Bimodal locomotion elicited by electrical stimulation of the midbrain in the salamander Notophthalmus viridescens

Abstract

The present experiments were designed to identify the mesencephalic locomotor region (MLR) in the salamander. An in vitro semi-intact preparation from a decerebrate adult salamander (Notophthalmus viridescens) was developed in which the locomotor activities were monitored from electromyographic and video recordings. The results show that the two locomotor modes exhibited by salamanders (i.e., stepping and swimming) were evoked by electrical microstimulation (5-15 Hz; 0.1-10 microA; 2 msec pulses) of a circumscribed region in the caudal mesencephalon. At threshold current strength (0.5-3.5 microA at 15 Hz), rhythmic limb movements and intersegmental coordination, such as during stepping, were induced. As the stimulation strength was subsequently increased, the frequency of stepping became more rapid, and, at 2.0-5.5 microA, the limbs were held back against the body wall and swimming movements of the trunk were induced. An additional increase of the stimulation strength induced an increase of the frequency and amplitude of the swimming movements. Anatomical studies conducted in parallel revealed the presence of choline acetyltransferase immunoreactive cells in the functionally identified MLR region. Together, the present results indicate that the MLR is present in salamanders and that its level of activation determines the mode of locomotion. Walking is induced at low activation levels, and swimming, which constitutes a faster mode of locomotion, requires stronger stimulation of the MLR. Furthermore, as in other vertebrates, the MLR contains cholinergic cells.

Fig. 1.

Stepping (A₁,A₂) and swimming (B₁,B₂) elicited by electrical stimulation of the same midbrain site in an adult salamander.A₁, EMG activity recorded at 0.53 SVL (i Rostral) and 0.76 SVL (i Mid) on the right side and at 0.46 SVL (co Rostral) on the left side during stepping evoked by midbrain microstimulation (2 msec pulses at 15 Hz, 2.8 μA). In-phase activation of ipsilateral myomeres is clearly visible (dashed line).A₂, Drawings illustrating the body contours every 10th successive frame (video sampling at 60 Hz) during one step cycle taken from the same stepping episode.B₁, EMG activity recorded at the same segmental levels during a swimming episode evoked by microstimulation of the same midbrain site (2 msec pulses at 15 Hz, 3.8 μA). A time delay between EMG bursts of ipsilateral myomeres is clearly visible (dashed line). B₂, Body movements during one swimming cycle taken from the same swimming episode.

Fig. 2.

Initiation and termination of MLR-induced locomotion. A₁, A period of postural adjustment began (arrow) a few seconds after the onset of MLR stimulation and lasted ∼25 sec before the locomotion started (locomotor activity). The gray bar atbottom indicates the period during which the MLR was stimulated (2 msec pulses at 15 Hz, 2.5 μA).A₂, Expansion of a part of the locomotor episode shown in A₁. The rostrocaudal phase lag between EMG bursts of ipsilateral myomeres (dashed line) and the alternation between ipsilateral and contralateral EMG bursts are clearly visible. Same voltage amplification in A₁ andA₂. B₁, Relationship between the onset delay and the stimulus intensity. B₂, Relationship between the end (“offset”) delay and the stimulation intensity. In B₁ andB₂, data are from the same four animals. The gray areas indicate the range of stimulus intensities that evoked stepping and/or swimming.

Fig. 3.

MLR stimulation induces two modes of locomotion in salamander. A, Traces are, from top tobottom, EMG activity recorded at 0.39 SVL (i Rostral) and 0.74 SVL (i Mid) on the right side and 0.41 SVL (co Rostral) on the left side. Electrical stimulation of the MLR (2 msec pulses at 15 Hz, 1.25 μA) started 2.2 sec before recordings and was maintained 30 sec. Note the abrupt switching from stepping to swimming and back to stepping.B₁, Effect of increasing the stimulus intensity (2 msec pulses at 15 Hz) on the frequency of swimming.B₂, Effect of increasing the stimulus frequency (2 msec at 6 μA) on the frequency of swimming.Bars are mean values, and error bars are SEM.

Fig. 4.

ChAT-IR cells around the MLR-coagulated site.A, Transverse section (40 μm thick) at the isthmic level showing the coagulated site (right) and the intact side (left). Note the dense plexus of ChAT-IR fibers in the interpeduncular nucleus. B, Magnification of theboxed area in A showing ovoid ChAT-IR cells (arrows) and numerous ChAT-IR fibers around the coagulated site. C, Transverse section (20 μm thick) at approximately the same level as in A. The interpeduncular nucleus shows intense labeling. D, Magnification of the boxed area in Cshowing two distinct groups of ChAT-IR cells, the LDT and the NI. Note the axonal tract arising from the caudal LDT. Scale bars:A, C, 200 μm; B,D, 100 μm. IP, Interpeduncular nucleus;OT, optic tectum.

Fig. 5.

Schematic representation of the distribution of ChAT-IR cells at the isthmic level. Each schematic represents three consecutive 20-μm-thick sections. Total distance, 240 μm. Scale bars: whole brain representation, 1 mm; brainstem transverse sections, 200 μm. IP, Interpeduncular nucleus;OT, optic tectum.