Role of neuronal positioning in jaw motor circuit function in zebrafish

Development of the vertebrate nervous system involves substantial cell migration, where immature neurons move to specific locations to generate functional circuits. Precise neuronal migration and positioning are essential for proper brain architecture and function. Abnormal neuronal migration can contribute to neurological disorders such as lissencephaly, autism and schizophrenia. However, the consequences of abnormal neuronal migration for circuit organization and functional output are poorly understood. To provide some insight, I used the facial branchiomotor (FBM) neurons in zebrafish as a model system to analyze the effects of aberrant neuronal migration on circuit function. The FBM neurons are a subset of the branchiomotor neurons, which are generated in the vertebrate hindbrain and innervate facial and jaw muscles. During development in zebrafish and mice, FBM neurons migrate caudally from rhombomere 4 (r4) to r6 to form the facial motor nucleus and innervate jaw and gill muscles (in fish). In order to examine the consequences of aberrant neuronal migration, one must first characterize the normal functional output of the FBM circuit that drives jaw movements. In collaboration with colleagues in the MU Department of Computer Science, we developed an automated image analysis system to extract motion features from video recordings of jaw movement, enabling rapid and accurate high-throughput analysis. We used this software to examine the emergence of jaw movement in zebrafish larvae between 3-9 days post fertilization (dpf). While gape, the displacement of the lower jaw to form the mouth opening, was minimal at 3 dpf, gape frequency increased sharply by 5 dpf, and stabilized by 7 dpf. A detailed analysis of branchiomotor axons and neuromuscular junctions (NMJs) on jaw muscles suggest that this "maturation" of branchiomotor circuit output may be driven by changes in presynaptic structures at the jaw NMJs. To evaluate the consequences of defective neuronal migration on circuit output, I examined whether jaw movement was affected in the zebrafish off-limits (olt) mutant in which FBM neurons fail to migrate out of r4. In olt mutants, the increase in gape frequency occurred normally between 3-5 dpf. However, the average gape frequency was [approximately] 50 [percent] lower than wildtype siblings from 5-9 dpf while gape amplitude was unaffected. Given the jaw movement defect in olt mutants, I evaluated food intake, an independent measure of jaw movement and another functional output of the branchiomotor circuit. Olt mutants ate poorly compared to their wildtype siblings, consistent with their reduced jaw movement. I then tested several potential mechanisms that could generate the functional deficits in olt mutants. While fzd3a, the gene inactivated in olt mutants, is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles were unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, FBM axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data suggest that aberrant positioning of FBM neurons in olt mutants results in subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs. In the future, we will examine modulatory inputs from other brain regions to the branchiomotor neurons and examine their roles in impacting circuit output in olt mutants.

Functional brain stem circuits for control of nose motion

Rodents shift their nose from side to side when they actively explore and lateralize odors in the space. This motor action is driven by a pair of muscles, the deflector nasi. We studied the premotor control of this motion. We used replication-competent rabies virus to transsynaptically label inputs to the deflector nasi muscle and find putative premotor labeling throughout the parvocellular, intermediate, and gigantocellular reticular formations, as well as the trigeminal nuclei, pontine reticular formation, midbrain reticular formation, red nucleus, and superior colliculus. Two areas with extensive labeling were analyzed for their impact on nose movement. One area is in the reticular formation caudal to the facial motor nucleus and is denoted the nose retrofacial area. The second is in the caudal part of the intermediate reticular region near the oscillator for whisking (the nose IRt). Functionally, we find that optogenetic activation of glutamatergic cells in both areas drives deflection of the nose. Ablation of cells in the nose retrofacial area, but not the nose IRt, impairs movement of the nose in response to the presentation of odorants but otherwise leaves movement unaffected. These data suggest that the nose retrofacial area is a conduit for a sensory-driven orofacial motor action. Furthermore, we find labeling of neurons that are immediately upstream of premotor neurons in the preBötzinger complex that presumably synchronizes a small, rhythmic component of nose motion to breathing. NEW & NOTEWORTHY We identify two previously undescribed premotor areas in the medulla that control deflection of the nose. This includes a pathway for directed motion of the nose in response to an odorant.

Differential Innervation of Tissues Located at Traditional Acupuncture Points in the Rat Forehead and Face

Objectives To compare the neural pathways associated with the tissues located at different traditional acupuncture points in the rat forehead and face using the cholera toxin B subunit (CTB) neural tracing technique. Methods After injection of CTB into the tissues at GB14, ST2 and ST6 in the rat, the neural labelling associated with each acupuncture point was revealed by fluorescent immunohistochemistry of the nervous system, including the trigeminal ganglion (TRG), cervical dorsal root ganglia (DRG), spinal cord and brain. Results The CTB labelling included sensory neurons and their transganglionic axonal terminals, as well as motor neurons. The labelled sensory neurons associated with GB14, ST2 and ST6 were distributed in both the TRG and cervical DRG, and their centrally projected axons terminated in an orderly fashion at their corresponding targets in the spinal trigeminal nucleus and cervical spinal dorsal horn. In addition, labelled motor neurons were observed in the facial motor nucleus, trigeminal motor nucleus and cervical spinal ventral horn, in which facial motor neurons projected to the tissues located at all three acupuncture points. Trigeminal motor neurons innervated both ST2 and ST6, while spinal motor neurons only correlated with ST6. Conclusions These results indicate that the tissues located at each of these three traditional acupuncture points in the rat forehead and face has its own sensory and motor connection with the nervous system in a region-specific pattern through distinct neural pathways. Understanding the neuroanatomical characteristics of acupuncture points from the peripheral nervous system to the central nervous system should help inform acupuncture point selection according to the demands of the clinical situation.

Sciatic nerve stimulation activates the retrotrapezoid nucleus in anesthetized rats

Retrotrapezoid nucleus (RTN) neurons sustain breathing automaticity. These neurons have chemoreceptor properties, but their firing is also regulated by multiple synaptic inputs of uncertain function. Here we test whether RTN neurons, like neighboring presympathetic neurons, are excited by somatic afferent stimulation. Experiments were performed in Inactin-anesthetized, bilaterally vagotomized, paralyzed, mechanically ventilated Sprague-Dawley rats. End-expiratory CO2 (eeCO2) was varied between 4% and 10% to modify rate and amplitude of phrenic nerve discharge (PND). RTN and presympathetic neurons were recorded extracellularly below the facial motor nucleus with established criteria. Sciatic nerve stimulation (SNstim, 1 ms, 0.5 Hz) slightly increased blood pressure (6.6 ± 1.6 mmHg) and heart rate and, at low eeCO2 (<5.5%), entrained PND. Ipsi- and contralateral SNstim produced the known biphasic activation of presympathetic neurons. SNstim evoked a similar but weaker biphasic response in up to 67% of RTN neurons and monophasic excitation in the rest. At low eeCO2, RTN neurons were silent and responded more weakly to SNstim than at high eeCO2. RTN neuron firing was respiratory modulated to various degrees. The phasic activation of RTN neurons elicited by SNstim was virtually unchanged at high eeCO2 when PND entrainment to the stimulus was disrupted. Thus RTN neuron response to SNstim did not result from entrainment to the central pattern generator. Overall, SNstim shifted the relationship between RTN firing and eeCO2 upward. In conclusion, somatic afferent stimulation increases RTN neuron firing probability without altering their response to CO2. This pathway may contribute to the hyperpnea triggered by nociception, exercise (muscle metabotropic reflex), or hyperthermia.

Face to face with the social brain

Recent comparative evidence suggests that anthropoid primates are the only vertebrates to exhibit a quantitative relationship between relative brain size and social group size. In this paper, I attempt to explain this pattern with regard to facial expressivity and social bonding. I hypothesize that facial motor control increases as a secondary consequence of neocortical expansion owing to cortical innervation of the facial motor nucleus. This is supported by new analyses demonstrating correlated evolution between relative neocortex size and relative facial nucleus size. I also hypothesize that increased facial motor control correlates with enhanced emotional expressivity, which provides the opportunity for individuals to better gauge the trustworthiness of group members. This is supported by previous evidence from human psychology, as well as new analyses demonstrating a positive relationship between allogrooming and facial nucleus volume. I suggest new approaches to the study of primate facial expressivity in light of these hypotheses.