Monosynaptic premotor circuit tracing reveals neural substrates for oro-motor coordination
- Duke University Medical Center, United States
Figures
Figure 1 with 1 supplement
Schematics detailing the premotor circuit tracing strategy.
(A) Illustration of viral injection sites used in this study. Left, the jaw-closing masseter muscle; right, the genioglossus: a muscle of the tongue controlling protrusion. (B) Genetic cross used in …
Figure 1—figure supplement 1
Extremely rare labeling of ChAT+ premotor neurons in masseter and genioglossus premotor tracing studies.
The brains from monosynaptic rabies tracing experiments were immunostained for choline acetyltransferase (ChAT). Examples of rarely labeled ChAT+ premotor neurons in the masseter (A) and …
Figure 2 with 1 supplement
Representative images of labeled jaw premotor neurons after ΔG-RV injection into the left masseter muscle.
(A) Caudal brainstem showed labeling primarily in the caudal intermediate reticular formation (IRt-c) and medullary reticular formation (MdRt). (B) Rostral brainstem at the level of the facial motor …
Figure 2—figure supplement 1
The masseter premotor circuit contains more LPGi neurons in old pups.
(A) Schematic illustrating monosynaptic rabies-mediated tracing of the masseter premotor circuit in older pups (P8→P15 tracing). Rabies containing mCherry (red) was injected into the left masseter …
Figure 3
Evidence for the presence of bilateral-projecting masseter premotor neurons.
(A–F) Simultaneous tracing of left (ΔG-RV-EGFP, green) and right (ΔG-RV-mCherry, red) masseter premotor neurons. Yellow cells, which indicate bilaterally projecting premotor neurons, were observed …
Figure 4
Representative images of labeled tongue premotor neurons after ΔG-RV injection into the left genioglossus muscle.
(A) Caudal brainstem at the level of the hypoglossal motor nucleus (MoXII), illustrating primary infection of the left MoXII, and extensive labeling in IRt-c, in the spinal trigeminal nucleus …
Figure 5
Premotor axon boutons onto ChAT+ motoneurons revealing direct premotor control of multiple motor groups.
(A) ChAT-immunostained (red) jaw-opening digastric (Dig) motoneurons showing innervation from labeled genioglossus premotor axons (green). (B) The boxed region in A, and a line scan (right) of an …
Figure 6
Representative images from experiments simultaneously tracing both genioglossus (green) and left masseter (red) premotor neurons.
(A) Caudal brainstem at the level of MoXII, showing a rough spatial segregation between the two premotor populations in the IRt-c, with masseter premotor neurons more ventrally situated as compared …
Figure 7
Neurotransmitter phenotypes of labeled premotor neurons.
In situ hybridization in combination with rabies tracing showing glycinergic (A and E) GABAergic (B and F), and glutamatergic (C and G) premotor neurons to masseter and genioglossus motoneurons. …
Figure 8
Summary of premotor circuit tracing results and models for circuit mechanisms underlying several aspects of motor coordination.
(A) Models for ensuring bilaterally symmetric jaw movements. (A1) Previous model based on interactions of independent CPGs on each side of the brainstem. (A2) A model based on this study; signals …
Author response image 1
Videos
Video 1
Representative complete premotor circuit labeling after injection of ΔG-RV-EGFP into the left masseter muscle.
Sections were obtained from the brainstem of an 8-day-old pup 7 days after peripheral rabies injection. 80-µm serial sections are shown in sequence from caudal to the hypoglossal motor nucleus …
Video 2
Representative complete premotor circuit labeling after injection of ΔG-RV-EGFP into the genioglossus muscle.
Sections were obtained from the brainstem of an 8-day-old pup 7 days after peripheral rabies injection. 80-µm serial sections are shown in sequence from caudal to the hypoglossal motor nucleus …
Video 3
Comparison of masseter (ΔG-RV-mCherry) and genioglossus (ΔG-RV-EGFP) premotor circuitry.
Sections were obtained from the brainstem of an 8-day-old pup 7 days after peripheral rabies injection. 80-µm serial sections are shown in sequence from caudal to the hypoglossal motor nucleus …
Video 4
Pseudocolored movie of masseter (ΔG-RV-mCherry) and genioglossus (ΔG-RV-EGFP) premotor circuitry.
Sections were obtained from the brainstem of an 8-day-old pup 7 days after peripheral rabies injection. 80-µm serial sections are shown in sequence from caudal to the hypoglossal motor nucleus …
Tables
Table 1
Description and quantification of the distribution of masseter premotor neurons
| Masseter premotor neurons | ||
|---|---|---|
| Premotor region | % Ipsilateral | % Contralateral |
| Reticular regions | ||
| Medullary reticular formation, caudal intermediate reticular formation | 5.65 ± 0.76 | 5.45 ± 0.69 |
| Rostral intermediate reticular formation | 19.41 ± 1.61 | 14.68 ± 1.22 |
| Parvocellular reticular formation | 13.43 ± 0.98 | 5.32 ± 0.32 |
| Lateral paragigantocellular nucleus | 0.44 ± 0.03 | 0.17 ± 0.02 |
| Trigeminal sensory regions | ||
| Mesencephalic sensory nucleus | 16.81 ± 3.98 | 1.11 ± 0.35 |
| Peri-trigeminal zone | 8.16 ± 0.59 | 1.91 ± 0.22 |
| Dorsal principal trigeminal sensory nucleus | 2.47 ± 0.70 | 1.31 ± 0.37 |
| Spinal trigeminal nucleus, Oralis | 1.78 ± 0.21 | 0.31 ± 0.08 |
| Descending control regions | ||
| Dorsal midbrain reticular formation | 0.45 ± 0.21 | 0.08 ± 0.03 |
| Deep cerebellar nuclei | 0.18 ± 0.10 | 0.40 ± 0.06 |
| Red nucleus | 0.01 ± 0.01 | 0.48 ± 0.12 |
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Extensive bilateral labeling in both caudal (level of MoXII; MdRt, IRt-c) and rostral (rostral to MoXII to caudal MoV; IRt-r, PCRt) reticular regions was observed. Trigeminal sensory-related nuclei labeling primarily included MesV, comprised of jaw muscle proprioceptive and periodontal sensory neurons, and rostral trigeminal sensory nuclei (SpVo, dPrV, and PeriV). Labeling in MesV and SpVo showed a strong ipsilateral bias. Nuclei implicated in descending control were labeled, consisting of contralateral DCN and RN, and ipsilateral dMRf, as well as LPGi. We also found scattered and sparse labeling of premotor neurons in the Gi, interneuron labeling in the contralateral MoV, lateral reticular formation, pre-Bötzinger complex (pre-BötC), medial vestibular nucleus, raphe magnus nucleus, raphe pallidus nucleus, dorsal medial tegmental nucleus, and pontine reticular nucleus. However the labeling pattern and number of neurons in these nuclei were few and not consistent across animals. Percentage of total premotor neurons in a region was calculated within sample (thereby normalizing values to tracing efficacy), and subsequent values were averaged across five mice. All values are averages ± SEM.
Table 2
Description and quantification of the distribution of genioglossus premotor neurons
| Genioglossus premotor neurons | ||
|---|---|---|
| Premotor region | % Ipsilateral | % Contralateral |
| Reticular regions | ||
| Caudal intermediate reticular formation | 18.84 ± 1.79 | 8.99 ± 0.78 |
| Rostral intermediate reticular formation, parvocellular reticular formation | 27.85 ± 0.45 | 24.23 ± 1.78 |
| Lateral paragigantocellular nucleus | 0.85 ± 0.23 | 0.73 ± 0.18 |
| Trigeminal sensory regions | ||
| Spinal trigeminal sensory nucleus, caudalis | 4.21 ± 1.44 | 2.19 ± 0.70 |
| Peri-trigeminal zone | 2.66 ± 0.47 | 1.88 ± 0.30 |
| Mesencephalic sensory nucleus | 1.53 ± 0.31 | 0.59 ± 0.10 |
| Spinal trigeminal sensory nucleus, oralis | 0.95 ± 0.20 | 0.56 ± 0.06 |
| Dorsal principal trigeminal sensory nucleus | 0.73 ± 0.25 | 0.52 ± 0.13 |
| Descending control regions | ||
| Dorsal midbrain reticular formation | 1.18 ± 0.38 | 1.21 ± 0.33 |
| Deep cerebellar nuclei | 0.11 ± 0.03 | 0.08 ± 0.02 |
| Red nucleus | 0.06 ± 0.04 | 0.05 ± 0.04 |
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Extensive bilateral labeling was observed in a concentrated band within the IRt from the medulla to the caudal border of MoVII (IRt-c, IRt-r), after which it spread slightly into the PCRt (IRt-r, PCRt). Labeling in trigeminal sensory related nuclei was primarily in the caudal sensory nuclei, particularly in bilateral SpVc. Additional sparse labeling of neurons in trigeminal sensory-related regions was found in SpVi, dPrV, PeriV, and MesV, with the MesV labeling occurring as far rostral as dorsal to the PAG. Nuclei implicated in descending control were labeled, consisting of contralateral DCN, bilateral dMRf, and bilateral LPGi. We also found scattered and sparse labeling of premotor neurons in the Gi, nucleus of the solitary tract (NTS), rostral ventral respiratory group, lateral reticular nucleus, pre-BötC, midline raphe nuclei, superior vestibular nucleus, pontine reticular nucleus, and dorsal medial tegmental area. However, the labeling pattern and number of neurons in these nuclei were few and not consistent across animals. Percentage of total premotor neurons in a region was calculated within sample (thereby normalizing values to tracing efficacy), and subsequent values were averaged across five samples. All values are averages ±SEM.
