Hyperexcitability of muscle spindle afferents in jaw-closing muscles in experimental myalgia: Evidence for large primary afferents involvement in chronic pain.

The goals of this review are to improve understanding of the aetiology of chronic muscle pain and identify new targets for treatments. Muscle pain is usually associated with trigger points in syndromes such as fibromyalgia and myofascial syndrome, and with small spots associated with spontaneous electrical activity that seems to emanate from fibers inside muscle spindles in EMG studies. These observations, added to the reports that large-diameter primary afferents, such as those innervating muscle spindles, become hyperexcitable and develop spontaneous ectopic firing in conditions leading to neuropathic pain, suggest that changes in excitability of these afferents might make an important contribution to the development of pathological pain. Here, we review evidence that the muscle spindle afferents (MSAs) of the jaw-closing muscles become hyperexcitable in a model of chronic orofacial myalgia. In these afferents, as in other large-diameter primary afferents in dorsal root ganglia, firing emerges from fast membrane potential oscillations that are supported by a persistent sodium current (INaP ) mediated by Na+ channels containing the α-subunit NaV 1.6. The current flowing through NaV 1.6 channels increases when the extracellular Ca2+ concentration decreases, and studies have shown that INaP -driven firing is increased by S100ß, an astrocytic protein that chelates Ca2+ when released in the extracellular space. We review evidence of how astrocytes, which are known to be activated in pain conditions, might, through their regulation of extracellular Ca2+ , contribute to the generation of ectopic firing in MSAs. To explain how ectopic firing in MSAs might cause pain, we review evidence supporting the hypothesis that cross-talk between proprioceptive and nociceptive pathways might occur in the periphery, within the spindle capsule.

Functional Connectivity Between the Trigeminal Main Sensory Nucleus and the Trigeminal Motor Nucleus.

The present study shows new evidence of functional connectivity between the trigeminal main sensory (NVsnpr) and motor (NVmt) nuclei in rats and mice. NVsnpr neurons projecting to NVmt are most highly concentrated in its dorsal half. Their electrical stimulation induced multiphasic excitatory synaptic responses in trigeminal MNs and evoked calcium responses mainly in the jaw-closing region of NVmt. Induction of rhythmic bursting in NVsnpr neurons by local applications of BAPTA also elicited rhythmic firing or clustering of postsynaptic potentials in trigeminal motoneurons, further emphasizing the functional relationship between these two nuclei in terms of rhythm transmission. Biocytin injections in both nuclei and calcium-imaging in one of the two nuclei during electrical stimulation of the other revealed a specific pattern of connectivity between the two nuclei, which organization seemed to critically depend on the dorsoventral location of the stimulation site within NVsnpr with the most dorsal areas of NVsnpr projecting to the dorsolateral region of NVmt and intermediate areas projecting to ventromedial NVmt. This study confirms and develops earlier experiments by exploring the physiological nature and functional topography of the connectivity between NVsnpr and NVmt that was demonstrated in the past with neuroanatomical techniques.

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions. Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest. This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Functional rhythmogenic domains defined by astrocytic networks in the trigeminal main sensory nucleus.

Stimuli that induce rhythmic firing in trigeminal neurons also increase astrocytic coupling and reveal networks that define the boundaries of this particular population. Rhythmic firing depends on astrocytic coupling which in turn depends on S100ß. In many nervous functions that rely on the ability of neuronal networks to generate a rhythmic pattern of activity, coordination of firing is an essential feature. Astrocytes play an important role in some of these networks, but the contribution of astrocytic coupling remains poorly defined. Here we investigate the modulation and organization of astrocytic networks in the dorsal part of the trigeminal main sensory nucleus (NVsnpr), which forms part of the network generating chewing movements. Using whole-cell recordings and the dye coupling approach by filling a single astrocyte with biocytin to reveal astrocytic networks, we showed that coupling is limited under resting conditions, but increases importantly under conditions that induce rhythmic firing in NVsnpr neurons. These are: repetitive electrical stimulation of the sensory inputs to the nucleus, local application of NMDA and decrease of extracellular Ca2+ . We have previously shown that rhythmic firing induced in NVsnpr neurons by these stimuli depends on astrocytes and their Ca2+ -binding protein S100ß. Here we show that extracellular blockade of S100ß also prevents the increase in astrocytic coupling induced by local application of NMDA. Most of the networks were small and remained confined to the functionally distinct area of dorsal NVsnpr. Disrupting coupling by perfusion with the nonspecific gap junction blocker, carbenoxolone or with GAP26, a selective inhibitor of connexin 43, mostly expressed in astrocytes, abolished NMDA-induced rhythmic firing in NVsnpr neurons. These results suggest that astrocytic coupling is regulated by sensory inputs, necessary for neuronal bursting, and organized in a region specific manner.

Ion Homeostasis in Rhythmogenesis: The Interplay Between Neurons and Astroglia.

Proper function of all excitable cells depends on ion homeostasis. Nowhere is this more critical than in the brain where the extracellular concentration of some ions determines neurons' firing pattern and ability to encode information. Several neuronal functions depend on the ability of neurons to change their firing pattern to a rhythmic bursting pattern, whereas, in some circuits, rhythmic firing is, on the contrary, associated to pathologies like epilepsy or Parkinson's disease. In this review, we focus on the four main ions known to fluctuate during rhythmic firing: calcium, potassium, sodium, and chloride. We discuss the synergistic interactions between these elements to promote an oscillatory activity. We also review evidence supporting an important role for astrocytes in the homeostasis of each of these ions and describe mechanisms by which astrocytes may regulate neuronal firing by altering their extracellular concentrations. A particular emphasis is put on the mechanisms underlying rhythmogenesis in the circuit forming the central pattern generator (CPG) for mastication and other CPG systems. Finally, we discuss how an impairment in the ability of glial cells to maintain such homeostasis may result in pathologies like epilepsy and Parkinson's disease.

Neonatal low-protein diet reduces the masticatory efficiency in rats.

Little is known about the effects of undernutrition on the specific muscles and neuronal circuits involved in mastication. The aim of this study was to document the effects of neonatal low-protein diet on masticatory efficiency. Newborn rats whose mothers were fed 17% (nourished (N), n 60) or 8% (undernourished (U), n 56) protein were compared. Their weight was monitored and their masticatory jaw movements were video-recorded. Whole-cell patch-clamp recordings were performed in brainstem slice preparations to investigate the intrinsic membrane properties and N-methyl-d-aspartate-induced bursting characteristics of the rhythmogenic neurons (N, n 43; U, n 39) within the trigeminal main sensory nucleus (NVsnpr). Morphometric analysis (N, n 4; U, n 5) were conducted on masseteric muscles serial cross-sections. Our results showed that undernourished animals had lower numbers of masticatory sequences (P=0·049) and cycles (P=0·045) and slower chewing frequencies (P=0·004) (N, n 32; U, n 28). Undernutrition reduced body weight but had little effect on many basic NVsnpr neuronal electrophysiological parameters. It did, however, affect sag potentials (P<0·001) and rebound firing (P=0·005) that influence firing pattern. Undernutrition delayed the appearance of bursting and reduced the propensity to burst (P=0·002), as well as the bursting frequency (P=0·032). Undernourished animals showed increased and reduced proportions of fibre type IIA (P<0·0001) and IIB (P<0·0001), respectively. In addition, their fibre areas (IIA, P<0·001; IIB, P<0·001) and perimeters (IIA, P<0·001; IIB, P<0·001) were smaller. The changes observed at the behavioural, neuronal and muscular levels suggest that undernutrition reduces chewing efficiency by slowing, weakening and delaying maturation of the masticatory muscles and the associated neuronal circuitry.

An astrocyte-dependent mechanism for neuronal rhythmogenesis.

Communication between neurons rests on their capacity to change their firing pattern to encode different messages. For several vital functions, such as respiration and mastication, neurons need to generate a rhythmic firing pattern. Here we show in the rat trigeminal sensori-motor circuit for mastication that this ability depends on regulation of the extracellular Ca(2+) concentration ([Ca(2+)]e) by astrocytes. In this circuit, astrocytes respond to sensory stimuli that induce neuronal rhythmic activity, and their blockade with a Ca(2+) chelator prevents neurons from generating a rhythmic bursting pattern. This ability is restored by adding S100ß, an astrocytic Ca(2+)-binding protein, to the extracellular space, while application of an anti-S100ß antibody prevents generation of rhythmic activity. These results indicate that astrocytes regulate a fundamental neuronal property: the capacity to change firing pattern. These findings may have broad implications for many other neural networks whose functions depend on the generation of rhythmic activity.

Generation of the masticatory central pattern and its modulation by sensory feedback.

The basic pattern of rhythmic jaw movements produced during mastication is generated by a neuronal network located in the brainstem and referred to as the masticatory central pattern generator (CPG). This network composed of neurons mostly associated to the trigeminal system is found between the rostral borders of the trigeminal motor nucleus and facial nucleus. This review summarizes current knowledge on the anatomical organization, the development, the connectivity and the cellular properties of these trigeminal circuits in relation to mastication. Emphasis is put on a population of rhythmogenic neurons in the dorsal part of the trigeminal sensory nucleus. These neurons have intrinsic bursting capabilities, supported by a persistent Na(+) current (I(NaP)), which are enhanced when the extracellular concentration of Ca(2+) diminishes. Presented evidence suggest that the Ca(2+) dependency of this current combined with its voltage-dependency could provide a mechanism for cortical and sensory afferent inputs to the nucleus to interact with the rhythmogenic properties of its neurons to adjust and adapt the rhythmic output. Astrocytes are postulated to contribute to this process by modulating the extracellular Ca(2+) concentration and a model is proposed to explain how functional microdomains defined by the boundaries of astrocytic syncitia may form under the influence of incoming inputs.

Modulation of rhythmogenic properties of trigeminal neurons contributing to the masticatory CPG.

Increasing evidence suggests that the dorsal part of the principal sensory nucleus of the trigeminal nerve (NVsnpr) contains a significant core of the central pattern generator (CPG) circuitry required for mastication (Tsuboi et al., 2003). Like many trigeminal brainstem neurons, those of NVsnpr are rhythmically active in phase with fictive mastication in vivo (Tsuboi et al., 2003) and project directly to the trigeminal motoneurons (Kolta et al., 2000), but in contrast with the others, they are the only neurons with intrinsic bursting abilities (Sandler et al., 1998; Brocard et al., 2006) within the minimal area of the brainstem necessary to produce rhythmic activity in trigeminal nerves (Bourque and Kolta, 2001). Development of bursting in NVsnpr neurons closely follows the development of mastication. It is mediated by a persistent Na(+) current (I(NaP)) that is expressed only within a certain membrane potential range and that is modulated by the extracellular Ca(2+) concentration ([Ca(2+)](e)), the lower the concentration, the larger the magnitude of I(NaP). Under physiological [Ca(2+)](e), bursting can also be induced in vitro by repetitive electrical stimulation of the trigeminal sensory tract, which projects massively to NVsnpr or by local applications of N-methyl-d-aspartic acid. Both types of stimuli also depolarize glial cells recorded in NVsnpr and increase coupling between them. Glial cells play a determinant role in setting [Ca(2+)](e) and hence are in a key position to influence NVsnpr neuronal firing pattern.

Electrical properties of interneurons found within the trigeminal motor nucleus.

The trigeminal motor nucleus contains the somata of motoneurons innervating the jaw muscles, but also those of interneurons that we have characterized morphologically and immunohistochemically previously. Here we compare their basic physiological characteristics and synaptic inputs from the peri-trigeminal area (PeriV) to those of motoneurons using whole-cell recordings made with pipettes containing biocytin in brainstem slices of rats that had a tracer injected into their masseters. Values for input resistance, spike duration and overall duration of afterhyperpolarization (AHP) were greater for interneurons than for motoneurons. Some interneurons (44%) and motoneurons (33%) had an outward rectification during depolarization. Hyperpolarization-induced inward rectification was seen predominantly in interneurons (85% vs. 31% for motoneurons). Few interneurons (15%) showed depolarization and time-dependent firing frequency accommodation, while half (52%) of the motoneurons did. Rebound excitation at the offset of hyperpolarization was more common in interneurons than in motoneurons (62% vs. 34%). Both populations received synaptic inputs from PeriV. These inputs were predominantly excitatory and were mediated by non-N-methyl-d-aspartate glutamatergic receptors. Response latencies and rise times of the evoked potentials were longer in interneurons than in motoneurons, suggesting that some of the inputs to interneurons could be polysynaptic and/or occurring at distal dendritic locations. Miniature synaptic events could be seen in about half of the neurons in both populations. These results suggest that interneurons can be clearly distinguished from motoneurons on the basis of some electrophysiological properties like the input resistance and spike and AHP durations, and the kinetics of their synaptic inputs from adjacent areas.

A review of burst generation by trigeminal main sensory neurons.

In this paper, we present evidence that neurons in the dorsal part of the trigeminal main sensory nucleus participate in the patterning of mastication. These neurons have special membrane properties that allow them to generate rhythmical bursts of action potentials in the frequency range of natural mastication even when cut off from synaptic inputs. These properties mature during the third postnatal week in rats at the same time as mastication begins. Finally, we present evidence that a reduction on extracellular calcium concentration is an important step in the initiation of mastication.

Emergence of intrinsic bursting in trigeminal sensory neurons parallels the acquisition of mastication in weanling rats.

There is increasing evidence that a subpopulation of neurons in the dorsal principal sensory trigeminal nucleus are not simple sensory relays to the thalamus but may form the core of the central pattern generating circuits responsible for mastication. In this paper, we used whole cell patch recordings in brain stem slices of young rats to show that these neurons have intrinsic bursting abilities that persist in absence of extracellular Ca(2+). Application of different K(+) channel blockers affected duration and firing rate of bursts, but left bursting ability intact. Bursting was voltage dependent and was abolished by low concentrations of Na(+) channel blockers. The proportion of bursting neurons increased dramatically in the second postnatal week, in parallel with profound changes in several electrophysiological properties. This is the period in which masticatory movements appear and mature. Bursting was associated with the development of an afterdepolarization that depend on maturation of a persistent sodium conductance (I(NaP)). An interesting finding was that the occurrence of bursting and the magnitude of I(NaP) were both modulated by the extracellular concentration of Ca(2+). Lowering extracellular [Ca(2+)] increased both I(NaP) and probability of bursting. We suggest that these mechanisms underlie burst generation in mastication and that similar processes may be found in other motor pattern generators.

Synaptic inputs to trigeminal primary afferent neurons cause firing and modulate intrinsic oscillatory activity.

In this paper, we investigated the influence of synapses on the cell bodies of trigeminal muscle spindle afferents that lie in the trigeminal mesencephalic nucleus (NVmes), using intracellular recordings in brain stem slices of young rats. Three types of synaptic responses could be evoked by electrical stimulation of the adjacent supratrigeminal, motor, and main sensory nuclei and the intertrigeminal area: monophasic depolarizing postsynaptic potentials (PSPs), biphasic PSPs, and all or none action potentials without underlying excitatory PSPs (EPSPs). Many PSPs and spikes were abolished by bath-application of 6,7-dinitroquinoxaline (DNQX) alone or combined with D,L-2-amino-5-phosphonovaleric acid (APV), suggesting that they are mediated by non-N-methyl-D-aspartate (NMDA) and NMDA glutamatergic receptors, while some action potentials were sensitive to bicuculline, indicating involvement of GABAA receptors. A number of cells showed spontaneous membrane potential oscillations, and stimulation of synaptic inputs increased the amplitude of the oscillations for several cycles, which often triggered repetitive firing. Furthermore, the oscillatory rhythm was reset by the stimulation. Our results show that synaptic inputs to muscle primary afferent neurons in NVmes from neighboring areas are mainly excitatory and that they cause firing. In addition, the inputs synchronize intrinsic oscillations, which may lead to sustained, synchronous firing in a subpopulation of afferents. This may be of importance during rapid biting and during the mastication of very hard or tough foods.

GABAergic control of action potential propagation along axonal branches of mammalian sensory neurons.

The main axons of mammalian sensory neurons are usually viewed as passive transmitters of sensory information. However, the spindle afferents of jaw-closing muscles behave as if action potential traffic along their central axons is phasically regulated during rhythmic jaw movements. In this paper, we used brainstem slices containing the cell bodies, stem axons, and central axons of these sensory afferents to show that GABA applied to the descending central (caudal) axon often abolished antidromic action potentials that were elicited by electrical stimulation of the tract containing the caudal axons of the recorded cells. This effect of GABA was most often not associated with a change in membrane potential of the soma and was still present in a calcium-free medium. It was mimicked by local applications of muscimol on the axons and was blocked by bath applications of picrotoxin, suggesting activation of GABA(A) receptors located on the descending axon. Antidromic action potentials could also be blocked by electrical stimulation of local interneurons, and this effect was prevented by bath application of picrotoxin, suggesting that it results from the activation of GABA(A) receptors after the release of endogenous GABA. We suggest that blockage is caused mainly by shunting within the caudal axon and that motor command circuits use this mechanism to disconnect the rostral and caudal compartments of the central axon, which allows the two parts of the neuron to perform different functions during movement.