Sandrine Bertrand

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.

GABAB receptor- and metabotropic glutamate receptor- dependent cooperative long-term potentiation of rat hippocampal GABAA synaptic transmission

Repetitive stimulation of Schaffer collaterals induces activity‐dependent changes in the strength of polysynaptic inhibitory postsynaptic potentials (IPSPs) in hippocampal CA1 pyramidal neurons that are dependent on stimulation parameters. In the present study, we investigated the effects of two stimulation patterns, theta‐burst stimulation (TBS) and 100 Hz tetani, on pharmacologically isolated monosynaptic GABAergic responses in adult CA1 pyramidal cells. Tetanization with 100 Hz trains transiently depressed both early and late IPSPs, whereas TBS induced long‐term potentiation (LTP) of early IPSPs that lasted at least 30 min. Mechanisms mediating this TBS‐induced potentiation were examined using whole‐cell recordings. The paired‐pulse ratio of monosynaptic inhibitory postsynaptic currents (IPSCs) was not affected during LTP, suggesting that presynaptic changes in GABA release are not involved in the potentiation. Bath application of the GABAB receptor antagonist CGP55845 or the group I/II metabotropic glutamate receptor antagonist E4‐CPG inhibited IPSC potentiation. Preventing postsynaptic G‐protein activation or Ca2+ rise by postsynaptic injection of GDP‐β‐S or BAPTA, respectively, abolished LTP, indicating a G‐protein‐ and Ca2+‐dependent induction in this LTP. Finally during paired‐recordings, activation of individual interneurons by intracellular TBS elicited solely short‐term increases in average unitary IPSCs in pyramidal cells. These results indicate that a stimulation paradigm mimicking the endogenous theta rhythm activates cooperative postsynaptic mechanisms dependent on GABABR, mGluR, G‐proteins and intracellular Ca2+, which lead to a sustained potentiation of GABAA synaptic transmission in pyramidal cells. GABAergic synapses may therefore contribute to functional synaptic plasticity in adult hippocampus.

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.

Coupling between lumbar and sacral motor networks in the neonatal rat spinal cord.

We have studied the rhythm‐generating capabilities of the lumbar, sacral and coccygeal (Co) areas using an isolated spinal cord preparation of the newborn rat. The bath‐application of a mixture of N‐methyl‐ d‐l‐aspartate (NMA) and serotonin (5‐HT) on the whole spinal cord induced a coordinated rhythmic activity that could be recorded from the lumbar to the coccygeal ventral roots. The phase relationships and mean burst duration between the activity in the rostral lumbar segments and the activity in the sacral segments was analysed. The direct activation of the sacral network, by using sections or by selective pharmacological activation, showed that these caudal segments possess their own rhythmogenic capability. By combining section experiments and compartmentation of the spinal cord, we demonstrated that a strong coupling exists between the lumbar and sacral motor networks. In addition, we found that in an intact spinal cord the activity of the sacral networks is driven by the lumbar networks. We have found that different modes of coordination between the lumbar and the sacral activity may occur. Finally, we have shown that the coupling between the lumbar and sacral networks can be modified by sensory inputs, suggesting that the spinal machinery could modulate and adapt the coupling of these two spinal networks.

The respective contribution of lumbar segments to the generation of locomotion in the isolated spinal cord of newborn rat

Various studies on isolated neonatal rat spinal cord have pointed to the predominant role played by the rostral lumbar area in the generation of locomotor activity. In the present study, the role of the various regions of the lumbar spinal cord in locomotor genesis was further examined using compartmentalization and transections of the cord. We report that the synaptic drive received by caudal motoneurons following N‐methyl‐d‐l‐aspartate (NMA)/5‐HT superfusion on the entire lumbar cord is different from that triggered by the same compounds specifically applied on the rostral segments. These differences appear to be due to the direct action of NMA/5‐HT on motoneuron membrane potential, rather than on premotoneuronal input activation. In order to assess the possible participation of the caudal lumbar segments in locomotor rhythm generation, the segments were over‐stimulated with high concentrations of NMA or K+. We find that significant variations in motor cycle period occurred during the over‐activation of the rostral segments. Over‐activation of caudal segments only si+gnificantly increased the caudal ventral roots burst amplitude. We find that low 5‐HT concentrations were unable to induce fictive locomotion under our experimental conditions. When a hemi‐transection of the cord was performed between the L2–L3 segments, rhythmic bursting in the ipsilateral L5 disappeared while rhythmicity persisted on the contralateral side. Sectioning of the remaining L2–L3 side totally suppressed rhythmic activity in both L5 ventral roots. These results show that the thoracolumbar part of the cord constitutes the key area for locomotor pattern generation.

Ubiquity of motor networks in the spinal cord of vertebrates

In a recent paper, we found that it is possible to record motor activity in sacral segments in the in vitro neonatal rat spinal cord preparation. This motor activity recorded in segments that are not innervating hindlimbs is driven by the lumbar locomotor network. Indeed, compartimentalizations of the cord with Vaseline walls or section experiments, reveals that the sacral segments possess their own rhythmogenic capabilities but that in an intact spinal cord they are driven by the lumbar locomotor network. In this review, these recent findings are placed in the context of spinal motor network interactions. As previously suspected, the motor networks do not operate in isolation but interact with each other according to behavioural needs. These interactions provide some insight into the discrepancies observed in several studies dealing with the localization of the lumbar locomotor network in the neonatal rat spinal cord. In conclusion, the spinal cord of quadrupeds appears as an heterogeneous structure where it is possible to identify neuronal networks that are crucial for the genesis of locomotor-related activities.

Gabaergic Control of Spinal Locomotor Networks in the Neonatal Rat

Abstract: We studied the GABAergic control of the spinal locomotor network using an isolated brain stem/spinal cord from newborn rats, in which locomotor‐like activity was recorded. We demonstrate that endogenously released GABA controls the locomotor network, by decreasing or completely abolishing all locomotor‐like activity. At first, we investigated the role played by GABA in the control of the locomotor period. By separately superfusing various compartments of the lumbar cord, we identified the targets of GABA. When bath‐applied on the upper lumbar segments (L1/L2), GABA or its agonists (muscimol, baclofen) modulated the locomotor period, whereas it had no effects when bath‐applied on the caudal lumbar cord (L3/L6). In the second step we studied how GABA may presynaptically control the locomotor drive arising from the locomotor network located in L1/L2. By use of the partitioned spinal cord, intracellular recordings from the caudal pool motoneurons (L4/L5) were performed, while initiating locomotor‐like activity in L1/L2. We found that GABA or its agonists decreased the monosynaptic locomotor drive that the motoneurons received from the L1/L2 network, and we found a presynaptic effect exerted through the activation of GABAB receptors. In conclusion, this study emphasizes the role played by GABA at various levels in the control of the locomotor network in mammals.

Unitary synaptic currents between lacunosum-moleculare interneurones and pyramidal cells in rat hippocampus

1 Unitary inhibitory postsynaptic currents (uIPSCs) were characterised between 23 synaptically coupled interneurones at the border of stratum radiatum and lacunosum‐moleculare (LM) and CA1 pyramidal cells (PYR) using dual whole‐cell recordings and morphological identification in rat hippocampal slices. 2 LM interneurones presented a morphology typical of stellate cells, with a fusiform soma as well as dendritic and axonal arborisations in stratum radiatum and lacunosum‐moleculare. 3 Single spikes in interneurones triggered uIPSCs in pyramidal cells that were blocked by the GABAA antagonist bicuculline and mediated by a chloride conductance. The latency, rise time, duration and decay time constant of uIPSCs were a function of amplitude in all pairs, suggesting a homogeneity in the population sampled. 4 During paired pulse stimulation, individual LM‐PYR connections exhibited facilitation or depression. The paired pulse ratio was inversely related to the amplitude of the first response. The transition from facilitation to depression occurred at 26 % of the maximal amplitude of the first uIPSC. Paired pulse depression was not modified by CGP 55845 and thus was GABAB receptor independent. 5 CGP 55845 failed to modify the amplitude of uIPSCs, suggesting an absence of tonic presynaptic GABAB inhibition at LM‐PYR connections. 6 Increasing GABA release by repetitive activation of interneurones failed to induce GABAB IPSCs. With extracellular minimal stimulation, increasing stimulation intensity above threshold, or repetitive activation, evoked GABAB IPSCs, probably as a result of coactivation of several GABAergic fibres. 7 Thus, dendritic inhibition by LM interneurones involves GABAA uIPSCs with kinetics dependent on response amplitude and subject to GABAB‐independent paired pulse plasticity.

Presynaptic GABAergic control of the locomotor drive in the isolated spinal cord of neonatal rats

The in vitro newborn rat isolated brain stem/spinal cord preparation was used to study the involvement of presynaptic inhibition in the control of the synaptic locomotor drive. The recording chamber was partitioned with Vaseline walls to separate the L1–L2 locomotor network from the motoneurons in the lower segments. When locomotor like activity was induced by bath applying a mixture of N‐methyl‐d‐l‐aspartate and serotonin to the L1–L2 segments, intracellular recordings of L3–L5 motoneurons show an alternating pattern of monosynaptic excitatory glutamatergic and inhibitory glycinergic inputs known as the locomotor drive. Gamma‐aminobutyric acid (GABA), baclofen and muscimol (respectively GABAB and GABAA agonists) superfused on the L3–L5 segments depressed the synaptic locomotor drive of motoneurons during the ongoing activity. On the contrary, the GABAB receptor antagonist CGP35348 enhanced the locomotor drive, which suggests that an endogenous release of GABA occurs during locomotor‐like activity. Baclofen, unlike muscimol and GABA, did not affect the passive membrane properties and the firing discharge of synaptically isolated motoneurons. Baclofen and muscimol acted on the two phases (inhibitory and excitatory) of the synaptic drive. The effects of GABAergic agonists on the whole locomotor activity were tested. When superfused on the L3–L5 part of the cord, they affected only the L5 burst amplitude. When bath‐applied to the L1–L2 network, GABA and muscimol decreased the amplitude of the L2 and L5 bursts and increased the locomotor period while baclofen had significant effects only on the period. It was concluded that GABA modulates the information conveyed by the L1–L2 network to its target motoneurons presynaptically via GABAB and possibly GABAA receptors and postsynaptically, via GABAA receptors.

Plateau potentials and membrane oscillations in parasympathetic preganglionic neurones and intermediolateral neurones in the rat lumbosacral spinal cord

Whole‐cell patch recordings were made from parasympathetic preganglionic neurones (P‐PGNs) and unidentified intermediolateral (IML) neurones in thick slices of the lower lumbar and sacral spinal cord of 14‐ to 21‐day‐old rats. The P‐PGNs and IML neurones examined were similar in terms of soma sizes, input resistance and capacitance, and displayed a sag conductance as well as rebound firing. In the absence of drugs, the neurones responded with either tonic or adapting firing to depolarizing current steps. However, in the presence of the group I metabotropic glutamate receptor agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG), almost half of the neurones displayed accelerating firing rates during the constant current injection, followed by a sustained after‐discharge. In the presence of TTX, plateau potentials were observed. The firing changes and plateaux were blocked by nifedipine, an L‐type Ca2+ channel blocker, and (S)‐(−)‐Bay K8644 was able to produce these firing changes and plateaux in the absence of DHPG, demonstrating the involvement of an L‐type Ca2+ conductance. Ca2+‐activated nonspecific cationic conductances also appear to contribute to the firing changes. A few neurones displayed membrane oscillations and burst firing in the presence of DHPG. The results suggest that the firing characteristics of both P‐PGNs and other neurones likely to be involved in caudal spinal reflex control are not static but, rather, quite dynamic and under metabotropic glutamate receptor modulatory control. Such changes in firing patterns may be involved in normal pelvic parasympathetic reflex function during micturition, defaecation and sexual reflexes, and may contribute to the abnormal output patterns seen with loss of descending brainstem input and visceral or perineal sensory disturbances.