleus (Buchan Cells in the paralemniscal zone of the lateral tegmentum (LT) are also and Baimbridge, '88; Resibois et al., '89). Moreover, using cabp(+). Cabp(+f fibers can be seen running through both the INLL (B) and VNLLv (I)).B. Small cabp(+) cells found in the dorsal tip of in situ hybridization in the chick, Rogers ('87) has shown that NL and its source of bilateral input, nucleus magnocelINLL with an immunopositive fiber appearing to end on four of them. C. Lack of staining seen in DNLL. D. Cahp(+) cell columns of VNLLv lularis, contain calretinin, not calbindin. Since the more stacked along the lengths of ascending lemniscal fibers. The cells recent studies of cross-reactivity of these two proteins in additionally form two sheets in the transverse plane of the 50-km mammals has shown that calretinin is confined to the cochsection. One sheet is in focus; the other underlying sheet appears lear nucleus and auditory nerve (Rogers et al., '89; Resibois out-of-focus as a group of blurred gray spots. Very fine cabp(+) fibers et al., '89), one must conclude that birds and mammals can be seen in close association with the cells. E. Drawing of section differ in the distribution of calretinin in the brainstem. (note that micrographs C and D were made on side opposite to A and B).
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Fig. 6. Cabp immunoreactivity within the inferior colliculus (IC). Low power view in A shows the three divisions of the central nucleus (ICc) definable morphologically and physiologically in the mustached bat. The ALD and MD are tonotopically organized, respectively representing frequencies from about 10 to 59 kHz, and 63 to 120 kHz.DPD represents the bat’s reference frequency around 60 kHz and can be considered an hypertrophied isofrequency lamina. Cabp(+) cells are
M.L. ZETTEL ET AL.
scattered among cabp(-) cells in each of the three divisions. Staining of the neuropil is moderately strong in DPD (D) and MD (B),very strong in ALD (0. (Note: photos in B and C were taken from correspondingly labeled regions shown in A; that in D is from a different section.) In ALD, the perimeters of cabp(-) cells are heavily invested with cabp(+) endings (C).E.Drawing of section.
CALBINDIN IN THE BAT AUDITORY SYSTEM
13
Fig. 7. Auditory thalamus at the level of medial geniculate body. A-C. Low power views progressing from rostral (A) to caudal (C); “D,” “W,”“M” = dorsal, ventral and medial divisions, respectively, of the MGB. The numerous intensely stained cells and fibers of all three divisions of the MGB contrast greatly with the lack of staining of SG and PO. D. Heavy Iabeling of cells and fibers in the dorsal division (from area “D” in section shown in B). E. Drawing of section at the level of MGB shown in A.
Calbindin immunoreactivity occurs in pathways potentially important in echolocating animals for processing temporal information. In contrast with the LSO and MSO, the principal cells of the MNTB were strongly cabp(+). The MNTB is a monaural nucleus that receives inputs (typically via calyces of Held) from immunopositive globular cells of the contralateral AVp, PVl, and the octopus cell zone (PVo). The large afferents of MNTB cells were
cabp( +), and presumably arise from these groups of labeled cells of the cochlear nucleus. In vitro slice experiments have shown that the collaterals of the individual d e r e n t s as well as their target MNTB principal cells project in a precise convergent pattern onto the same individual cells in VMPO, DMPO, and perhaps most interestingly,the VNLLv (Kuwabara et al.,’89). The latter nuclei are also cabp(+) in other mammals (cat, Matsubara, ’90; chinchilla, Kelley et al.,
14
M.L. ZETTEL ET AL.
Fig. 8. Cabp immunoreactivity in superior colliculus and pontine nuclei. Labeled cells are sparsely scattered in the deep layers of the SC, as shown in A, B, and D. B.Small labeled cells in the intermediate gray layer; D shows the giant cells of the deep layer of SC. C. Drawing of section from which photomicrographs are taken. E. In the pons, both lateral (LPN) and medial nucleus (MPN) cells are strongly cabp(+).
submitted; rat, Celio, '90; Webster et al., '90). The VNLLv is especially notable because it is hypertrophied and much more clearly columnar in echolocating bats and dolphins than in nonecholocating mammals (Zook and Casseday, '82a; Covey and Casseday, '86; Zook et al., '88). The cabp(+) endings we found associated with VNLLv cells
could therefore be attributable to the precisely convergent cabp( +) projections from AVp and MNTB. Zook and Casseday ('851, Covey and Casseday ('861, and Zook et al. ('88) have suggested that this direct/indirect, parallel projection to the highly organized VNLLv may play an important role in echo-ranging. Bats estimate the dis-
CALBINDIN IN THE BAT AUDITORY SYSTEM tance to a target from the time difference between the emitted pulse and returning echo, and under optimal conditions can detect time differences at least as small as 0.5 p s (Simmons, '79; Moss and Schnitzler, '89). The VNLLv has anatomical features suitable for pulse compression and pulse-echo cross-correlation, processes implicated in range estimation by the auditory system (for review of range estimation in bats, see Simmons, '89). Neurophysiological studies in bats have found intensity tolerant, timepreserving neurons ("constant latency responders") in the lateral lemniscus (E. Covey, pers. comm.; O'Neill, Holt, and Zettel, unpubl. observ.) and IC (Suga, '71; Pollak et al., '77; O'Neill, '85). Such cells can encode the occurrence of particular frequencies within a frequency sweep with a precision of r250 p s or less, functioning as time markers for the same spectral components in both the emitted pulse and returning echo. Thus calbindin may be associated with a monaural system whose cells are specialized to preserve temporal information thought to be important for echoranging. Distinct species differencesoccur in the distribution of calbindin above the level of the ZC. Calbindin-likeimmunoreactivity in the cells of the IC is relatively sparse when contrasted with nuclei in the brainstem, not only in the mustached bat, but in all other mammalian species so far examined (Garcia-Segura et al., '84; Celio, '90; Kelley et al., submitted). There are also few unlabeled cells surrounded by cabp(+) terminals, except in the ventral region of the ALD. This result is perhaps not too surprising, since a significant proportion of the afferents to the ICC arise from nuclei with cells that are cabp(-), namely, the MSO, LSO, and DNLL. The remaining afferent input, however, derives in large part from cabp(+) nuclei of the lateral lemniscus, the cochlear nucleus, the periolivary nuclei, and nuclei of the trapezoid body (Frisina et al., '89; Ross and Pollak, '89). Consistent with this widespread cabp(+) afferent input is the observation that the neuropil of the IC is stained well above the background by a very fine plexus of immunopositive fibers, especially in certain areas where fibers are found in large numbers (e.g., Fig. 6C). Nevertheless, the lack of labeled cells in IC, and the even more puzzling lack of labeled terminals, stands in contrast not only to the brainstem origins of many of its afferents, but also to the nearly ubiquitous cell staining found in the MGB, the principal target of ICC efferents. Consistent with the results in the rat reported by Celio ('90) and Garcia-Segura et al. ('84), the antibody used in this study labeled all divisions of the medial geniculate body. By contrast, labeling in the macaque (using an antibody that cross reacts with calretinin) was mainly confined to the magnocellular components of the medial nucleus (Jones and Hendry, '89). Further species differences appear when comparing the density of labeling in the three principal nuclei of the MGB. Celio ('90) reports that in the rat, the density of labeling was strongest in the ventral nucleus, weakest in the medial nucleus, and intermediate in the dorsal nucleus. We found that all three divisions were equally densely labeled in the mustached bat. Similarly, whereas in both the rat and macaque the suprageniculate and posterior group of thalamic nuclei were cabp(+), we found that cellular labeling was conspicuously absent from these regions of the auditory thalamus of the mustached bat. Regarding the apparent association of calbindin immunoreactivity and monaural nuclei in the brainstem, there is no anatomical evidence for such an association at the mid- or
15
forebrain levels in any species so far examined. Thus strong cross-species similarities are found in the distribution of calbindin in the brainstem, whereas beyond the level of the IC, differences are apparent that may prove interesting in regard to function. Conclusions. Using an antibody specific to calbindin D-28k, we found that cabp immunoreactivity is widespread in the subcortical auditory system of the mustached bat. Especially striking is the selective labeling of monaural pathways in the subcollicular brainstem. In the nuclei receivingbilateral input, only the marginal cells of the MSO were cabp(+). The majority of cells in the core of MSO, as well as the entire LSO and DNLL, were cabp(-), as seen in cat and chinchilla. Since the evidence appears to be mounting that calretinin and calbindin are not found in the same cellular compartments above the cochlear nucleus level, there remains the intriguing question of whether different calcium binding proteins are specific for functionally different pathways. Specifically, is a different type of calcium binding protein found in the binaural system? Complementary studies with antibodies against mammalian calretinin and other calcium binding proteins (such as parvalbumin) would be worth pursuing to answer this question.
ACKNOWLEDGMENTS We are especially grateful to Dr. Kenneth Baimbridge both for the primary antiserum against Cabp and for his invaluable advice and time. We thank Dr. Robert Sloviter for the protein A, Dr. John Olschowka and Ms. Sally Brown for technical advice, and Dr. John Zook and Dr. Robert Frisina for assistance in identifying anatomical divisions. This research was supported by grants from the National Science Foundation (BNS-8617152)and the National Institute for Deafness and Other Communicative Disorders (R01-DC00267)to W.E.O.
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