Morphology and axonal projection patterns of auditory neurons in the midbrain of the painted frog, Discoglossus pictus

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Abstract

Acoustic signals are extensively used for guiding various behaviors in frogs such as vocalization and phonotaxis. While numerous studies have investigated the anatomy and physiology of the auditory system, our knowledge of intrinsic properties and connectivity of individual auditory neurons remains poor. Moreover, the neural basis of audiomotor integration still has to be elucidated. We determined basic response patterns, dendritic arborization and axonal projection patterns of auditory midbrain units with intracellular recording and staining techniques in an isolated brain preparation. The subnuclei of the torus semicircularis subserve different tasks. The principal nucleus, the main target of the ascending auditory input, has mostly intrinsic neurons, i.e., their dendrites and axons are restricted to the torus itself. In contrast, neurons of the magnocellular and the laminar nucleus project to various auditory and non-auditory processing centers. The projection targets include thalamus, tegmentum, periaqueductal gray, medulla oblongata, and – in the case of laminar neurons – the spinal cord. Additionally, tegmental cells receive direct auditory input and project to various targets, including the spinal cord. Our data imply that both auditory and premotor functions are implemented in individual toral and tegmental neurons. Their axons constitute parallel descending pathways to several effector systems and might be part of the neural substrate for differential audiomotor integration.

Introduction

The auditory system of anuran amphibians is a model system for the analysis of the neural basis of acoustically mediated behavior. In various studies, the connectivity of the auditory pathway (overview in Neary, 1988; Wilczynski, 1988) and the physiology of auditory neurons in different nuclei (overview in Fuzessery, 1988; Walkowiak, 1988a; Hall, 1994) have been analyzed. In addition to studies of pure sensory processing, questions about sensorimotor integration are attracting more attention (Walkowiak and Luksch, 1994).

A particularly important neural structure in this respect is the largest auditory nucleus, the midbrain torus semicircularis, which comprises three auditory subnuclei, the principal, the magnocellular, and the laminar nuclei. This area is organized tonotopically (Pettigrew et al., 1981; Hermes et al., 1982; Mohneke, 1982; Walkowiak and Luksch, 1994). Compared to neurons of primary auditory nuclei, neurons in the torus show highly differentiated and, in many cases, selective response patterns concerning complex frequency processing and coding of fine temporal structures like amplitude modulation rates (Walkowiak, 1980; Fuzessery and Feng, 1983; Diekamp and Schneider, 1988; Gooler and Feng, 1992; Hall, 1994). Additionally, brain areas rostral to the midbrain seemingly are not involved in auditory responses with a short latency, such as antiphonal calling (Walkowiak, 1992; Mohr and Schneider, 1993), since auditory responses in the thalamus and telencephalon have rather long latencies (Mudry et al., 1977) and/or habituate very rapidly (Mudry and Capranica, 1987). The toral subnuclei have been shown to possess distinct connection patterns (Feng and Lin, 1991; Walkowiak and Luksch, 1994). Briefly, the principal nucleus receives afferents mainly from primary auditory nuclei and projects back to its afferent sources as well as to different mesencephalic targets. The magnocellular nucleus receives additional input from various mes- and diencephalic areas and projects to nuclei in the di- and mesencephalon as well as to targets in the medulla oblongata. Afferents to the laminar nucleus arise from various areas in the tel-, di- and mesencephalon as well as from the medulla oblongata, and its efferents innervate target nuclei throughout the entire brain including the spinal cord. The widespread connectivity of the laminar nucleus has led to the hypothesis that neurons of this nucleus might play a prominent role in the process of interfacing between sensory evoked excitation and (pre-)motor networks (Walkowiak and Luksch, 1994). Additionally, this nucleus gets massive modulatory input and is sensitive to sex steroids (Kelley et al., 1978; Luksch and Walkowiak, 1992), indicating a role of this structure in the short- and long-term modulation of auditory evoked behavior. Besides the torus, the tegmentum seems also to be involved in several behavioral reactions based on audio-motor integration (Schmidt, 1988, Schmidt, 1990).

The auditory pathway is organized in both a hierarchical and a parallel fashion with divergent and convergent projections. However, it is not known whether this connectivity is implemented at the population level or at the individual cell level. For example, projections from the torus to several targets might arise from different neuronal populations within the torus, or might stem from the divergent axonal projection of an individual neuron. It is therefore of interest to gain insight into how the connectivity of the auditory pathway is organized on the single cell level. In addition, the morphology of the neurons that give rise to certain projections could provide further insight into the functional anatomy of the nucleus in question. We therefore recorded and stained neurons of the auditory midbrain intracellularly to describe their detailed morphology and to learn whether the different connections of a subnucleus are constituted by different cellular subtypes or whether individual neurons innervate several target nuclei simultaneously. In addition, we attempted to align the anatomical characteristics of a neuron to its physiological response parameters.

To address these questions, we conducted intracellular recordings and subsequent dye injections in an isolated brain preparation. This type of preparation was developed for anuran amphibians several years ago and is currently being applied to answer various anatomical, physiological and pharmacological questions (Schaffer, 1982; Straka and Dieringer, 1993; McLean et al., 1995; Luksch et al., 1996). The major advantage of this in vitro approach is the stability of intracellular recording because tissue pulsations caused by the circulation are avoided. In our study, we replaced the acoustic stimulation by electrical stimulation of the branchlets of the auditory nerve that innervate the sensory epithelia. To test whether this stimulation leads to an activation of the entire auditory pathway comparable to the situation in an intact animal, we additionally recorded neurons intracellularly in an in vivo preparation.

Section snippets

Materials and methods

Brains of 34 male and female Discoglossus pictus (body size 34–64 mm) were isolated. Animals were deeply anesthetized with 0.2% MS 222 (tricaine-methanesulfonate; Ohr, 1976), cooled down to a body temperature of 5°C and perfused transcardially with 40 ml ice cold Ringer's solution (Na+ 100 mM, K+ 2 mM, Ca2+ 2 mM, Mg2+ 0.5 mM, Cl 82 mM, HCO3 25 mM, glucose 11 mM, pH 7.3). The Ringer's solution was prepared after Straka and Dieringer (1993). The subsequent dissection was carried out on a Sylgard

Results

The stimulation current necessary to elicit responses depended on the quality of the preparation of the branchlets; if the nerves had been bruised or strained during the preparation, the stimulus current had to be increased to up to 50 μA, whereas in good preparations responses could be elicited with currents as low as 2–10 μA. Responses could be recorded intracellularly in the isolated brain for up to 4 days without noticeable loss of activity; however, the stimulation current had to be

Methodological considerations

In the last two decades, a variety of in vitro approaches to the study of amphibian neural tissue has been reported in the literature, ranging from brain slices (Holohean et al., 1990), isolated brainstem (Schmidt, 1976; Schaffer, 1982; Straka and Dieringer, 1993; McLean et al., 1995) and whole-brain preparations (Luksch and Walkowiak, 1993; Dicke and Roth, 1994; Wiggers and Roth, 1994) to combined spinal cord-musculature preparations (Sagawa et al., 1987; Wheatley et al., 1992). The

Acknowledgements

We thank M. Grosmann, I. Markus and M. Dohms for technical assistance. We greatly appreciate critical reading of the manuscript and helpful comments by H. Carl Gerhardt, Joshua J. Schwartz, and the anonymous reviewers. This research was supported by a grant of the Deutsche Forschungsgemeinschaft (Wa 446/3-2).

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    Present address: Institut für Biologie II, RWTH Aachen, Kopernikusstraße 16, D-52074 Aachen, Germany.

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