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Key points Inputs impinging on layer 5 pyramidal neurons perform essential operations as these cells represent one of the most important output carriers of the cerebral cortex. However, the contribution of astrocytes, a type of glial cell, to these operations is poorly documented. Here we found that optogenetic activation of astrocytes in the vicinity of layer 5 in the mouse primary visual cortex induces spiking in local pyramidal neurons through Nav1.6 ion channels and prolongs the responses elicited in these neurons by stimulation of their distal inputs in cortical layer 1. This effect partially involved glutamatergic signalling but relied mostly on the astrocytic calcium-binding protein S100β, which regulates the concentration of calcium in the extracellular space around neurons. These findings show that astrocytes contribute to the fundamental computational operations of the cortex by acting on the ionic environment of neurons. Abstract The most complex cerebral functions are performed by the cortex, whose most important output is carried out by its layer 5 pyramidal neurons. Their firing reflects integration of the sensory and contextual information that they receive. There is evidence that astrocytes influence cortical neuron firing through the release of gliotransmitters such as ATP, glutamate or GABA. These effects have been described at the network and at the synaptic levels, but it is still unclear how astrocytes influence neuron input-output transfer function at the cellular level. Here, we used optogenetic tools coupled with electrophysiological, imaging and anatomical approaches to test whether and how astrocytic activation affected processing of distal inputs to layer 5 pyramidal neurons (L5PNs). We show that optogenetic activation of astrocytes near L5PN cell body prolonged firing induced by distal inputs to L5PNs and potentiated their ability to trigger spikes. The observed astrocytic effects on L5PN firing involved glutamatergic transmission to some extent but relied mostly on release of S100β, an astrocytic Ca2+ -binding protein that decreases extracellular Ca2+ once released. This astrocyte-evoked decrease in extracellular Ca2+ elicited firing mediated by activation of Nav1.6 channels. Our findings suggest that astrocytes contribute to the cortical fundamental computational operations by controlling the extracellular ionic environment.

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.

KEY POINTS: GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing. ABSTRACT: Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red‐eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABA(A) receptors. By positioning the sensor recorded in the whole‐cell patch‐clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca(2+), we concluded that they originated from non‐neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca(2+) increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca(2+) in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs.

A population of neurons in the trigeminal principal sensory nucleus (NVsnpr) fire rhythmically during fictive mastication induced in the in vivo rabbit. To elucidate whether these neurons form part of the central pattern generator (CPG) for mastication, we performed intracellular recordings in brainstem slices taken from young rats. Two cell types were defined, nonbursting (63%) and bursting (37%). In response to membrane depolarization, bursting cells, which dominated in the dorsal part of the NVsnpr, fired an initial burst followed by single spikes or recurring bursts. Non-bursting neurons, scattered throughout the nucleus, fired single action potentials. Microstimulation applied to the trigeminal motor nucleus (NVmt), the reticular border zone surrounding the NVmt, the parvocellular reticular formation or the nucleus reticularis pontis caudalis (NPontc) elicited a postsynaptic potential in 81% of the neurons tested for synaptic inputs. Responses obtained were predominately excitatory and sensitive to glutamatergic antagonists DNQX and/or APV. Some inhibitory and biphasic responses were also evoked. Bicuculline methiodide or strychnine blocked the IPSPs indicating that they were mediated by GABA(A) or glycinergic receptors. About one-third of the stimulations activated both types of neurons antidromically, mostly from the masseteric motoneuron pool of NVmt and dorsal part of NPontc. In conclusion, our new findings show that some neurons in the dorsal NVsnpr display both firing properties and axonal connections which support the hypothesis that they may participate in masticatory pattern generation. Thus, the present data provide an extended basis for further studies on the organization of the masticatory CPG network.

Neurons of the dorsal nucleus reticularis pontis caudalis (nPontc) fire rhythmically during fictive mastication, while neurons of the ventral half tend to fire tonically (Westberg et al., 2001). This paper describes the changes in the pattern of rhythmical mastication elicited by stimulation of the sensorimotor cortex during inhibition or excitation of neurons in this nucleus and adjacent parts of nucleus reticularis gigantocellularis (Rgc) in the anaesthetized rabbit. Masticatory movements and electromyographic (EMG) activity of the masseter and digastric muscles produced by cortical stimulation were recorded before, during and after injections of a local anaesthetic (lidocaine) or excitatory amino acid N-methyl-d-aspartate (NMDA) into nPontc and Rgc through a microsyringe with attached microelectrode to record neuronal activity. Lidocaine inhibited local neurons and modified the motor program, and the effects varied with the site of injection. Most injections into the ventral half of nPontc increased cycle duration, digastric burst duration and burst area. The action of lidocaine in dorsal nPontc was more variable, although burst duration and area were often decreased. The effects on the muscle activity were always bilateral. Lidocaine block of the rostromedial part of Rgc had no effect on movements or on EMGs. Injections of NMDA excited local neurons and when injected into ventral nPontc, it completely blocked mastication. Dorsal injections either had no effect or increased cycle frequency, while decreasing burst duration and area. No increases in EMG burst duration or area were observed with NMDA. Our findings suggest that neurons of ventral nPontc tonically inhibit other parts of the central pattern generator during mastication, while dorsal neurons have mixed effects. We incorporated these findings into a new model of the masticatory central pattern generator.

The trigeminal principal sensory nucleus (NVsnpr) contains both trigemino-thalamic neurons and interneurons projecting to the reticular formation and brainstem motor nuclei. Here we describe the inputs and patterns of firing of NVsnpr neurons during fictive mastication in anaesthetized and paralysed rabbits to determine the role that NVsnpr may play in patterning mastication. Of the 272 neurons recorded in NVsnpr, 107 changed their firing patterns during repetitive stimulation of the left or right sensorimotor cortex to induce fictive mastication. Thirty increased their firing tonically. Seventy-seven became rhythmically active, but only 31 fired in phase with mastication. The others discharged in bursts at more than twice the frequency of trigeminal motoneurons. Most rhythmic masticatory neurons were concentrated in the dorsal part, and those which fired during the jaw closing phase of the cycle were confined to the anterior pole of the nucleus. Most of these cells had inputs from muscle spindle afferents, whereas most of those firing during jaw opening had inputs from periodontal receptors. Non-masticatory rhythmical neurons had receptive fields on the lips and face. The majority of rhythmical masticatory units were modulated during fictive mastication evoked by both the left and right cortices and only four changed their phase of firing when switching from one cortex to the other. When coupled with the finding that NVsnpr neurons exhibit spontaneous bursting in vitro[Sandler et al. (1998) Neuroscience, 83, 891], the results described here suggest that neurons of dorsal NVsnpr may form the core of the central pattern generator for mastication.

Primary afferent neurons innervating muscle spindles in jaw-closing muscles have cell bodies in the trigeminal mesencephalic nucleus (NVmes) that are electrically coupled and receive synapses. Each stem axon gives rise to a peripheral branch and a descending central branch. It was previously shown that some spikes generated by constant muscle stretch fail to enter the soma during fictive mastication. The present study examines whether the central axon is similarly controlled. These axons were functionally identified in anaesthetized and paralysed rabbits, and tonic afferent firing was elicited by muscle stretch. For the purpose of comparison, responses were recorded extracellularly both from the somatic region and from the central axon in the lateral brainstem. Two types of fictive masticatory movement patterns were induced by repetitive stimulation of the masticatory cortex and monitored from the trigeminal motor nucleus. Field potentials generated by spike-triggered averaging of action potentials from the spindle afferents were employed to determine their postsynaptic effects on jaw-closing motoneurons. Tonic firing of 32% NVmes units was inhibited during the jaw-opening phase, but spike frequency during closing was almost equal to the control rate during both types of fictive mastication. A similar inhibition occurred during opening in 83% of the units recorded along the central branch. However, firing frequency in these was significantly increased during closing in 94%, probably because of the addition of antidromic action potentials generated by presynaptic depolarization of terminals of the central branch. These additional spikes do not reach the soma, but do appear to excite motoneurons. The data also show that the duration and/or frequency of firing during the bursts varied from one pattern of fictive mastication to another. We conclude that the central axons of trigeminal muscle spindle afferents are functionally decoupled from their stem axons during the jaw-closing phase of mastication. During this phase, it appears that antidromic impulses in the central axons provide one of the inputs from the masticatory central pattern generator (CPG) to trigeminal motoneurons.