In vivo recording of nerve conduction velocity of spinal CNS fibers in the mouse.

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.

Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.

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.

Long lasting activity of nociceptive muscular afferents facilitates bilateral flexion reflex pattern in the feline spinal cord.

Chronic muscular limb pain requires the adoption of motor patterns distinct from the classic ipsilateral flexion, crossed extension and corresponding reciprocal inhibitions to acute exteroceptive stimulation. Using selective chemical activation of group III/IV afferents in gastrocnemius-soleus (GS) muscles we investigated bilaterally their reflex responses conditioned by (a) acute 'myositis' induced by intramuscular carrageenan; and (b) sub-acute 'myositis' induced by infusion of complete Freund's adjuvant (CFA). Reflex transmission was detected by monosynaptic testing and c-fos staining used to identify increased neuronal activity. In all control experiments with chemical stimulation of group III/IV afferents, ipsilateral responses conformed to the flexor reflex pattern. However, the expected contralateral facilitation of GS motoneurones occurred in fewer than 50% trials while only 9% of trials induced contralateral inhibition of flexor posterior-biceps-semitendinosus (PBSt) motoneurones. During carrageenan acute myositis contralateral PBSt was transiently facilitated by selective activation of group III/IV afferents. During CFA-induced myositis, contralateral only inhibition of GS motoneurones occurred instead of any facilitation, while bidirectionally a crossed facilitation of PBST dominated. These reflex changes were mirrored in an enhanced number of neurones with enhanced c-fos expression. Muscle pain, particularly if chronically persistent, requires another behavioural response pattern than acute exteroceptive pain.

In vivo measurement of conduction velocities in afferent and efferent nerve fibre groups in mice.

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 (Agamma: 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.

Role of L-DOPA in spinal nociceptive reflex activity: higher sensitivity of Aδ versus C fibre-evoked nociceptive reflexes to L-DOPA.

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.

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction.

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. Here, we investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, we show that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, we tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. Our data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses.

Rhythmic phrenic, intercostal and sympathetic activity in relation to limb and trunk motor activity in spinal cats.

During L-DOPA-induced fictive spinal locomotion rhythmic activities in nerves to internal intercostal and external oblique abdominal muscles and in phrenic and sympathetic nerves were observed which were always coordinated with locomotor activity in forelimb and hindlimb muscle nerves. A periodicity with longer lasting tonic phases could be induced by cutaneous nerve stimulation or asphyxia. This activity was observed in limb motor nerves as well as in respiratory motor and sympathetic nerves. A slow independent activity of the phrenic and intercostal nerves or the sympathetic nerves, which could be related to a normal respiratory rhythm or independent sympathetic rhythms was not observed. The findings indicate that during fictive spinal locomotion the activity of spinal rhythm generators for locomotion also projects onto respiratory and sympathetic spinal neurones.

Phrenic, intercostal and sympathetic activity related to fictive locomotor activity of limb muscles in spinal cats.

During L-DOPA induced fictive spinal locomotion co-ordinated rhythmic activities in external and internal intercostal, phrenic and sympathetic nerves were observed which were always co-ordinated with locomotor activity in forelimb and hindlimb muscle nerves. If long lasting tonic activity was induced by cutaneous nerve stimulation or asphyxia this activity was observed in limb motor nerves as well as in respiratory motor and sympathetic nerves. A slow independent activity of the phrenic and intercostal nerves or the sympathetic nerves which could be related to a normal respiratory rhythm or independent sympathetic rhythms was not observed. The findings indicate that during fictive spinal locomotion the activity of spinal rhythm generators for locomotion irradiates onto respiratory and sympathetic spinal neurones.

Two types of motor modulation underlying human stepping evoked by spinal cord electrical stimulation (SCES).

The EMG-activity during stepping-like movements evoked by spinal cord electrical stimulation (SCES) was studied in paraplegic patients. Both typical locomotor EMG-pattern with rhythmically, bilaterally alternating flexor-extensor activity ("locomotor stepping") as well as a rhythmic, bilaterally alternating modulation of short latency stimulus coupled reflexes ("reflexogenic stepping") and mixed pattern were observed. While the frequency of "locomotor stepping" could be independent from the stimulus frequency and the stepping was continuing after the end of stimulation for several cycles, the "reflexogenic stepping" was largely somehow coupled to the stimulus frequency and ended with the end of stimulation. It is assumed that both types of rhythmic motor activity reflect the activity of a spinal locomotor generator, but that the activity of the locomotor generator is subthreshold for fully activating the motoneuronal pools in cases with "reflexogenic stepping".

Nociceptive reflexes evoked by TTX-resistant C-fibre afferents and their sensitivity to opioids in the cat.

The contribution of nociceptive Adelta-fibres and C-fibres of the central pad of the foot to nociceptive spinal flexor reflex pathways (FRA-type) and to nociceptive excitatory reflex pathways to foot extensors (non-FRA type) was investigated in high spinal cats by blocking A-fibres completely by TTX; effects persisting after TTX were attributed to nociceptive C-fibres. The results revealed that both Adelta- and C-fibre afferents contributed to nociceptive reflexes of an FRA pattern and of a non-FRA pattern, the effects of Adelta-fibres being evoked with a distinctly shorter delay than those of C-fibres. Partly Adelta-fibres exerted a significant inhibitory influence on the C-fibre action in FRA pathways. A distinct part of the opioid action on nociceptive reflex pathways of the FRA-type and of the non-FRA-type was evidently exerted via C-fibres.

Parallel nociceptive reflex pathways with negative and positive feedback functions to foot extensors in the cat.

1. Nociceptive reflex pathways to foot extensors were investigated with particular attention given to those not following a flexor reflex (FRA) or withdrawal pattern. 2. In anaemically decapitated, high spinal paralysed cats nociceptive afferents of the foot pad were activated by noxious radiant heat (48-60 degrees C), while for comparison non-nociceptive afferents were activated by weak mechanical stimulation of the skin or graded electrical nerve stimulation. The reflex action of the afferents on hindlimb motoneurones, innervating plantaris and intrinsic foot extensors (tibial nerve), was investigated by intracellular recording, by monosynaptic reflex testing and by recording of neurograms during fictive locomotion. A possible descending control of the nociceptive and non-nociceptive pathways was tested by application of opioidergic and monoaminergic compounds. 3. Beside the typical FRA pattern evoked in the majority of hindlimb motoneurone pools by nociceptive afferents from different skin areas of the foot, the results revealed parallel excitatory and inhibitory nociceptive reflex pathways from the central pad and partly from the toe pads to foot extensors. The excitatory pathways, which did not follow the FRA pattern, were predominantly to plantaris and intrinsic foot extensors. They were distinctly less depressed by opioids and monoaminergic compounds than FRA pathways. 4. While the nociceptive FRA pathways have a general nocifensive withdrawal function, the nociceptive excitatory non-FRA pathway to the foot extensors causes a movement of the affected area towards the stimulus or at least a resistance against the stimulus, i.e. it mediates a positive feedback.

Nociceptive input to ascending tract neurones forwarding information from low threshold cutaneous and muscle afferents in cats.

Effects of noxious skin stimulation (central foot pad and foot dorsum) by radiant heat were tested on neurones of ascending tracts with a main input from non-nociceptors. The dominating effect on ventral spinocerebellar tract neurones was a depression (mainly from the pad). Responses of spinocervical tract neurones were either facilitated (predominantly from the foot dorsum) or depressed (predominantly from the pad). The dominating effect on neurones tentatively classified as dorsal horn dorsal spinocerebellar tract neurones was facilitatory from both skin areas. Similar effects were evoked by selective actions of C-fibres when A-delta fibres were blocked by TTX.

Contribution of TTX-resistant C-fibres and Adelta-fibres to nociceptive flexor-reflex and non-flexor-reflex pathways in cats.

The contribution of Adelta-fibres and C-fibres activated by noxious heat stimulation of the central pad of the foot to nociceptive spinal flexor reflex pathways (FRA-type) and to nociceptive excitatory reflex pathways to foot extensors (non-FRA type) was investigated in high spinal cats. A-fibres were completely blocked by tetrodotoxin (TTX), leaving C-fibre conduction intact. Thus, effects persisting after TTX were attributed to nociceptive C-fibres while the contribution of nociceptive Adelta-fibres was defined by the difference between those effects and the control effects before TTX. The initial action of noxious stimulation on both types of reflex action was mediated predominantly by Adelta-fibres, while the later action was mainly mediated by C-fibres. In two (out of seven) experiments Adelta-fibres exerted a significant inhibitory influence on the C-fibre action in FRA pathways, but such an inhibitory interaction between the two fibre groups was absent in the non-FRA reflex pathways. The technique of TTX application at the peripheral nerve proved to be a reliable method for a long-lasting selective investigation of C-fibre effects. The results revealed that both Adelta- and C-fibres contributed to nociceptive FRA and non-FRA reflex pathways.

Neither a general flexor nor a withdrawal pattern of nociceptive reflexes evoked from the human foot.

In humans motor reactions to noxious radiant heat stimulation of the sole and the dorsum of the foot do not resemble a locally specific pattern of multiple modular withdrawal reflexes but rather a general flexion reflex pattern with a few exceptions which did neither fit a withdrawal nor a flexion reflex pattern. The partly observed excitatory feed back to foot extensors from nociceptive afferents of the foot sole is functionally discussed as a foot stabilizing mechanism under particular conditions.

Nociceptive input to spinal interneurones in reflex pathways from group II muscle afferents in cats.

Effects of noxious stimulation of the skin by radiant heat were tested on responses of first order interneurones in reflex pathways from group II muscle afferents in mid-lumbar, lower-lumbar and sacral segments of the spinal cord. In mid- and lower-lumbar segments both background discharges and monosynaptically evoked responses of intermediate zone interneurones were facilitated. Those of mid-lumbar dorsal horn interneurones were also facilitated suggesting that both these interneuronal populations contribute to the facilitation of flexion reflexes by nociceptors. In contrast, the dominating effects of noxious heat on sacral dorsal horn group II interneurones were inhibitory. The effects evoked by selective activation of C fibres, after A-delta fibres had been blocked by TTX, were similar to those obtained before TTX application.

Contribution of group III and IV muscle afferents to multisensorial spinal motor control in cats.

The contribution of group III and IV muscle afferents to multisensorial segmental reflex pathways was investigated by testing for spatial facilitation between these afferents and non-nociceptive segmental afferents from skin, muscles and joints on postsynaptic potentials (PSPs) in alpha-motoneurones recorded in anaemically decapitated high spinal cats. Group III and IV muscle afferents were activated by intraarterial injection of potassium chloride (320 mM) or bradykinin triacetate (81 microM). Skin, joint and group I-II muscle afferents were stimulated by graded electrical stimulation of various nerves. Conditioning by stimulation of group III and IV muscle afferents spatially facilitated the transmission in segmental reflex pathways from low- to medium-threshold cutaneous and joint afferents as well as from lb and group II muscle afferents. Both excitatory and inhibitory pathways from these afferents were facilitated. Monosynaptic excitation and disynaptic antagonistic inhibition from group Ia afferents remained unaffected. It is concluded that the spatial facilitation observed between group III and IV muscle afferents and the other afferents indicate a convergence from group III and IV muscle afferents and the other afferents on common interneurones in segmental flexor reflex pathways. Under physiological conditions they thus contribute to the multisensorial feedback of the flexor reflex pathways. Pathophysiologically, the observed convergence may aggravate muscle weakness and atrophy of muscles induced by group III and IV muscle afferents.

Flexor reflex afferents reset the step cycle during fictive locomotion in the cat.

The generation of locomotor-like spinal rhythms has been proposed to involve two neural centres with mutual reciprocal inhibition (Graham Brown's "half-centre" hypothesis). Much later a particular set of segmental flexor reflex pathways were described as being organized in accordance with this half-centre hypothesis. As these pathways became operative following injection of monoaminoxidase inhibitors and L-3,4-dihydroxyphenylalanine (L-dopa), i.e. under the same conditions under which a spontaneous locomotor activity may develop, it was assumed that these particular pathways and spinal rhythm generators involve the same neuronal networks. In order to give further evidence to this hypothesis, we investigated whether short trains to "flexor reflex afferents" (FRA) reset the spinal locomotor rhythm, i.e. shorten or lengthen the stimulated cycle after which the regular rhythm is resumed with step cycles of the original duration. The experiments were performed in anaemically decapitated, high-spinal curarized cats. A steady locomotor rhythm was induced by injection of nialamide and L-dopa and the influence of electrical stimulation (trains of 50-1000 ms) of FRA (joint, cutaneous, and group II and III muscle afferents) onto this rhythm was tested. Stimulation of FRA induced a clear resetting of the locomotor rhythm, which was mainly characterized by a flexion reflex pattern: during the extension phase the extensor activity was interrupted and a flexion phase was initiated; during the late flexion phase mainly a prolongation of that phase with a variable change of the following extension phase was induced. In addition to this prevailing pattern, stimulation of some nerves (in particular nerves to more distal extensors and the sural nerve) could often prolong extension, when stimulated during the late extension, or terminate the flexor burst and initiate a new extension phase, when stimulated during the late flexion phase. This pattern is probably due to the concomitant stimulation of group I afferents in the case of the muscle nerves and to separate non-FRA pathways in the case of the sural nerve. The results demonstrate that the interneurones of the FRA pathways, which are operative during L-dopa-induced locomotion in spinal animals, can be considered as neuronal elements of the rhythm-generating network for locomotion.

Comparative analysis of L-DOPA actions on nociceptive and non-nociceptive spinal reflex pathways in the cat.

The actions of L-DOPA (40-100 mg/kg i.v.) on nociceptive and non-nociceptive spinal reflex pathways were investigated in anaemically decapitated high spinal cats. The results revealed a differential pattern of effects of L-DOPA on monosynaptic and oligo-orpolysynaptic nociceptive and non-nociceptive reflexes. (1) L-DOPA depressed monosynaptic reflexes of flexors without affecting those of the extensors. (2) Excitatory pathways from flexor reflex afferents (FRA) were distinctly depressed by L-DOPA, pathways from group II muscle afferents reacted with greater sensitivity than pathways from non-nociceptive cutaneous and joint afferents. (3) Inhibitory FRA pathways were distinctly less affected by L-DOPA than excitatory ones. (4) Transmission in nociceptive excitatory FRA pathways was depressed to the same high degree as that in pathways from group II muscle afferents. (5) Effects on transmission in non-FRA pathways such as the group Ib inhibitory pathway and the excitatory nociceptive pathway from the foot pad to plantaris and intrinsic foot extensors were either minor or absent. (6) L-DOPA increased the delay in the reaction to noxious stimulation. (7) The effects of L-DOPA could not be specifically antagonised by naloxone. Thus, mainly excitatory FRA pathways, irrespective of a nociceptive or non-nociceptive origin, are under strong depressive dopaminergic influences. These effects are similar to those evoked by opioids.

A leu-enkephalin depresses transmission from muscle and skin non-nociceptors to first-order feline spinal neurones.

1. The effects of an opioid (D-Ser-Leu-enkephalin-Thr; DSLET) were tested on synaptic actions of non-nociceptive afferents: group I and II muscle afferents and low-threshold skin afferents. They were tested on population EPSPs (field potentials) evoked in the dorsal horn and the intermediate zone of mid-lumbar segments, and on monosynaptically evoked responses of single interneurones at the same location. DSLET was applied locally (ionophoretically) at locations at which the field potentials were maximal and close to the selected neurones. 2. DSLET potently depressed transmission from group II muscle afferents and from low-threshold skin afferents. Transmission to neurones located in the dorsal horn or in the intermediate zone was depressed to a similar extent. The depression was readily antagonized by naloxone. Transmission from group Ia or Ib muscle afferents to neurones located in the intermediate zone was not affected, or was facilitated by DSLET. 3. The results show that DSLET has similar depressive actions on spinal neurones to monoamines, but its actions are more widespread. Like monoamines it affects transmission from nociceptors and group II muscle afferents, but in addition it gates transmission from low-threshold cutaneous afferents. Furthermore its effects do not appear to be restricted to interneurones at particular locations since it depressed responses of dorsal horn interneurones (gated by serotonin) as well as intermediate zone interneurones (gated by noradrenaline).

The Hoffmann reflex of human plantar foot muscles.

Electrical stimulation of the tibial nerve in the popliteal fossa evoked an M wave (10.9 ms) and a late reflex response (38.1 ms) in the plantar foot muscles of all 10 volunteers. The late response had a somewhat lower electrical threshold than the corresponding M wave (8.5 versus 9 mA), and reached a maximum of amplitude when the stimulus intensity was increased, but was strongly suppressed by further increased intensity. A more distal stimulation of the tibial nerve at the ankle shortened the onset latency of the M wave and lengthened that of the late response. The reflex was facilitated by activation of synergists and inhibited by activation of antagonists. We showed that the late response was contaminated neither by volume conducted activity from the soleus muscle, as shown by intramuscular recordings from the abductor hallucis muscle, nor by a F wave, as shown by double stimulation. In summary, we conclude that this late response in human plantar foot muscles corresponded to an H reflex, which may be used to assess alterations of distal motoneuronal excitability.