Frédéric Nagy

Expression of vesicular glutamate transporters in rat lumbar spinal cord, with a note on dorsal root ganglia

Three vesicular glutamate transporters (VGLUTs) have been recently identified and their distribution has been mapped in various brain areas. In the present study, we used morphological approaches to investigate their expression in the rat lumbar spinal cord and dorsal root ganglia. Our results show a complementary distribution of VGLUT‐expressing fibers in the spinal cord, with no overlapping in nerve endings. In the dorsal horn, VGLUT1 is most abundant in mechanosensory/proprioceptive deep afferent fibers. VGLUT2 and VGLUT3 are expressed only at moderate levels in primary sensory afferent fibers and are not used by central projections of nociceptive neurons. VGLUT1 and VGLUT2 mRNAs are mainly segregated in superficial laminae but colocalized in deeper laminae. Weak expression of VGLUT3 mRNA is only detected in deep laminae. The colocalization of VGLUT1 and VGLUT2 transcripts in most sensory neurons of the dorsal root ganglia is not in agreement with the clear segregation between the proteins in their spinal projections. Such a discrepancy suggests targeting mechanisms specific for each transporter and/or a distinct regulation of their translation. In the ventral horn, the expression of VGLUT1 and VGLUT2 mRNAs in motoneuron perikarya suggests the possible unexpected role of glutamate in the vertebrate neuromuscular junction. These results demonstrate the existence of different subpopulations of glutamate nerve terminals in the rat lumbar spinal cord and suggest that functionally distinct subsets of excitatory glutamatergic neuronal networks are involved in sensory processing and motor control. J. Comp. Neurol. 468:380–394, 2004.

Metabotropic receptors for glutamate and GABA in pain.

Glutamate and gamma-amino butyric acid (GABA) are respectively two major excitatory and inhibitory neurotransmitters of the adult mammalian central nervous system. These neurotransmitters exert their action through two types of receptors: ionotropic and metabotropic receptors. While ionotropic receptors are ligand gated ion channels involved in fast synaptic transmission, metabotropic receptors belong to the superfamily of G-protein coupled receptors (GPCRs) and are responsible for the neuromodulatory effect of glutamate and GABA. Metabotropic glutamate receptors (mGluRs) and metabotropic GABA receptors (GABA-B) are present at different levels of the pain neuraxis where they regulate nociceptive transmission and pain. The present review will focus on the role of these receptors in the modulation of pain perception.

Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states.

Sensory relay structures in the spinal cord dorsal horn are now thought to be active processing structures that function before supraspinal sensory integration. Dorsal horn neurons directly receive nociceptive (pain) signals from the periphery, express a high degree of functional plasticity and are involved in long-term sensitization and chronic pain. We show here that deep dorsal horn neurons (DHNs) in Wistar rats can switch their intrinsic firing properties from tonic to plateau or endogenous bursting patterns, depending upon the balance of control by metabotropic glutamate (mGlu) and GABAB receptors. We further show that this modulation acts on at least one common target, the inwardly rectifying potassium channel (Kir3). Finally, we found that these firing modes correspond to specific functional states of information transfer in which dorsal horn neurons can faithfully transmit, greatly enhance or block the transfer of nociceptive information.

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

Chronic pain states are characterized by long‐term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up‐regulation of Cav1.2‐comprising L‐type calcium channel (Cav1.2‐LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR‐103, simultaneously regulates the expression of the three subunits forming Cav1.2‐LTC in a novel integrative regulation. This regulation is bidirectional since knocking‐down or over‐expressing miR‐103, respectively, up‐ or down‐regulate the level of Cav1.2‐LTC translation. Functionally, we show that miR‐103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR‐103 is down‐regulated in neuropathic animals and that miR‐103 intrathecal applications successfully relieve pain, identifying miR‐103 as a novel possible therapeutic target in neuropathic chronic pain.

The pyloric central pattern generator in Crustacea: a set of conditional neuronal oscillators

SummaryIn the stomatogastric nervous system of the Cape lobster,Jasus lalandii, all the neurons comprising the pyloric central pattern generator are cellular oscillators. This was shown by isolating in situ pyloric neurons from all other elements in the pyloric network. These neuronal oscillators are conditional and require modulatory inputs from anterior centers in order to express their oscillatory capabilities.1.Pyloric neurons were isolated from their counterparts in the network by photoinactivation of most of their presynaptic neurons (Miller and Selverston 1979, 1982a), and completed by pharmacological blockade of the remaining presynaptic influences. Under these conditions, all pyloric neurons display spontaneous bursting and underlying oscillations of their membrane potential.2.Both period and amplitude of oscillation in all isolated pyloric neurons vary when their membrane potential is modified by current injection. Furthermore, in isolated constrictor neurons (LP, PY and IC) the duration of the rhythmic regenerative depolarizations is also dependent on membrane potential.3.After isolation in situ, each type of pyloric neuron manifests a particular oscillatory behavior. The isolated interneuron AB has the fastest oscillation period with the shortest depolarizing phase. These oscillations closely resemble AB oscillations in the intact network. At the other extreme are the constrictor neurons (LP, PY and IC) which, when isolated, display spontaneous oscillations rather different from their oscillations in the intact network. Oscillations of isolated pyloric constrictor neurons are rhythmical plateaus whose duration and frequency are fairly irregular.4.When axonal conduction is blocked in the single input nerve to the stomatogastric ganglion, none of the isolated pyloric neurons is able to oscillate whether at rest or when their membrane potential is modified by current injection. Under these conditions, electrical stimulation of the input nerve on the ganglionic side of the block restores oscillations in isolated pyloric neurons.5.In conclusion, the pyloric network can be considered as a set of conditional oscillatory neurons, one of which, the interneuron AB, has a pacemaker function.

Rapid optical control of nociception with an ion-channel photoswitch

Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium–azobenzene–quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.

Plateau potential-dependent windup of the response to primary afferent stimuli in rat dorsal horn neurons

In the spinal cord, repetitive stimulation of nociceptive afferent fibres induces a progressive build‐up of dorsal horn neuron (DHN) responses. This ‘action potential windup’ is used as a cellular model of central sensitization to pain. It partly relies on synaptic plasticity, being reduced after blocking NMDA and neurokinin receptors. Using intracellular recordings in a slice preparation of the rat spinal cord, we have analysed the implication of an additional non‐synaptic component of windup. Primary afferent fibres were electrically stimulated in the dorsal root. Of 47 responding deep DHNs, 17 (36%) produced action potential windup and afterdischarge during consecutive periods of repeated stimuli (0.4–1 Hz) activating high‐ (n = 13 neurons) and low‐threshold (n = 6 neurons) afferent fibres. When the NMDA receptors were blocked, the rate of windup did not change. In all neurons, there was an absolute correlation between expression of windup and the production of calcium‐dependent plateau potentials. Sensitization of the DHN response, similar to the synaptically induced windup, was obtained by repetitive intracellular injection of depolarizing current pulses. This intracellularly induced windup had the same pharmacology as the plateau potential. Synaptically induced windup was also abolished by nifedipine, an L‐type calcium‐channel blocker. Expression of plateau properties in DHNs is therefore a critical component of windup, operating downstream of synaptic processes. Being associated with calcium influx, generation of plateau potentials could be a link between short‐term plasticity and the long‐term modification of DHN excitability associated with central sensitization.

Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain

The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation–transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes CaV1.2 and CaV1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of CaV1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-CaV1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of CaV1.2 mRNA and protein in neuropathic animals concomitant to specific CaV1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on CaV1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising CaV1.2.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato‐dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2‐ and VGLUT1‐positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

Nociceptive integration in the rat spinal cord: role of non‐linear membrane properties of deep dorsal horn neurons

Deep dorsal horn neurons (DHNs) involved in nociception can relay long‐lasting inputs and generate prolonged afterdischarges believed to enhance the transfer of nociceptive responses to the brain. We addressed the role of neuronal membrane properties in shaping these responses, by recording lamina V DHNs in a slice preparation of the rat cervical spinal cord. of 256 neurons, 102 produced accelerating discharges in response to depolarizing current pulses, whereas the other neurons showed spike frequency adaptation. Two mechanisms mediated the firing acceleration: a slow inactivation of a K+ current expressed upon activation of the neuron from hyperpolarized holding potentials, and the expression of a regenerative plateau potential activating around resting membrane potential. The increase in firing frequency was much stronger when sustained by the plateau potential (71 DHNs, 28%). A few neurons produced adaptation and both types of acceleration, in different membrane potential domains, showing that the firing pattern of a deep DHN is not a rigid characteristic. Plateau potentials could be elicited by stimulation of nociceptive primary afferent fibres. The bistability associated with plateau potentials permitted afterdischarges. Because plateau potentials had slow activation kinetics and were voltage‐dependent, the neurons had non‐linear input–output relationships in both the amplitude and time domains. Nociceptive primary afferent stimulation elicited intense and prolonged responses in plateau‐generating DHNs, while brief bursts of spikes were evoked otherwise. These results indicate that in a population of deep DHNs, intense firing and prolonged afterdischarges in response to nociceptive stimulation depend on non‐linear intrinsic membrane properties.