We developed appropriate surgical procedures for single and repetitive multi-photon imaging of spinal cord in vivo. By intravenous anesthesia, artificial ventilation and laminectomy, acute experiments were performed in the dorsal and lateral white matter. By volatile anesthesia and minimal-invasive surgery, chronic repetitive imaging up to 8 months were performed in the dorsal column through the window between two adjacent spines. Transgenic mouse technology enabled simultaneous imaging of labeled axons, astrocytes and microglia. Repetitive imaging showed positional shifts of microglia over time. These techniques serve for investigations of cellular dynamics and cell-cell interactions in intact and pathologically changed spinal tissue.
- MeSH
- White Matter cytology diagnostic imaging MeSH
- Microscopy, Confocal methods MeSH
- Laser Scanning Cytometry methods MeSH
- Spinal Cord cytology diagnostic imaging MeSH
- Mice, Inbred C57BL MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Organ Culture Techniques MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Anesthetic and surgical procedures and an electrophysiological method were developed for recording nerve conduction velocity (NCV) of CNS fibers in the murine spinal cord. Under intravenous anesthesia and artificial ventilation the lumbar spinal cord segments L1 to L4 and dorsal roots L3 to L5 on the left side were exposed by laminectomy. After stimulation of the dorsal root L4, a compound action potential (CAP) was recorded at the ipsilateral left fasciculus gracilis at the spinal cord level L1. The latency from stimulation to the CAP together with the measured distance between the electrodes was used for the determination of the NCV. NCV of the fastest fibers in the fasciculus gracilis was observed to be approximately 28 m/s. Reversible decrease of the NCV was measured, in vivo, under general hypothermia. The technique described serves for in vivo electrophysiological investigations of spinal central fibers in wildtype and mutant mice.
Heterologous expression of Kir channels offers a tool to modulate excitability of neurons which provide insight into Kir channel functions in general. Inwardly-rectifying K+ channels (Kir channels) are potential candidate proteins to hyperpolarize neuronal cell membranes. However, heterologous expression of inwardly-rectifying K+ channels has previously proven to be difficult. This was mainly due to a high toxicity of the respective Kir channel expression. We investigated the putative role of a predominantly glial-expressed, weakly rectifying Kir channel (Kir4.1 channel subunit; KCNJ10) in modulating electrophysiological properties of a motoneuron-like cell culture (NSC-34). Transfection procedures using an EGFP-tagged Kir4.1 protein in this study proved to have no toxic effects on NSC-34 cells. Using whole cell-voltage clamp, a substantial increase of inward rectifying K+ currents as well as hyperpolarization of the cell membrane was observed in Kir4.1-transfected cells. Na+ inward currents, observed in NSC-34 controls, were absent in Kir4.1/EGFP motoneuronal cells. The Kir4.1-transfection did not influence the NaV1.6 sodium channel expression. This study demonstrates the general feasibility of a heterologous expression of a weakly inward-rectifying K+ channel (Kir4.1 subunit) and shows that in vitro overexpression of Kir4.1 shifts electrophysiological properties of neuronal cells to a more glial-like phenotype and may therefore be a candidate tool to dampen excitability of neurons in experimental paradigms.
- MeSH
- Potassium Channels, Inwardly Rectifying genetics metabolism MeSH
- Membrane Potentials MeSH
- Patch-Clamp Techniques MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Spinal Cord Neoplasms genetics metabolism MeSH
- NAV1.6 Voltage-Gated Sodium Channel metabolism MeSH
- Neuroblastoma genetics metabolism MeSH
- Recombinant Fusion Proteins metabolism MeSH
- Transfection MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Electrophysiological investigations in mice, particularly with altered myelination, require reference data of the nerve conduction velocity (CV). CVs of different fibre groups were determined in the hindlimb of anaesthetized adult mice. Differentiation between afferent and efferent fibres was performed by recording at dorsal roots and stimulating at ventral roots, respectively. Correspondingly, recording or stimulation was performed at peripheral hindlimb nerves. Stimulation was performed with graded strength to differentiate between fibre groups. CVs of the same fibre groups were different in different nerves of the hindlimb. CVs for motor fibres were for the tibial nerve (Tib) 38.5±4.0 m/s (Aγ: 16.7±3.0 m/s), the sural nerve (Sur) 39.3±3.1 m/s (12.0±0.8 m/s) and the common peroneal nerve (Per) 46.7±4.7 m/s (22.2±4.4 m/s). CVs for group I afferents were 47.4±3.1 m/s (Tib), 43.8±3.8 m/s (Sur), 55.2±6.1 m/s (Per) and 42.9±4.3 m/s for the posterior biceps (PB). CVs of higher threshold afferents, presumably muscle and cutaneous, cover a broad range and do not really exhibit nerve specific differences. Ranges are for group II 22-38 m/s, for group III 9-19 m/s, and for group IV 0.8-0.9 m/s. Incontrovertible evidence was found for the presence of motor fibres in the sural nerve. The results are useful as references for further electrophysiological investigations particularly in genetically modified mice with myelination changes.
- MeSH
- Action Potentials physiology MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Nerve Fibers physiology MeSH
- Neural Conduction physiology MeSH
- Synaptic Transmission MeSH
- Neurons, Afferent physiology MeSH
- Neurons, Efferent physiology MeSH
- Hindlimb innervation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
The role of L-DOPA in spinal nociceptive reflex activity has been re-evaluated. In high spinal cats, with supraspinal loops being excluded, the onset of reflex facilitation induced by noxious radiant heat is delayed after injection of L-DOPA by 4 to 10 s, i.e. the early component of nociceptive reflex facilitation is blocked, while the late component persisted. Further investigations have shown that the early component of reflex facilitation induced by noxious radiant heat is mediated by Adelta-fibres and the late component by C-fibres. Therefore, it can be assumed that L-DOPA, like opioids, preferentially blocks the transmission in nociceptive reflex pathways from Adelta-fibres.
- MeSH
- Cats MeSH
- Levodopa pharmacology MeSH
- Pain Measurement methods drug effects MeSH
- Spinal Cord physiology drug effects MeSH
- Nerve Fibers, Myelinated physiology drug effects MeSH
- Nerve Fibers, Unmyelinated physiology drug effects MeSH
- Nociceptors physiology MeSH
- Reflex physiology drug effects MeSH
- Animals MeSH
- Check Tag
- Cats MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
