Movement- and behavioral state-dependent activity of pontine reticulospinal neurons

Abstract

Forty-five years ago Shik and colleagues were the first to demonstrate that electrical stimulation of the dorsal pontine reticular formation induced fictive locomotion in decerebrate cats. This supraspinal motor site was subsequently termed the "mesencephalic locomotor region (MLR)". Cholinergic neurons of the pedunculopontine tegmental nucleus (PPT) have been suggested to form, or at least comprise in part, the neuroanatomical basis for the MLR, but direct evidence is lacking. In an effort to clarify the location and activity profiles of pontine reticulospinal neurons supporting locomotor behaviors, we employed in the present study a retrograde tracing method in combination with single-unit recordings and antidromic spinal cord stimulation as well as characterized the locomotor- and behavioral state-dependent activities of both reticulospinal and non-reticulospinal neurons. The retrograde labeling and antidromic stimulation responses suggested a candidate group of reticulospinal neurons that were non-cholinergic and located just medial to the PPT cholinergic neurons and ventral to the cuneiform nucleus (CnF). Unit recordings from these reticulospinal neurons in freely behaving animals revealed that the preponderance of neurons fired in relation to motor behaviors and that some of these neurons were also active during rapid eye movement sleep. By contrast, non-reticulospinal neurons, which likely included cholinergic neurons, did not exhibit firing activity in relation to motor behaviors. In summary, the present study provides neuroanatomical and electrophysiological evidence that non-cholinergic, pontine reticulospinal neurons may constitute the major component of the long-sought neuroanatomic MLR in mammals.

Figure 1

A representative figure showing phasic (A) and tonic (B) firing neurons and their inter-spike interval histograms (C & D).

Figure 2

A. Histological image showing retrogradely labeled CTb cells (CTb injection at the level of C8-T1, the spinal ventral horn) in the lateral pontine tegmentum (LPT) (black color) region and ChAT–positive cells (light brown color & green arrows) just below the ventrolateral periaquaductal gray (vlPAG) and the cuneiform nucleus (CnF) and medial to the pedunculopontine tegmental nucleus (PPT) cholinergic neurons marked by the box. Inset: A larger magnification of the boxed area. B. Schematic images illustrate the reconstruction of the location of the recorded neurons on the background of neutral red stained sections. The placement of the recorded neurons is based on the Prussian blue that marks electrode tip. Each neuron is represented by a symbol designating their firing pattern and activity across behavioral states (AW and REM sleep). Symbols: Filled blue (Phasic AW/REM); Open blue (Tonic AW/REM); Filled green (Phasic REM); Filled red (Phasic AW); Open green (Tonic REM); AW- Active wake active; AW/REM- Active wake and REM sleep active, REM-REM sleep active; Phasic – Phasic firing neurons show intermittent higher activity with occasional burst firing (usually preceded or followed by silent or low activity); Tonic – Tonic firing neurons show low or high firing activity but with regular interval. Numbers in B represent approximate AP distance (in mm) from bregma (Paxinos & Watson, 1998). The yellow circled region shows neurons with no relation to motor behavior nor antidromic response to spinal cord stimulation. Spinally-projecting neurons depicted here are CTb labeled cells (green circle) and neurons (red circle) with higher activity during motor behavior with antidromic projection to spinal cord. Bar in A. 50μm.

Figure 3

A. Schematic images illustrate the reconstruction of the location of the stimulating electrode in the neutral red stained spinal cord sections. The placement of the tip of the electrode at the level approx. the T1 level, in all 7 (marked by filled blue star) rats. Axial muscles (Ax9); forearm ext (FEx9); forearm flex (FFI9); intercostals (ICo9); lamina 7 (7sp); lamina 8 (8sp); latiss dorsi (LD9); triceps (Tr9). B. & C. Representative histological images from rat (n=2) spinal cord sections showing the stimulating electrode (arrow).

Figure 4

A. Histological image showing the track of the recording electrode targeted to the lateral pontine tegmentum (LPT) where a phasic active wake (AW) spinally-projecting neuron was recorded. The blue mark (ferrous deposits) track (blue arrow) is from the recording electrode and is the result of the Pearl's Prussian-blue reaction seen against the background of the neutral red stain. B. Image shows an enlarged view of the area shown (box) in the figure 2A. Recording were done from the cells (green arrows) close to the electrode track. Bar in A. 50μm; B. 10 μm.

Figure 5. Neuronal firing characteristics of LPT neurons

Individual [upper trace; spikes/second)] and group rate [lower trace; mean firing rate / second)] across sleep-wake states [active wake (AW), quiet wake (QW), non-REM (NREM) sleep and REM sleep]. These neurons were classified into three groups based on their antidromic response to spinal cord stimulation and further sub-divided based upon their firing pattern (tonic/phasic) and rate during period of high cortical EEG activity (active wake and REM sleep). * indicates a significant difference (p<0.05) between behavioral states.

Figure 6. An example of a phasic firing AW active neuron

The neuron exhibits a higher firing rate during motor activity (higher EMG amplitude) in AW state as compared to QW, NREM sleep and REM sleep. This neuron was within the MLR region as verified by the electrode track.

Figure 7. AW active neuron and their response to spinal cord stimulation

A, shows antidromic activation with a short latency response time of 3.5 ms. (10 overlap tracing); B, shows repetitive responses to higher frequency (300Hz) stimulation trains; C, collision test showing that the antidromic response of this neuron is blocked with a shorter spike-to-stimulus interval. Large arrow: stimulation artifact, A: antidromic cell response; S: spontaneous cell firing; C: collision test blocking antidromic cell response. Bar in B and C, 5ms

Figure 8. An example of a tonic firing AW and REM active neuron

The neuron exhibits a higher firing rate irrespective of motor behavior in AW (higher EMG amplitude) or the REM sleep state (loss of motor activity) compared to QW and NREM sleep. This neuron was within the PPT region as verified by the electrode track.

Figure 9. A REM-ON neuron in the PPT region

This neuron showed burst firing (phasic activity) during REM sleep but was nearly silent during AW, QW and NREM sleep.

Figure 10. REM-ON phasic firing neuron in the PPT region showing orthodromic response to spinal cord stimulation

A, “Broad” spike shape (presumably a cholinergic neuron); B, orthodromic response to spinal cord stimulation (latency response time of 17ms); C, no response to trains of high frequency stimulation (300Hz). Large arrow: stimulation artifact, S: spontaneous cell firing

Figure 11. Pontine neural circuitry regulating motor behaviors and arousal

PPT cholinergic neurons regulate arousal via ascending projections to the thalamus and basal forebrain and motor function via descending projections to the RVM (green color). The reticulospinal neurons (red color) regulate motor behavior via a direct projection to the spinal cord (red color).