Identification of brainstem interneurons projecting to the trigeminal motor nucleus and adjacent structures in the rabbit

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Abstract

Neurons of several nuclei within the medial pontomedullar reticular formation are active during mastication, but their relationship with other elements of the pattern generating circuits have never been clearly defined. In this paper, we have studied the connection of this area with the trigeminal motor nucleus and with pools of last-order interneurons of the lateral brainstem. Retrograde tracing techniques were used in combination with immunohistochemistry to define populations of glutamatergic and GABAergic neurons. Injections of tracer into the Vth motor nucleus marked neurons in several trigeminal nuclei including the ipsilateral mesencephalic nucleus, the contralateral Vth motor nucleus, the dorsal cap of the main sensory nucleus and the rostral divisions of the spinal nucleus bilaterally. Many last-order interneurons formed a bilateral lateral band running caudally from Regio h (the zone surrounding the Vth motor nucleus), through the parvocellular reticular formation and Vth spinal caudal nucleus. Injections of tracer into Regio h, an area rich in last-order interneurons, marked, in addition to the areas listed above, a large number of neurons in the medial reticular formation bilaterally. The major difference between injection sites was that most neurons projecting to the Vth motor nucleus were located laterally, whereas most of those projecting to Regio h were found medially. Both populations contained glutamatergic and GABAergic neurons intermingled. Our results indicate that neurons of the medial reticular formation that are active during mastication influence Vth motoneurons output via relays in Regio h and other adjacent nuclei.

Introduction

The basic network responsible for generation of rhythmic jaw movements is located in the brain stem. The most recent evidence suggests that this network lies between the rostral poles of the trigeminal (N.V.mt) and facial (N.VII) motor nuclei, but the medio-lateral limits are not yet defined. Although Tanaka et al. (1999) have recently implied that the lateral reticular formation extending 200–400 μm medial to N.V.mt suffices to generate rhythmic motor activity, this evidence is not conclusive, because the activity was induced by a pharmacological mixture that produces rhythmical membrane potential fluctuations in motoneurons deprived of synaptic input (Kim and Chandler, 1995). Nevertheless, it is clear from other studies that the circuitry for generation of rhythmic jaw movements does include the lateral brainstem surrounding N.V.mt (Donga and Lund, 1991, Inoue et al., 1992, Westberg et al., 1998). This region contains Regio h (the reticular zone surrounding N.V.mt that is composed of the supratrigeminal area dorsally, the intertrigeminal area laterally and the juxtatrigeminal area medially), the rostral part of the V spinal nucleus (N.V.spo γ) and the parvocellular reticular formation adjacent to it (R.pc α). Neurons in all these regions project to N.V.mt of both sides (Mizuno et al., 1978, Mizuno et al., 1983, Travers and Norgren, 1983, Li et al., 1995) and change their firing frequency during mastication (Moriyama, 1987, Donga and Lund, 1991, Inoue et al., 1992, Westberg et al., 1995).

However, there are also neurons in the medial reticular formation that appear to participate in the genesis of mastication. In guinea pigs, neurons in and around the rostral pole of nucleus giganto-cellularis (Rgc) fire tonically during mastication induced by cortical stimulation, and a group of neurons lying dorsal to these are rhythmically active (Chandler and Tal, 1986, Nozaki et al., 1986a, Nozaki et al., 1986b, Chandler et al., 1990). In rabbits, similar groups of neurons have been described in the ventral and dorsal nucleus pontis caudalis (nPC) (Lund et al., 1998). We hypothesize that these medial groups act as a rhythm generator, while the lateral groups generate motoneuron bursts by integrating sensory information and descending commands with the masticatory rhythm generated medially (Lund et al., 1998). This is similar to some models that have been proposed for other complex rhythmic behaviours in mammals (e.g. respiration, Feldman et al., 1988).

In an attempt to provide anatomical support for this model, we have compared populations of neurons projecting to V motoneurons of rabbits to those projecting to a component of the putative burst generator, namely Regio h. To gain insight on how these areas are functionally linked we also attempted to identify subpopulations of excitatory and inhibitory neurons by combining the tract tracing technique with immunohistochemistry against glutamate and GABA.

Section snippets

Materials and methods

Six male New Zealand white rabbits were used for the experiments. The animals were anaesthetized with urethane (1 g kg−1, i.v.). The trachea was intubated and the femoral vein was cannulated for administration of drugs and for fluid replacement. The ECG was recorded. The masseter and inferior alveolar nerves were prepared for electrical stimulation as described previously (Olsson et al., 1986a). Wounds were closed, the animal was transferred to a stereotaxic frame, and an opening was made in

Results

One of the six injected animals was excluded from analysis because the retrograde and immunochemical labelling were very faint and suggested that there was a problem either during the survival period or the subsequent perfusion. The five remaining injection sites are shown in Fig. 1. In two cases (R1 and R3), small injections were made within the boundaries of N.V.mt, although some spread was seen dorsally along the electrode track in R1. In a third (R5), the core of a larger injection was also

Immunoreactivity of retrogradely labelled neurons

Fig. 6 shows two retrogradely labelled cells in nPC of R5 that were immunoreactive to glutamate and GABA antibodies (left and right panels respectively). Table 3 gives, for R1, 3, 4 and 5, the number of double labelled neurons for the nuclei containing most of the retrograde labelling (listed in Table 2). These numbers are also expressed as the percentage of total number of labelled cells in the structure identified in the first column. The bottom line (total) gives the total number of

Discussion

In the present study, we attempted to identify the anatomical pathway linking areas of the caudal pontine medial reticular formation (nPC) that play a role in mastication to trigeminal motoneurons using retrograde tracing techniques. We also tried to identify among these retrogradely labelled neurons those that were inhibitory and excitatory on the basis of their immunoreactivity to GABA and glutamate respectively. The results show that neurons of nPC have virtually no projections to N.V.mt,

Conclusion

The present study provides anatomical support for the hypothesis that the medial reticular formation has a role in the control of trigeminal motoneurons via direct projections from the caudal gigantocellular nucleus, and through a more important indirect connection from nucleus pontis caudalis via relays in Regio h. Regio h and the more caudal parts of the lateral parvocellular reticular formation are rich in last-order interneurons, many of which are also immunoreactive to GABA and glutamate.

Acknowledgements

The authors wish to sincerely thank Professor E.G. Jones and T. Hashikawa for their help with the immunohistochemical work, which was carried out at the RIKEN Institute, Wako, Japan. We are also grateful to Danielle Veilleux and Julie Martin for their technical assistance. This work was supported by a Group Grant from the Canadian MRC (#GR-176), the Canadian natural sciences and engineering research council (# OGP0172682) and the Swedish MRC (B93-04X- 00045-29A; K93-24P-10133-02A).

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