Antidromic discharges in dorsal roots of decerebrate cats. II: studies during treadmill locomotion

In a previous companion paper [Brain Res. 846 (1999) 87-105] we have shown that the dorsal root activity of a decerebrate cat is composed of both orthodromic and antidromic discharges as determined by spike triggered averaging (STA). Furthermore we have shown that, during fictive locomotion in decerebrate and paralyzed cats, antidromic discharges peak in different parts of locomotion cycle but mainly in the flexion phase. In the present study, we have recorded unit potentials from dorsal rootlets during treadmill locomotion in order to understand better the role of movement-related feedback in the generation of antidromic potentials. The unitary activity of 92 antidromically discharging units was recorded in proximal stumps of cut dorsal roots, and that of 20 such units was recorded in uncut roots using two bipolar Ag/AgCl electrodes in both cases. The activity of 80% (74/92) units in cut filaments and of 70% (14/20) units in uncut ones was phasewise related to stepping movements. The peaks of activity of different units occurred during different phases of the step cycle both in cut and uncut filaments. In most cases, the peak of activity was superimposed upon a background of sustained discharge. After blocking the orthodromic flow in a filament (local anesthesia or distal section), the antidromic discharges continued to peak during the same phase but the rate of the discharges increased. We conclude that movement-related afferent feedback significantly modulates the antidromic discharges in dorsal roots during treadmill locomotion. We suggest that these antidromic discharges have a role in controlling afferent feedback during movement.     

Spontaneous and locomotor-related GABAergic input onto primary afferents in the neonatal rat

The in vitro brain stem-spinal cord preparation of neonatal rats (0-5 days old) was used to examine the contribution of GABAA (gamma-aminobutyric acid) receptors to the spontaneous and locomotor-related antidromic firing in the dorsal roots of neonatal rats. Spontaneous bursts of antidromic discharges were generated by the underlying afferent terminal depolarizations reaching spiking threshold. The number of antidromic action potentials increased significantly in saline solution with Cl- concentration reduced to 50% of control. Bath application of the GABAA receptor antagonist bicuculline, at low concentrations (1-2 microM), or picrotoxin blocked the antidromic discharges in the dorsal roots almost completely. The increase in Cl- conductance was therefore mediated by an activation of GABAA receptors. Increasing the concentration of bicuculline to 10-20 microM never blocked these discharges further. On the contrary, in half of the preparations, the number of antidromic action potentials was higher in the presence of high concentrations of bicuculline (10-20 microM) than in the presence of picrotoxin or low concentrations of bicuculline. This suggests that bicuculline, at high concentrations, may have other effects, in addition to blocking GABAA receptors. Dorsal root firing was observed during fictive locomotion induced by bath application of excitatory amino acids and serotonin. A rhythmical pattern was often demonstrated. Bicuculline at low concentrations caused a decrease of the antidromic discharge whereas, at high concentrations, bursts of discharges appeared. A double-bath with a barrier built at the L3 level was then used to separate the mechanisms which generate locomotion from those mediating primary afferent depolarizations. Excitatory amino acids and serotonin were perfused in the rostral pool only. Decreasing the concentration of chloride in the caudal bath caused a sharp increase in the number of antidromic action potentials recorded from the L5 dorsal root. These discharges, which were modulated in phase with the locomotor rhythm, were blocked by bicuculline. These data demonstrate the existence of a locomotor-related GABAergic input onto primary afferent terminals in the neonatal rat.     

The effects of antidromic discharges on orthodromic firing of primary afferents in the cat

This study investigated the effects of antidromically conducted nerve impulses on the transmission of orthodromic volleys in primary afferents of the hindlimb in decerebrated paralyzed cats. Two protocols were used: (A) Single skin and muscle afferents (N=20) isolated from the distal part of cut dorsal rootlets (L7-S1) were recorded while stimulation was applied more caudally. The results showed that during the trains of three to 20 stimuli, the orthodromic firing frequency decreased or ceased, depending on the frequency of stimulation. Remarkably, subsequent to these trains, the occurrence of orthodromic spikes could be delayed for hundreds of ms (15/20 afferents) and sometimes stopped for several seconds (10/20 afferents). Longer stimulation trains, simulating antidromic bursts reported during locomotion, caused a progressive decrease, and a slow recovery of, orthodromic firing frequency (7/20 afferents), indicating a cumulative long-lasting depressing effect from successive bursts. (B) Identified stretch-sensitive muscle afferents were recorded intra-axonally and antidromic spikes were evoked by the injection of square pulses of current through the micropipette. In this case, one to three antidromic spikes were sufficient to delay the occurrence of the next orthodromic spike by more than one control inter-spike interval. If the control inter-spike interval was decreased by stretching the muscle, the delay evoked by antidromic spikes decreased proportionally. Overall, these findings suggest that antidromic activity could alter the mechanisms underlying spike generation in peripheral sensory receptors and modify the orthodromic discharges of afferents during locomotion.     

The effects of 4-aminopyridine on the cat spinal cord: rhythmic antidromic discharges recorded from the dorsal roots

In a previous paper, we have reported that 4-aminopyridine (4-AP, i.v., 10 mg/kg) induces in decerebrate spinal and paralyzed cats, a sustained rhythmic activity (2.5-8.5 Hz) in various muscle nerves. We describe here that similar discharges are recorded from the proximal stump of cut cutaneous nerves. The latter rhythmic activity arises from intense antidromic discharges in the dorsal roots. The rhythmic discharges are recorded from dorsal roots of both spinal cord enlargements as well as from thoracic roots. The rhythmic activity is highly synchronous among adjacent dorsal roots. Bilateral activity is also highly cross-correlated, but may be dissociated by unilateral stimulation of one dorsal root. It is not yet possible to determine the precise site where the antidromic discharges recorded from the dorsal roots are generated. 4-AP could act directly at the terminal level of the primary afferents or could activate interneurons impinging upon the terminals.     

Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys

The aim of this study was to determine whether the transmission from sensory inputs to reticulospinal neurons is modulated during fictive locomotion in lampreys. Reticulospinal neurons play a key role in the control of locomotion; modulation of sensory transmission to these neurons might be of importance for the adaptation of the control they exert during locomotion. In this series of experiments, intracellular synaptic responses of reticulospinal neurons of the posterior rhombencephalic reticular nucleus elicited by electrical stimulation of vestibular nerves on each side were studied during fictive locomotion induced by 50 microM N-methyl-D-aspartate (NMDA). Interestingly, shortly after NMDA had reached the bath and much before locomotor discharges were apparent in the recorded ventral roots, there was a significant depression of the synaptic transmission from vestibular nerves. The effect was reversed by washing out the NMDA and persisted in the isolated brainstem after spinal transection at the first segmental level. As locomotor discharges appeared in the ventral roots, synaptic responses elicited by vestibular nerve stimulation showed a clear phasic modulation of their amplitude during the locomotor cycle. Responses to stimulation of the ipsilateral vestibular nerve were smaller during the ipsilateral burst discharge than during the contralateral activity, whilst responses to stimulation of the contralateral vestibular nerve were minimal during contralateral activity and maximal during ipsilateral activity. This opposite pattern of modulation observed in the same reticulospinal neuron suggests that the phasic modulation of vestibular transmission is not due to changes in the membrane properties of the reticulospinal cell but is produced at a pre-reticular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unitary discharges in dorsal and ventral roots after the administration of 4-aminopyridine in the cat

The administration of 4-aminopyridine (4-AP) in decerebrate paralyzed cats induces centrifugal rhythmic discharges in both ventral and dorsal roots. This study describes the mode of discharge of individual primary afferents as well as some ventral root fibers. Several patterns of antidromic discharge have been observed in primary afferents after the administration of 4-AP. A large proportion of the units (n = 96; 53%) showed rhythmic bursts of discharge related (n = 41) or not (n = 55) to the ongoing rhythmic activity in the peripheral nerves. Other units (n = 86; 47%) discharged either tonically, sporadically or had no antidromic activity at all. The conduction velocity of the non-bursting units was significantly higher (89.7 +/- 18.4 m/s) than that of the bursting units (70.6 +/- 15.4 m/s; P less than 0.01). Ventral roots showed rhythmic activity although less intense than that of the dorsal roots. As in dorsal roots, some fibers showed a rhythmical pattern of discharge related to the mass activity recorded from whole dorsal roots or peripheral nerves, while other units were not related. It is concluded that bursting activity which occurs in peripheral nerves after the administration of 4-AP is mainly the result of the antidromic activation of medium to small size primary afferent fibers.     

The effect of 4-aminopyridine on the spinal locomotor rhythm induced byl-DOPA

In high spinal curarized cats rhythmic motor output similar to locomotion (‘fictive locomotion’) of all 4 limbs was obtained after intravenous application of the noradrenergic precursor,l-DOPA, and nialamide combined with 4-aminopyridine (4-AP). The activity was recorded from muscle nerves.In the presence of 4-AP, which enhances transmission at various excitatory and inhibitory synapses, reduced amounts of DOPA were sufficient to evole fictive locomotion. 4-AP alone did not elicit locomotion.The burst rate increased up to 6 Hz with the amount of 4-AP given (0.5–50 mg/kg).The cycle frequency of high spinal cats exhibiting either fictive locomotion or walking on a treadmill was accelerated by 4-AP.After a supplementary transection of the spinal cord at the upper lumbar level both fore- and hindlimbs generally continued to show fictive locomotion with similar frequencies.In the presence of high doses of clonidine (alpha-receptor-activator, > 4mg/kg), the locomotor pattern was replaced by regular (2 Hz) synchronous discharges in all flexors and extensors.     

Phase-modulated antidromic action potentials in the dorsal root of the rat during locomotion

Discharges of antidromic spike potentials (ASP) were recorded from the rat dorsal root during locomotion. Tonic and phasic components were revealed in discharges of ASP. The phase-dependent modulation of phasic component was observed during locomotion. The role of ASP as a possible mechanism for restriction of afferent input was discussed.     

The bed nucleus of the stria terminalis: electrophysiological properties and responses to amygdaloid and hypothalamic stimulation

Single unit activity was recorded in the bed nucleus of the stria terminalis (BNST) in response to amygdaloid and hypothalamic stimulation. BNST neurons were identified by their spontaneous discharge rate, spike duration, orthodromic and antidromic responses and convergence properties. The results indicate that these neurons have characteristic electrophysiological features and that they are organized to receive amygdaloid and hypothalamic inputs with increasing convergence along a dorsal to ventral continuum.     

Ectopic impulse generation in focally demyelinated trigeminal nerve

In five cats, microgranulomas containing areas of focal demyelination were produced in the trigeminal nerve near the dorsal root entry zone. Two types of ectopic spike initiation were evoked: extra action potentials from orthodromic stimuli and prolonged high-frequency after discharges following short priming trains of antidromic or orthodromic stimuli.     

Ectopic discharges and adrenergic sensitivity of sensory neurons after spinal nerve injury

At various times after spinal nerve injury, dorsal root ganglia (DRGs) from injured segments were removed with attached dorsal roots and spinal nerves. In an in vitro recording chamber, spontaneously active units were recorded from teased dorsal root fascicles. Sustained spontaneous activity could first be recorded at 13 h after the ligation, but adrenergic sensitivity did not develop until 24 h after the injury. Almost all recorded activity originated from the DRG. Thus, the DRG is the most common site for ectopic discharge generation after spinal nerve injury and separate mechanisms seem to be involved in the development of ectopic discharges and adrenergic sensitivity.     

Characterization of synaptic potentials in hindlimb extensor motoneurons during L-DOPA-induced fictive locomotion in acute and chronic spinal cats

Intracellular recordings were carried out in extensor hindlimb motoneurons during L-DOPA-induced fictive locomotion from acute and chronically spinalized cats. It was demonstrated that motoneurons in chronic animals exhibited irregularly occurring, high frequency discharges within a given burst of a spike train, and sporadic membrane potential depolarizations and hyperpolarizations correlated with extensor and flexor nerve filament activity, respectively, during the locomotor cycle. In contrast, motoneurons recorded from acute cats demonstrated smooth membrane potential fluctuation and regularly occurring low frequency spike discharges. These results indicate that the pharmacologically activated central pattern generator (CPG) for locomotion in young adult chronic cats spinalized at two weeks of age produces disorganized locomotor-like patterns in the absence of sensory feedback. It is suggested that the above-mentioned characteristics of membrane potential fluctuations and spike discharges are not due to alterations in the motoneuron membrane properties, but instead are due to changes in the inputs to the motoneurons.     

Axon-to-axon transmission in tullidora (buckthorn) neuropathy

Oral administration of ether extracts of the tullidora (Karwinskia humboldtiana) fruit, which contains an identified neurotoxin, produced flaccid hind limb paralysis in cats after a latency of 4 to 7 weeks. Acute experiments were conducted after the paralysis was evident. Spinal roots of lumbar and sacral segments were transected as close as possible to the spinal cord and divided into several filaments. Stimulation of some filaments distal to the transection evoked action potentials in other filaments (axon-to-axon transmission or cross talk) after a latency of at least 8 ms. Cross-talk responses frequently consisted of multiple discharges. Axon-to-axon transmission was seen only between motor axons and disappeared when hind limb nerves were transected 10 to 15 cm from the spinal cord. Twin pulses were applied to a filament at various intervals; the pulse intensity was adjusted so that the conditioning pulse was subthreshold to elicit cross talk, but the test pulse frequently elicited it (temporal facilitation). In three fully studied fibers the facilitation was prolonged to 50 to 80 ms. In some cases, no cross talk was evoked in a given filament by individual stimulation of two other filaments, but simultaneous stimulation of the same filaments did evoke cross talk (spatial facilitation). Series of periodic bursts of activity spontaneously occurred in those axons responding with multiple discharges to single stimulation of other axons. At low temperatures (about 30°C) the stimulus could trigger essentially similar series of bursts. Single motoneurons were intracellularly stimulated by brief depolarizing pulses. The action potential elicited by the stimulus was followed after several msec by a secondary train of discharges generated at the periphery (“back firing”).     

Phasic modulation of vestibulospinal neuron activity during fictive locomotion in lampreys

This study was aimed at characterizing the activity of vestibulospinal neurons recorded intracellularly during fictive locomotion in lampreys. The majority (78%) of identified vestibulospinal neurons showed rhythmic fluctuations of their membrane potential correlated with locomotor discharges recorded in pairs of rostral ventral roots. Of the rhythmically modulated vestibulospinal cells, most (72%) were maximally depolarized during ipsilateral ventral root discharges and showed a minimum during contralateral activity. Other cells (20%) showed an opposite pattern, that is their peak of depolarization occurred during contralateral activity. Finally, a third category of cells (8%) showed a more complex pattern of activity. Two waves of depolarization could occur per locomotor cycle, one during each burst discharge. The pattern of fluctuation recorded in vestibulospinal neurons appears to be related to the side of the spinal cord onto which the cells are projecting.     

Depression of afferent-induced primary afferent depolarization at the lumbar spinal cord following concussive head injury

Afferent-induced primary afferent depolarization (PAD) was depressed for 2-5 min following concussive head injury in the cat, as assessed by dorsal root potentials and augmentation of antidromic dorsal root potentials, both evoked by stimulation of adjacent dorsal roots. These changes in PAD were abolished by spinal cord transection but not affected by midpontine transection. Spontaneous dorsal root potentials, resting amplitudes of antidromic dorsal root potentials and reductions of antidromic dorsal root potentials following tetanic root stimulation were not substantially altered by injury. These findings suggest that concussive head injury depresses spinal interneuronal transmission by neurally mediated processes involving the bulbar brainstem.     

Geniculate interactions with a penicillin discharge in visual cortex

Spontaneous activity of principal cells in the dorsal nucleus of the lateral geniculate body was studied 1 sec before and 1 sec after penicillin discharges in the striate cortex. Many cells exhibited increased activity prior to the penicillin discharge suggesting that corticopetal activity from the geniculate is capable of triggering the penicillin discharge. Many cells also exhibited increased activity for as long as 1 sec after the discharge. Some cells displayed both phenomena. Two patterns of transient depression were also observed in the postdischarge period. The latter phenomena are believed to be related to the inhibitory process associated with after-positivity which follows orthodromic or antidromic stimulation of the geniculate body.     

The neuronal adhesion protein D2 in differentiating aggregates of brain cells

The preparation and maintenance of a novel slice of the rat gracile nucleus is described. The slice includes both gracile nuclei as well as an intact afferent input from the dorsal columns. Extracellular recording revealed that a compound tract action potential (CAP) could be recorded from the gracile nucleus following stimulation of the ipsilateral dorsal column. The CAP was followed by slower field potentials which are thought to be dependent on synaptic activity. Four consequences of stimulating the dorsal columns were observed: (1) a subsequent CAP was conducted more rapidly along the afferents whether it travelled in an orthodromic or antidromic direction; (2) the amplitude of a subsequent orthodromic CAP was reduced; (3) the amplitude of a subsequent submaximal antidromic CAP was increased; and (4) a slow positive potential could be recorded from the dorsal columns. All 4 phenomena had comparable time-courses and were similarly sensitive to agents which reduce synaptic transmission. Pharmacological evidence indicated that all 4 phenomena were mediated by GABA. It is suggested that a GABA-mediated depolarization of the gracile afferents can be evoked in this slice.     

Studies of locomotor network neuroprotection by the selective poly(ADP-ribose) polymerase-1 inhibitor PJ-34 against excitotoxic injury to the rat spinal cord in vitro

Delayed neuronal destruction after acute spinal injury is attributed to excitotoxicity mediated by hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) that induces 'parthanatos', namely a non-apoptotic cell death mechanism. With an in vitro model of excitotoxicity, we have previously observed parthanatos of rat spinal cord locomotor networks to be decreased by a broad spectrum PARP-1 inhibitor. The present study investigated whether the selective PARP-1 inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide.HCl (PJ-34) not only protected networks from kainate-evoked excitotoxicity, but also prevented loss of locomotor patterns recorded as fictive locomotion from lumbar (L) ventral roots (VRs) 24 h later. PJ-34 (60 μm) blocked PARP-1 activation and preserved dorsal, central and ventral gray matter with maintained reflex activity even after a large dose of kainate. Fictive locomotion could not, however, be restored by either electrical stimulation or bath-applied neurochemicals (N-methyl-D-aspartate plus 5-hydroxytryptamine). A low kainate concentration induced less histological damage that was widely prevented by PJ-34. Nonetheless, fictive locomotion was observed in just over 50% of preparations whose histological profile did not differ (except for the dorsal horn) from those lacking such a rhythm. Our data show that inhibition of PARP-1 could amply preserve spinal network histology after excitotoxicity, with return of locomotor patterns only when the excitotoxic stimulus was moderate. These results demonstrated divergence between histological and functional outcome, implying a narrow borderline between loss of fictive locomotion and neuronal preservation. Our data suggest that either damage of a few unidentified neurons or functional network inhibition was critical for ensuring locomotor cycles.     

The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey

Lamprey reticulospinal neurons are rhythmically modulated during fictive swimming. The present study examines the possibility that this modulation may originate from the spinal cord locomotor networks rather than from the brainstem. To test this, the in vitro preparation of the lamprey brainstem-spinal cord was separated into two compartments which could be exposed to different chemical environments. Locomotor activity was induced pharmacologically in the caudal spinal cord compartment and reticulospinal (RS) neurons from the posterior rhombencephalic reticular nucleus (PRRN) were recorded intracellularly in the rostral compartment containing normal lamprey Ringer. Under these conditions, the membrane potential of RS neurons showed clear rhythmic oscillations which are correlated with the ongoing locomotor activity in the caudal spinal cord bath, although no locomotor discharges were present in the ventral roots of the rostral bath. Such oscillations were not present in the absence of locomotion. These results indicate that the spinal cord locomotor networks can contribute to the rhythmic oscillations which occur in RS neurons during fictive locomotion. Moreover, the latter oscillations of membrane potential are due to both phasic excitation and Cl- -dependent inhibition in the opposite phase.