- Email: [email protected]
Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus
ANNALS OF ANATOMY
Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus Lorenzo Aiibardi Dipartimento di Biologia evoluzionistica spefimentale, University of Bologna, via Selmi 3; 1-40126, Bologna, Italy
Summary. The neural tracer wheat germ agglutinin conjugated to horse radish peroxidase was injected into the rat dorsal cochlear nucleus and acoustic stria. Some labelled neurons in the ipsilateral ventral cochlear nucleus were found as a result. These neurons were studied at the ultrastructural level, and their axo-somatic synaptic profile and glycine immunoreactivity were determined. Most neurons were glycine negative and classified as type I multipolar neurons. The latter showed a different synaptic profile from that of neurons projecting to the contralateral inferior colliculus or cochlear nucleus. This suggests the presence of differing populations of multipolar cells based on their synaptic profile. Few labelled multipolar neurons of type II were found, which appeared glycine negative and, rarely, glycine positive. The latter show an ultrastructure and axo-somatic profile similar to that of glycinergic commissural neurons in the dorsal and ventral cochlear nucleus. In particular, about one-third of boutons contained round synaptic vesicles, which are believed to contain an excitatory neurotransmitter. The ultrastructural analysis of the synaptic boutons in the cochlear nucleus confirms the presence of numerous cases of colocalization of glycine and GABA where flat and pleomorphic synaptic vesicles are mixed. The present study is in accordance with previous tract-tracing light microscopic studies which have indicated that large glycinergic neurons in the ventral cochlear nucleus act as broad-band inhibitory neurons in microcircuits of the dorsal cochlear nucleus and contralateral cochlear nucleus.
Key words: Rat - Ipsilateral cochlear nuclei - Connecting neurons - Glycine - Ultrastructure Correspondence to: L. Alibardi E-mail: [email protected] Ann Anat (2001) 183:459-469 © Urban & Fischer Verlag
http:llwww.urbanfischer.deljournalslannanat
Introduction The cochlear nuclear nuclei receive the terminals of the acoustic nerve and their intrinsic connections form the basic network that modulates the electrical activity of higher brainstem auditory nuclei (Moore 1986; Helfert et al. 1991). The three main subdivisions of the chochlear nucleus are the dorsal cochlear nucleus (DCN), and the ventral nucleus (VCN), the latter subdivided into a posteroventral cochlear nucleus (PVCN) and anteroventral cochlear nucleus (AVCN). Aside from interneurons, various projective neurons (pyramidal, bushy, multipolar and octopus) are also present in the 3 main subdivisions of the cochlear nucleus and their intrinsic and extrinsic connections are largely known (Kane 1973, Cant and Morest 1979; Cant 1982, 1992; Tolbert et al. 1982; Caspary 1986; Smith and Rhode 1987, 1989; Ostapoff and Morest 1991; Ostapoff et al. 1994; Helfert et al. 1991; Morest 1993). In the VCN, bushy neurons directly transmit the information from the acoustic nerve (primary-like discharge pattern) to higher auditory nuclei (superior olivary complex) by excitatory neurotransmitters, and this is believed to produce timing information coded in the lower auditory system which is important for sound localization. Multipolar neurons participate in intrinsic and extrinsic circuits of the cochlear nuclei, where they send both inhibitory and excitatory terminals (Oliver 1987; Smith and Rhode 1989; Caspary 1991). Multipolar cells may be involved in regulatory circuits within the acoustic system, which permits signalling of the different frequency components of sound (spectral detection). Multipolar neurons project to the ipsilateral and contralateral inferior colliculus (Adams 1983; Oliver 1987; Schofield and Cant 1996 b), contralateral cochlear nucleus 0940-9602/01/183/5-459 $15.00/0
(Cant and Gaston 1982; Wenthold 1987; Shore et al. 1992; Schofield and Cant 1996 a), and from the VCN to the ipsilateral DCN (Snyder and Leake 1988; Oertel et al. 1990; Shore and Godfrey 1994; Doucet and Ryugo 1997; Doucet et al. 1999; Ostapoff et al. 1999). These studies have characterized different multipolar cells and have shown that some subtypes project to different acoustic areas as excitatory or inhibitory cells (Oertel et al. 1990; Schofield and Cant, 1996 a, b; Doucet et al., 1999). Although multipolar neurons have been divided into types I and II based on cytological and ultrastructural characteristics (Cant 1981), it has become apparent that together with their specific neurotransmitter, the synaptic profile of the cell body and dendritic three shapes their electrical response and determines the specific stimulus to the connected acoustic areas (Cant 1982; Smith and Rhoe 1989; Ostapoff and Morest 1991; Ostapoff et al. 1994; Alibardi 1998 a, b; Josephson and Morest 1998). In the VCN, T-stellate cells (projecting into the trapezoid body and directed to the inferior colliculus) correspond to type I multipolar neurons, and D-stellate neurons (projecting to the ipsilateral D C N and perhaps exiting through the dorsal acoustic striae as commissural axons) correspond to type II multipolar neurons (Cant 1981; Adams 1983; Smith and Rhode 1989; Oertel et al. 1990; Kolston et al. 1992). Both T-stellate and D-stellate cells send collateral axons to the ipsilateral DCN. Three main types of projective neurons, planar, marginal and radiate, have been identified to project from the AVCN and PVCN to the ipsilateral DCN (Doucet and Ryugo 1997; Doucet et al. 1999). The radiate cells appear glycinergic and therefore inhibitory, and it has been suggested that these neurons correspond to commissural neurons that send collateral axons to the D C N before projecting their main axon to the contralateral cochlear nucleus (Doucet et al. 1999). This identity can be revealed by comparison of their fine structure, synaptology and immunocytochemistry for amino acidic neurotransmitters between neurons projecting ipsilaterally (DCN) with those projecting to the contralateral CN (Alibardi 1988 a, 2000). The study of the synaptic profile of neurons is a modern morphological approach useful to understand the function of single neurons within the interconnected acoustic networks (Cant 1992; Morest 1993). It has also been shown that, aside from neurons (Osen et al. 1990; Kolston et al. 1992; Winer et al. 1995; Juiz et al. 1996 b; Moore et al 1996; Kemmer and Vater 1997), synaptic terminals with a specific ultrastructure also store different amino acidic neurotransmitters (Otterson and Storm Mathisen 1990; Altschuler et al. 1993; Juiz et al. 1993, 1996 a). These studies have shown that glycine and G A B A are variably localized, or even colocalized, within specific synaptic terminals of different areas of the acoustic systems of rats and guinea pigs. Three main types of synaptic boutons are recognized: 1) R containing pleomorphic vesicles and making symmetric pre- and post-synaptic thickenings, 2) FR similar to the former but containing a high or even prevalent proportion of fiat ve-
sides, and 3) R, containing prevalently round vesicles and making asymmetric pre- and post-synaptic vesicles. The present study is a continuation of previous ultrastructural analyses of identified cochlear nucleus neurons in different neuronal populations in rat and guinea pig cochlear nuclei (Alibardi 1998 a, b; 1999 a, b; 2000 a, b, c, d). In this report we aim to determine the heterogeneity of synaptic profiles, and make an ultrastructural and immunocytochemical comparison between ipsilateral projecting neurons from VCN to DCN (Doucet and Ryugo 1997; Doucet et al. 1999) with commissural neurons projecting to the contralateral cochlear nuclei.
Materials and methods The animals used in the present study on ipsilateral connections between VCN and DCN were previously studied for their contralateral (commissural) projections (Alibardi 1998 b, 2000 b). Therefore the comparison between ipsilateral and commissural projecting neurons has been done among the same 3 rats previously used after DCN injection. Surgery, tracer injection, perfusion, fixation, and immunocytochemical techniques have previously been reported in detail (see above). Briefly, wheat germ agglutinin conjugated to horse radish peroxidase (WGA-HRP) was injected into the DCN of 3 rats, and the ipsilateral VCN was studied for tracer localization in labelled cells. The tracer, using the method of Weinberg and Van Eyck (1991), appeared in the form of electron-dense needles. The immunolabelling for GABA and glycine was revealed under the light microscope by a peroxydase-antiperoxidase reaction (PAP), and for the electron microscope by post-embedding immunogold labelling with silver-enhancement of gold particles. Quantification of cell parameters measured on eletron micrographs (perimeter, percentage of boutons etc., see Table 1) was done using a computer program (Leica Qwin Software, Leica Imaging Systems, Ltd., UK).
Results Retrograde labelled neurons in the VCN. The large injections performed, although mainly confined within the DCN, showed upon light microscopic examination that the molecular, fusiform and polymorphic layers of the DCN received the tracer (Fig. 1). Although mainly confined within the DCN some tracer was also seen spreading into part of the dorsal acoustic striae, which is located at the base of the DCN. Examination of the labelled areas showed that pyramidal cells and smaller cells, including tuberculoventral neurons, were labelled with their surrounding neuropile (Fig. 2). Labelled cells were also seen around the injection site, while more distant regions in the DCN showed few or no labelled cells. Most cells in the octopus cell area which is close to the injection site, were not labelled in the injected cases, and only occasional cells (octopus?) showed weak labelling (1-3 trace needles/cell). This indicated that the intermedi-
460
Fig. 1. Semthin section of injected DCN showing the spreading of the tracer (arrow) into the molecular (1), fusiform (2) and polymorphic (3) layer, including the acoustic striae (arrowhead). 100 x. Fig. 2. Higher magnification of labelled cells within the injection site. The arrow indicates a pyramidal cell. The arrowhead points a likely tuberculoventral neuron. 200 x. Fig. 3. A-B are serial sections showing two labelled cells (L) in ipsilateral PCVN which are glycine (GLY) negative (arrowhead) and positive (arrow). 750 x. Fig. 4. Labelled multipolar neuron of type II in ipsilateral PVCN with some labelled axo-somatic terminals (arrowheads). v, indicating blood vessels also visible in the semithin sections of this labelled cell (inset L, 800 x) which is glycine positive (inset GLY, 800 x). 5000 x.
461
Fig. 5. Labelled multipolar neuron of type I in ipsilateral PVCN, which is contacted by few synapses (arrows). E, rough endoplasmic reticulum, v, referring blood vessel also visible in semithin sections of this labelled cell (inset L, 700 x) which is glycine negative (inset GLY, 700 x). 7000 x. Fig. 6. Other elongated multipolar neuron of type I (D, dendrite) showing a few axo-somatic synapses (arrows). 6400 x.
462
Table. 1. Synaptic profile of retrograde labelled multipolar neurons in the ipsilateral ventral cochlear nucleus. n = number of cells or of axo-somatic synaptic boutons. +, Standard deviation. Cell type
Cell diameter 0tm)
Percentage coverage
Boutons/cell
Types of boutons/cell
Type I
15.38 + 2.49 (n = 20)
26.60 + 12.04
8.35 + 4.18 (n = 167 total)
FP 29.34% (n = 49)
Type II
21.40 + 3.14 (n = 7)
64.46 + 10.41
23.0 + 5.16 (n = 161 total)
21.38% (n = 34)
ate acoustic stria was little or not at all influenced by the spreading of the injected tracer. Labelled multipolar (non-globular) cells were seen in the multipolar area of the PVCN and AVCN near the acoustic nerve root region. The injections were large, and not localized to produce a tonotopic labelling in the VCN (Doucet and Ryugo 1997; Doucet et al. 1999). However, in some cross sections of VCN, no or rare cells were seen; in other sections of different VCN areas, labelled neurons were more numerous in the magnocellular (central) part. Most of these cells, whose shape varied from oval and elongated to roundish, were glycine negative but a few were glycine positive (Fig. 3). Out of 92 retrograde labelled cells (14-24 ~tm large), 82 were glycine-negative and 10 where glycine positive. Of the latter, 3 were also characterized ultrastructurally (Fig. 4). They were medium to large sized cells (20-24 gm) with short peripheral ergastoplasm (Nissl bodies), sparce pale areas occupied by microtubules and neurofilaments, and surrounded by numerous (51-79%) axo-somatic boutons (see Table 1). Most of the boutons contained pleomorphic vesicles (P boutons) but about 30% were represented by round vesicles (R boutons), while the less frequent type contained pleomorphic vesicles enriched by flat vesicles (FP boutons). These characteristic cytological features identify thse cells as multipolar neurons of type II (Cant 1981; Smith and Rhode 1989). Most glycine negative cells, despite their shape (roundish to fusiform), and position in the VCN were ultrastructurally recognizable as type I multipolar neurons (Figs. 5, 6). Nissl bodies were less numerous but with longer endoplasmic cisternae, and axo-somatic boutons were less frequent than the former type (9-48%, Table1). Most boutons were also of the P type, followed by less than half by those of FP type, and by few R boutons.
Ultrastructural comparison with commissural neurons. Few large glycinergic neurons in the D C N were found in the polymorphic layer. The ultrastructural study of the few, large glycinergic neurons in the DCN (Fig. 7) that were labelled after injection in the contralateral cochlear nucleus, showed small peripheral Nissl bodies and numerous neurofilaments like multipolar type II cells in the VCN. These neurons were mostly surrounded by axo-somatic boutons (28-33%) mainly P, then by R, and less frequently by FP boutons. While P and FP boutons stored
R 5.99% (n = 10)
P 64.67% (n = 108)
33.33% (n = 53)
45.28% (n = 72)
G A B A or glycine to different degrees, the R boutons were negative for both (Figs. 8, 9, 10). The analysis of commissural neurons in the VCN showed that they had similar ultrastructural features, immunoreactivity and synaptology as the above cells. They were surrounded by 28-30% axo-somatic boutons and were glycine positive multipolar cells of type If (Fig. 11). As glycinergic commissural neurons, also glycinergic multipolar neurons of type II in the ipsilateral VCN, received 28-33% of the axo-somatic boutons in form of R boutons. This relatively high R boutons input seems to be characteristic of all these glycinergic cells. Immunogold labelling of boutons. The immunogold observations on R FP and R boutons present in the D C N and VCN, both axo-somatic or in the neuropile (axo-dendritic), revealed that no labelling was present in R boutons. Glycine was more commonly seen in FP boutons than GABA, where it was scarse or absent in P boutons. In the latter G A B A was instead more frequently present (Figs. 8-10). Numerous cases of colocalization of G A B A with glycine in P or FP boutons were also seen (Figs. 1214). The intensity of immunolabelling to glycine often increased where numerous flat vesicles were seen, while G A B A immunolabelling prevailed only where pleomorphic vesicles were seen.
Discussion Injection site. As a result of the large injections performed in this study, we have not been able to label tonotopically neurons in the ipsilateral VCN (Shore and Godfrey 1994; Doucet and Ryugo 1997; Doucet et al. 1999). The microscopical study has shown that, aside from the DCN, our injections effected part of the dorsal acoustic striae but little or none of the intermediate acoustic striae. Since the dorsal stria, particularly the medial-anterior part, also collects exiting commissural axons which are glycinergic (Oertel et al. 1990; Kolston et al. 1992; Ottersen et al. 1995), we cannot exclude the possibility that the axons of our few glycinergic cells have taken-up the tracer into axons en passage within the striae. In fact, in contrast to previous studies (Doucet et al. 1999), we were not able to confine our injections to the DCN, although this
463
Fig. 7. vessel gative Fig. 8. Fig. 9.
Labelled (arrow) commissural multipolar neurons of type II in DCN surrounded by axo-somatic boutons, v, referring blood also visible in semithin sections of this labelled cell (inset L, 700 x) which is glycine positive (inset GLY, 700 x) and G A B A ne(inset GABA, 700 x). 4000 x. Glycine (GLY) immunolabelled bouton (F) containing numerous fiat vesicles in DCN. 28800 x. G A B A (GABA) immunolabelled bouton (P) containing pleomorphic vesicles in DCN. 53600 x.
464
Fig. 10. A - B are serial sections of two synaptic terminals in DCN immunolabelled for G A B A (A) or glycine (GLY). A, shows labelled terminals containing pleomorphic vesicle (P) which is not labelled for glycine (B). Boutouns containing round vesicles (R) are not labelled with either antibodys. 24100 ×. Fig. 11. Labelled (arrows) commissural multipolar neuron of type II in PVCN surrounded by numerous axo-somatic boutons, some also labelled (arrowheads). The inset (700 x) shows that this cell is glycine positive (GLY). v, referring blood vessel. 4300 x.
465
Fig. 12-14. Examples of GABA-glycine colocalization in VCN neuropile. R pleomorphic boutons. F, flat and pleomorphic boutons. R, round boutons. In Figures 12 (40300 x) and 13 (36900x) GABA labelling is more prevalent than glycine labelling in P boutons. In Figure 14 (27000 x) glycine labelling is more prevalent in F boutons.
466
was by far the nervous area that most received of the tracer. However tracer spreading into the stria was not as dense as inside the DCN, resulting in a diffuse labelling which is considered less specific than granular labelling in retrograde tract-tracing studies. This means that retrograde labelled neurons do not necessarily project to the area where the diffuse labelling is located (Tolbert et al. 1982; Snyder and Leake 1998). Besides, previous studies did not find retrograde labelled neurons in the ipsilateral VCN after injection into the DAS (Adam and Warr 1976; Adams 1983). The fact that occasional cells in the octopus cell area (octopus cells or other types) were weakly labelled indicates that most of the intermediate acoustic striae was not influenced by these larger injections. Based on the above considerations, we believe that the ipsilateral retrograde labelled neurons seen in this study are projecting through a specific route to the ipsilateral DCN (Adams 1983; Oertel et al. 1990; Ostapoff et al. 1999). Ipsilateral projecting neurons. In their study on neurons sending axons through the dorsal acoustic striae in cats, Adams and Warr (1976) rarely found retrograde labelled cells in the ipsilateral VCN after Horseradish Peroxidaseapplication in the severed dorsal acoustic stria. A following study (Adams 1983) instead found frequent retrograde labelled stellate neurons in the VCN after injection in the ipsilateral DCN, indicating a direct connection between the two nuclei by a route different from that of the dorsal acoustic striae. It is possible that most, if not all, multipolar cells projecting to the inferior colliculus or outside the VCN through the ventral acoustic striae also send collaterals to the DCN (Ostapoff et al. 1999). This intrinsic circuit has been implicated with the formation of a more refined sensitivity of target neurons in the DCN (pyramidal neurons) to acoustic signals that shape their specific electric response directed outside the DCN (Zhang and Oertel 1994; Doucet et al. 1999). Therefore it is hypothesised that interpreting frequency components of sounds by pyramidal cells can produce more precise electrical signals to transmit to the inferior colliculus. According to previous studies it is known that the glycine-negative multipolar cells in the VCN that project to the ipsilateral DCN do not use the dorsal acoustic striae as the exiting route but instead use the ventral acoustic striae in the trapezoid body (Adams 1983; Oertel et al. 1990) or remain confined within the cochlear nucleus (Doucet and Ryugo 1997; Doucet et al. 1999; Ostapoff et al. 1999). We confirm that most of these neurons, identified as type I multipolar, are glycine negative (Doucet et al. 1999). The analysis of the synaptic profile in type I multipolar neurons projecting to the contralateral inferior colliculus (Alibardi 1998a) or cochlear nucleus (Alibardi 1998b, 2000) shows some significant differences to those projecting to the ipsilateral DCN. The latter are presumably the planar and marginal neurons as indicated by the study of Doucet and Ryugo (1997). Specifically, the multipolar
neurons of type I, projecting to the inferior colliculus, receive more P boutons and less R boutons, and are slighly larger on the average (Alibardi 1998a). Instead, the multipolar neurons of type I projecting to the contralateral cochlear nucleus receive a little more axo-somatic boutons which are of P type, and less of R type, and their diameter is larger (Alibardi 1998 b). From the result of the present study it seems possible that multipolar cells of type I are different in their synaptic profiles and localization of excitatory and inhibitory synapes, which influence the spike generator located in the axonal hillox (Josephson and Morest 1998). Therefore multipolar cells projecting to different areas, such as ipsilateral or contralateral inferior colliculus, or cochlear nucleus may have a different synaptic profile and may correspond to the different populations of previously described multipolar cells in the VCN (Schofield and Cant 1996 a). The few large glycine positive neurons probably correspond to radiate neurons, cells hypothesized to exert a broad band inhibitory stimulation to excitatory neurons (expecially pyramidal cells) located in the DCN (Doucet et al. 1999). Comparison with glycinergic commissural neurons. The present study supports previous results which indicated, at least for some D-stellate neurons (Oertel et al. 1990) or radiate neurons (Doucet and Ryugo 1997; Doucet et al. 1999) in VCN, that they coincide with commissural neurons. However, since we were not able to completely restrict our injections to the DCN, and part of the dorsal acoustic stria also received the tracer, we cannot exclude the possibility that we have caused retrograde labelling of glycine positive commissural neurons from the ipsilateral side through their initial axons (Wenthold 1987; Kolston et al. 1992). As a result of the tract-tracing study we cannot be completely sure that the identified glycinergic cells are radiate neurons. Also commissural neurons have a similar ultrastructure and synaptology of our presumed radiate glycinergic neurons, and in particular a high percentage of axo-somatic R-boutons (Alibardi 1998b, 2000). The relatively high number of R (excitatory) vesicles may be put in relation to the onset firing activity of type II muJtipolar cells. This indicates that all these neurons belong to the same category and that they might derive from the same precursors during embryogenesis. It has been reported that a high number of excitatory boutons converging onto the cell body of a multipolar cell, especially near the axon hillox (where the spike generator is located), transforms the discharge rate from regular (chopper-type) to irregular (onset-type) (Smith and Rhode 1989; Oertel et al. 1990; Ostapoff et al. 1994; Josephson and Morest 1998). Glycinergic commissural/radiate neurons could therefore inhibit both the excitatory neurons (mostly pyramidal cells) in the ipsilateral and contralateral DCN and excitatory neurons in the contralateral VCN (mostly bushy and T-stellate cells) (Cant and Gaston 1982; Oertel
467
et al. 1990; Schofield and Cant 1996a, b; Doucet and Ryugo 1997; Alibardi 1998 a, b, 2000; D o u c e t et al. 1999). In the guinea pig this inhibition may also be tonotopic to the contralateral cochlear nucleus (Shore et al. 1994). Neurotransmitters localization and colocalization. Four main amino acids (aspartate, glutamate, glycine and G A B A ) , putative neurotransmitters, are present at high levels in the D C N - V C N (Godfrey et al. 1978). Of these, glutamate is excitatory while glycine and G A B A are inhibitory and specifically localized in cochlear nucleus neurons (Osen et al. 1990; Kolston et al. 1992; Ottersen et al. 1995; Wirier et al. 1995; Juiz et al. 1996b; Moore et al. 1996; K e m m e r and Vater 1997). These studies have shown that m a n y interneurons in the D C N and V C N colocalize glycine and G A B A . Aside from G A B A , both glutamate and glycine may be used for metabolic or neurotransmission purposes. In the second case they are stored in synaptic vesicles which are transported to the synaptic terminals, merged with the plasmalemma near the synaptic thickenings, and release the amino acids into the synaptic cleft (Davanger 1996). The immunogold labelling at the ultrastructural level (Ottersen et al 1990; Storm-Mathisen and Ottersen 1990) has allowed correlation of vesicle morphology to a specific amino acidic neurotransmitter in synaptic boutons within different areas of the acoustic systems (Helfert et al. 1992; Altschuler et al. 1993; Juiz et al. 1993, 1996a; Alibardi 2000a, b, c, d). The present qualitative study confirms previous results that have also shown that glycine and G A B A are colocalized in synaptic boutons of the guinea pig V C N (Altschuler et al. 1993; Alibardi 1999 a, 2000 c) and rat (Juiz et al. 1993; Alibardi 1999 b, 2000 a, b, d). This strongly supports the notion that, at least in the auditory system, morphology of synaptic boutons is correlated to their neurotransmitter. Examples of aminoacidic neurotransmitters colocalization have been found in other nervous areas other than in the acoustic system, and include both inhibitory (glycine/ G A B A ) or inhibitory and excitatory neurotransmitters (GABA/glutamate) (Ottersen et al. 1995; D a v a n g e r 1996). One of the suggested effects in the case of G A B A / glycine colocalization is that G A B A may bind to the glycine receptor, induce some conformational variation of the receptor, and modulate the action of glycine. A n o t h e r G A B A effect may be its presynaptic bind to prevent release of GABA-glycine into the synaptic cleft in order to bind to their receptor/s. Prevalent G A B A terminals (P) or glycine terminals (FP) may therefore have a different post-synaptic action than terminals colocalizing the two amino acids. Acknowledgements. The study of the dorsal cochlear nucleus was initiated at the University of Connecticut, Storrs, USA, in Prof. E. Mugnaini's laboratory, and was mainly carried on at the University of Bologna (60% grant). Mrs. L. Dipietrangelo made the photographic and computer-reproduction with Adobe-Photoshop program. Mr. M. Crowther (University of Sydney) proof read the manuscript.
References Adams JC (1983) Multipolar cells in the ventral cochlear nucleus project to the dorsal cochlear nucleus and the inferior colliculus. Neurosci Lett 37:205-208 Adams JC, Warr BW (1976) Origin of axons in the cat's acoustic striae determined by injection of horseradish peroxidase into severed tracts. J Comp Neurol 170:107-122 AlibardiL (1998a) Ultrastructural and immunocytochemical characterization of neurons in the rat ventral cochlear nucleus projecting to the inferior colliculus. Ann Anat 180:415-426 AlibardiL (1998b) Ultrastructural and immunocytochemical characterization of commissural neurons in the ventral cochlear nucleus of the rat. Ann Anat 180:427-438 Alibardi L (1999a) Characterization of tuberculo-ventral neurons in the dorsal cochlear nucleus of the guinea pig. J Submicrosc Cytol Pathol 31:295-300 Alibardi L (1999 b) Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus. J Brain Res 39:429-439 Alibardi L (2000 a) Identification of tuberculo-ventral neurons in the polymorphic layer of the rat dorsal cochlear nucleus. Eur J Morphol 38:153-166 Alibardi L (2000 b) Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat. Ann Anat 182: 207-220 Alibardi L (2000 c) Cytology of large neurons in the guinea pig dorsal cochlear nucleus contacting the inferior colliculus. Eur J Histochem 44:365-375 Alibardi L (2000 d) Putative commissural and collicular axo-somatic terminals on neurons of the rat ventral cochlear nucleus. J Submicr Cytol Pathol 32:555-566 Altschuler RA, Juiz JM, Shore SE, Bledsoe SC, Helfert RH, Wenthold EJ (1993) Inhibitory amino acid synapses and pathways in the ventral cochlear nucleus. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series. A Life Sciences, vol 239, pp 211-224 Cant NB (1981) The fine structure of two types of stellate cells in the anterior division of the anteroventral cochlear nucleus of the cat. Neuroscience 6:2643-2655 Cant NB (1982) Identification of cell types in the anteroventral cochlear nucleus that project to the inferior colliculus. Neurosci Let 32:241-246 Cant NB (1992) The cochlear nucleus. Neuronal types and their synaptic organization. In: Webster DB, Popper AN, Fay RR (Eds) The mammalian auditory pathway: Neuroanatomy. Handbook of Auditory Research, vol 1. Springer: New York, pp 66-116 Cant NB, Morest GK (1979) The bushy cells in the anteroventral cochlear nucleus of the cat. A study with the electron microscope. Neurosci 4:1925-1945 Cant NB, Gaston KG (1982) Pathways connecting the right and left cochlear nuclei. J Comp Neurol 212:313-326 Caspary DM (1986) Cochlear nuclei: functional neuropharmacology of the principal cell types. In: RA Altschuler, DW Hoffmann and RP Bobbin (Eds) Neurobiology of hearing: the Cochlea. Raven Press: New York, pp 303-332 Davenger S (1996) Colocalization of amino acid signal molecules in neurons and endocrine cells. Anat Embryol 194:1-12 Doucet JR, Ryugo DK (1997) Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats. J Comp Neurol 385:245-264
468
Doucet JR, Ross AT, Gillespie MB, Ryugo DK (1999) Glycine immunoreactivity of multipolar neurons in the ventral cochlear nucleus which project to the dorsal cochlear nucleus. J Comp Neurol 408:515-531 Godfrey DA, Carter JA, Lowry OH, Matschinsky FM (1978) Distribution of gamma-aminobutyric acid, glycine glutamate and aspartate in the cochlear nucleus of the rat. J Histochem Cytochem 26:118-126 HelfertRH, SneadCR, AltschulerRA (1991) The ascending auditory pathways. In: AltschulerRA, Bobbin RR Clopton BM and Hoffman DW (Eds) Neurobiology of hearing: The central auditory system. Raven Press: New York, pp 1-25 Helfert RH, Juiz JM, Bledsoe SC, Bonneau JM, Wenthold RJ, AltschulerRA (1992) Patterns of glutamate, glycine, and G A B A immunolabelling in four synaptic terminal classes in the lateral superior olive of the guinea pig. J Comp Neurol 323:305-325 Josephson EM, Morest DK (1998) A quantitative profile of the synapses on the stellate cell body and axon in the cochlear nucleus of the chinchilla. J Neurocytol 27:841-864 Juiz JM, Rubio ME, Helfert RH, Altschuler R A (1993) Localizing putative excitatory endings in the cochlear nucleus by quantitative immunocytochemistry. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series, A Life Sciences, vol 239, pp 167-177 Juiz JM, Helfert RH, Bonneau JM, Wenthol RJ, Altschuler R A (1996 a) Three classes of inhibitory amino acid terminals in the cochlear nucleus of the guinea pig. J Comp Neuro1373:11-26 Juiz JM, Helfert RH, Campos ML, Altschuler R A (1996b) Distribution of glycine and GABA immunoreactivities in the cochlear nucleus: quantitative patterns of putative inhibitory inputs on three cell types. J Brain Res 37:561-574 Kane E (1973) Octopus cells in the cochlear nucleus of the cat: heterotypic synapses upon homeotypic neurons. Int J Neurosci 5:251-279 Kemmer M, Vater M (1997) The distribution of GABA and glycine immunostaining in the cochlear nucleus of the mustached bat (Pteronotus parnelli). Cell Tissue Res 287:487-506 Kolston J, Osen KK, Hackney CM, Ottersen OR Storm-Mathisen J (1992) An atlas of glycine and GABA-like immunoreactivity and colocalization in the cochlear nuclear complex of the guinea pig. Anat Embryol 186:443-465 Moore JK (1986) Cochlear nuclei: relationship to the auditory nerve. In: Altschuler RA, Hoffmann DW, Bobbing RP (Eds) Neurobiology of hearing: the cochlea. Raven Press: New York, pp 283-301 Moore JK, Osen KK, Storm-Mathisen J, Ottersen OP (1996) gamma-aminobutyric acid and glycine in the baboon cochlear nuclei: an immunocytochemical colocalization study with reference to interspecies differences in inhibitory systems. J Comp Neurol 369:497-519 Morest KD (1993) The cellular basis for signal processing in the mammalian cochlear nuclei. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series, A Life Sciences, vo1239, Plenum Press: New York, pp 1-18 Oertel D, Wu SH, Garb MW, Dizack C (1990) Morphology and physiology of cells in slice preparations of posteroventral cochlear nucleus of mice. J Comp Neurol 295:136-154 Oliver DL (1987) Projections to the inferior colliculus from the aventeroventral cochlear nucleus in the cat: possible substrate for binaural interaction. J Comp Neurol 264:24-46 Osen KK, Ottersen OR Storm-Mathiesen J (1990) Colocalization
of glycine-like and GABA-like immunoreactivities: a semiquantitative study of individual neurons in the dorsal cochlear nucleus of cat. In: Ottersen OR Storm-Mathiesen J (Eds) Glycine neurotransmission. John Wyley and Sons: Chicester, pp 417-451 Ostapoff EM, Morest K (1991) Synaptic organization of globular bushy cells in the ventral cochlear nucleus of the cat: a quantitative study. J Comp Neurol 314:598-613 OstapoffEM, Feng JJ, Morest DK (1994) A physiological and structural study of neuron types in the cochlear nucleus. II. Neuron types and their structural correlation with response properties. J Comp Neurol 346:19-42 Ostapoff EM, Morest KD, Parham K (1999) Spatial organization of the reciprocal connections between the cat dorsal and anteroventral cochlear nuclei. Hear Res 130:75-93 Ottersen OR Storm-Mathisen J, Laake JH (1990) Cellular and subcellular localization of glycine studied by quantitative electron microscopic immunocytochemistry. In: OttersenOR Storm-Mathisen J (Eds). Glycine neurotransmission. John Wyley and Sons: Chicester, pp 303-328 Ottersen OP, Hjelle OR Osen KK, Laake JH (1995) Amino acid transmitters. In: Paxinos G (Ed) The rat nervous system, IInded. Academic Press, pp 1017-1037 Schofield BR, Cant NB (1996 a) Origin and targets of commissural connections between the cochlear nuclei in guinea pig. J Comp Neurol 375:128-146 Schofield BT, Cant NB (1996b) Projections from the ventral to the inferior colliculus and the contralateral cochlear nucleus in guinea pig. Hear Res 102:1-14 Shore SE, Godfrey DA (1994) Intrinic and contralateral connections of rat and guinea pig dorsal cochlear nucleus. Ass Res Otolaryngxol (Abstract) Shore SE, Godfrey DA, Helfert RH, Altschuler RA, Bledsoe SC (1992) Connections between the cochlear nuclei in guinea pig. Hear Res 62:16-26 Smith PH, Rhode WS (1987) Characterization of HRP-labelled globular bushy cells in the cat anteroventral cochlear nucleus. J Comp Neurol 266:360-375 Smith PH, Rhode WS (1989) Structural and functional properties distinguish two types of multipolar cells in the ventral cochlear nucleus. J Comp Neurol 282:595-616 Snyder RL, Leake PA (1988) Intrinsic connections within and between cochlear nucleus subdivisions in cat. J Comp Neurol 278:209-225 Storm-Mathisen J, Ottersen OP (1990) Immunocytochemistry of glutamate at the synaptic level. J Histochem Cytochem 38: 1733-1743 Tolbert LP, Morest DK, Yurgelun-Todd DA (1982) The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: horseradish peroxidase labelling of identified cell types. Neuroscience 7:3031-3052 Wenthold RJ (1987) Evidence for a glycinergic pathway connecting the two cochlear nuclei: an immunocytochemical and retrograde transport study. Brain Res 415:183-187 Weinberg RJ, Van Eyck J (1991) A tetramethylbenzydine/tungstate reaction for horseradish peroxidase histochemistry. J Histochem Cytochem 39:1143-1148 Winer JA, Larue DT, Pollack GD (1995) GABA and glycine in the central auditory system of the mustache bat: structural substrate for inhibitory neuronal organization. J Comp Neurol 335:317-353 Zhang SU, Oertel D (1994) Neuronal circuits associated with the output of the dorsal cochlear nucleus through fusiform cells. J Neurophysiol 71:914-930 Accepted May 16, 2001
469
Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus Lorenzo Aiibardi Dipartimento di Biologia evoluzionistica spefimentale, University of Bologna, via Selmi 3; 1-40126, Bologna, Italy
Summary. The neural tracer wheat germ agglutinin conjugated to horse radish peroxidase was injected into the rat dorsal cochlear nucleus and acoustic stria. Some labelled neurons in the ipsilateral ventral cochlear nucleus were found as a result. These neurons were studied at the ultrastructural level, and their axo-somatic synaptic profile and glycine immunoreactivity were determined. Most neurons were glycine negative and classified as type I multipolar neurons. The latter showed a different synaptic profile from that of neurons projecting to the contralateral inferior colliculus or cochlear nucleus. This suggests the presence of differing populations of multipolar cells based on their synaptic profile. Few labelled multipolar neurons of type II were found, which appeared glycine negative and, rarely, glycine positive. The latter show an ultrastructure and axo-somatic profile similar to that of glycinergic commissural neurons in the dorsal and ventral cochlear nucleus. In particular, about one-third of boutons contained round synaptic vesicles, which are believed to contain an excitatory neurotransmitter. The ultrastructural analysis of the synaptic boutons in the cochlear nucleus confirms the presence of numerous cases of colocalization of glycine and GABA where flat and pleomorphic synaptic vesicles are mixed. The present study is in accordance with previous tract-tracing light microscopic studies which have indicated that large glycinergic neurons in the ventral cochlear nucleus act as broad-band inhibitory neurons in microcircuits of the dorsal cochlear nucleus and contralateral cochlear nucleus.
Key words: Rat - Ipsilateral cochlear nuclei - Connecting neurons - Glycine - Ultrastructure Correspondence to: L. Alibardi E-mail: [email protected] Ann Anat (2001) 183:459-469 © Urban & Fischer Verlag
http:llwww.urbanfischer.deljournalslannanat
Introduction The cochlear nuclear nuclei receive the terminals of the acoustic nerve and their intrinsic connections form the basic network that modulates the electrical activity of higher brainstem auditory nuclei (Moore 1986; Helfert et al. 1991). The three main subdivisions of the chochlear nucleus are the dorsal cochlear nucleus (DCN), and the ventral nucleus (VCN), the latter subdivided into a posteroventral cochlear nucleus (PVCN) and anteroventral cochlear nucleus (AVCN). Aside from interneurons, various projective neurons (pyramidal, bushy, multipolar and octopus) are also present in the 3 main subdivisions of the cochlear nucleus and their intrinsic and extrinsic connections are largely known (Kane 1973, Cant and Morest 1979; Cant 1982, 1992; Tolbert et al. 1982; Caspary 1986; Smith and Rhode 1987, 1989; Ostapoff and Morest 1991; Ostapoff et al. 1994; Helfert et al. 1991; Morest 1993). In the VCN, bushy neurons directly transmit the information from the acoustic nerve (primary-like discharge pattern) to higher auditory nuclei (superior olivary complex) by excitatory neurotransmitters, and this is believed to produce timing information coded in the lower auditory system which is important for sound localization. Multipolar neurons participate in intrinsic and extrinsic circuits of the cochlear nuclei, where they send both inhibitory and excitatory terminals (Oliver 1987; Smith and Rhode 1989; Caspary 1991). Multipolar cells may be involved in regulatory circuits within the acoustic system, which permits signalling of the different frequency components of sound (spectral detection). Multipolar neurons project to the ipsilateral and contralateral inferior colliculus (Adams 1983; Oliver 1987; Schofield and Cant 1996 b), contralateral cochlear nucleus 0940-9602/01/183/5-459 $15.00/0
(Cant and Gaston 1982; Wenthold 1987; Shore et al. 1992; Schofield and Cant 1996 a), and from the VCN to the ipsilateral DCN (Snyder and Leake 1988; Oertel et al. 1990; Shore and Godfrey 1994; Doucet and Ryugo 1997; Doucet et al. 1999; Ostapoff et al. 1999). These studies have characterized different multipolar cells and have shown that some subtypes project to different acoustic areas as excitatory or inhibitory cells (Oertel et al. 1990; Schofield and Cant, 1996 a, b; Doucet et al., 1999). Although multipolar neurons have been divided into types I and II based on cytological and ultrastructural characteristics (Cant 1981), it has become apparent that together with their specific neurotransmitter, the synaptic profile of the cell body and dendritic three shapes their electrical response and determines the specific stimulus to the connected acoustic areas (Cant 1982; Smith and Rhoe 1989; Ostapoff and Morest 1991; Ostapoff et al. 1994; Alibardi 1998 a, b; Josephson and Morest 1998). In the VCN, T-stellate cells (projecting into the trapezoid body and directed to the inferior colliculus) correspond to type I multipolar neurons, and D-stellate neurons (projecting to the ipsilateral D C N and perhaps exiting through the dorsal acoustic striae as commissural axons) correspond to type II multipolar neurons (Cant 1981; Adams 1983; Smith and Rhode 1989; Oertel et al. 1990; Kolston et al. 1992). Both T-stellate and D-stellate cells send collateral axons to the ipsilateral DCN. Three main types of projective neurons, planar, marginal and radiate, have been identified to project from the AVCN and PVCN to the ipsilateral DCN (Doucet and Ryugo 1997; Doucet et al. 1999). The radiate cells appear glycinergic and therefore inhibitory, and it has been suggested that these neurons correspond to commissural neurons that send collateral axons to the D C N before projecting their main axon to the contralateral cochlear nucleus (Doucet et al. 1999). This identity can be revealed by comparison of their fine structure, synaptology and immunocytochemistry for amino acidic neurotransmitters between neurons projecting ipsilaterally (DCN) with those projecting to the contralateral CN (Alibardi 1988 a, 2000). The study of the synaptic profile of neurons is a modern morphological approach useful to understand the function of single neurons within the interconnected acoustic networks (Cant 1992; Morest 1993). It has also been shown that, aside from neurons (Osen et al. 1990; Kolston et al. 1992; Winer et al. 1995; Juiz et al. 1996 b; Moore et al 1996; Kemmer and Vater 1997), synaptic terminals with a specific ultrastructure also store different amino acidic neurotransmitters (Otterson and Storm Mathisen 1990; Altschuler et al. 1993; Juiz et al. 1993, 1996 a). These studies have shown that glycine and G A B A are variably localized, or even colocalized, within specific synaptic terminals of different areas of the acoustic systems of rats and guinea pigs. Three main types of synaptic boutons are recognized: 1) R containing pleomorphic vesicles and making symmetric pre- and post-synaptic thickenings, 2) FR similar to the former but containing a high or even prevalent proportion of fiat ve-
sides, and 3) R, containing prevalently round vesicles and making asymmetric pre- and post-synaptic vesicles. The present study is a continuation of previous ultrastructural analyses of identified cochlear nucleus neurons in different neuronal populations in rat and guinea pig cochlear nuclei (Alibardi 1998 a, b; 1999 a, b; 2000 a, b, c, d). In this report we aim to determine the heterogeneity of synaptic profiles, and make an ultrastructural and immunocytochemical comparison between ipsilateral projecting neurons from VCN to DCN (Doucet and Ryugo 1997; Doucet et al. 1999) with commissural neurons projecting to the contralateral cochlear nuclei.
Materials and methods The animals used in the present study on ipsilateral connections between VCN and DCN were previously studied for their contralateral (commissural) projections (Alibardi 1998 b, 2000 b). Therefore the comparison between ipsilateral and commissural projecting neurons has been done among the same 3 rats previously used after DCN injection. Surgery, tracer injection, perfusion, fixation, and immunocytochemical techniques have previously been reported in detail (see above). Briefly, wheat germ agglutinin conjugated to horse radish peroxidase (WGA-HRP) was injected into the DCN of 3 rats, and the ipsilateral VCN was studied for tracer localization in labelled cells. The tracer, using the method of Weinberg and Van Eyck (1991), appeared in the form of electron-dense needles. The immunolabelling for GABA and glycine was revealed under the light microscope by a peroxydase-antiperoxidase reaction (PAP), and for the electron microscope by post-embedding immunogold labelling with silver-enhancement of gold particles. Quantification of cell parameters measured on eletron micrographs (perimeter, percentage of boutons etc., see Table 1) was done using a computer program (Leica Qwin Software, Leica Imaging Systems, Ltd., UK).
Results Retrograde labelled neurons in the VCN. The large injections performed, although mainly confined within the DCN, showed upon light microscopic examination that the molecular, fusiform and polymorphic layers of the DCN received the tracer (Fig. 1). Although mainly confined within the DCN some tracer was also seen spreading into part of the dorsal acoustic striae, which is located at the base of the DCN. Examination of the labelled areas showed that pyramidal cells and smaller cells, including tuberculoventral neurons, were labelled with their surrounding neuropile (Fig. 2). Labelled cells were also seen around the injection site, while more distant regions in the DCN showed few or no labelled cells. Most cells in the octopus cell area which is close to the injection site, were not labelled in the injected cases, and only occasional cells (octopus?) showed weak labelling (1-3 trace needles/cell). This indicated that the intermedi-
460
Fig. 1. Semthin section of injected DCN showing the spreading of the tracer (arrow) into the molecular (1), fusiform (2) and polymorphic (3) layer, including the acoustic striae (arrowhead). 100 x. Fig. 2. Higher magnification of labelled cells within the injection site. The arrow indicates a pyramidal cell. The arrowhead points a likely tuberculoventral neuron. 200 x. Fig. 3. A-B are serial sections showing two labelled cells (L) in ipsilateral PCVN which are glycine (GLY) negative (arrowhead) and positive (arrow). 750 x. Fig. 4. Labelled multipolar neuron of type II in ipsilateral PVCN with some labelled axo-somatic terminals (arrowheads). v, indicating blood vessels also visible in the semithin sections of this labelled cell (inset L, 800 x) which is glycine positive (inset GLY, 800 x). 5000 x.
461
Fig. 5. Labelled multipolar neuron of type I in ipsilateral PVCN, which is contacted by few synapses (arrows). E, rough endoplasmic reticulum, v, referring blood vessel also visible in semithin sections of this labelled cell (inset L, 700 x) which is glycine negative (inset GLY, 700 x). 7000 x. Fig. 6. Other elongated multipolar neuron of type I (D, dendrite) showing a few axo-somatic synapses (arrows). 6400 x.
462
Table. 1. Synaptic profile of retrograde labelled multipolar neurons in the ipsilateral ventral cochlear nucleus. n = number of cells or of axo-somatic synaptic boutons. +, Standard deviation. Cell type
Cell diameter 0tm)
Percentage coverage
Boutons/cell
Types of boutons/cell
Type I
15.38 + 2.49 (n = 20)
26.60 + 12.04
8.35 + 4.18 (n = 167 total)
FP 29.34% (n = 49)
Type II
21.40 + 3.14 (n = 7)
64.46 + 10.41
23.0 + 5.16 (n = 161 total)
21.38% (n = 34)
ate acoustic stria was little or not at all influenced by the spreading of the injected tracer. Labelled multipolar (non-globular) cells were seen in the multipolar area of the PVCN and AVCN near the acoustic nerve root region. The injections were large, and not localized to produce a tonotopic labelling in the VCN (Doucet and Ryugo 1997; Doucet et al. 1999). However, in some cross sections of VCN, no or rare cells were seen; in other sections of different VCN areas, labelled neurons were more numerous in the magnocellular (central) part. Most of these cells, whose shape varied from oval and elongated to roundish, were glycine negative but a few were glycine positive (Fig. 3). Out of 92 retrograde labelled cells (14-24 ~tm large), 82 were glycine-negative and 10 where glycine positive. Of the latter, 3 were also characterized ultrastructurally (Fig. 4). They were medium to large sized cells (20-24 gm) with short peripheral ergastoplasm (Nissl bodies), sparce pale areas occupied by microtubules and neurofilaments, and surrounded by numerous (51-79%) axo-somatic boutons (see Table 1). Most of the boutons contained pleomorphic vesicles (P boutons) but about 30% were represented by round vesicles (R boutons), while the less frequent type contained pleomorphic vesicles enriched by flat vesicles (FP boutons). These characteristic cytological features identify thse cells as multipolar neurons of type II (Cant 1981; Smith and Rhode 1989). Most glycine negative cells, despite their shape (roundish to fusiform), and position in the VCN were ultrastructurally recognizable as type I multipolar neurons (Figs. 5, 6). Nissl bodies were less numerous but with longer endoplasmic cisternae, and axo-somatic boutons were less frequent than the former type (9-48%, Table1). Most boutons were also of the P type, followed by less than half by those of FP type, and by few R boutons.
Ultrastructural comparison with commissural neurons. Few large glycinergic neurons in the D C N were found in the polymorphic layer. The ultrastructural study of the few, large glycinergic neurons in the DCN (Fig. 7) that were labelled after injection in the contralateral cochlear nucleus, showed small peripheral Nissl bodies and numerous neurofilaments like multipolar type II cells in the VCN. These neurons were mostly surrounded by axo-somatic boutons (28-33%) mainly P, then by R, and less frequently by FP boutons. While P and FP boutons stored
R 5.99% (n = 10)
P 64.67% (n = 108)
33.33% (n = 53)
45.28% (n = 72)
G A B A or glycine to different degrees, the R boutons were negative for both (Figs. 8, 9, 10). The analysis of commissural neurons in the VCN showed that they had similar ultrastructural features, immunoreactivity and synaptology as the above cells. They were surrounded by 28-30% axo-somatic boutons and were glycine positive multipolar cells of type If (Fig. 11). As glycinergic commissural neurons, also glycinergic multipolar neurons of type II in the ipsilateral VCN, received 28-33% of the axo-somatic boutons in form of R boutons. This relatively high R boutons input seems to be characteristic of all these glycinergic cells. Immunogold labelling of boutons. The immunogold observations on R FP and R boutons present in the D C N and VCN, both axo-somatic or in the neuropile (axo-dendritic), revealed that no labelling was present in R boutons. Glycine was more commonly seen in FP boutons than GABA, where it was scarse or absent in P boutons. In the latter G A B A was instead more frequently present (Figs. 8-10). Numerous cases of colocalization of G A B A with glycine in P or FP boutons were also seen (Figs. 1214). The intensity of immunolabelling to glycine often increased where numerous flat vesicles were seen, while G A B A immunolabelling prevailed only where pleomorphic vesicles were seen.
Discussion Injection site. As a result of the large injections performed in this study, we have not been able to label tonotopically neurons in the ipsilateral VCN (Shore and Godfrey 1994; Doucet and Ryugo 1997; Doucet et al. 1999). The microscopical study has shown that, aside from the DCN, our injections effected part of the dorsal acoustic striae but little or none of the intermediate acoustic striae. Since the dorsal stria, particularly the medial-anterior part, also collects exiting commissural axons which are glycinergic (Oertel et al. 1990; Kolston et al. 1992; Ottersen et al. 1995), we cannot exclude the possibility that the axons of our few glycinergic cells have taken-up the tracer into axons en passage within the striae. In fact, in contrast to previous studies (Doucet et al. 1999), we were not able to confine our injections to the DCN, although this
463
Fig. 7. vessel gative Fig. 8. Fig. 9.
Labelled (arrow) commissural multipolar neurons of type II in DCN surrounded by axo-somatic boutons, v, referring blood also visible in semithin sections of this labelled cell (inset L, 700 x) which is glycine positive (inset GLY, 700 x) and G A B A ne(inset GABA, 700 x). 4000 x. Glycine (GLY) immunolabelled bouton (F) containing numerous fiat vesicles in DCN. 28800 x. G A B A (GABA) immunolabelled bouton (P) containing pleomorphic vesicles in DCN. 53600 x.
464
Fig. 10. A - B are serial sections of two synaptic terminals in DCN immunolabelled for G A B A (A) or glycine (GLY). A, shows labelled terminals containing pleomorphic vesicle (P) which is not labelled for glycine (B). Boutouns containing round vesicles (R) are not labelled with either antibodys. 24100 ×. Fig. 11. Labelled (arrows) commissural multipolar neuron of type II in PVCN surrounded by numerous axo-somatic boutons, some also labelled (arrowheads). The inset (700 x) shows that this cell is glycine positive (GLY). v, referring blood vessel. 4300 x.
465
Fig. 12-14. Examples of GABA-glycine colocalization in VCN neuropile. R pleomorphic boutons. F, flat and pleomorphic boutons. R, round boutons. In Figures 12 (40300 x) and 13 (36900x) GABA labelling is more prevalent than glycine labelling in P boutons. In Figure 14 (27000 x) glycine labelling is more prevalent in F boutons.
466
was by far the nervous area that most received of the tracer. However tracer spreading into the stria was not as dense as inside the DCN, resulting in a diffuse labelling which is considered less specific than granular labelling in retrograde tract-tracing studies. This means that retrograde labelled neurons do not necessarily project to the area where the diffuse labelling is located (Tolbert et al. 1982; Snyder and Leake 1998). Besides, previous studies did not find retrograde labelled neurons in the ipsilateral VCN after injection into the DAS (Adam and Warr 1976; Adams 1983). The fact that occasional cells in the octopus cell area (octopus cells or other types) were weakly labelled indicates that most of the intermediate acoustic striae was not influenced by these larger injections. Based on the above considerations, we believe that the ipsilateral retrograde labelled neurons seen in this study are projecting through a specific route to the ipsilateral DCN (Adams 1983; Oertel et al. 1990; Ostapoff et al. 1999). Ipsilateral projecting neurons. In their study on neurons sending axons through the dorsal acoustic striae in cats, Adams and Warr (1976) rarely found retrograde labelled cells in the ipsilateral VCN after Horseradish Peroxidaseapplication in the severed dorsal acoustic stria. A following study (Adams 1983) instead found frequent retrograde labelled stellate neurons in the VCN after injection in the ipsilateral DCN, indicating a direct connection between the two nuclei by a route different from that of the dorsal acoustic striae. It is possible that most, if not all, multipolar cells projecting to the inferior colliculus or outside the VCN through the ventral acoustic striae also send collaterals to the DCN (Ostapoff et al. 1999). This intrinsic circuit has been implicated with the formation of a more refined sensitivity of target neurons in the DCN (pyramidal neurons) to acoustic signals that shape their specific electric response directed outside the DCN (Zhang and Oertel 1994; Doucet et al. 1999). Therefore it is hypothesised that interpreting frequency components of sounds by pyramidal cells can produce more precise electrical signals to transmit to the inferior colliculus. According to previous studies it is known that the glycine-negative multipolar cells in the VCN that project to the ipsilateral DCN do not use the dorsal acoustic striae as the exiting route but instead use the ventral acoustic striae in the trapezoid body (Adams 1983; Oertel et al. 1990) or remain confined within the cochlear nucleus (Doucet and Ryugo 1997; Doucet et al. 1999; Ostapoff et al. 1999). We confirm that most of these neurons, identified as type I multipolar, are glycine negative (Doucet et al. 1999). The analysis of the synaptic profile in type I multipolar neurons projecting to the contralateral inferior colliculus (Alibardi 1998a) or cochlear nucleus (Alibardi 1998b, 2000) shows some significant differences to those projecting to the ipsilateral DCN. The latter are presumably the planar and marginal neurons as indicated by the study of Doucet and Ryugo (1997). Specifically, the multipolar
neurons of type I, projecting to the inferior colliculus, receive more P boutons and less R boutons, and are slighly larger on the average (Alibardi 1998a). Instead, the multipolar neurons of type I projecting to the contralateral cochlear nucleus receive a little more axo-somatic boutons which are of P type, and less of R type, and their diameter is larger (Alibardi 1998 b). From the result of the present study it seems possible that multipolar cells of type I are different in their synaptic profiles and localization of excitatory and inhibitory synapes, which influence the spike generator located in the axonal hillox (Josephson and Morest 1998). Therefore multipolar cells projecting to different areas, such as ipsilateral or contralateral inferior colliculus, or cochlear nucleus may have a different synaptic profile and may correspond to the different populations of previously described multipolar cells in the VCN (Schofield and Cant 1996 a). The few large glycine positive neurons probably correspond to radiate neurons, cells hypothesized to exert a broad band inhibitory stimulation to excitatory neurons (expecially pyramidal cells) located in the DCN (Doucet et al. 1999). Comparison with glycinergic commissural neurons. The present study supports previous results which indicated, at least for some D-stellate neurons (Oertel et al. 1990) or radiate neurons (Doucet and Ryugo 1997; Doucet et al. 1999) in VCN, that they coincide with commissural neurons. However, since we were not able to completely restrict our injections to the DCN, and part of the dorsal acoustic stria also received the tracer, we cannot exclude the possibility that we have caused retrograde labelling of glycine positive commissural neurons from the ipsilateral side through their initial axons (Wenthold 1987; Kolston et al. 1992). As a result of the tract-tracing study we cannot be completely sure that the identified glycinergic cells are radiate neurons. Also commissural neurons have a similar ultrastructure and synaptology of our presumed radiate glycinergic neurons, and in particular a high percentage of axo-somatic R-boutons (Alibardi 1998b, 2000). The relatively high number of R (excitatory) vesicles may be put in relation to the onset firing activity of type II muJtipolar cells. This indicates that all these neurons belong to the same category and that they might derive from the same precursors during embryogenesis. It has been reported that a high number of excitatory boutons converging onto the cell body of a multipolar cell, especially near the axon hillox (where the spike generator is located), transforms the discharge rate from regular (chopper-type) to irregular (onset-type) (Smith and Rhode 1989; Oertel et al. 1990; Ostapoff et al. 1994; Josephson and Morest 1998). Glycinergic commissural/radiate neurons could therefore inhibit both the excitatory neurons (mostly pyramidal cells) in the ipsilateral and contralateral DCN and excitatory neurons in the contralateral VCN (mostly bushy and T-stellate cells) (Cant and Gaston 1982; Oertel
467
et al. 1990; Schofield and Cant 1996a, b; Doucet and Ryugo 1997; Alibardi 1998 a, b, 2000; D o u c e t et al. 1999). In the guinea pig this inhibition may also be tonotopic to the contralateral cochlear nucleus (Shore et al. 1994). Neurotransmitters localization and colocalization. Four main amino acids (aspartate, glutamate, glycine and G A B A ) , putative neurotransmitters, are present at high levels in the D C N - V C N (Godfrey et al. 1978). Of these, glutamate is excitatory while glycine and G A B A are inhibitory and specifically localized in cochlear nucleus neurons (Osen et al. 1990; Kolston et al. 1992; Ottersen et al. 1995; Wirier et al. 1995; Juiz et al. 1996b; Moore et al. 1996; K e m m e r and Vater 1997). These studies have shown that m a n y interneurons in the D C N and V C N colocalize glycine and G A B A . Aside from G A B A , both glutamate and glycine may be used for metabolic or neurotransmission purposes. In the second case they are stored in synaptic vesicles which are transported to the synaptic terminals, merged with the plasmalemma near the synaptic thickenings, and release the amino acids into the synaptic cleft (Davanger 1996). The immunogold labelling at the ultrastructural level (Ottersen et al 1990; Storm-Mathisen and Ottersen 1990) has allowed correlation of vesicle morphology to a specific amino acidic neurotransmitter in synaptic boutons within different areas of the acoustic systems (Helfert et al. 1992; Altschuler et al. 1993; Juiz et al. 1993, 1996a; Alibardi 2000a, b, c, d). The present qualitative study confirms previous results that have also shown that glycine and G A B A are colocalized in synaptic boutons of the guinea pig V C N (Altschuler et al. 1993; Alibardi 1999 a, 2000 c) and rat (Juiz et al. 1993; Alibardi 1999 b, 2000 a, b, d). This strongly supports the notion that, at least in the auditory system, morphology of synaptic boutons is correlated to their neurotransmitter. Examples of aminoacidic neurotransmitters colocalization have been found in other nervous areas other than in the acoustic system, and include both inhibitory (glycine/ G A B A ) or inhibitory and excitatory neurotransmitters (GABA/glutamate) (Ottersen et al. 1995; D a v a n g e r 1996). One of the suggested effects in the case of G A B A / glycine colocalization is that G A B A may bind to the glycine receptor, induce some conformational variation of the receptor, and modulate the action of glycine. A n o t h e r G A B A effect may be its presynaptic bind to prevent release of GABA-glycine into the synaptic cleft in order to bind to their receptor/s. Prevalent G A B A terminals (P) or glycine terminals (FP) may therefore have a different post-synaptic action than terminals colocalizing the two amino acids. Acknowledgements. The study of the dorsal cochlear nucleus was initiated at the University of Connecticut, Storrs, USA, in Prof. E. Mugnaini's laboratory, and was mainly carried on at the University of Bologna (60% grant). Mrs. L. Dipietrangelo made the photographic and computer-reproduction with Adobe-Photoshop program. Mr. M. Crowther (University of Sydney) proof read the manuscript.
References Adams JC (1983) Multipolar cells in the ventral cochlear nucleus project to the dorsal cochlear nucleus and the inferior colliculus. Neurosci Lett 37:205-208 Adams JC, Warr BW (1976) Origin of axons in the cat's acoustic striae determined by injection of horseradish peroxidase into severed tracts. J Comp Neurol 170:107-122 AlibardiL (1998a) Ultrastructural and immunocytochemical characterization of neurons in the rat ventral cochlear nucleus projecting to the inferior colliculus. Ann Anat 180:415-426 AlibardiL (1998b) Ultrastructural and immunocytochemical characterization of commissural neurons in the ventral cochlear nucleus of the rat. Ann Anat 180:427-438 Alibardi L (1999a) Characterization of tuberculo-ventral neurons in the dorsal cochlear nucleus of the guinea pig. J Submicrosc Cytol Pathol 31:295-300 Alibardi L (1999 b) Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus. J Brain Res 39:429-439 Alibardi L (2000 a) Identification of tuberculo-ventral neurons in the polymorphic layer of the rat dorsal cochlear nucleus. Eur J Morphol 38:153-166 Alibardi L (2000 b) Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat. Ann Anat 182: 207-220 Alibardi L (2000 c) Cytology of large neurons in the guinea pig dorsal cochlear nucleus contacting the inferior colliculus. Eur J Histochem 44:365-375 Alibardi L (2000 d) Putative commissural and collicular axo-somatic terminals on neurons of the rat ventral cochlear nucleus. J Submicr Cytol Pathol 32:555-566 Altschuler RA, Juiz JM, Shore SE, Bledsoe SC, Helfert RH, Wenthold EJ (1993) Inhibitory amino acid synapses and pathways in the ventral cochlear nucleus. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series. A Life Sciences, vol 239, pp 211-224 Cant NB (1981) The fine structure of two types of stellate cells in the anterior division of the anteroventral cochlear nucleus of the cat. Neuroscience 6:2643-2655 Cant NB (1982) Identification of cell types in the anteroventral cochlear nucleus that project to the inferior colliculus. Neurosci Let 32:241-246 Cant NB (1992) The cochlear nucleus. Neuronal types and their synaptic organization. In: Webster DB, Popper AN, Fay RR (Eds) The mammalian auditory pathway: Neuroanatomy. Handbook of Auditory Research, vol 1. Springer: New York, pp 66-116 Cant NB, Morest GK (1979) The bushy cells in the anteroventral cochlear nucleus of the cat. A study with the electron microscope. Neurosci 4:1925-1945 Cant NB, Gaston KG (1982) Pathways connecting the right and left cochlear nuclei. J Comp Neurol 212:313-326 Caspary DM (1986) Cochlear nuclei: functional neuropharmacology of the principal cell types. In: RA Altschuler, DW Hoffmann and RP Bobbin (Eds) Neurobiology of hearing: the Cochlea. Raven Press: New York, pp 303-332 Davenger S (1996) Colocalization of amino acid signal molecules in neurons and endocrine cells. Anat Embryol 194:1-12 Doucet JR, Ryugo DK (1997) Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats. J Comp Neurol 385:245-264
468
Doucet JR, Ross AT, Gillespie MB, Ryugo DK (1999) Glycine immunoreactivity of multipolar neurons in the ventral cochlear nucleus which project to the dorsal cochlear nucleus. J Comp Neurol 408:515-531 Godfrey DA, Carter JA, Lowry OH, Matschinsky FM (1978) Distribution of gamma-aminobutyric acid, glycine glutamate and aspartate in the cochlear nucleus of the rat. J Histochem Cytochem 26:118-126 HelfertRH, SneadCR, AltschulerRA (1991) The ascending auditory pathways. In: AltschulerRA, Bobbin RR Clopton BM and Hoffman DW (Eds) Neurobiology of hearing: The central auditory system. Raven Press: New York, pp 1-25 Helfert RH, Juiz JM, Bledsoe SC, Bonneau JM, Wenthold RJ, AltschulerRA (1992) Patterns of glutamate, glycine, and G A B A immunolabelling in four synaptic terminal classes in the lateral superior olive of the guinea pig. J Comp Neurol 323:305-325 Josephson EM, Morest DK (1998) A quantitative profile of the synapses on the stellate cell body and axon in the cochlear nucleus of the chinchilla. J Neurocytol 27:841-864 Juiz JM, Rubio ME, Helfert RH, Altschuler R A (1993) Localizing putative excitatory endings in the cochlear nucleus by quantitative immunocytochemistry. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series, A Life Sciences, vol 239, pp 167-177 Juiz JM, Helfert RH, Bonneau JM, Wenthol RJ, Altschuler R A (1996 a) Three classes of inhibitory amino acid terminals in the cochlear nucleus of the guinea pig. J Comp Neuro1373:11-26 Juiz JM, Helfert RH, Campos ML, Altschuler R A (1996b) Distribution of glycine and GABA immunoreactivities in the cochlear nucleus: quantitative patterns of putative inhibitory inputs on three cell types. J Brain Res 37:561-574 Kane E (1973) Octopus cells in the cochlear nucleus of the cat: heterotypic synapses upon homeotypic neurons. Int J Neurosci 5:251-279 Kemmer M, Vater M (1997) The distribution of GABA and glycine immunostaining in the cochlear nucleus of the mustached bat (Pteronotus parnelli). Cell Tissue Res 287:487-506 Kolston J, Osen KK, Hackney CM, Ottersen OR Storm-Mathisen J (1992) An atlas of glycine and GABA-like immunoreactivity and colocalization in the cochlear nuclear complex of the guinea pig. Anat Embryol 186:443-465 Moore JK (1986) Cochlear nuclei: relationship to the auditory nerve. In: Altschuler RA, Hoffmann DW, Bobbing RP (Eds) Neurobiology of hearing: the cochlea. Raven Press: New York, pp 283-301 Moore JK, Osen KK, Storm-Mathisen J, Ottersen OP (1996) gamma-aminobutyric acid and glycine in the baboon cochlear nuclei: an immunocytochemical colocalization study with reference to interspecies differences in inhibitory systems. J Comp Neurol 369:497-519 Morest KD (1993) The cellular basis for signal processing in the mammalian cochlear nuclei. In: Merchant MA, Juiz JM, Godfrey DA, Mugnaini E (Eds) The mammalian cochlear nuclei: organization and function. NATO ASI series, A Life Sciences, vo1239, Plenum Press: New York, pp 1-18 Oertel D, Wu SH, Garb MW, Dizack C (1990) Morphology and physiology of cells in slice preparations of posteroventral cochlear nucleus of mice. J Comp Neurol 295:136-154 Oliver DL (1987) Projections to the inferior colliculus from the aventeroventral cochlear nucleus in the cat: possible substrate for binaural interaction. J Comp Neurol 264:24-46 Osen KK, Ottersen OR Storm-Mathiesen J (1990) Colocalization
of glycine-like and GABA-like immunoreactivities: a semiquantitative study of individual neurons in the dorsal cochlear nucleus of cat. In: Ottersen OR Storm-Mathiesen J (Eds) Glycine neurotransmission. John Wyley and Sons: Chicester, pp 417-451 Ostapoff EM, Morest K (1991) Synaptic organization of globular bushy cells in the ventral cochlear nucleus of the cat: a quantitative study. J Comp Neurol 314:598-613 OstapoffEM, Feng JJ, Morest DK (1994) A physiological and structural study of neuron types in the cochlear nucleus. II. Neuron types and their structural correlation with response properties. J Comp Neurol 346:19-42 Ostapoff EM, Morest KD, Parham K (1999) Spatial organization of the reciprocal connections between the cat dorsal and anteroventral cochlear nuclei. Hear Res 130:75-93 Ottersen OR Storm-Mathisen J, Laake JH (1990) Cellular and subcellular localization of glycine studied by quantitative electron microscopic immunocytochemistry. In: OttersenOR Storm-Mathisen J (Eds). Glycine neurotransmission. John Wyley and Sons: Chicester, pp 303-328 Ottersen OP, Hjelle OR Osen KK, Laake JH (1995) Amino acid transmitters. In: Paxinos G (Ed) The rat nervous system, IInded. Academic Press, pp 1017-1037 Schofield BR, Cant NB (1996 a) Origin and targets of commissural connections between the cochlear nuclei in guinea pig. J Comp Neurol 375:128-146 Schofield BT, Cant NB (1996b) Projections from the ventral to the inferior colliculus and the contralateral cochlear nucleus in guinea pig. Hear Res 102:1-14 Shore SE, Godfrey DA (1994) Intrinic and contralateral connections of rat and guinea pig dorsal cochlear nucleus. Ass Res Otolaryngxol (Abstract) Shore SE, Godfrey DA, Helfert RH, Altschuler RA, Bledsoe SC (1992) Connections between the cochlear nuclei in guinea pig. Hear Res 62:16-26 Smith PH, Rhode WS (1987) Characterization of HRP-labelled globular bushy cells in the cat anteroventral cochlear nucleus. J Comp Neurol 266:360-375 Smith PH, Rhode WS (1989) Structural and functional properties distinguish two types of multipolar cells in the ventral cochlear nucleus. J Comp Neurol 282:595-616 Snyder RL, Leake PA (1988) Intrinsic connections within and between cochlear nucleus subdivisions in cat. J Comp Neurol 278:209-225 Storm-Mathisen J, Ottersen OP (1990) Immunocytochemistry of glutamate at the synaptic level. J Histochem Cytochem 38: 1733-1743 Tolbert LP, Morest DK, Yurgelun-Todd DA (1982) The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: horseradish peroxidase labelling of identified cell types. Neuroscience 7:3031-3052 Wenthold RJ (1987) Evidence for a glycinergic pathway connecting the two cochlear nuclei: an immunocytochemical and retrograde transport study. Brain Res 415:183-187 Weinberg RJ, Van Eyck J (1991) A tetramethylbenzydine/tungstate reaction for horseradish peroxidase histochemistry. J Histochem Cytochem 39:1143-1148 Winer JA, Larue DT, Pollack GD (1995) GABA and glycine in the central auditory system of the mustache bat: structural substrate for inhibitory neuronal organization. J Comp Neurol 335:317-353 Zhang SU, Oertel D (1994) Neuronal circuits associated with the output of the dorsal cochlear nucleus through fusiform cells. J Neurophysiol 71:914-930 Accepted May 16, 2001
469