Functional Organization of Locomotor Interneurons in the Ventral Lumbar Spinal Cord of the Newborn Rat

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca2+ indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat

Contrôle sérotoninergique de la locomotion (étude de la restauration de la fonction chez le rat paraplégique et des propriétés intrinsèques des motoneurones spinaux )

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Sediment toxicity assessment in the Lagoon of Venice (Italy) using Paracentrotus lividus (Echinodermata: Echinoidea) fertilization and embryo bioassays.

The capacity of two toxicity bioassays (fertilization and embryo toxicity tests) to discriminate sediment toxicity using the sea urchin Paracentrotus lividus was tested in five stations with different levels of pollution in the Lagoon of Venice. Two stations were located in estuarine sites, two in the industrial zone, and one in a site at the top of our quality gradient (reference). Elutriate was chosen as sediment matrix to assess the potential effects of bioavailable pollutants in the water column as a consequence of sediment resuspension (dredging and dumping, fishing gear, etc.). An experimental design based on Quality Assurance/Quality Control procedures (QA/QC) was adopted in order to set the methodological basis for an effective use of these bioassays in monitoring programs. Results revealed both higher embriotoxicity than spermiotoxicity in all stations and the efficacy of combined use of both toxicity bioassays in discriminating differing pollution/ bioavailability between stations and periods. The good representativeness of the integrated sampling scheme and the standardization of all experimental phases yielded high precision of results. Clear Toxicity Fingerprints were evidenced for the investigated sites through the combined use of both bioassays. A good fit between ecotoxicological data and chemical contamination levels was found, except for unnatural sediment texture

Supra-spinal FAAH is required for the analgesic action of paracetamol in an inflammatory context

International audienceParacetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 μg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol

Ca²⁺ imaging experiments.

<p>A, Photomicrographs showing Fluo-4 AM labeling in the <i>in vitro</i> ventral SC preparation at the T13-L1 level (A1, magnification ×4; scale bar = 200 µm). Right photomicrographs were taken from areas delineated by grey squares 1 and 2 (A2, A3, magnification ×10; Scale bar = 100 µm). B, Extracellular recordings of left L2 VR and its rectified and low-pass filtered trace illustrating a typical rhythmic activity. Grey bars are aligned with r L2 VR bursts and reveal three cells examples tending to have Ca²⁺ peaks in phase (cell1), out of phase (cell2) or mixed (cell3) with lL2 VR activity. C, Circular plots illustrating the Ca²⁺ peak phases (black circles) for each cell in relation to the timing of L2 VRs bursts. The mean VR bursts over a 60 s recording bout range from spans phases 0 to 0.4 in the circular plot and is illustrated in grey. Vectors show the mean phase values and r values. Vector orientation indicates preferred phase of firing; vector length is proportional to coupling strength. D, Dot diagram shows distribution of all cells with phase relationship with motor output from T12 to L5. Cells were illustrated in phase (red dots), out of phase (black dots) and mixed (grey dots). E, Density plot of in phase and out of phase cells from T12 to L5 segments (n = 4 experiments; cc: central canal; scale bar = 100 µm).</p

Lesion experiments.

<p>A, Photomicrographs of a ventral SC preparation (top) and its corresponding transverse section (bottom) showing an example of electrocoagulation of a restricted region located at the T13 segment level (magnification 4×; scale bar = 300 µm). B, Schematic drawing presenting all microlesions performed from T12 to L2 segments. The thick circles represent the first lesion (n = 21) whereas the thin circles represent the second lesion n = 16/21). The right and left dashed lines delineate the grey and white matter. The lesion size is expressed as a percentage of SC size (central canal set to 0). C, Lesion effects on locomotor activity. (C1), Extracellular recordings of L2 and L5 VRs before and after a lesion at the L1 level. The arrow represents the onset of locomotor recovery. (C2), Time course of the motor period 1 min before (control) and after a lesion at the T13 level (circles), the L1 level (triangles) and L2 level (squares). The onset of recovery is indicated by successive arrows for each lesion. D, Lesion effect on duration of locomotor activity disruption (D1), motor period (D2), and burst area (D3) of microlesions performed at different SC level (T13-L1-L2). The black circles indicate the first microlesions and the grey triangles illustrate the second microlesions.</p

Experimental setup.

<p>A, Schematic drawing of the <i>in vitro</i> ventral SC preparation with VRs recording electrodes positioned at L2 and L5 (r = right; l = left). B, Transverse section of an intact SC (B1) and of two SC preparations with a horizontal cut at different dorso-ventral levels (B2, B3). Scale bars = 300 µm. The bottom panels show the motor activity recorded in VRs. F = Flexor activity, E = Extensor activity. C, Schematic drawing of transverse sections of the SC illustrating horizontal cuts at different levels (light grey bar and dark grey bar). The histogram plots the data of the mean period ± SEM of the different horizontal cuts.</p

Lamina specific postnatal development of PKCγ interneurons within the rat medullary dorsal horn

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Subpopulations of PKCγ interneurons within the medullary dorsal horn revealed by electrophysiologic and morphologic approach

International audienceMechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH) through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Specific interneurons located within inner lamina II (IIi) and expressing the gamma isoform of protein kinase C (PKCγ⁺) have been shown to be key elements for such circuits. However, their morphologic and electrophysiologic features are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized such lamina IIi PKCγ⁺ interneurons and compared them with neighboring PKCγ⁻ interneurons. Our results reveal that PKCγ⁺ interneurons display very specific activity and response properties. Compared with PKCγ⁻ interneurons, they exhibit a smaller membrane input resistance and rheobase, leading to a lower threshold for action potentials. Consistently, more than half of PKCγ⁺ interneurons respond with tonic firing to step current. They also receive a weaker excitatory synaptic drive. Most PKCγ⁺ interneurons express Ih currents. The neurites of PKCγ⁺ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, at least 2 morphologically and functionally different subpopulations of PKCγ⁺ interneurons can be identified: central and radial PKCγ⁺ interneurons. The former exhibit a lower membrane input resistance, rheobase and, thus, action potential threshold, and less PKCγ⁺ immunoreactivity than the latter. These 2 subpopulations might thus differently contribute to the gating of dorsally directed circuits within the MDH underlying mechanical allodynia