Neural circuits engaged in mastication and orofacial nociception

Abstract: A deeper understanding of both movement control and the effects of nociceptor inputs on our motor systems is critical for proper clinical diagnosis of musculo-skeletal dysfunctions and for development of novel rehabilitation schemes. In the jaw system, masticatory movements are produced by a central pattern generator (CPG) located in the brainstem. Considerable efforts have been made in deciphering this neuronal network. The present thesis contributes towards an increasingly detailed understanding of its essential elements, and presents a hypothesis of how deep somatic pain (i.e. muscle pain) may be evoked and interferes with the masticatory CPG circuitry.In Paper I, the expression of c-Fos-like protein was used as a molecular marker to visualize brainstem neurons that were active during induced fictive mastication in the anesthetized and paralyzed rabbit. Our findings provide a previously lacking detailed record of the neuronal populations that form the masticatory motor pattern. Certain cells were located in brainstem areas previously suggested to be involved in the masticatory CPG. However, it was a new finding that neurons in the dorsal part of the trigeminal main sensory nucleus (NVsnpr-d) may belong to this circuitry. Paper II focused on the discovered neurons in NVsnpr in an in vitro slice preparation from young rats.  Intracellular recordings allowed us to define two cell types based on their response to depolarizing current. Microstimulation applied to the trigeminal motor nucleus, its reticular border, the parvocellular reticular formation and the nucleus reticularis pontis caudalis, elicited postsynaptic potentials in 81% of the neurons tested. Responses obtained were predominately excitatory and sensitive to gluta-matergic antagonists DNQX or/and APV. Some inhibitory and biphasic responses were also evoked. Bicuculline methiodide or strychnine blocked the IPSPs indicating that they were mediated by GABAA or glycinergic receptors. About one third of the stimulations activated both types of neurons antidromically. Neurons in NVsnpr-d seem to gather all the conditions that can theoretically account for a role in masticatory rhythm generation.In Paper III, the masticatory model system was used to investigate the possible role of muscle spindle primary afferents in development of persistent musculoskeletal pain. Following intramuscular acidic (pH 4.0) saline injections of rat masseter muscles, in vitro whole cell recordings were done from jaw closing muscle spindle somata located in the trigeminal mesencephalic nucleus (NVmes). Compared to control neurons, the somata of afferents exposed to acid had more hyperpolarized membrane potentials, more hyperpolarized thresholds for firing, high frequency membrane oscillations and ectopic bursting of action potentials. These changes in membrane properties lasted for up to 35 days. Within the same time frame experi-mental animals showed hypersensitivity to touch on the skin covering the injected muscle. Similar saline injections also resulted in a significant increase of activity dependent c-Fos expression in NVmes neurons compared to controls. Immuno-fluorescence and lectin binding studies indicated that small-caliber muscle afferents containing known nociceptor markers (CGRP, SP, P2X3, TRPV1 and IB4) and expressing glutamate receptors are found close to the annulo-spiral endings of the NVmes afferents. Combined, our new observations support the hypothesis that excessive release of glutamate, within muscle spindles due to ectopically evoked antidromic action potentials, could lead to development of persistent musculoskeletal pain by activation and/ or sensitization of adjacent muscle afferent nociceptors.