Development of a topographically organized auditory network in slice culture is calcium dependent

Development of a Topographically OrganizedAuditory Network in Slice CultureIs Calcium Dependent

Christian Lohmann, Vesna Ilic, Eckhard Friauf

Zentrum der Physiologie, Klinikum der Johann-Wolfgang-Goethe Universitat, Theodor-Stern-Kai 7,60590 Frankfurt, Germany

Received 5 August 1997; accepted 16 September 1997

able from that in vivo. The addition of calcium channelABSTRACT: Inhibitory and excitatory connec-blockers (MgCl2 and nifedipine) to the high-KCl me-tions of remarkably precise topographic order aredium reduced organotypicity drastically, indicatingcharacteristic features of the mammalian auditorythat a depolarization-induced increase of intracellularsystem, particularly within the superior olivary com-calcium is indispensable. Furthermore, the temporalplex (SOC). Little is known about the requirementscourse of the expression of the calcium-binding pro-for the correct development of these specific connec-tein parvalbumin in culture under high KCl mimicstions. Previous in vivo experiments have demonstratedthat in vivo, demonstrating developmental processesa high expression of calcium-binding proteins in thisduring incubation. The need for calcium influx intosystem during development, pointing to the need forneurons of this auditory network in vitro (which is

precise calcium regulation. Here, we have employed not seen in other slice culture systems) strengthensan organotypic slice culture from the above neuronal the hypothesis that an optimal calcium concentrationnetwork and analyzed the requirements for the main- is exceptionally important in auditory neurons. Thetenance and development of this system in vitro. When effect of KCl in the slice cultures may substitute forslices from neonatal rats were incubated in standard input activity regulating intracellular calcium in audi-culture medium for up to 7 days, we found no organo- tory neurons in vivo. q 1998 John Wiley & Sons, Inc. Jtypic features. Only if 25 mM KCl was added to the Neurobiol 34: 97–112, 1998culture medium, the cytoarchitecture of the nuclei, the Keywords: superior olivary complex; lateral superiorneuronal morphology, and the specificity and topogra- olive; medial nucleus of trapezoid body; topography;phy of internuclear connections were indistinguish- rat

INTRODUCTION receive inhibitory glycinergic input from the contra-lateral ear via the medial nucleus of the trapezoidbody (MNTB) and excitatory glutamatergic inputInhibitory as well as excitatory projections arefrom the ipsilateral ear via the ventral cochlear nu-highly topographically ordered within the mamma-cleus (VCN) (Irvine, 1986; Feng, 1992) [Fig.lian auditory system. Within the superior olivary1(A,B)] . The mechanisms that lead to the estab-complex (SOC), which is involved in sound local-lishment of this topographic network are largelyization, neurons in the lateral superior olive (LSO)unknown. It was shown that afferent activity fromthe cochlea is indispensable for the maintenance anddevelopment of auditory neurons and their projec-Correspondence to: C. Lohmann

Contract grant sponsor: DFG; contract grant number: SFB tions (Pasic et al., 1994; Moore et al., 1995; Sanes269, B5 and Hafidi, 1996). Afferent activity from the co-Contract grant sponsor: Dr. Paul and Cilli Weill-Stiftung,

chlea determines the intracellular calcium concen-Frankfurtq 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/020097-16 tration in auditory neurons (Zirpel and Rubel,

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98 Lohmann, Ilic, and Friauf

times a week. Initially, the medium contained 24% horse1996). Therefore, an optimal intracellular calciumserum, 48% minimum essential medium, 24% Earl’s bal-concentration, probably depending on cochlear ac-anced salt solution, 1% glutamine solution (200 mM) ,tivity, may be crucial for neuron survival and devel-and 2.75% glucose solution (200 g/L; all compoundsopment. Interestingly, calcium-binding proteins areobtained from Gibco). This composition was essentiallyhighly and transiently expressed in auditory brainequivalent to standard solutions previously used by other

stem nuclei during development (Friauf, 1993; Loh- investigators (e.g., Gahwiler, 1981; Stoppini et al., 1991;mann and Friauf, 1996). These data further corrob- Hafidi et al., 1995). To improve the viability of the SOCorate the idea that the intracellular calcium concen- cultures, we employed a variety of different substances,tration has to be kept in an optimal range during such as somatostatin (Sandoz), glycine (Sigma), and adevelopment. high concentration of KCl. As the viability was greatly

improved when KCl was added (see below), the afore-To further elucidate what mechanisms determinementioned culture medium plus 25 mM KCl was subse-neuron survival and topographic connectivity, wequently used throughout most of the experiments.reveal the necessities for maintenance and develop-

ment of organotypicity in slice cultures of the devel-oping SOC of rats, particularly within the gly- Histologycinergic projection from the MNTB to the LSO.

Nissl Staining (n Å 13 animals) . Slices were fixed withThe slice culture technique has become very popular4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4,and is frequently used to analyze developing synap-PFA) in the refrigerator overnight. After cryoprotectiontic circuitry (e.g., Gahwiler, 1981; Bolz et al.,with 30% sucrose (in the refrigerator for 24 h), they were1990).resectioned on a freezing microtome (30 mm). SectionsOur results show that SOC nuclei appear to bewere mounted on slides and stained with thionine.

unusual compared with other central nervous system(CNS) structures kept in vitro, because a raised

Biocytin Tracing (n Å 102 animals) . Small crystals ofextracellular KCl concentration in the culture me-biocytin (Sigma) were applied to slices with the sharp

dium is necessary to maintain the viability of their tip of an injection cannula (27 gauge). Slices were incu-neurons and the topography of the MNTB–LSO bated for another 3–6 h to allow for biocytin uptake andprojection. Furthermore, we provide evidence that transport. Thereafter, the slices were fixed, cryoprotected,the beneficial effect of KCl is mediated via K/- and resectioned as described above. Sections were incu-induced depolarizations which most probably acti- bated in 0.2% Triton X-100 for 30 min and subsequently

transferred into 0.5% H2O2 for 20 min to quench endoge-vate voltage-gated Ca2/ channels, resulting in annous peroxidases. After rinsing in Tris-buffered salineincrease of the intracellular free Ca2/ concentration.(TBS), the slices were transferred to a blocking solution[2% bovine serum albumin (BSA) and 0.1% Triton X-100 in TBS] for 1 h and then to peroxidase-conjugatedMATERIALS AND METHODSavidin (NeutraLiteTM; Molecular Probes; dilution 1:2000in the blocking solution) for 2 h at room temperature.

Culture After rinsing, the biocytin-labeled structures were visual-ized with diaminobenzidine (Sigma, 0.02%), ammo-The static culture method, introduced by Stoppini et al.nium-nickel-sulfate (Sigma, 0.3%), and H2O2 (Sigma,(1991), was applied. Newborn Sprague–Dawley rats0.006%) in TBS for about 10 min. Finally, the sections(P3–5, n Å 132) received an overdose of ketamine (0.3were mounted on gelatin-coated slides, air-dried, Nisslg/kg, intramuscularly). After decapitation, the brain wascounterstained, dehydrated in graded ethanol, and cov-removed from the skull under sterile conditions. The brainered in Entellan (Merck).stem area of interest was dissected out and the dura was

removed under a dissecting microscope in ice-cold prepa-ration ringer [recipe modified from Forsythe (1994): DiI/DiA Tracing (n Å 12 animals) . To further charac-

terize the topography of the MNTB–LSO projection un-containing (mM) : 2.5 KCl, 1 MgCl2 , 260 D-glucose, 26NaHCO3, 1.25 NaHPO4, 2 Na-pyruvate, 3 myo-inositol, der culture conditions, the lipophilic dyes DiI (1,1-diocta-

decyl-3,3,3 *,3 *-tetramethylinocarbocyanine perchlorate;1 kynurenic acid, 2 CaCl2 , pH 7.4] . Coronal slices (300mm) were obtained by cutting the tissue with a vibrating Molecular Probes) and DiA [4-(4-dihexadecylaminost-

yryl)-N-methylpyridinium iodide; Molecular Probes]microtome (Vibracut 3; FTB, Bensheim, Germany). Se-lected slices containing the SOC and, in about 80% of were applied into fixed slices to the MNTB or the LSO as

retrograde or anterograde tracers. For retrograde tracing, athe cases the VCN, were placed on Millipore CM mem-branes in six-well plates; 1 mL culture medium was added DiI crystal was implanted into the lateral part of the LSO,

while a DiA crystal was implanted into the medial part.to each well; and the slices were incubated at 36.57C forup to 7 days (DIV7) in a humidified atmosphere con- For anterograde tracing, DiI and DiA were applied to the

medial and lateral part of the MNTB, respectively. Thetaining 5% CO2. The culture medium was changed three


Slice Culture of Topographic Network 99

slices were stored in PFA at room temperature for a mini- rons: Rietzel and Friauf, 1998; for MNTB neurons: Som-mer et al., 1993; and for CN neurons: Hackney et al.,mum of 10 days and a maximum of 2 months.1990).

Immunohistochemistry (n Å 5 animals) . Slices werefixed, cryoprotected, and resectioned as described. En-dogenous peroxidases were quenched and the avidin– RESULTSbiotin–peroxidase (ABC) method was applied. Afterrinsing, the sections were preincubated for 1 h in a Slice Cultures Maintained Underblocking solution of 10% goat serum and 2.5% BSA in

Conventional Conditionsphosphate-buffered saline (PBS) with 0.3% Triton X-100and then incubated with the primary antiserum: mouse In the initial experiments, we used a culture mediummonoclonal antibodies against parvalbumin (PV) which was very similar to that routinely used by(Sigma; clone PA-235; dilution of 1:5000 in PBS con-

others ( i.e., a medium without additional KCl)taining 1% goat serum, 1% BSA, and 0.3% Triton X-(Stoppini et al., 1991; Hafidi et al., 1995). This100) in the refrigerator overnight. Thereafter, the sectionsresulted in remarkably deformed brain stem slices,were washed and transferred to biotinylated anti-mousewhich appeared to be atrophied mainly in the dorso-immunoglobulin G (IgG) (1:1000; Jackson) and incu-ventral direction compared to those obtained in vivobated for 1.5 h at room temperature. Sections were then

washed again and incubated for 1.5 h in a horseradish [Fig. 1(C–F)] . Moreover, the typical outlines ofperoxidase–coupled avidin–biotin complex (1:50; Vec- the main SOC nuclei [LSO, MNTB, and superiortor: Elite-Kit) . After washing, the biocytin-labeled struc- paraolivary nucleus (SPN)] could not be identified.tures were visualized with diaminobenzidine (Sigma, For example, the well-known ‘‘S’’-shaped structure0.05%), and H2O2 (Sigma, 0.01%) in PBS for about 10 of the LSO was absent in all 24 slice cultures keptmin. Finally, the sections were mounted on gelatin-coated under these conditions. These data indicated thatslides, air-dried, dehydrated, and covered in Entellan.

our slice cultures were compromised when we usedstandard culture media and that they required furtherfactors to maintain organotypicity.Data Analysis

Representative sections were photographed with a lightmicroscope at different magnifications (12.5–100). To Addition of KCl to Culture Medium Isquantify the maintenance of the MNTB–LSO projection, Necessary to Preserve Organotypicitythe density of retrogradely biocytin-labeled MNTB neu-

We tested several protocols to increase the viabilityrons was determined from camera lucida drawings ofslices from 89 animals. We calculated the density rather of the slice cultures. In an attempt to depolarize thethan the absolute number of retrogradely labeled neurons, neurons, we elevated the extracellular KCl concen-because the density value was less dependent on the size tration by 25 mM ( in the following referred to asof the biocytin application area and the size of the area high [K/]o) (Koike et al., 1989). This resulted incontaining labeled neurons. In each animal, the analysis the maintenance of the overall shape of brain stemwas performed exclusively in the 30-mm section that ap- slices [Fig. 1(G)] . Moreover, the characteristic cy-parently contained the highest density of labeled neurons,

toarchitecture of the SOC nuclei, such as the typicaland only one brain stem side (left or right) was selected‘‘S’’-shaped LSO, was present after DIV7 [Fig.for counting the number of retrogradely labeled somata1(H)] . It should be noted, however, that the size(thus, the number of counts amounted to 89). If the num-of the SOC, especially of the MNTB (maybe be-ber of labeled MNTB neurons was less than three, thecause of its very high density of cell bodies) , wasdensity was defined as zero. Otherwise, the size of the

area containing labeled MNTB neurons was estimated by increased in vitro, similar to findings obtained forcutting and weighing the piece of drawing paper which other brain areas in slice cultures (Stoppini et al.,contained neurons. The weight of this piece was divided 1991; Gotz and Bolz, 1992). Nonetheless, this didby the weight of a piece of paper corresponding to 1 not impair the position of the individual SOC nucleimm2, and the density of MNTB neurons per square milli- as well as the arrangement of the somata. Further-meter was finally deduced. For statistical analysis, treat- more, in low [K/]o the cross-sectional area of neu-ment and control groups were compared and significance

ronal somata in the MNTB (n Å 34) was decreasedwas calculated with the Mann–Whitney U test.by 23% compared to slices incubated in high [K/]oThe morphology of representative retrogradely labeled(n Å 42), indicating that low [K/]o leads to shrink-neurons in the MNTB, VCN, and LSO was describedage of MNTB neurons. When several other sub-from biocytin experiments. The shape of the somata andstances such as glycine (n Å 4) (cf. Kandler andthe principal arrangement of the dendritic trees were com-

pared with data from in vivo experiments (for LSO neu- Friauf, 1995a) and somatostatin (nÅ 5) (cf. Kungel



Figure 1 Cytoarchitecture of the developing superior olivary complex (SOC) in vivo and inslice cultures. (A) Schematic diagram of a coronal brain stem section illustrating the locationand shape of the lateral superior olive (LSO), the medial nucleus of the trapezoid body(MNTB), and the ventral nucleus of the cochlear nuclear complex (VCN). (B) LSO neuronsreceive glutamatergic input from the ipsilateral VCN and glycinergic input from the MNTB,which in turn receives glutamatergic input from the contralateral VCN. All inputs are highlytopographically organized. For example, neurons in medial and lateral parts of the MNTBproject into medial and lateral aspects of the LSO, respectively. (C,D) Nissl-stained brainstem section from a rat at postnatal day (P) 4 at different magnifications. Note characteristiccytoarchitecture and boundaries of the nuclei. (E,F) Nissl-stained brain stem section at thelevel of the SOC, which was incubated for 7 days (P5/DIV7) in a culture medium without



and Friauf, 1995) were added to the slice cultures, [Fig. 2(C)] . As MNTB projections into the SPNhave been described in vivo (Sommer et al., 1993),this did not improve the viability of the SOC nuclei

(data not shown). Finally, we coincubated SOC our data show a normal projection pattern of MNTBneurons after several days in culture. Third, thenuclei with slices of the inferior colliculus (n Å 8),

the major target nucleus of LSO neurons (Helfert number of retrogradely labeled MNTB neurons wasconsiderably higher compared to normal [K/]o , re-et al., 1991). This was done under the assumption

that this target might release trophic factors and thus sulting in a significantly increased density of thesecells [Fig. 2(D)] ( low [K/]o : 249 { 104 mm02 ,help to maintain a normal cytoarchitecture. Unfortu-

nately, this treatment was also unsuccessful. n Å 11; high [K/]o : 938 { 103 mm02 ; n Å 18, põ 0.01). Fourth, somata in the ipsilateral VCNAlthough the normal cytoarchitecture of the SOC

nuclei was lost in medium without high [K/]o , it could also be retrogradely labeled [Fig. 2(E)] , re-flecting the normal situation. We never saw projec-was still possible that other characteristics such as

connectivity were preserved under these conditions. tions from the SPN or the medial superior olive tothe LSO, indicating that aberrant projections fromTo evaluate whether afferent connections to the

LSO also strongly depend on high [K/]o , biocytin auditory nuclei which are absent in vivo did notdevelop in vitro. Finally, the somato-dendritic ap-crystals were applied to the site where the LSO is

normally located, to retrogradely label projection pearance of SOC neurons appeared to be healthy asevidenced by the typical orientation of bipolar LSOneurons. In the experiments performed under stan-

dard conditions, the labeling never revealed the neurons [Fig. 2(F)] . Taken together, all of thesecriteria show that the organotypicity of SOC slicescharacteristic ‘‘S’’-shaped appearance of the LSO

[Fig. 2(A)] . Furthermore, the morphology of bio- is well preserved when culture medium with high[K/]o is used.cytin-labeled objects resembled glia cells or dying

neurons. The axons of only a few MNTB neurons The beneficial effect of high KCl was not due toan increased osmolarity. This was shown in controlreached the presumed LSO area. In contrast to the

SOC, however, nonauditory neurons in the ventral experiments (n Å 12) in which 50 mM mannitol(an osmotically active but chemically inert sub-brain stem survived well in our slice cultures. They

could be retrogradely labeled and displayed somato- stance) was added to the medium instead of 25 mMKCl, resulting in badly conserved neural structuredendritic morphologies that appeared normal [Fig.

2(B)] . All these data indicate that SOC nuclei were and internuclear connections.The differences in biocytin labeling betweenspecifically compromised when we used standard

culture medium. This assumption was further cor- slices treated with high or low [K/]o were notcaused by differences in active uptake and/or trans-roborated by the fact that slices which we obtained

from other brain structures (cortex or midbrain) port mechanisms. This could be demonstrated byapplication of the lipophilic fluorescent dyes DiIlived well under standard conditions.

In contrast to our negative findings on slice cul- and DiA into fixed slices. The labeling with thesetracers in dead tissue showed the same results astures kept in standard medium, biocytin tracing in

slice cultures incubated with high [K/]o revealed a biocytin labeling. Whereas many MNTB neuronsprojecting into the LSO were labeled when highwell-preserved projection from the MNTB to the

LSO [Fig. 2(C)] . Striking differences to the situa- [K/]o was present, there were almost none whenculture medium without additional KCl was usedtion of normal [K/]o were apparent in several as-

pects. First, the shape of the LSO and the MNTB (data not shown). Therefore, all subsequent experi-ments described below were performed with highcould be easily estimated. Second, a densely labeled

axonal field was obvious in the SPN, presumably [K/]o . As more than 90% of the slices exhibitedorganotypic features when treated with high [K/]o ,representing axon terminals from MNTB neurons

additional KCl. After DIV7, the dorsoventral extent of the slice had decreased and the typicalstructure of the SOC nuclei was lost. (G,H) Nissl-stained brain stem section (P4/DIV7), whichwas incubated in a culture medium to which 25 mM KCl had been added (high [K/]o) . Thelocation, shape, and cytoarchitecture of the SOC nuclei were mainly preserved, indicative oforganotypicity. The outline of the LSO is indicated by dots. Note, however, that the area ofthe SOC, particularly of the MNTB, was increased compared to in vivo. In this and all subse-quent figures, dorsal is toward the top and lateral to the right. Scale bars Å 1 mm in (G) [alsoapplies to (C,E)] , and 200 mm in (H) [also applies to (D,F)] .



Figure 2 Normal connectivity is preserved in slice cultures incubated with high [K/]o . (A)SOC slice (P4/DIV4) incubated without additional KCl. A biocytin crystal was implanted intothe presumptive LSO area to reveal the connectivity and morphology of neurons. Only oneretrogradely labeled MNTB neuron can be seen (arrow). Most LSO neurons appear to be dead;yet, in the same slice neurons outside the SOC look healthy (B). (C) SOC (P4/DIV6) incubatedwith high [K/]o . SOC nuclei can be identified and many MNTB neurons are retrogradelylabeled (density Å 790 mm02) . Labeled neuropil, presumably representing axon terminals ofMNTB neurons, can be seen in the superior paraolivary nucleus (SPN). (D) Quantitativeanalysis showing that high [K/]o increases the density of retrogradely labeled MNTB neurons(mean { S.E.M.; **p õ 0.01). (E) Axons in the ventral acoustic stria ( triangles) and somata



beling experiments, in which small crystals of theit appears that these culture conditions provide afluorescent tracers DiI and DiA were implantedvery successful means for maintaining the devel-within the same slice, yet into different parts ofoping SOC in vitro.the MNTB or the LSO, further demonstrated thepresence of topography. This was shown both by

Topography of the MNTB–LSO retrograde tracing [Fig. 4(B,C)] as well as antero-Projection Is Maintained in Culture grade tracing [Fig. 4(D–F)] .The glycinergic projection from the MNTB to theLSO is topographically organized (Irvine, 1986) Somato-Dendritic Neuron Morphology[Fig. 1(B)] . To further elucidate which organo- Resembles That In Vivotypic features were present in our culture system,

Because not only somata but also dendritic arborswe investigated whether this topography was main-could be retrogradely labeled by the biocytin tech-tained. Application of tracers into the medial or lat-nique, we were able to investigate the morphologyeral parts of the LSO (for retrograde tracing) or theof labeled LSO, MNTB, and VCN neurons (Fig.MNTB (for anterograde tracing) indicated that the5). In all nuclei, the neuronal morphology showedtopography of the MNTB–LSO projection was in-remarkable similarities to that obtained in vivodeed preserved. Application of biocytin into the lat-(Rietzel and Friauf, 1998; Sommer et al., 1993;eral limb of the LSO resulted in retrogradely labeledHackney et al., 1990). LSO neurons were mainlyneurons in the lateral part of the MNTB [Fig.bipolar-fusiform or multipolar [Fig. 5(A)] . Most3(A)] . On the other hand, application into the me-neurons in the VCN were multipolar and displayeddial limb of the LSO labeled neurons throughoutrather complex dendritic trees [Fig. 5(B)] . Somatathe whole MNTB [Fig. 3(B)] . This finding wasof MNTB neurons were usually round, with primarynot surprising because it can be attributed to thedendrites often originating from the dorsal and ven-fact that fibers of passage, originating from neuronstral poles of the soma [Fig. 5(C)] .in the lateral part of the MNTB, took up the biocytin

in addition to fibers of medially situated MNTBneurons. Further evidence that the connection from Tetrodotoxin Does Not Impair thethe medial part of the MNTB to medial part of Beneficial Effect of High [K/ ]othe LSO was present in vitro was obtained fromanterograde tracing experiments in which biocytin The finding that the survival and organotypicity of

the SOC slices depend on elevated [K/]o was sur-was implanted into the medial part of the MNTB.In these experiments, labeled axon terminals were prising, because such a dependence has not been

reported for slice cultures of other brain areas (Gah-restricted to the medial limb of the LSO [Fig.3(C)] . Unfortunately, the technique of biocytin la- wiler, 1981; Frotscher et al., 1995; Hafidi et al.,

1995). It prompted us to ask which effects can bebeling applied here did not allow us to reconstructsingle axons, because the density of labeled axons induced by high [K/]o , finally improving neuronal

survival in the SOC nuclei. First, K/-induced depo-was too high. Thus, the innervation pattern of indi-vidual MNTB axons remains to be described in the larization may increase the open probability of Na/

channels, and therefore increase spike activity. Tofuture.Figure 4(A) shows camera lucida drawings from test whether Na/ channel-mediated spike activity

affected survival, we added tetrodotoxin (TTX)three different slices which had received biocytincrystals into different parts of the LSO. The draw- (0.5 mM, Sigma) to the incubation medium. This

concentration has been shown to block spike activ-ings were digitized, color coded, and superimposedto demonstrate the spatial correlation between the ity effectively in other systems (Annis et al., 1994;

Seil and Drake-Baumann, 1994; Wilkemeyer andapplication site and the area of retrogradely labeledMNTB neurons. From these drawings it becomes Angelides, 1996). TTX treatment did not alter the

MNTB–LSO topography [Fig. 6(A,B)] or the den-obvious that the labeling pattern systematicallyshifted from lateral to medial when the application sity of retrogradely labeled MNTB neurons [Fig.

6(C)] (1067 { 139 mm02 , n Å 12, p Å 0.47),site was moved from lateral to medial. Double-la-

of VCN neurons (arrows) are labeled after a biocytin injection into the LSO (P4/DIV3; high[K/]o) . (F) Following biocytin labeling, LSO neurons display their typical arrangement anddendritic orientation (P4/DIV7; high [K/]o , the outline of the LSO is indicated by dots) .Scale bars Å 150 mm in (A–C), 500 mm in (E), 100 mm in (F).



Figure 3 The topography of the MNTB–LSO projection is preserved with high [K/]o . (A)Biocytin application into the lateral limb of the LSO retrogradely labeled neurons in the lateralpart of the MNTB (P4/DIV7). Insert shows a camera lucida drawing with the application site,labeled MNTB neurons, and the contours of the nuclei. (B) Biocytin application into the mediallimb of the LSO retrogradely labeled neurons in medial and lateral parts of the MNTB (P4/DIV7). Labeling in lateral parts can be attributed to fibers of passage [cf. Fig. 1(B)] . (C)Biocytin application into the medial MNTB anterogradely labeled axons in the medial part ofthe LSO and SPN (P3/DIV7). The lateral part of the LSO remained unlabeled. Scale barsÅ 200 mm.

indicating that spike activity is not required for neu- voltage-gated Ca2/ channels (VGCCs) and a subse-ron survival and the maintenance of organotypicity quent Ca2/ influx, thus increasing the viability ofin SOC slices cultured under high [K/]o . SOC slices. We tested this hypothesis by adding

Mg2/ (applied as MgCl2) , a nonselective Ca2/

Calcium Channel Blockers Reduce the channel blocker (Bessho et al., 1994; Seil andViability of the SOC Drake-Baumann, 1994), to the slice cultures in the

presence of high [K/]o . Under these conditions,The beneficial effects of high [K/]o could also bemediated via depolarization-induced opening of both the structure of the SOC and the typical appear-



ance of LSO neurons were lost [Fig. 7(A)] . Fur- DISCUSSIONthermore, the density of retrogradely labeled MNTB

The present study demonstrates that a neuronal net-neurons decreased in a dose-dependent mannerwork of inhibitory and excitatory connections from[Fig. 7(B)] (5.5 mM Mg2/ : 230 { 141 mm02 , nthe auditory brain stem of newborn rats can be main-Å 10, p õ 0.01; 11 mM Mg2/ : 38 { 38 mm02 , ntained in organotypic slice culture for at least 1Å 13, p õ 0.001). These results are indicative ofweek, provided that 25 mM KCl is added to thea role of Ca2/ for the survival and organotypicityincubation medium. Under these conditions, manyof the MNTB–LSO projection.features that are present in vivo, such as the cytoar-To further characterize the involvement ofchitecture, the specificity and topography of in-VGCCs, we incubated slices in high [K/]o with theternuclear projections, and the neuron morphology,L-type–specific Ca2/ channel antagonist nifedipinecan be preserved. In addition, the dynamics of PV(Tsien et al., 1988; Snutch and Reiner, 1992). SOCexpression are the same as in vivo. Experimentalslices treated with the solvent ethanol (0.7%) servedevidence suggests that a high [K/]o may improveas controls and yielded a density of 701 { 177neuron survival via a depolarization-induced Ca2/

retrogradely labeled MNTB neurons per square mil-influx through L-type VGCCs.limeter (n Å 12). These controls did not differ sig-

nificantly from slices that were incubated withoutethanol (pÅ 0.18). In contrast, nifedipine treatment Afferent Input into the SOC Is(10 mM; Research Biochemicals International) Necessary for Neuron Survivaldrastically changed the structure of the SOC [Fig.

Organotypicity of CNS slices maintained in vitro8(A)] and significantly reduced the density of ret-has been demonstrated for several brain structures,rogradely labeled MNTB neurons [Fig. 8(B)] (263e.g., the cortex (Bolz et al., 1990), the hippocampus{ 150 mm02 , n Å 13, p õ 0.05). These findings(Frotscher and Heimrich, 1993), the cerebellumsuggest that the K/-induced effect on neuronal sur-(Mouginot and Gahwiler, 1995), and the inferiorvival in the SOC is at least partially mediated bycolliculus (Hafidi et al., 1995). However, a neces-

L-type VGCCs.sity for an elevated extracellular KCl concentrationhas not been demonstrated in these structures. Onthe other hand, such a need has been observed forperipheral nervous system neurons and for dissoci-

Expression of Parvalbumin Increases ated cerebellar granule cells (summarized in Frank-Massively during Incubation lin and Johnson, 1992). Thus, it appears that the

SOC nuclei, particularly the LSO, are unusualThe calcium-binding protein PV is highly expressed among CNS structures in that they are exceptionallyin the SOC of adult rats and is rapidly up-regulated vulnerable and prone to cell death in vitro. Theduring the second postnatal week (Lohmann and following important question arises from theseFriauf, 1996), a few days before the onset of hear- findings: What is missing in cultured SOC neuronsing. We asked whether PV is also up-regulated dur- and can be substituted by the addition of KCl to theing incubation in vitro. Immunohistochemical ex- culture medium? One possibility is that much of theperiments revealed no PV immunoreactivity (ir) afferent input is lost in the slice cultures, whichwithin the SOC of rats at P4 [Fig. 9(A)] , the age normally exerts a trophic influence. Although thewhen slices were obtained (DIV0). At DIV7, how- main input stations to the LSO—namely the VCNever, the MNTB was densely packed with PV-ir and the MNTB, which together form about 95% ofsomata, the LSO contained PV-ir neuropil and some the afferents to the LSO (Glendenning et al.,PV-ir somata, and heavily labeled neuropil was ob- 1985) —are present in our slices, the number ofvious within the SPN [Fig. 9(C)] . This pattern of VCN neurons projecting into the LSO appears toPV-ir was very similar to that found in P10 animals be substantially reduced. Furthermore, the input to[Fig. 9(B)] . Therefore, the time course of PV ex- the VCN from the periphery, i.e., from the cochlea,pression is virtually the same in vitro as in vivo. is inevitably lost during the slicing procedure. Re-These results demonstrate that developmentally re- cent studies have demonstrated spontaneous in vivolated dynamic processes do take place within the activity in the developing auditory brain stem, oftenslices during incubation, although the network is occurring in bursts (Lippe, 1994; Kotak and Sanes,separated from the periphery, i.e., devoid of input 1995). The cochlea appears to be the driving source

of this activity (Lippe, 1994). Cochlea removal ex-from the cochlea.



Figure 4 Topography in the MNTB–LSO projection. (A) Results from three culture experi-ments (coded in different colors) , in which biocytin was applied into different parts of theLSO. Note shift of retrogradely labeled MNTB neurons from lateral to medial when the injectionsite in the LSO was moved from lateral to medial. (B) Double-labeling experiment (DiIapplication into the lateral LSO; DiA application into the medial LSO, P4/DIV6). DiI-labeledneurons (red) are seen in lateral parts of the MNTB, whereas DiA-labeled neurons (green,arrows) are located further medially. (C) Part of the MNTB shown in (B) at higher magnifica-tion and different exposure times to emphasize DiA-labeled neurons in the medial part of theMNTB. Arrows point to the same neurons as in (B). (D–F) The topography was also seen



periments performed in vivo have emphasized the Kelly, 1992; Helfert et al., 1992; Kandler and Fri-auf, 1995a), and glutamate application onto the de-importance of a functional synaptic input for the

normal development. Cochlea removal in neonatal veloping LSO has indeed been shown to result inCa2/ influx (Schmanns and Friauf, 1994). Like-gerbils resulted in a cell loss in the VCN, amounting

to almost 60% (Hashisaki and Rubel, 1989). Simi- wise, it is also possible that the activation of theMNTB–LSO input triggers Ca2/ influx. Althoughlar experiments done in ferrets induced transsynap-

tic neuronal death of about 30% and an abnormal this input is glycinergic, it is depolarizing duringperinatal life, having a reversal potential positiveoutline of the LSO (Moore, 1992). In our slice

cultures without high [K/]o , the outline and cytoar- enough (about 022 mV at P0, about 045 mV atP4) (Kandler and Friauf, 1995a) to open VGCCs,chitecture of the LSO appeared to be even more

deteriorated than in these in vivo experiments. Thus, whose threshold is within this range (Bean, 1989;Tsien et al., 1991). Preliminary results from cur-it is highly probable that because of the considerable

reduction of functional synaptic input to the audi- rent-clamp studies in our laboratory have demon-strated that SOC neurons become tonically depolar-tory nuclei in our slice cultures, LSO neurons lack

trophic and survival-promoting factors which are ized to about 030 mV, when 25 mM KCl is addedto the extracellular solution (personal communica-released by afferent neurons and crucial for their

integrity. Neurotrophins may be good candidates tion, Dr. Martin Kungel and Dr. Stefan Lohrke);these empirical data fit well with the theoreticalfor such survival-promoting effects because of their

well-known ability to protect those neurons from value obtained by the Goldman equation. Thus, itis probable that the neurons in slice cultures withcell death which have lost their input (reviewed in

Davies, 1994; Cellerino and Maffei, 1996). Indeed, high [K/]o display membrane potentials around030 mV.two types of neurotrophin receptors (TrkB and

TrkC) have been described in LSO neurons of de- Taken together, it is conceivable that the conver-gent input to the developing LSO provides a crucialveloping gerbils (Hafidi et al., 1996). They were

reported to be present at moderate and weak levels, signal during normal in vivo development that isadequate to increase the opening probability ofrespectively, around P7, indicating that brain-de-

rived neurotrophic factor, neurotrophin-3, and/or VGCCs and to raise the intracellular Ca2/ concen-tration to a level that guarantees neuron survival. Ifneurotrophin-4, known to bind to these receptors

with high affinity, may exert trophic effects in the so, one would expect that electrical and/or pharma-cological activation of the LSO input in slice cul-LSO. Whether these neurotrophins are indeed in-

volved in the development of the SOC has to be tures could also elicit a Ca2/ influx, thereby causingeffects that make an elevated [K/]o unnecessary.determined.Recently, Sanes and Hafidi (1996) reported an or-ganotypic slice culture of the MNTB–LSO pathwayIntracellular Ca2/ Concentrationprepared from P6–7 gerbils. The authors appliedIs Importantthe Na/-channel agonist veratridine (10 mM) to theculture medium to increase neuronal activity; byOur pharmacological data with blockers against

VGCCs (Mg2/ , nifedipine) indicate that the K/- doing so, they could obtain organotypicity. Thus,their data also point to the importance of electricalinduced improvement of neuronal survival in the

SOC is mediated via an increased [Ca2/]i . In vivo, activity in the development of the glycinergic inputto the LSO.proper Ca2/ influx into SOC neurons may be

achieved when spontaneous activity leads to the ac- In the present study, TTX treatment did not affectLSO survival and topography. This result could sug-tivation of glutamate receptors, which themselves

may be permeable to Ca2/ (Wisden and Seeburg, gest that spike activity is not required for normalSOC development, thus calling in question our1993) or cause a depolarization, and thus open

VGCCs. Glutamatergic input is present on MNTB aforementioned assumption. However, this conclu-sion may be misleading because our TTX experi-neurons (Borst et al., 1995; Barnes-Davies and For-

sythe, 1995) as well as LSO neurons (Wu and ments were performed in the presence of high

by anterograde tracing when DiI and DiA were placed into medial and lateral parts of theMNTB, respectively (P4/DIV5). (D) Double exposure; (E) DiI labeling in the medial LSO;(F) DiA labeling throughout the LSO. Again, labeling in medial aspects of the LSO can beattributed to fibers of passage. Scale bars Å 100 mm in (B) and (D–F), 50 mm in (C).



Figure 5 The morphology of auditory neurons remains normal in the slice culture as shownby biocytin labeling. (A) LSO neurons (the reconstructed neuron on the left is also shown ina photomicrograph, P4/DIV7). (B) VCN neuron that projects into the LSO (P4/DIV6). (C)MNTB neuron that projects into the LSO (P4/DIV7).

[K/]o . Therefore, it is probable that the beneficial being required. This idea is supported by previousfindings which have shown that the survival of dis-effects were achieved directly by the K/ ions which

substituted for the role of spike activity normally sociated rat sympathetic neurons does not depend



onstrate the transient presence of calcium-bindingproteins which are abundantly expressed in LSOneurons between E20 and P8 (Friauf, 1993; Loh-mann and Friauf, 1996). It is quite possible thatcalcium-binding proteins act in maintaining an opti-mal Ca2/ homeostasis in LSO neurons and thushelp to protect them from cell death caused by over-activation (Oppenheim, 1991).

Conclusions

The culture system introduced here should enableus to address pivotal questions regarding the matu-ration of topographically organized projections—inparticular, the glycinergic connection between theMNTB and the LSO. One pitfall of our system could

Figure 6 Tetrodotoxin (TTX) (0.5 mM) has no effecton the MNTB–LSO projection. (A,B) A high numberof topographically projecting MNTB neurons could beretrogradely labeled after TTX treatment [(A) biocytinapplication into the lateral limb of the LSO, P3/DIV7;(B) biocytin application into the medial limb of the LSO,P4/DIV7]. The labeling pattern is indistinguishable fromthat seen in controls [cf. Fig. 3(A,B)] . Scale bar Å 200mm. (C) There was no significant difference betweentreated and control cultures in the density of retrogradely

Figure 7 Effect of additional MgCl2 (5.5 and 11 mM)labeled MNTB neurons.on the MNTB–LSO projection. MgCl2 was applied tounspecifically block voltage-gated Ca2/ channels. (A)After 5.5 mM MgCl2 (P4/DIV7), the typical structure

on spike activity, provided that a high [K/]o is of the LSO is not apparent and only few MNTB neuronsmaintained (Koike et al., 1989). are retrogradely labeled. Scale bar Å 200 mm. (B) MgCl2

Interestingly, results from immunohistochemis- reduces the density of retrogradely labeled MNTB neu-try reinforce the idea that Ca2/ ions play a special rons in a dose-dependent manner (5.5 mM, **p õ 0.01;

11 mM, ***p õ 0.001).role in developing LSO neurons. These results dem-



be that the afferents to the rat’s LSO are laid downprenatally (Kandler and Friauf, 1993) and becomefunctional by E18 (Kandler and Friauf, 1995a), i.e.,about a week earlier than when we prepare our slicecultures (at P3–P5). Thus, several developmentalprocesses of synapse formation have already takenplace in the animals that we use in our experiments.However, other processes of differentiation takeplace postnatally, such as the regulation of calcium-binding proteins (e.g., PV, as shown here) , the re-finement of axonal and dendritic morphology(Sanes and Siverls, 1991; Sanes et al., 1992; Rietzeland Friauf, 1998), and the maturation of neurotrans-mission (Kandler and Friauf, 1995a,b) . In futureexperiments we will try to induce axonal regrowthby cutting the glycinergic MNTB-LSO projection,to investigate the capacity and the necessities for

Figure 9 The developmental increase of parvalbumin(PV) immunoreactivity (ir) in vitro is indistinguishablefrom that in vivo. (A) SOC nuclei are devoid of PV-irat P4, when slices are obtained. (B) By P10, many MNTBneurons have become PV-ir in vivo. Within the LSO,some neurons are PV-ir and neuropil is heavily labeled.(C) By DIV7, the pattern of PV-ir is virtually the sameas seen at P10 in vivo, indicating that dynamic processestake place in the slice cultures. Scale bar Å 200 mm.

the (re)establishment of the topographic projectionFigure 8 Effect of nifedipine (10 mM) on the MNTB–in this slice culture system.LSO projection. Nifedipine was applied to block voltage-

gated Ca2/ channels of the L-type. (A) As in the situationseen after MgCl2 treatment, both the structure of the LSO The authors thank Beate Piksa for excellent technical

help. CL is indebted to Kim McAllister, Kirstin Obst,and the number of retrogradely labeled MNTB neuronsare compromised (P5/DIV7). Scale bar Å 200 mm. (B) and Claudius Griesinger for teaching him the secrets of

brain slice culturing. The expert photographic help ofThe density of retrogradely labeled MNTB neurons isreduced by nifedipine (*p õ 0.05). Ulrike Doring and H.-J. Lohmann is acknowledged.



Many thanks to Dr. Stefan Lohrke and Dr. Martin Kungel tures of nervous tissue. J. Neurosci. Methods 4:329–for valuable comments and suggestions. This work was 342.supported by the DFG (SFB 269, B5) and Dr. Paul and GLENDENNING, K. K., HUTSON, K. A., NUDO, R. J., andCilli Weill-Stiftung, Frankfurt (CL). MASTERTON, R. B. (1985). Acoustic chiasm II: ana-

tomical basis of binaurality in the lateral superior oliveof the cat. J. Comp. Neurol. 232:261–285.

GOTZ, M. and BOLZ, J. (1992). Formation and preserva-REFERENCES tion of cortical layers in slice cultures. J. Neurobiol.

23:783–802.HACKNEY, C. M., OSEN, K. K., and KOLSTON, J. (1990).

ANNIS, C. M., ODOWD, D. K., and ROBERTSON, R. T. Anatomy of the cochlear nuclear complex of guinea(1994). Activity-dependent regulation of dendritic pig. Anat. Embryol. 182:123–149.spine density on cortical pyramidal neurons in organo-

HAFIDI, A., MOORE, T., and SANES, D. H. (1996). Re-typic slice cultures. J. Neurobiol. 25:1483–1493.gional distribution of neurotrophin receptors in the de-BARNES-DAVIES, M. and FORSYTHE, I. D. (1995). Pre-veloping auditory brainstem. J. Comp. Neurol.and postsynaptic glutamate receptors at a giant excit-367:454–464.atory synapse in rat auditory brainstem slices. J. Phys-

HAFIDI, A., SANES, D. H., and HILLMAN, D. E. (1995).iol. Lond. 488:387–406.Regeneration of the auditory midbrain intercommissu-BEAN, B. P. (1989). Classes of calcium channels in verte-ral projection in organotypic culture. J. Neurosci.brate cells. Annu. Rev. Physiol. 51:367–384.15:1298–1307.BESSHO, Y., NAWA, H., and NAKANISHI, S. (1994). Selec-

HASHISAKI, G. T. and RUBEL, E. W. (1989). Effects oftive up-regulation of an NMDA receptor subunitunilateral cochlea removal on anteroventral cochlearmRNA in cultured cerebellar granule cells by K/-in-nucleus neurons in developing gerbils. J. Comp. Neu-duced depolarization and NMDA treatment. Neuronrol. 283:465–473.12:87–95.

HELFERT, R. H., JUIZ, J. M., BLEDSOE, S. C., BONNEAU,BOLZ, J., NOVAK, N., GOTZ, M., and BONHOEFFER, T.J. M., WENTHOLD, R. J., and ALTSCHULER, R. A.(1990). Formation of target-specific neuronal projec-(1992). Patterns of glutamate, glycine, and GABA im-tions in organotypic slice cultures from rat visual cor-munolabeling in four synaptic terminal classes in thetex. Nature 346:359–362.lateral superior olive of the guinea pig. J. Comp. Neu-BORST, J. G. G., HELMCHEN, F., and SAKMANN, B.rol. 323:305–325.(1995). Pre- and postsynaptic whole-cell recordings in

the medial nucleus of the trapezoid body of the rat. J. HELFERT, R. H., SNEAD, C. R., and ALTSCHULER, R. A.Physiol. Lond. 489:825–840. (1991). The ascending auditory pathways. In: Neuro-

CELLERINO, A. and MAFFEI, L. (1996). The action of biology of Hearing: The Central Auditory System.neurotrophins in the development and plasticity of the R. A. Altschuler, R. P. Bobbin, B. M. Clopton, andvisual cortex. Progr. Neurobiol. 49:53–71. D. W. Hoffman, Eds. Raven Press, New York, pp. 1–

DAVIES, A. M. (1994). The role of neurotrophins in the 25.developing nervous system. J. Neurobiol. 25:1334– IRVINE, D. R. F. (1986). Progress in Sensory Physiology.1348. Vol. 7: The Auditory Brainstem. Springer, New York.

FENG, A. S. (1992). Information processing in the audi- KANDLER, K. and FRIAUF, E. (1993). Pre- and postnataltory brainstem. Curr. Opin. Neurobiol. 2:511–515. development of efferent connections of the cochlear

FORSYTHE, I. D. (1994). Direct patch recording from nucleus in the rat. J. Comp. Neurol. 328:161–184.identified presynaptic terminals mediating gluta- KANDLER, K. and FRIAUF, E. (1995a). Development ofmatergic EPSCs in the rat CNS in vitro. J. Physiol.

glycinergic and glutamatergic synaptic transmission inLond. 479:381–387.

the auditory brainstem of perinatal rats. J. Neurosci.FRANKLIN, J. L. and JOHNSON, E. M. (1992). Suppression

15:6890–6904.of programmed neuronal death by sustained elevation

KANDLER, K. and FRIAUF, E. (1995b). Development ofof cytoplasmic calcium. Trends Neurosci. 15:501–508.electrical membrane properties and discharge charac-FRIAUF, E. (1993). Transient appearance of calbindin-teristics of superior olivary complex neurons in fetalD28k-positive neurons in the superior olivary complexand postnatal rats. Eur. J. Neurosci. 7:1773–1790.of developing rats. J. Comp. Neurol. 334:59–74.

KOIKE, T., MARTIN, D. P., and JOHNSON, E. M. (1989).FROTSCHER, M. and HEIMRICH, B. (1993). Formation ofRole of Ca2/ channels in the ability of membrane depo-layer-specific fiber projections to the hippocampus inlarization to prevent neuronal death induced by trophic-vitro. Proc. Natl. Acad. Sci. USA 90:10400–10403.factor deprivation: evidence that levels of internal cal-FROTSCHER, M., SAFIROV, S., and HEIMRICH, B. (1995).cium determine nerve growth factor dependence ofDevelopment of identified neuronal types and of spe-sympathetic ganglion cells. Proc. Natl. Acad. Sci. USAcific synaptic connections in slice cultures of rat hippo-86:6421–6425.campus. Progr. Neurobiol. 45:143.

GAHWILER, B. H. (1981). Organotypic monolayer cul- KOTAK, V. C. and SANES, D. H. (1995). Synaptically



evoked prolonged depolarizations in the developing au- SANES, D. H., SONG, J., and TYSON, J. (1992). Refine-ment of dendritic arbors along the tonotopic axis ofditory system. J. Neurophysiol. 74:1611–1620.

KUNGEL, M. and FRIAUF, E. (1995). Somatostatin and the gerbil lateral superior olive. Dev. Brain Res. 67:47–55.leu-enkephalin in the rat auditory brainstem during fe-

tal and postnatal development. Anat. Embryol. SCHMANNS, H. and FRIAUF, E. (1994). K/- and transmit-ter-induced rises of [Ca2/]i in auditory neurons of de-191:425–443.

LIPPE, W. R. (1994). Rhythmic spontaneous activity in veloping rats. Neuroreport 5:2321–2324.SEIL, F. J. and DRAKE-BAUMANN, R. (1994). Reducedthe developing avian auditory system. J. Neurosci.

14:1486–1495. cortical inhibitory synaptogenesis in organotypic cere-bellar cultures developing in the absence of neuronalLOHMANN, C. and FRIAUF, E. (1996). Distribution of the

calcium-binding proteins parvalbumin and calretinin in activity. J. Comp. Neurol. 342:366–377.SNUTCH, T. P. and REINER, P. B. (1992). Ca2/ channels:the auditory brainstem of adult and developing rats. J.

Comp. Neurol. 367:90–109. diversity of form and function. Curr. Opin. Neurobiol.2:247–253.MOORE, D. R. (1992). Trophic influences of excitatory

and inhibitory synapses on neurones in the auditory SOMMER, I., LINGENHOHL, K., and FRIAUF, E. (1993).Principal cells of the rat medial nucleus of the trapezoidbrain stem. Neuroreport 3:269–272.

MOORE, D. R., RUSSELL, F. A., and CATHCART, N. C. body: an intracellular in vivo study of their physiologyand morphology. Exp. Brain Res. 95:223–239.(1995). Lateral superior olive projections to the infe-

rior colliculus in normal and unilaterally deafened fer- STOPPINI, L., BUCHS, P.-A., and MULLER, D. (1991). Asimple method for organotypic cultures of nervous tis-rets. J. Comp. Neurol. 357:204–216.

MOUGINOT, D. and GAHWILER, B. H. (1995). Character- sue. J. Neurosci. Methods 37:173–182.TSIEN, R. W., ELLINOR, P. T., and HORNE, W. A. (1991).ization of synaptic connections between cortex and

deep nuclei of the rat cerebellum in vitro. Neuroscience Molecular diversity of voltage-dependent Ca2/ chan-nels. Trends Pharmacol. Sci. 12:349–354.64:699–712.

OPPENHEIM, R. W. (1991). Cell death during develop- TSIEN, R. W., LIPSCOMBE, D., MADISON, D. V., BLEY,K. R., and FOX, A. P. (1988). Multiple types of neu-ment of the nervous system. Annu. Rev. Neurosci.

14:453–501. ronal calcium channels and their selective modulators.Trends Neurosci. 11:431–437.PASIC, T. R., MOORE, D. R., and RUBEL, E. W. (1994).

Effect of altered neuronal activity on cell size in the WILKEMEYER, M. F. and ANGELIDES, K. J. (1996). Addi-tion of tetrodotoxin alters the morphology of thalamo-medial nucleus of the trapezoid body and ventral co-

chlear nucleus of the gerbil. J. Comp. Neurol. cortical axons in organotypic cocultures. J. Neurosci.Res. 43:707–718.348:111–120.

RIETZEL, H.-J. and FRIAUF, E. (1998). Neuron types in WISDEN, W. and SEEBURG, P. H. (1993). Mammalianionotropic glutamate receptors. Curr. Opin. Neurobiol.the rat lateral superior olive and development changes

in the complexity of their dendritic arbors. J. Comp. 3:291–298.WU, S. H. and KELLY, J. B. (1992). Synaptic pharmacol-Neurol., to appear.

SANES, D. H. and HAFIDI, A. (1996). Glycinergic trans- ogy of the superior olivary complex studied in mousebrain slice. J. Neurosci. 12:3084–3097.mission regulates dendrite size in organotypic culture.

J. Neurobiol. 31:503–511. ZIRPEL, L. and RUBEL, E. W. (1996). Eighth nerve activ-ity regulates intracellular calcium concentration ofSANES, D. H. and SIVERLS, V. (1991). Development and

specificity of inhibitory terminal arborizations in the avian cochlear nucleus neurons via a metabotropic glu-tamate receptor. J. Neurophysiol. 76:4127–4139.central nervous system. J. Neurobiol. 8:837–854.


Development of a topographically organized auditory network in slice culture is calcium dependent

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