Dorly Verdier

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.

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 Ca2+ concentration ([Ca2+]e) by astrocytes. In this circuit, astrocytes respond to sensory stimuli that induce neuronal rhythmic activity, and their blockade with a Ca2+ chelator prevents neurons from generating a rhythmic bursting pattern. This ability is restored by adding S100β, an astrocytic Ca2+-binding protein, to the extracellular space, while application of an anti-S100β 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.

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.

Inputs to nucleus pontis caudalis from adjacent trigeminal areas.

Recent studies suggest that the nucleus pontis caudalis (nPontc) plays a role in patterning mastication through interactions with the adjacent lateral tegmentum. In this study, we used in vitro intracellular recording and staining to describe the basic membrane properties and morphology of nPontc neurones and to further explore interactions with adjacent structures, using coronal sections of the brainstem of 78 rats, aged 9–28 days. Neurones were large, with dendrites that spread in all directions, and about 64% fired tonically even in the absence of synaptic inputs. Tonic neurones were predominant in the centre of the nucleus. Electrical stimulation of all regions of the nPontc produced mixed excitatory and inhibitory effects on interneurones of lateral tegmental nuclei. Focal inactivation of the dorsal nPontc with injections of tetrodotoxin also had mixed effects on the spontaneous firing of both interneurones and motoneurones but similar injections in the ventral nPontc produced mostly increases of firing. Sixty‐five percent of nPontc neurones received synaptic inputs from the lateral tegmental areas and most of these (68%) were excitatory and mediated by glutamatergic receptors. Inhibitory postsynaptic potentials were mediated by GABAA or glycinergic receptors. Although most responses occurred at relatively long latencies (> 2 ms), they could follow relatively high‐frequency stimulation (> 50 Hz). Excitatory and inhibitory connections between ipsi‐ and contralateral nPontc neurones were also documented, which could contribute to bilateral coordination of jaw movements. This study provides evidence that the nPontc exerts both tonic and phasic influences on the premotor components of the masticatory central pattern generator.

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.

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.

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 S100β. 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 S100β. Here we show that extracellular blockade of S100β 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.

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.