Synaptic excitation in the dorsal nucleus of the lateral lemniscus

Progress in Neurobiology Vol. 57, pp. 357 to 375, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/98/$ - see front matter

PII: S0301-0082(98)00058-6

SYNAPTIC EXCITATION IN THE DORSAL NUCLEUS OF THE LATERAL LEMNISCUS SHU HUI WU* Laboratory of Sensory Neuroscience, Institute of Neuroscience, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 (Received 16 April 1998) AbstractÐThe dorsal nucleus of the lateral lemniscus (DNLL) is a distinct auditory neuronal group located ventral to the inferior colliculus (IC). It receives excitatory and inhibitory a€erent inputs from various structures of the auditory lower brainstem and sends GABAergic inhibitory e€erents mainly to the contralateral DNLL and the bilateral IC. The synaptic excitation in DNLL neurons consists of two components, an early fast depolarization and a later long lasting one. Glutamate is the probable excitatory neurotransmitter for DNLL neurons. a-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate the early part of the excitation while N-Methyl-D-aspartate (NMDA) receptors mediate the long lasting component. The long lasting NMDA receptor-mediated component in the DNLL may contribute to a prolonged inhibition in the IC. The DNLL is thought to be a structure for processing binaural information. Most DNLL neurons in rat and bat are sensitive to interaural intensity di€erences (IIDs). They are excited by stimulation of the contralateral ear and inhibited by stimulation of the ipsilateral ear, showing an excitatory/inhibitory (EI) binaural response pattern. The EI pattern can be attributed to synaptic inputs that originate from various structures in the lower auditory brainstem and impinge on the DNLL neurons. In cat some DNLL neurons are sensitive to IIDs and some are sensitive to interaural time di€erences. In addition, DNLL neurons exhibit di€erent temporal response patterns to contralateral tonal stimulation and respond to amplitude modulated tones, implying that DNLL may contribute to processing temporally complex acoustic information. DNLL neurons shape binaural responses in the contralateral inferior colliculus and auditory cortex through their inhibitory brainstem projections and contribute to the accuracy with which animals localize sounds in space. # 1998 Elsevier Science Ltd. All rights reserved

CONTENTS 1. Introduction 2. A€erent projections to the dorsal nucleus of the lateral lemniscus 2.1. Cochlear nucleus 2.2. Superior olivary complex 2.3. Nuclei of the lateral lemniscus 3. E€erent projections of the dorsal nucleus of the lateral lemniscus 4. Physiological studies in vitro 4.1. Brief view of brain slice methods 4.2. Excitatory synaptic responses 4.3. Excitatory synaptic neurotransmitters and receptors 5. Physiological response properties of DNLL neurons in vivo 6. Functional role of the dorsal nucleus of the lateral lemniscus in auditory processing Acknowledgements References

ABBREVIATIONS AMPA a-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid APV D,L-2-Amino-5-phosphonovaleric acid AVCN Anteroventral cochlear nucleus CN Cochlear nucleus CNQX 6-Cyano-7-nitroquinoxaline-2,3-dione CPP (2)-3(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid

DCN DNLL DNQX EE EI EM EPSP

Dorsal cochlear nucleus Dorsal nucleus of the lateral lemniscus 6, 7-Dinitroquinoxaline-2, 3-dione Excitatory-excitatory (binaural cell type) Excitatory-inhibitory (binaural cell type) Electron microscopy Excitatory postsynaptic potential

* Tel.: (613) 520-2600, ext. 3953 (oce) or 2671 (lab); fax: (613) 520-4052; e-mail: [email protected]. 357

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ABBREVIATIONS EPSC GAD IC IID INLL IPSP ITD LSO LNTB MNTB MSO

Excitatory postsynaptic current Glutamic acid decarboxylase Inferior colliculus Interaural intensity di€erence Intermediate nucleus of the lateral lemniscus Inhibitory postsynaptic potential Interaural time di€erence Lateral superior olive Lateral nucleus of the trapezoid body Medial nucleus of the trapezoid body Medial superior olive

1. INTRODUCTION The dorsal nucleus of the lateral lemniscus (DNLL) is located in the auditory pathway just below the midbrain inferior colliculus (IC) and is one of three prominent groups (dorsal, intermediate and ventral nuclei of the lateral lemniscus) of neurons embedded within the ®bers of the lateral lemniscus. The DNLL receives monaural and binaural inputs from the major structures in the auditory lower brainstem, such as the cochlear nucleus (CN), the superior olivary complex (SOC), and the intermediate and ventral nuclei of the lateral lemniscus (INLL and VNLL) through the lateral lemniscus, and from the contralateral DNLL through the commissure of Probst (Glendenning et al., 1981; Kudo, 1981; Shneiderman et al., 1988; Oliver and Shneiderman, 1989; Hutson et al., 1991; Hu€man and Covey, 1995; Yang et al., 1996). Therefore, the DNLL appears to be an essential site of neural integration as well as a neural substrate for binaural interaction. The DNLL projects to the DNLL of the opposite side of the brain and bilaterally to the IC which is an obligatory relay in the auditory midbrain for nearly all ascending auditory ®bers (Beyerl, 1978; Adams, 1979; Brunso-Bechtold et al., 1981; Kudo, 1981; Zook and Casseday, 1982; Tanaka et al., 1985; Coleman and Clerici, 1987; Ross et al., 1988; Shneiderman et al., 1988; Oliver and Shneiderman, 1989; Shneiderman and Oliver, 1989; Hutson et al., 1991; Bajo et al., 1993; MerchaÂn et al., 1994; Hu€man and Covey, 1995; Vater et al., 1995; Kelly et al., 1998a). Immunocytochemical and in situ hybridization histochemical studies have shown that the vast majority of DNLL neurons synthesize GABA (Adams and Mugnaini, 1984; Thompson et al., 1985; Moore and Moore, 1987; Roberts and Ribak, 1987; Glendenning and Baker, 1988; Vater et al., 1992; Winer et al., 1995; Wynne et al., 1995). Synaptic endings of DNLL neurons mediate release and high-anity uptake of GABA in the IC (Shneiderman et al., 1993). Studies using retrograde tract tracing combined with immunocytochemical staining also provide direct evidence of a GABAergic projection from the DNLL to the IC (GonzaÂlez-HernaÂndez et al., 1996; Zhang et al., 1998). All these results suggest that the DNLL is a signi®cant source of inhibition in the auditory midbrain. Despite the potential importance of the DNLL in auditory processing, it received little attention com-

NBQX 1,2,3,4-Tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium NMDA N-Methyl-D-aspartate PVCN Posteroventral cochlear nucleus SAM Sinusoidally amplitude modulated SOC Superior olivary complex SPN Superior paraolivary nucleus THIP 4,5,6,7-Tetrahydroisoxazolo-[5,4-c]pyridin-3-ol VCN Ventral cochlear nucleus VNLL Ventral nucleus of the lateral lemniscus VNTB Ventral nucleus of the trapezoid body

pared to the other major structures in the central auditory pathway until the 1990s. Recent research including neuroanatomical, electrophysiological and behavioral studies indicates that the DNLL plays an active and important role in shaping binaural responses and localizing sounds in space. This paper will present evidence regarding the identity of the excitatory neurotransmitter and receptors that serve physiological and auditory perceptual function of the DNLL. 2. AFFERENT PROJECTIONS TO THE DORSAL NUCLEUS OF THE LATERAL LEMNISCUS Several studies have employed anterograde and retrograde transport methods to trace connections from the structures in the lower auditory brainstem to the DNLL (Glendenning et al., 1981; Shneiderman et al., 1988; Hu€man and Covey, 1995; Labelle and Kelly, 1996). The primary a€erent inputs to the DNLL are from the ventral cochlear nucleus (VCN), the SOC, the VNLL and INLL, and from the DNLL on the opposite side of the brain. 2.1. Cochlear Nucleus The projection of the VCN to the DNLL comes mainly from the contralateral side. Glendenning et al. (1981) examined retrograde transport of HRP after local injections into the DNLL of the cat and found that almost all the labeled VCN neurons, which were nearly evenly divided between the anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN), were on the contralateral side. The study of retrograde transport after injections of the ¯uorescent dye, Fluorogold, into the DNLL of the rat shows a prominent labeling in the contralateral AVCN and PVCN (Labelle and Kelly, 1996). In both cat and rat only a few neurons in the contralateral DCN are found to project to the DNLL (Glendenning et al., 1981; Labelle and Kelly, 1996). A similar pattern of a€erent inputs from the CN to the DNLL has been demonstrated by retrograde labeling in the big brown bat (Hu€man and Covey, 1995) and the mustache bat (Yang et al., 1996). Although it is agreed that the projections from the CN to the DNLL originate mainly from the contralateral VCN, a slight projection from the ipsilateral VCN to the DNLL has also been reported.

Synaptic Excitation in the Dorsal Nucleus of the Lateral Lemniscus

Using both anterograde degeneration and retrograde tracing a very sparse projection from the CN to the ipsilateral DNLL has been found in the cat (Glendenning et al., 1981). Another study using anterograde tract-tracing methods in the cat has shown that the AVCN sends projections to the DNLL bilaterally (Shneiderman et al., 1988). A tonotopic projection from the CN to the DNLL has been reported. An anterograde labeling study in the cat shows that the dorsomedial high frequency part of the AVCN projects to the contralateral ventral DNLL, and the ventrolateral low frequency area projects to the dorsal DNLL bilaterally (Shneiderman et al., 1988). In the rat anterograde labeling also shows a tonotopic projection from the AVCN to the DNLL, but in a di€erent fashion. The high frequency areas of the AVCN project to the outer margins of the DNLL forming a ring-like pattern and low frequency areas project to the middle of the DNLL (Labelle, 1997). These results are consistent with the suggestion of MerchaÂn et al. (1994) that the DNLL in the rat has an onion-like tonotopic concentric organization, with high frequencies represented in the periphery and low frequencies in the center of the nucleus.

2.2. Superior Olivary Complex It is well established that the SOC projects heavily to the DNLL with collateral projections to the central nucleus of the IC (Elverland, 1978; Glendenning et al., 1981; Glendenning and Masterton, 1983; Hutson et al., 1991; Hu€man and Covey, 1995; Vater et al., 1995; Labelle and Kelly, 1996; Yang et al., 1996). After placement of HRP in the cat's DNLL, Glendenning et al. (1981) found that 80% of the retrogradely labeled cells in the SOC were ipsilateral and 20% contralateral to the injection site. These authors also demonstrated that all of the contralaterally labeled cells were located in the lateral superior olive (LSO) or periolivary nuclei, whereas the majority of the ipsilaterally labeled cells were located in the medial superior olive (MSO). Several other studies using retrograde tract-tracing methods have shown that the LSO projects to the DNLL bilaterally in various species of mammals [bat: Hu€man and Covey (1995); Yang et al. (1996); cat: Elverland (1978); rat: Labelle and Kelly (1996)]. Two studies with anterograde tract-tracing techniques in the cat con®rm the bilateral projections from the LSO to the DNLL. One study by Glendenning and Masterton (1983) shows substantial projections from the LSO to the DNLL on both sides of the brain. A second study by Shneiderman et al. (1988) further demonstrates a topographic projection from the LSO to the DNLL. The lateral limb of LSO (the low-frequency area) projects to the dorsal DNLL, whereas the medial limb of LSO (high frequency area) projects to the ventral DNLL. The projection to the dorsal DNLL was found to be evenly distributed whereas the projection to the ventral DNLL exhibited a mediolateral gradient. The ipsilateral LSO had a heavier projection to the lateral part of the ventral DNLL whereas the contralateral LSO projected more heavily to the medial

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part of the ventral DNLL (Shneiderman et al., 1988). The MSO is a major source of ipsilateral projections to the DNLL. Retrograde and anterograde axonal transport studies have shown that the MSO projects heavily to the DNLL ipsilaterally [bat: Yang et al. (1996); cat: Elverland (1978); Glendenning et al. (1981); Henkel and Spangler (1983); Shneiderman et al. (1988); rat: Labelle and Kelly (1996)]. Glendenning et al. (1981) compared the projections from MSO and LSO to DNLL in the same HRP retrograde transport case and found almost twice as many labeled cells in the ipsilateral MSO than in the ipsilateral LSO. This led them to conclude that the projection from MSO to DNLL is substantial in the cat. The projections from the MSO to the DNLL are found to be topographically organized (Henkel and Spangler, 1983). Shneiderman et al. (1988) described the topography of the MSO input to the DNLL in the cat; the dorsal low-frequency part of the MSO projected to the dorsal DNLL, and the ventral portion of the MSO projected to the middle of DNLL. They found no projections from the MSO to the ventral DNLL. These results are consistent with the tonotopic organization of these two nuclei. In the cat's DNLL, low frequencies are represented dorsally and high frequencies ventrally (Aitkin et al., 1970). The MSO is a predominantly low frequency nucleus (Irvine, 1986). Thus, the lack of a ventral input to DNLL may be explained by the relatively slight high-frequency representation in the MSO (Shneiderman et al., 1988). The projection of the MSO, unlike that of the LSO, does not exhibit any mediolateral gradient within the DNLL (Shneiderman et al., 1988). A recent anterograde tract tracing study by Henkel (1997) demonstrated that, in the cat lateral lemniscal axons arising from neurons in the MSO gave rise to collaterals ending in the DNLL before entering the IC. These axonal collaterals terminated in thin, horizontal bands forming laminae that extended throughout the DNLL from rostral to caudal. Vater et al. (1995) investigated convergence and divergence of ascending pathways from the MSO and LSO to the DNLL in the mustached bat. Terminal ®elds in the DNLL were found to be largely overlapping following injections of two di€erent tracers in tonotopically matched areas of the MSO and LSO. The results suggest that projections of the MSO and LSO overlap extensively at the DNLL. The superior paraolivary nucleus (SPN) is a prominent member of the periolivary group of nuclei in the SOC of rodents. Although the functional role of the SPN in audition is not clearly understood, it is known that the SPN sends projections to the DNLL ipsilaterally. Colombo et al. (1996) found labeled terminals in the ipsilateral DNLL following injections of anterograde tract tracer into the SPN of the rat. Labelle and Kelly (1996) obtained similar results in a recent retrograde and anterograde tract tracing study. The other two recognizable nuclei of the periolivary group, the lateral nucleus of the trapezoid body (LNTB) and the ventral nucleus of the trape-

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zoid body (VNTB), send minor projections to the DNLL ipsilaterally as shown in tract tracing studies [cat: Glendenning et al. (1981); rat: Labelle and Kelly (1996)]. Yang et al. (1996) also found projections from the periolivary nuclei to the ipsilateral DNLL in the bat. 2.3. Nuclei of the Lateral Lemniscus The DNLL receives projections from both INLL and VNLL on the same side of the brain [bat: Yang et al. (1996); cat: Glendenning et al. (1981); rat: Labelle and Kelly (1996)]. It is very likely that some of these projections are collaterals of the axons that course further to the IC (Iwahori, 1986; Zhao and Wu, 1998). Many studies have shown that the DNLL sends substantial projections to the contralateral DNLL through the commissure of Probst [bat: Hu€man and Covey (1995); Yang et al. (1996); cat: Goldberg and Moore (1967); Glendenning et al. (1981); Kudo (1981); Shneiderman et al. (1988); Hutson et al. (1991); rat: Bajo et al. (1993); Labelle (1997)]. The projection of the commissure of Probst appears to be topographically organized in the cat (Shneiderman et al., 1988). Injection of anterograde tracer into the dorsal or ventral part of the DNLL results in labeling in the dorsal or ventral region of the contralateral DNLL, respectively. Anterograde axonal transport studies have shown a similar symmetry in DNLL to DNLL projections in the rat (Bajo et al., 1993; Labelle, 1997). Recently, Yang et al. (1996) demonstrated that the DNLL of the mustache bat consists of two divisions, anterior and posterior, based on distinct physiological response patterns, frequency representation and a€erent inputs. The CN, LSO, INLL and DNLL send projections to both divisions, whereas the VNLL projects predominantly to the anterior, and the MSO projects to the posterior division of the DNLL. In summary, the DNLL receives prominent ascending a€erents from various structures in the lower auditory brainstem, including the contralateral CN, bilateral LSO, and ipsilateral MSO, SPN, INLL and VNLL. There is also a major projection to the DNLL from its counterpart on the opposite side of the brain (Fig. 1). Many of the same structures that project to DNLL also project to IC probably via axon collaterals. The projections from various nuclei of the auditory lower brainstem to the DNLL appear to be topographic and tonotopically organized. The anatomical arrangement of a€erent inputs to the DNLL suggests considerable convergence of a€erent inputs and synaptic integration within the DNLL. The results of immunocytochemical studies suggest several possible sources of glutamatergic excitation through a€erent projections to the DNLL. Glendenning et al. (1992) reported that cells giving rise to the projection to the IC from the contralateral LSO were glutamate immunoreactive and may provide glutamatergic excitation to the IC. Since the ®bers from LSO are believed to send collateral projections to DNLL on the way to IC, it follows that the LSO is a likely source of excitatory projections

to DNLL as well. Many neurons in MSO may be glutamatergic (Godfrey et al., 1988) and they send ipsilateral projections to the DNLL (Henkel and Spangler, 1983; Henkel, 1997). Thus, it is considered to be a signi®cant source of excitation of DNLL neurons. Although the ipsilateral SPN has a substantial projection to the DNLL in the rat, there is insucient data to determine whether the projection is predominantly excitatory or inhibitory. The CN sends projections to the IC that have morphology (round vesicles and asymmetrical contacts) associated with excitation (Oliver, 1984, 1985, 1987). Thus, it is likely that there is an excitatory projection from the CN to the IC with collaterals projecting to the DNLL. Immunocytochemical studies suggest that a substantial proportion of the ipsilateral projection from the LSO may be inhibitory. In the cat, the central nucleus of the IC receives a heavy glycinergic projection from the ipsilateral LSO (Saint Marie et al., 1989; Saint Marie and Baker, 1990; Glendenning et al., 1992) and it is probable that parallel or collateralized projections are sent to DNLL as well. In addition, many cells in the INLL and VNLL are reported to be glycinergic and/or GABAergic (Thompson et al., 1985; Moore and Moore, 1987; Roberts and Ribak, 1987; Aoki et al., 1988; Vater et al., 1992; Saint Marie, 1993; Winer et al., 1995; Oliver and Bishop, 1998; Riquelme et al., 1998). Many GABAergic neurons in the INLL and VNLL are found to project to the IC (GonzaÂlez-HernaÂndez et al., 1996; Zhang et al., 1998). Therefore the INLL and VNLL may be additional sources of inhibition in the lemniscal projection to the DNLL. As shown in Fig. 2 the ipsilateral MSO, and contralateral CN and LSO provide excitatory inputs to the DNLL whereas the ipsilateral LSO, INLL and VNLL, and contralateral DNLL provide inhibitory inputs to the DNLL.

3. EFFERENT PROJECTIONS OF THE DORSAL NUCLEUS OF THE LATERAL LEMNISCUS The main targets of the e€erents from the DNLL are the contralateral DNLL and bilateral IC (Beyerl, 1978; Adams, 1979; Brunso-Bechtold et al., 1981; Zook and Casseday, 1982; Coleman and Clerici, 1987; Shneiderman et al., 1988; Bajo et al., 1993; MerchaÂn et al., 1994; Kelly et al., 1998a). The DNLL gives rise to projections to the contralateral DNLL and IC through the commissure of Probst. Two separate populations of neurons send ipsilateral and contralateral projections to the IC. Retrograde labeling in the DNLL after injection of tracer into the IC shows that more neurons project contralaterally than ipsilaterally. The contralateral projections arise from ca 60% of the labeled neurons in the cat and ca 70% of the labeled neurons in the rat (Shneiderman et al., 1988; Ito et al., 1995). Less than 10% of neurons in cat's DNLL have collateralized projections to both sides of the brain (Hutson et al., 1991). Electron microscopy (EM) autoradiographic studies of the projections from the DNLL show that the axonal endings from the con-

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Fig. 1. Fluorogold injection into the DNLL of a rat results in retrograde labeled neurons in the nuclei of the lateral lemniscus, SOC and CN. Photomicrographs show Fluorogold injection of the left DNLL and labeling in the INLL and VNLL on the same side of the brain (A), in the opposite DNLL (B), in the ipsilateral LSO, MSO and SPN (C), in the contralateral LSO (D), and in the contralateral CN (F). Few neurons were labeled in the ipsilateral CN (E). Magni®cations: 130.

tralateral DNLL contain pleomorphic vesicles and make symmetrical synaptic contacts, a pattern that is associated with inhibitory synapses. Many endings from the ipsilateral DNLL also contain pleomorphic vesicles but some contain round vesicles that are associated with excitatory synapses (Oliver and Shneiderman, 1989; Shneiderman and Oliver, 1989). These authors suggested that the pathway from the

DNLL to the opposite DNLL and contralateral IC may have an inhibitory function as DNLL neurons exhibit immunoreactivity to glutamic acid decarboxylase (GAD) and GABA antibodies; the ipsilateral projection of the DNLL to the IC might serve both inhibitory and excitatory functions. The DNLL exerts a more powerful inhibitory in¯uence on the contralateral side of the brain than the ipsilateral one.

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Fig. 2. Schematic diagram of the major excitatory and inhibitory projections to the DNLL. R, Excitatory synapses; r, inhibitory synapses. It is not known whether the projection from the SPN to the DNLL is predominantly excitatory or inhibitory (indicated by `?'). - - -, Midline of the brain.

Another target of e€erents from the DNLL is the superior colliculus (Kudo, 1981; Tanaka et al., 1985; Covey et al., 1987; Bajo et al., 1993). The rostral DNLL sends projections to the deep layers of the superior colliculus bilaterally, although this projection appears to be weak compared with the DNLL± IC projection (Bajo et al., 1993).

4. PHYSIOLOGICAL STUDIES IN VITRO In order to study synaptic integration, probable neurotransmitters and synaptic receptors in the DNLL, we made intracellular and whole-cell patch clamp recordings in brain slice preparations of the rat's DNLL. 4.1. Brief View of Brain Slice Methods The techniques used in this laboratory have been described in detail in several previous papers (Wu and Kelly, 1995a,b, 1996; Fu et al., 1997). A brief account of the procedures used for intracellular and whole-cell patch clamp recordings in DNLL is given here. Twenty-one to 55-day-old rats (Albino Wistar) were used for making brain slices. Coronal slices ca 400 mm thick were cut through the DNLL while the tissue block was immersed in 308C oxygenated saline. The commissure of Probst, which enters the DNLL medially, was used as a landmark for locating the DNLL. The slice that contained the larger part of the DNLL was selected and transferred to a small recording chamber where a warm (33±348C) oxygenated saline solution was circulated continuously above and underneath the slice at a rate of 10±12 ml minÿ1. The saline solution consisted of (in mM) 129 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, and 10 glucose,

saturated with 95% O2±5% CO2, and had a pH of 7.4. The DNLL and the ®bers of the lateral lemniscus and the commissure of Probst were clearly visible under a dissecting microscope. Bipolar stimulating electrodes constructed of insulated tungsten wires were placed on the lateral lemniscus ventral to the DNLL and on the commissure of Probst medial to the DNLL. Intracellular recordings were made with glass micropipettes ®lled with 4 M potassium acetate. The electrode impedance was between 120 and 160 MO. Whole-cell patch clamp recordings were made with pipettes ®lled with an `intracellular' solution containing (in mM): 130 CsCl, 1 MgCl2, 5 EGTA and 10 HEPES. The impedance of the patch electrodes was ca 5 MO. In some experiments biocytin was added to the internal solution for intracellular labeling. Blind intracellular and patch clamp recordings were made from DNLL neurons. Current clamp experiments were carried out with sharp electrodes, and both current and voltage clamp experiments were carried out with patch clamp electrodes. Synaptic responses, that is, postsynaptic potentials and postsynaptic currents, were elicited by electrical stimulation of either the lateral lemniscus or the commissure of Probst. Experimental drugs and test solutions were administered by bath application. The physiological e€ects of various chemicals usually took place within 1±4 min. Negative responses were monitored for >5 min after bath application of the chemicals. 4.2. Excitatory Synaptic Responses In order to illustrate synaptic integration in the DNLL, I will ®rst present the general synaptic response pattern of DNLL neurons to stimulation of the lateral lemniscus. Three kinds of synaptic response pattern were obtained from a sample of 160 DNLL neurons: EPSPs only (67%), IPSPs only (6%) and combined EPSPs and IPSPs (27%) (Wu and Kelly, 1995a). Figure 3 shows several examples of these responses. It should be pointed out that the synaptic responses observed in DNLL brain slices are not necessarily identical to the synaptic responses elicited with acoustic stimulation in vivo. The failure to show one kind of postsynaptic potential might be due to the way in which the tissue was sectioned or the position of the stimulating electrodes along the course of ®bers entering the DNLL, and thus, does not necessarily imply a lack of underlying synaptic input. Another reason that one might fail to record a postsynaptic response is the possibility that either excitatory or inhibitory potentials might obscure or completely cancel potentials of the opposite sign. However, the data suggest that the lateral lemniscus makes excitatory synapses on the vast majority of DNLL neurons (94%). The intracellular data showing that both EPSPs and IPSPs could be observed in most cells after pharmacological manipulations (see Figs 6 and 7) indicate a high degree of convergence of synaptic excitation and inhibition on single DNLL neurons. As mentioned in Section 2, various structures in the lower auditory brainstem provide excitatory and inhibitory inputs to the DNLL via the lateral lemnis-

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Fig. 3. Postsynaptic responses of ®ve cells (A±E) in DNLL to electrical stimulation of the lateral lemniscus. The recordings show synaptic responses to electrical pulses of various strengths as indicated in volts at the left of each trace. (A) This neuron responded to lemniscal stimulation with a combined EPSP and IPSP. Near-threshold stimulation produced an IPSP with a latency of 0.8 msec. The amplitude of the IPSP increased substantially with greater stimulus strength. At higher stimulus levels an EPSP was apparent and a single action potential could be evoked. (B) In this neuron a short-latency IPSP was followed by a longer-latency EPSP. At low levels of stimulation a single shock evoked an IPSP with a latency of 1.0 msec. At higher levels of stimulation an EPSP emerged with a latency of ca 3.0 msec. At relatively high levels of stimulation the EPSP resulted in a single action potential. (C) For this neuron, the EPSP and IPSP had similar thresholds and latencies. Near-threshold stimulation evoked either an EPSP or an IPSP. As intensity was increased, the EPSP became dominant over the IPSP and a single action potential was elicited. (D) This neuron exhibited a short-latency EPSP at low stimulus levels. As the stimulus level was increased a short latency IPSP appeared in the record and dominated the response at the highest levels tested. For this cell no action potential could be elicited regardless of the level of the stimulus. (E) In this neuron an EPSP was apparent without any evidence of an IPSP. Increasing stimulus intensity produced a graded EPSP. The stepwise increase in EPSP amplitude in this case implies a recruitment of at least seven convergent excitatory inputs as stimulus strength was increased. Under no condition was an IPSP apparent in the response, although this does not rule out the possibility that some inhibitory component was present but obscured by the excitatory response. Arrows, time of the electrical stimulus and resulting stimulus artifact. From Wu and Kelly (1995a).

cus. The EPSPs elicited by stimulation of the lateral lemniscus in the DNLL brain slice may re¯ect excitatory inputs from the ipsilateral MSO, contralateral

CN or LSO. The IPSPs evoked by stimulation of the lateral lemniscus may re¯ect inhibitory inputs from the ipsilateral LSO, INLL or VNLL.

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Electrical stimulation of the commissure of Probst can elicit EPSPs and/or IPSPs (Wu and Kelly, 1995a). The most probable source of IPSPs is the well-established inhibitory projection from the contralateral DNLL. The vast majority of DNLL neurons are GABAergic and project to the opposite DNLL by way of the commissure of Probst (Goldberg and Moore, 1967; Kudo, 1981; Adams and Mugnaini, 1984; Thompson et al., 1985; Moore and Moore, 1987; Roberts and Ribak, 1987; Glendenning and Baker, 1988; Hutson et al., 1991; Vater et al., 1992; Bajo et al., 1993; Winer et al., 1995). Furthermore, the commissural IPSPs are blocked by the speci®c GABAA receptor antagonist, bicuculline, in the brain slice, con®rming that DNLL neurons receive GABAergic inputs from the contralateral DNLL via the commissure of Probst (Wu and Kelly, 1996). The source of the commissural EPSPs is not clear, as there are no anatomical data concerning probable excitatory pathways in the commissure of Probst or neighboring regions. The intracellular data from brain slice studies have shown that many DNLL neurons respond to both lemniscal and commissural stimuli, and that there is a convergence of inputs from the lateral lemniscus and commissure of Probst in at least 73% of neurons. This percentage is probably a conservative estimate of the amount of convergence occurring in the DNLL of an intact animal, because with the brain slice preparation the failure to show a response might be due to the plane of tissue section or location of the stimulating electrodes. Taking these factors into account, convergence between lemniscal and commissural inputs probably occurs on most DNLL neurons in the intact animal. The latencies for the EPSPs and IPSPs recorded from the DNLL neurons in brain slices were usually short, averaging ca 1 msec. EPSPs with longer latencies were also observed in many neurons. The latency of the action potential arising from the earlier EPSP at suprathreshold stimulation was highly regular and showed little variability. The longerlatency EPSP could generate a single or multiple action potentials with much longer and more variable latencies (Figs 4 and Fig. 7). The suprathreshold responses of DNLL neurons to repetitive stimulation of the lateral lemniscus showed a limited ability to follow high rates of stimulation above 10±100 Hz (Fig. 5) (Wu and Kelly, 1995a). A similar limited ability to follow high rates of stimulation was found in many LSO neurons (Wu and Kelly, 1993). These LSO neurons are incapable of reliable responding to stimulation of the trapezoid body at rates >125 Hz. As LSO neurons receive converging excitatory projections from the ipsilateral CN through the ventral acoustic stria, the trapezoid body, and inhibitory projections from the contralateral CN through the medial nucleus of the trapezoid body (MNTB) (Cant, 1991), acoustic stimulation of the ipsilateral ear excites the cells in LSO and contralateral stimulation inhibits activity in the same cells. The net response of LSO neurons is determined by the interplay between ipsilateral excitation and contralateral inhibition. The relative slow response of LSO neurons to repeated stimulation of the trapezoid

body probably re¯ects their role in the summation of synaptic inputs associated with binaural processing. In contrast, MNTB neurons which receive excitatory inputs from the contralateral CN through large, cup-shaped terminals, the calyces of Held, respond to repetitive stimulation faithfully at rates up to 667 Hz (Wu and Kelly, 1993). The ability of MNTB neurons to response reliably at very high rates of stimulation re¯ects their specialized role for converting excitation into inhibition. DNLL neurons are more like LSO neurons: they respond slowly to repetitive stimulation and are thought to be well adapted for integrating synaptic inputs from converging sources of excitation and inhibition. 4.3. Excitatory Synaptic Neurotransmitters and Receptors The excitatory neurotransmitter released from the contralateral CN and LSO, and ipsilateral MSO onto the DNLL neurons is probably glutamate. Immunocytochemical studies using antibodies against conjugates of glutamate and aspartate have revealed immunoreactive neurons in the CN and SOC, suggesting that the neurotransmitter of cells projecting from these structures to the DNLL may be an excitatory amino acid (Madl et al., 1986; Aoki et al., 1987; Glendenning et al., 1992). Physiological evidence from in vitro and in vivo studies supports the suggestion that synaptic excitation of DNLL neurons is mediated by an excitatory amino acid, probably glutamate. DNLL neurons are sensitive to excitatory amino acid agonists in brain slice preparations. Bath application of the excitatory amino acid agonists, L-glutamate, a-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid (AMPA) and Nmethyl-D-aspartate (NMDA), depolarizes the cell membrane, initiates spontaneous action potentials and reduces cell membrane resistance of DNLL neurons (Wu and Kelly, 1996). Iontophoretic application of glutamate also increases spontaneous discharge of DNLL neurons in vivo (Pollak, 1997). The receptors that mediate synaptic excitation in the DNLL have been studied by application of ionotropic glutamate receptor antagonists (Wu and Kelly, 1996; Fu et al., 1997). The short-latency EPSPs and action potentials were always blocked by the AMPA receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), but never by the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (APV) (Figs 6±8). The late EPSPs and action potentials were always blocked by the NMDA receptor antagonist APV, but never by CNQX or DNQX (Figs 7 and 8). The general amino acid antagonist, kynurenic acid, blocked both early and late EPSPs. These results suggest that excitation in the DNLL is mediated by glutamate, through AMPA and NMDA receptors. The earlier EPSPs are mediated by the AMPA receptors whereas the late EPSPs are mediated by the NMDA receptors. Furthermore, the late EPSPs appeared to be unaffected by complete pharmacological block of the early EPSPs with CNQX even when both long- and short-latency EPSPs were recorded from the same neuron (Figs 7 and 8). These data indicate the pre-

Synaptic Excitation in the Dorsal Nucleus of the Lateral Lemniscus

Fig. 4. Responses of two DNLL neurons to electrical stimulation of the lateral lemniscus with whole-cell patch recording. (A) Low level of stimulation (20 V) elicited an early EPSP, which was accompanied at slightly higher levels of stimulation (23 V) by a short-latency action potential. With further increase in stimulus strength (25 and 28 V) a long-latency action potential was also evoked. The latency of the initial action potential was highly reliable, but the latency of delayed action potentials was extremely variable. (B) Seven superimposed traces evoked at the same stimulus level (27 V) from a second DNLL neuron. At this level of stimulation the neuron generated two action potentials, a short-latency and a longlatency responses. Note the reliability of the short-latency action potential and the variability of the long-latency action potential recorded from the same neuron. Arrows point to stimulus artifact. From Fu et al. (1997).

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Fig. 5. Postsynaptic response of a DNLL neuron to repetitive stimulation of the lateral lemniscus. Stimulus artifacts are seen as transient pulses in the traces of membrane potential. Action potentials can be distinguished by their much larger amplitude. Each trace represents the response of the neuron to 10 stimuli presented at di€erent repetition rates as indicated above the record (167±25 Hz). Action potentials were elicited after electrical stimulation with a probability that was highly dependent on repetition rate. Recordings were obtained with the brain slice submerged in a saline solution containing 0.5 mM strychnine, which blocked inhibitory responses evoked by the lemniscal stimulation. From Wu and Kelly (1995a).

sence of two separate excitatory processes in the DNLL mediated by AMPA and NMDA receptors, respectively.

In our initial recordings from DNLL neurons with the brain slice bathed in normal saline, the early and late EPSPs were not always found in the

Fig. 6. Postsynaptic response of a DNLL neuron to electrical stimulation of the lateral lemniscus. (A) Under normal saline conditions, low-level stimulation (bottom trace, 4 V) elicited an EPSP, and slightly higher-level stimulation (top trace, 5.6 V) evoked an action potential. (B) Application of 5 mM CNQX totally eliminated the EPSP. Low-level stimulation (bottom trace, 4 V) failed to evoke any postsynaptic response, and substantially higher-level stimulation (top trace, 23 V) evoked an IPSP but no EPSP. (C) After the brain slice was perfused in normal saline the excitatory response returned to normal. (D) Application of 100 mM APV failed to a€ect the excitatory postsynaptic response to electrical stimulation (5.6 V). Arrow points to stimulus artifact. Modi®ed from Wu and Kelly (1996).

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Fig. 7. E€ects of strychnine, CNQX and APV on synaptic responses evoked by stimulation of the lateral lemniscus in a DNLL neuron. (A) Control responses to stimulation of the lateral lemniscus at four di€erent intensities from low to high as shown in the lower to upper traces, respectively. All responses were obtained with the brain slice in normal saline. At the lowest level of stimulation an IPSP was seen (bottom trace, 4 V). At a higher level of stimulation both early and late EPSPs as well as an IPSP were seen (second trace from the bottom, 5 V). At suprathreshold stimulus levels both short- and long-latency excitatory responses and action potentials were evoked (second from the top and top traces, 5.5 and 6 V, respectively). (B) Application of 1 mM strychnine abolished the IPSP at both lower (4 V) and higher (6 V) levels of stimulation (left and right traces, respectively). At higher levels of stimulation both early and late EPSPs and action potentials were always present in the same trace (right). (C) Application of 5 mM CNQX in a low-Mg2+ (0.3 mM) saline solution eliminated the early excitatory response but did not eliminate the long-latency spikes. The traces on the left and right show responses to low (8 V) and high (15 V) levels of stimulation, respectively. (D) 50 mM APV in 0-Mg2+ saline eliminated the longlatency action potentials but did not eliminate the early EPSP or action potential. Responses to low (8 V) and high (15 V) levels of stimulation are shown on the left and right, respectively. Arrow points to stimulus artifact. Modi®ed from Wu and Kelly (1996).

same neuron. Because large IPSPs could be elicited by lemniscal stimulation at the same locus as that for evoking EPSPs, the inhibitory potential often obscured the excitatory potential or vice versa. Appearance of the EPSPs or IPSPs depended on their relative thresholds for activation, and for many neurons the thresholds were similar, so that EPSPs and IPSPs could not be measured independently. Nevertheless, after blocking the early IPSP by application of the glycine receptor antagonist, strychnine, the presence of both early and late EPSPs could be readily revealed (Figs 7 and 8). To investigate synaptic excitation in the DNLL further, whole-cell patch recordings were made from DNLL neurons with the brain slice bathed in saline containing strychnine (0.5 mM), which eliminated all evidence

of synaptic inhibition. With this manipulation the early and late component of excitation were identi®ed in every DNLL neuron sampled (Fu et al., 1997). The early EPSP generated only one action potential whereas the late EPSP elicited one or multiple action potentials at suprathreshold levels of lemniscal stimulation (Figs 4 and 7). With whole-cell patch clamp recording it was con®rmed that the early and late components were pharmacologically distinct and mediated by AMPA and NMDA receptors, respectively (Fig. 8). Temporal and voltage-dependent properties of the NMDA receptors in DNLL neurons were investigated by voltage-clamp experiments. The rise time, decay time constant, time to peak and half-width of the excitatory postsynaptic currents (EPSCs)

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Fig. 8. The e€ects of CNQX and APV on suprathreshold excitatory postsynaptic responses in a DNLL neuron with whole-cell patch recording. Suprathreshold lemniscal stimulation elicited an early EPSP late action potentials. Application of 50 mM APV completely blocked the later action potential, but had no e€ect on the early response. CNQX (10 mM) blocked the early spike but had no e€ect on the later response. Arrow points to stimulus artifact. The levels of stimulation for the three conditions were the same. Modi®ed from Fu et al. (1997).

mediated by the AMPA receptors were all shorter than those of the EPSCs mediated by the NMDA receptors (Fu et al., 1997). The I±V curve for the AMPA component was linear over the whole voltage range, with a reversal potential near 0 mV, and that for the NMDA component was non-linear, with a region of negative slope and a reversal of membrane resistance at holding potentials between ÿ50 and ÿ100 mV (Fig. 9). The NMDA receptor-

mediated current in DNLL neurons was also subject to Mg2+ block. This blocking e€ect was voltagedependent and was overcome when the cell's membrane was suciently depolarized (Fig. 10). All these basic properties, including kinetics, voltage dependence and pharmacology, of the AMPA and NMDA receptor-mediated synaptic currents in DNLL neurons resemble those in other mammalian CNS neurons, including hippocampal and auditory

Fig. 9. The EPSC evoked by lemniscal stimulation with whole-cell patch recording under voltage-clamp mode from a DNLL neuron. (A) Individual traces of the EPSC at holding potential between +40 and ÿ100 mV. (B) The I±V curves for the EPSC plotted for the peak response (w) and 12 msec after the peak response (.), following the procedure of Isaacson and Walmsley (1995). The I±V curve for the later part of the EPSC exhibits voltage-dependent non-linearity. The portion of the waveform from which measurements were made is indicated in (A) by the symbols below the lowest trace. From Fu et al. (1997).

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369

Fig. 10. The e€ects of CNQX, APV and Mg2+ on EPSCs evoked by lemniscal stimulation recorded under voltage-clamp conditions. (A) With the holding potential held at +40 mV, 10 mM CNQX selectively reduced the magnitude of an early response (middle trace) and 50 mM APV selectively reduced a later response component. (B) With the brain slice in normal saline solution containing 1.3 mM MgSO4, APV had little or no e€ect on the evoked EPSC. In contrast, 10 mM CNQX had a marked blocking e€ect on the response. (C) With MgSO4 removed from the extracellular solution, both APV and CNQX had a pronounced blocking e€ect. APV preferentially blocked the later synaptic current and CNQX had a greater blocking e€ect on the early component. These data demonstrate that the NMDA-mediated EPSCs in the DNLL are sensitive to the presence of extracellular Mg2+ and are subject to a voltagedependent blockade. Modi®ed from Fu et al. (1997).

neurons (Forsythe and Westbrook, 1988; Hestrin et al., 1990; Forsythe and Barnes-Davies, 1993; Zhang and Trussell, 1994). It was further shown with whole-cell recordings that both the early and late components of the EPSP could be elicited in the same neuron near normal resting potential (ca ÿ60 mV) by stimulation of the lateral lemniscus with the slice bathed in saline containing normal Mg2+ concentration (1.3 mM) (Figs 4 and 8). The results suggest that the fast excitatory synaptic transmission is mediated by an excitatory amino acid via AMPA receptors whereas the late and longer-lasting synaptic excitation is mediated by NMDA receptors and can be brought into play near the resting potential of the cell membrane. Therefore, we suggest that both AMPA and NMDA components normally play a role in synaptic transmission in DNLL in vivo (Fu et al., 1997). Acoustic stimulation would cause an initial depolarization through activation of AMPA receptors. The initial depolarization would then be followed by activation of a longer lasting NMDA receptormediated excitation. Because the vast majority DNLL neurons are GABAergic and send e€erents to the IC and contralateral DNLL, this long lasting excitation would be expected to result in a long lasting inhibition of neurons in both structures. Therefore, synaptic excitation mediated by both AMPA and NMDA receptors probably contributes to sensory processing of acoustic information by controlling the pattern of inhibition exerted by DNLL neurons (Kelly and Kidd, 1997a).

5. PHYSIOLOGICAL RESPONSE PROPERTIES OF DNLL NEURONS IN VIVO Most DNLL neurons are sensitive to binaural stimulation. In rat and bat the vast majority of binaural DNLL neurons are sensitive to interaural intensity di€erences (IIDs) (Covey, 1993; Markovitz and Pollak, 1993, 1994; Yang and Pollak, 1994a,b; Kelly et al., 1998b). These neurons typically exhibit an excitatory/inhibitory (EI) response pattern: they are excited by contralateral stimulation and inhibited by simultaneous ipsilateral stimulation. The ®ring rate of such neurons is strongly a€ected by the relative balance of excitation and inhibition as determined by the level of acoustic stimulation at the two ears. An increase in the intensity of ipsilateral stimulation produces a sharp decrease in ®ring rate. Some DNLL neurons show an excitatory/excitatory (EE) pattern: they are driven by stimulation of either ear. More complex binaural properties, such as the facilitated EI (EI/F), inhibited EE (EE/I), and facilitated and inhibited EE (EE/FI) neurons are also observed in the bat and are probably present in other species as well (Markovitz and Pollak, 1994). Most neurons in the rat's DNLL are insensitive to binaural phase di€erences because of their relatively high characteristic frequencies compared to those of cats or other mammals with good low frequency hearing (Kelly and Masterton, 1977; Kelly et al., 1998b). However, the rat's DNLL neurons are sensitive to small interaural time di€erences (ITDs) between transients (e.g. clicks) presented to the two ears (Kelly et al., 1998b). In the cat, low frequency neurons in the

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dorsal part of the DNLL are sensitive to variation in ITD, and more ventrally located high frequency neurons are sensitive to changes in IID (Aitkin et al., 1970; Brugge et al., 1970). The binaural responses of DNLL neurons almost certainly re¯ect the properties of a€erent neurons, which are already binaural due to interaction in the SOC (e.g. LSO and MSO). Additional circuits from below the DNLL, that is, the CN, SPN, INLL, VNLL and the opposite DNLL may also make contribute to binaural response patterns (Markovitz and Pollak, 1993; Kelly, 1997). Unilateral destruction of the SOC by local injection of the excitatory neurotoxin, kainic acid, does not completely eliminate binaural excitatory-inhibitory responses as measured by single unit spikes in the rat's IC (Li and Kelly, 1992a; Sally and Kelly, 1992), which suggests the existence of an additional source of binaural interaction (Kelly and Li; 1997). Li and Kelly (1992b) have shown that injection of kynurenic acid into DNLL can alter the response to IIDs of single units in the contralateral IC. Kidd and Kelly (1996) have shown a similar e€ect on responses of IC neurons to ITDs. A recent study by Kelly and Kidd (1997b) shows that pharmacological blockade of the DNLL also reduces the strength of

binaural inhibition in the opposite DNLL, providing direct evidence of an active involvement of DNLL in binaural interaction of neurons in the opposite DNLL. Thus, both SOC and DNLL contribute to binaural response in DNLL. In order to understand the neural circuits that shape the binaural response of DNLL neurons to free ®eld sounds, we need to know the a€erent projections to the DNLL, the putative neurotransmitters of the projections, and their physiological responses. The schematic diagram shown in Fig. 11 illustrates the sources and neural circuits that might contribute to excitatory and inhibitory responses in the DNLL elicited by ipsilateral or contralateral acoustic stimulation. As mentioned above, the DNLL receives a€erent inputs from the CN, SOC (including LSO, MSO and SPN) and the nuclei of the lateral lemniscus (including DNLL, INLL and VNLL). Among these structures the LSO, MSO, CN and DNLL are well understood in terms of their projections and physiological responses. The vast majority of LSO neurons are excited by ipsilateral acoustic stimulation and inhibited by contralateral stimulation (Boudreau and Tsuchitani, 1968; Tsuchitani and Boudreau, 1969; Caird and Klinke, 1983). A large proportion

Fig. 11. Proposed neuronal circuits for generating an EI response pattern of DNLL neurons. A sound presented on the left side elicits excitatory responses (EPSPs and action potentials) in the right DNLL via glutamatergic projections from the left LSO and CN, and right MSO. A sound on the left also elicits inhibitory responses (IPSPs) in the left DNLL via glycinergic projections from the left LSO and GABAergic projections from the right DNLL. The left DNLL further disinhibits the right DNLL (- - -). The neuronal circuits of the lower auditory brainstem provide a converging interaction of excitation and inhibition in the DNLL for producing EI response pattern. See more details in the text. R, Excitatory synapses; r, inhibitory synapses.

Synaptic Excitation in the Dorsal Nucleus of the Lateral Lemniscus

of the ipsilateral projection from LSO to DNLL is glycinergic and the contralateral projection is thought to be excitatory, probably glutamatergic (Hutson et al., 1987; Saint Marie et al., 1989; Saint Marie and Baker, 1990; Glendenning et al., 1992; Ito et al., 1995). Thus, acoustic stimulation from a sound source on the left (i.e. ÿ908 azimuth) would result in activation of the left LSO, which would in turn cause excitation of the right DNLL by direct crossed excitatory projections. Excitation of DNLL neurons would result in a GABAergic inhibition of DNLL neurons on the opposite side of the brain. At the same time the ipsilateral DNLL would be inhibited by the uncrossed glycinergic projection from the LSO, and the contralateral DNLL would be disinhibited by a reduction in GABAergic input. Thus, the contralateral DNLL would receive a net excitatory in¯uence whereas the ipsilateral DNLL would receive inhibitory in¯uence from two sources: the uncrossed inhibitory glycinergic projections from the LSO and the GABAergic projections from the opposite DNLL. The MSO in mammals is also a binaural nucleus. Neurons in the MSO are typically excited by both ipsilateral and contralateral acoustic stimulation, and are sensitive to interaural time and phase di€erences (Goldberg and Brown, 1969; Yin and Chan, 1990; Irvine, 1992). Many MSO neurons in the cat are driven maximally by speci®c binaural time di€erences produced by sounds located in the contralateral spatial ®eld (Smith et al., 1993). Thus, neurons in the MSO would e€ectively be excited by sounds located in the contralateral ®eld, and would in turn activate the DNLL on the same side of the brain via their uncrossed excitatory projections. Although in the mustache bat the MSO is a monaural nucleus (Covey et al., 1991; Grothe et al., 1992; Covey and Casseday, 1995), it would probably be activated preferentially by a contralateral sound source, assuming that there is a substantial intensity di€erence between the two ears. Because most bats are specialized for high frequency hearing, a large IID would be expected. As the MSO projects to the ipsilateral DNLL, it would be expected to contribute primarily to excitation of the DNLL contralateral to the sound source. The CN sends a direct excitatory projection to the contralateral DNLL. Although the CN is a monaural nucleus, excited by stimulation of the ipsilateral ear, it could, nevertheless, make a preferential contribution to excitation of the contralateral DNLL at frequencies that results in level di€erences between the ears. At high frequencies, large IIDs are produced by the blocking e€ect of the head as a sound source is shifted to lateral positions in space. Of course, this blocking e€ect is most apparent in species with good high frequency hearing (e.g. bats and rats). Thus, in these species the DNLL would be excited by sound located in the contralateral sound ®eld via the crossed projection from the CN. In conclusion, the net result is that DNLL neurons are typically excited by contralateral sounds and inhibited by ipsilateral sounds (Fig. 11). DNLL neurons respond to contralateral tonal stimulation with di€erent temporal patterns. Onset and sustained are the two main response types

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(Aitkin et al., 1970; Covey, 1993; Markovitz and Pollak, 1993; Yang et al., 1996; Yang and Pollak, 1997; Kelly et al., 1998b). Markovitz and Pollak (1993) classi®ed responses of DNLL neurons into six categories: transient chopper; onset-chopper; primarylike; primarylike-with-notch; pause; and onset. Other authors used di€erent de®nitions to classify DNLL neurons into categories. Recently, neurons with sustained or onset response patterns in the DNLL of the mustache bat were found to be located in di€erent regions and to respond di€erently to sinusoidally amplitude modulated (SAM) signals (Yang et al., 1996). Neurons in the anterior one-third of DNLL responded to tone bursts with an onset discharge pattern and phaselocked to SAM signals with low modulation frequencies (<300 Hz), whereas neurons in the posterior two-thirds of DNLL responded to tone bursts with a sustained discharge and phase-locked to SAM signals with much higher modulation frequencies (400±800 Hz). With iontophoretic application of the GABAA receptor antagonist, bicuculline, the SAM evoked response pattern of onset neurons became more like that of sustained neurons, suggesting that the intrinsic properties and excitatory innervation of onset cells are similar to those of sustained cells (Yang and Pollak, 1997). These results are consistent with the observations from brain slice studies (Wu and Kelly, 1995b; Fu et al., 1996). Intracellular recordings from DNLL neurons in a rat brain slice show that various cell types have similar inherent electrical membrane characteristics and respond to depolarizing currents with a sustained train of action potentials, perhaps due to similar types of voltage sensitive ion channels. Furthermore, the synaptic excitatory responses elicited by stimulation of the lateral lemniscus are similar in all sampled DNLL. The distinction between onset and sustained neurons in vivo may be due to a di€erence in the degree and/or temporal pattern of inhibitory inputs to them (Yang and Pollak, 1997). These two types of neurons may serve separate functions in shaping the binaural properties of their targets in the IC and the temporal coding of complex sounds (Yang et al., 1996). The sustained neurons may have more profound inhibitory e€ects on the IC than the onset neurons. The onset neurons may convey more precise temporal information about amplitude ¯uctuations and may be involved in shaping the temporal coding of higher order collicular neurons in response to complex, amplitude modulated signals (Yang and Pollak, 1997). One distinct physiological feature of DNLL neurons is that the inhibition imposed on them by stimulation of the ipsilateral ear is long lasting, extending well beyond the period of acoustic stimulation. For example, in the rat's DNLL the responses evoked by a contralateral click are suppressed for 10±30 msec by presentation of a leading ipsilateral click stimulus (Kelly et al., 1998b). A similar pattern of inhibition that extends beyond the duration of a tone burst at the ipsilateral ear was found in the DNLL of the mustache bat (Yang and Pollak, 1994a,b). These authors suggested that this persistent inhibition might play a role in localizing multiple sound sources.

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Another striking feature of neurons in the rat's DNLL is that they tend to respond to a contralateral single click with multiple spikes (Kelly et al., 1998b). A train of three or four spikes was generated in response to each stimulus. The NMDA receptor mediated long lasting depolarization observed in DNLL neurons from brain slices may be responsible for the multiple spikes recorded in vivo (Wu and Kelly, 1996; Fu et al., 1997). Consequently, the prolonged excitation would be expected to extend the period of inhibition exerted by GABAergic neurons in the DNLL on the contralateral DNLL and IC (Kelly et al., 1998b).

6. FUNCTIONAL ROLE OF THE DORSAL NUCLEUS OF THE LATERAL LEMNISCUS IN AUDITORY PROCESSING Several in vivo physiological studies have demonstrated that DNLL exerts a powerful inhibitory in¯uence on binaural responses recorded from the IC or primary auditory cortex. Blockade of neuronal activity in the DNLL by local injection of pharmacological agents alters binaural responses of neurons in the contralateral IC (Li and Kelly, 1992b; Faingold et al., 1993; Kidd and Kelly, 1996; Kelly and Kidd, 1997a). Unilateral lesions of DNLL disrupt binaural responses in the contralateral auditory cortex (Glenn and Kelly, 1992). Li and Kelly (1992b) injected the non-speci®c excitatory amino acid antagonist, kynurenic acid, into the contralateral DNLL while recording single unit responses from the IC in the rat. Most neurons in the rat's IC were excited by stimulation of the contralateral ear and strongly inhibited by simultaneous stimulation of the ipsilateral ear. Following injection of kynurenic acid into the DNLL the inhibition normally produced by stimulation of the ear ipsilateral to the recording site was reduced. Faingold et al. (1993) found that electrical stimulation of the DNLL or local injection of the excitatory amino acid agonist, kainate, into DNLL reversibly enhanced binaural inhibition in the contralateral IC, and blockade of DNLL by local injection of lidocaine or the GABAA agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), reduced binaural inhibition in the contralateral IC. Results from these two studies suggest that binaural responses in the IC are shaped by the GABAergic projection from the contralateral DNLL. Pharmacological blockade of activity of DNLL neurons also markedly a€ects the ITD sensitivity of IC neurons. Kidd and Kelly (1996) demonstrated that EI neurons in the IC had the greatest probability of generating an action potential when a click presented to the contralateral ear led one presented to the ipsilateral ear. As the contralateral click was progressively delayed relative to the ipsilateral, the response probability was reduced. Following injection of the general amino acid antagonist, kynurenic acid, into the DNLL, stimulation of the ipsilateral ear was less e€ective in lowering the probability of responses evoked by stimulation presented to the contralateral ear. Similar results have been reported for slow evoked potentials

recorded from the rat's primary auditory cortex (Glenn and Kelly, 1992). Unilateral lesions of the DNLL produced by local injection of the excitatory neurotoxin, kainic acid, changed the ITD function and reduced the degree of binaural suppression in the auditory cortex contralateral to the lesion. Although the DNLL sends e€erents to the IC bilaterally, Kelly and his colleagues (Li and Kelly, 1992b; Kidd and Kelly, 1996) have shown only a contralateral inhibitory in¯uence on binaural responses of IC neurons. Injection of kynurenic acid into the DNLL released the inhibition of binaural responses of the neurons in the contralateral IC, but had no e€ect on the binaural response of the neurons in the ipsilateral IC. Transection of the commissure of Probst, which is the route for DNLL neurons projecting to the contralateral DNLL and IC, greatly reduced the extent of binaural inhibition of evoked responses in the IC (van Adel et al., 1997). These results con®rm the importance of the contralateral projection from the DNLL for shaping binaural responses in the IC (Kelly, 1997). As pointed out earlier, synaptic excitation in the DNLL is probably mediated by glutamate through both the AMPA and NMDA receptors. The AMPA receptors contribute to earlier fast responses and NMDA receptors contribute to late longer lasting responses (Wu and Kelly, 1996; Fu et al., 1997). As DNLL neurons are primarily GABAergic and project to the IC, the late longer lasting excitation would be expected to produce a prolonged inhibition in the IC. Long-lasting inhibition has been reported previously for single-unit recordings made from the IC of the cat and rabbit (Carney and Yin, 1989; Yin, 1994; Fitzpatrick et al., 1995) as well as the rat (Kidd and Kelly, 1996). These authors have suggested that the prolonged inhibitory responses in the IC might provide a neural mechanism for the active suppression of echoes. A recent physiological study has shown the contribution of speci®c receptor types underlying the inhibition in the IC (Kelly and Kidd, 1997a). These authors presented click pairs binaurally to the rat and manipulated the binaural time di€erences. An action potential was typically evoked with contralateral lead-times. As the binaural time di€erence was shifted in favor of the ipsilateral ear the probability of an action potential progressively decreased. The duration of the inhibitory e€ect could last 15±20 msec. Injection of speci®c AMPA or NMDA receptor antagonists, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX) or (2)-3(2-carboxypiperazin-4-yl)-propyl-1phosphonic acid (CPP), respectively, into the DNLL resulted in a reduction in the strength of binaural inhibition in the IC. NBQX preferentially a€ected the earlier component (<1 msec) and CPP a€ected the late longer lasting component (up to 30 msec). These results indicate that both receptors participate in central auditory processing; the AMPA receptors are responsible for the early inhibition and the NMDA receptors for the late inhibition in the IC. Behavioral studies further indicate that the DNLL plays a role in sound localization in the rat (Ito et al., 1996; Kelly et al., 1996). Unilateral or bilateral lesions of the rat's DNLL by local injection

Synaptic Excitation in the Dorsal Nucleus of the Lateral Lemniscus

of kainic acid, an excitatory neurotoxin which destroys nerve cell bodies but not the ®bers of passage coursing through the DNLL to the IC, caused marked de®cits in sound localization, that is, an elevation in minimum audible angles (Kelly et al., 1996). Surgical transection of the commissure of Probst in the rat also resulted in de®cits in sound localization comparable in severity to those found after lesions of the DNLL (Ito et al., 1996). Unlike the chemical lesions of the DNLL, which interrupt both ipsilateral and contralateral projections to the IC, the transection of the commissure of Probst selectively destroys the contralateral projection to the DNLL and IC. Thus, the contralateral projection from DNLL is shown to be important for normal auditory spatial acuity. AcknowledgementsÐThis research was supported by grants from the Natural Sciences and Engineering Research Council of Canada, and the Deafness Research Foundation (the United States). I extend my grateful appreciation to Dr Jack B. Kelly for many useful discussions and suggestions, and for careful and critical reading of the manuscript. I also thank Brian van Adel for providing Fig. 1 from his own work and Yan Zhang for preparation of illustrations.

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