Intra-axonal recordings in rat dorsal column axons: membrane hyperpolarization and decreased excitability precede the primary afferent depolarization

Intra-axonal recordings were obtained in the dorsal columns of the rat lumbosacral spinal cord. Dorsal root or dorsal column stimulation at levels subthreshold for the impaled axon elicited a prolonged depolarization corresponding to the primary afferent depolarization (PAD). The depolarization was preceded by a brief hyperpolarizing potential during which excitability was decreased. The hyperpolarization corresponds temporally to the extracellularly recorded DRP IV component of the dorsal root potential described by Lloyd and McIntyre, and may represent the intracellular correlate of this potential. Possible mechanisms for this hyperpolarization include electrical interactions between neuronal elements, a biphasic GABA response, or attenuation of background afferent axonal depolarization.     

The effect of intravenously administered gamma-aminobutyric acid on afferent fiber polarization.

(1) The effect of intravenously administered gamma-aminobutyric acid (GABA) on afferent fiber polarization in the feline spinal cord was ascertained from fluctuations induced in the DC level of dorsal root filaments. (2) A dose-related depolarization of the filament, with a concomitant reduction in the magnitude of the dorsal root potential, was observed after 50 and 100 mg/kg GABA. (3) GABA also depolarized filaments of preparations in which interneuronal activity was suppressed by pretreatment with tetrodotoxin. Since the magnitude of the depolarization induced in these preparations was equal to that observed in nonpretreated animals, it is likely that the depolarization in the latter preparations reflects a direct effect on afferent terminals or fibers rather than an action on interneurons. (4) GABA failed to depolarize filaments in animals pretreated with bicuculline. This suggests that intravenously administered GABA interacted with receptors that are identical with or similar to those involved in neurally evoked primary afferent depolarization (PAD). (5) The direct depolarization of afferent fibers by intravenous GABA and the blockade thereof by bicuculline are characteristics compatible with those of the endogenous axo-axonic transmitter operating in pathways mediating neurally evoked PAD. These data, therefore, support the involvement of GABA at this synapse in the mammalian spinal cord.     

The effects of bicuculline on the isolated spinal cord of the frog

An investigation has been made of the effects of topically applied bicuculline, a reported gamma-aminobutyric acid (GABA) antagonist, on the isolated, hemisected frog spinal cord by recording ventral and dorsal root potentials and reflexes evoked by volleys to various spinal cord inputs. Bicuculline had potent excitatory effects causing depolarization, spontaneous potentials in ventral and dorsal roots, and an increased polysynaptic ventral root reflex. More importantly, the alkaloid blocked presynaptic inhibition of orthodromic reflex activity produced by preceding ventral root stimulation and primary afferent depolarization. These effects were attributed to a demonstrated antagonism of the direct depolarizing effects of GABA on dorsal root terminals by the alkaloid. These actions of bicuculline suggest that GABA may be the transmitter responsible for primary afferent depolarization and presynaptic inhibition in the amphibian.     

Diminished dorsal root GABA sensitivity following chronic peripheral nerve injury

The depolarizing effect of gamma-aminobutyric acid (GABA) on rat lumbar dorsal roots was studied in a sucrose gap chamber following axotomy or crush injury of the sciatic nerve or dorsal root. The mean depolarization elicited by GABA on normal dorsal roots (3.96 +/- 0.71 mV, N = 14) was significantly reduced following chronic sciatic axotomy (2.02 +/- 0.99 mV, N = 15). Chronic sciatic crush injury had no significant effect on dorsal root GABA sensitivity. The amplitudes of the dorsal root compound action potentials were the same from rats with normal and injured sciatic nerves, indicating that axons proximal to the sciatic nerve lesion did not undergo appreciable degeneration. A marked loss of dorsal root GABA sensitivity was also seen following dorsal root axotomy or crush injury (1.02 +/- 0.98 mV (N = 10) and 0.69 +/- 0.70 mV (N = 9), respectively). These results indicate that GABA sensitivity of dorsal roots is attenuated following peripheral nerve lesions in which regeneration and functional reconnection with peripheral targets are prevented. Previous work indicates that the primary afferent depolarization is reduced under similar conditions. The reduction in GABA sensitivity of dorsal root fibers described here may have a contributory role in the reduced primary afferent depolarization that follows peripheral nerve transection, which has pathophysiologic implications in chronic pain syndromes.     

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.     

The development of responsiveness to substance P and glutamate in the spinal motoneurons of rat fetuses

The development of responsiveness of motoneurons to substance P (SP) and glutamate was investigated in the isolated spinal cord of the rat fetus at embryonic days 13.5-21.5. The motoneurons at embryonic day 14.5 first responded to SP with a slow depolarization. The responsiveness to SP increased by embryonic days 19.5-21.5. In a low Ca2+ solution, responsiveness was reduced after embryonic day 17.5. Glutamate caused a slow depolarization of motoneurons from embryonic day 13.5. The responsiveness increased until embryonic day 17.5 and decreased thereafter.     

The role of GABA and serotonin in the mediation of raphe-evoked spinal cord dorsal root potentials

The possible involvement of bulbospinal serotonergic systems in the mediation of analgesia has created a need for a better understanding of the influence this system has on neuronal mechanisms in the spinal cord. Therefore, these studies were designed to examine the effects of caudal raphe stimulation on primary afferent depolarization and to determine the role of serotonin (5-HT) and GABA in the mediation of these stimulation-produced effects. Stimulation of the raphe evoked two electrotonically conducted dorsal root potentials (DRP-1 and DRP-2) and two compound action potentials (VRP-1 and VRP-2) which were recorded from the dorsal and ventral roots, respectively. Length constant measurements indicated that DRP-1 was generated in group II and DRP-2 in group I primary afferent fibers. Histological determination of stimulation sites revealed that short-latency potentials (DRP-1 and VRP-1) were evoked from many sites within the caudal brain stem, while the long-latency potentials (DRP-2 and VRP-2) were evoked primarily from sites within the caudal raphe nuclei. The role of serotonin in mediating these evoked potentials was assessed by administering various antagonists of serotonin (cinanserin, methysergide and D-lysergic acid diethylamide). These agents consistently attenuated the long-latency potentials (DRP-2 and VRP-2) but increased the magnitude of DRP-1. The possibility of a GABAergic neuron in the descending systems projecting to primary afferent terminals was studied. Depletion of GABA by semicarbazide blocked DRP-1, but had only a modest effect of DRP-2. However, the putative GABA antagonist, bicuculline, inhibited both DRP-1, and DRP-2. These results suggest that a GABA interneuron is not involved in the bulbospinal serotonergic depolarization of primary afferent terminals. This system appears to constitute a presynaptic filter of afferent input, with the capacity to inhibit different fiber groups.     

The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K+ contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K+-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K+-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K+-rich solutions adequately models the localized K+ accumulation which occurs physiologically during afferent activity. K+-induced depolarizations were decreased in the presence of 20 mM Mg2+; this effect was due to a direct decrease in the membrane response to K+ and not to blockade of K+-induced transmitter release onto primary afferents. The depolarization caused by a K+ concentration comparable to a maximum estimate of the K+ accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K+ levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K+-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.     

The effects of naloxone, morphine and methionine enkephalinamide on Ia afferent terminations in the cat spinal cord

Naloxone, morphine, Met5-enkephalinamide (MENKA) and procaine were administered microelectrophoretically near extracellularly stimulated extensor muscle group Ia afferent fibres and terminations in the lumbar spinal cord of cats anaesthetized with pentobarbitone sodium. Observations were made of effects on the electrical threshold, on the depolarizing action of GABA or piperidine-4-sulphonate (P4S), and on bicuculline-sensitive primary afferent depolarization (PAD) generated by tetanic stimulation of flexor muscle low threshold afferents. All 4 agents reversibly elevated the threshold of Ia fibres in the dorsal column and Ia terminations in the ventral horn. The depolarizations of terminations by GABA or P4S were also reduced, an effect, which for all except MENKA, probably accounted for a concomitant reduction in PAD. In the absence of a consistent effect on either threshold or depolarization by GABAmimetics, MENKA reversibly diminished PAD, an action blocked by naloxone. Intravenously administered naloxone, in doses known to enhance spinal monosynaptic excitation in the cat, had no effect on GABAergic PAD and little or no effect on Ia termination threshold. The results are discussed in relation to a naloxone-sensitive effect of MENKA which reduces transmitter release from GABAergic axo-axonic synapses on Ia terminals, but which does not account for the enhancement of spinal reflexes by naloxone.     

Neurochemical and ionic mechanisms of dorsal root potentials in the spinal cord of the immature rat

Dorsal root potentials (DRP) recorded from spinal cord of 7-14-days old rats have two waves of depolarization. The fast wave of DRP is GABA-ergic in nature and the slow wave is evoked mainly by increasing of extracellular K+-ion concentration near the primary afferent terminals. The possible mechanisms of increasing extracellular K+-ion concentration evoked by dorsal root stimulation are discussed.     

The presynaptic effects of valproic acid in the isolated frog spinal cord

The effects of the anticonvulsant valproic acid (n-dipropylacetate, DPA) on frog primary afferent fibers was examined with sucrose gap recordings from the dorsal roots. Addition of DPA to the superfusate consistently reduced the amplitude and duration of the dorsal root potential. In contrast, DPA augmented the depolarization of dorsal roots produced by GABA, β-alanine and taurine. It also decreased afferent fiber ‘desensitization’ to GABA. DPA depressed the ability of K⁺ and the excitatory amino acids glutamate and aspartate to depolarize afferent fibers. In addition, the compounds decreased the amount of K⁺ released by tetanic stimulation of the dorsal root.

The K⁺-evoked release of tritiated GABA from cord slices was initially reduced by exposure to DPA, but was then unaffected after a longer application of the anticonvulsant. On the other hand, the high affinity uptake of tritiated GABA and glycine were almost totally blocked by the addition of DPA to the incubating medium.

In sum, DPA has complex effects on neuronal membranes. Some of these effects may serve to explain the anticonvulsant actions of this drug.     

Expression of the glycinergic system during the course of embryonic development in the mouse spinal cord and its co-localization with GABA immunoreactivity

To understand better the role of glycine and gamma-aminobutyric acid (GABA) in the mouse spinal cord during development, we previously described the ontogeny of GABA. Now, we present the ontogeny of glycine-immunoreactive (Gly-ir) somata and fibers, at brachial and lumbar levels, from embryonic day 11.5 (E11.5) to postnatal day 0 (P0). Spinal Gly-ir somata appeared at E12.5 in the ventral horn, with a higher density at the brachial level. They were intermingled with numerous Gly-ir fibers reaching the border of the marginal zone. By E13.5, at the brachial level, the number of Gly-ir perikarya sharply increased throughout the whole ventral horn, whereas the density of fibers declined in the marginal zone. In the dorsal horn, the first Gly-ir somata were then detected. From E13.5 to E16.5, at the brachial level, the density of Gly-ir cells remained stable in the ventral horn, and after E16.5 it decreased to reach a plateau. In the dorsal horn, the density of Gly-ir cells increased, and after E16.5 it remained stable. At the lumbar level, maximum expression was reached at E16.5 in both the ventral and dorsal horn. Finally, the co-localization of glycine and GABA was analyzed, in the ventral motor area, at E13.5, E15.5, and E17.5. The results showed that, regardless of developmental stage studied, one-third of the stained somata co-expressed GABA and glycine. Our data show that the glycinergic system matures 1 day later than the GABAergic system and follows a parallel spatiotemporal evolution, leading to a larger population of glycine cells in the ventral horn.     

Presynaptic mechanisms of the change in segmental responses accompanying the formation of the spinal locomotor generator in the cat

It is shown on unanesthetized immobilized decorticated cats that spinalization of the animal leads to depolarization of the central afferent terminals, decrease of early polysynaptic responses in motoneurons and dorsal root potentials (DRP) evoked by stimulation of the low-threshold cutaneous and muscle afferents, increase of early polysynaptic responses and DRP evoked by stimulation of high-threshold muscle afferents, reduction in the activity of intermediate nucleus interneurons mono- and polysynaptically connected with primary afferents, rise of the activity of interneurons di- and oligosynaptically connected with primary afferents. Injection of DOPA into spinal animals leads to opposite changes. Dependence between changes in the state of segmental neuronal apparatus and the level of spinal locomotor generator activity are discussed on the basis of the data obtained.     

Dorsal root potentials in the cat: effects of bicuculline

Bicuculline (0.2 1 mg/kg) administered intravenously depressed dorsal root potentials (DRPs) evoked by stimulation of mixed, pure muscle or pure cutaneous nerves which was clearly concurrent with enhanced background potentials in intact cat. Administration of sodium pentobarbitone (15-30 mg/kg i.v.) reduced the ability of bicuculline to enhance background potentials and to depress evoked DRPs. In spinalized preparations, bicuculline depression of evoked DRPs by bicuculline in intact cat may not result from its action at axo-axonic GABAergic synapses alone and occlusion may also play a part. However, the role of gamma-aminobutyric acid (GABA) in primary afferent depolarization is confirmed in the spinalized preparations.     

Functions of primary afferents and responses of extracellular K+ during spinal epileptiform seizures.

Paroxysmal activity in ventral roots induced by penicillin in decapitate cat spinal cords is associated with waves of depolarization of primary afferent fiber terminals. These paroxysmal depolarizations can be detected as spontaneously occurring negative dorsal root potentials (DRPs) and are associated with antidromic discharge of nerve impulses in dorsal root fibers; they can also be detected by testing the excitability of afferent nerve terminals by focal stimulation. Negative DRPs evoked by afferent nerve volleys are altered in waveform but not in amplitude during seizures induced by penicillin, although they are blocked by the administration of picrotoxin. While blocking afferent-evoked DRPs, picrotoxin does not interfere with paroxysmal DRP'S, INDICATING DIFFERENCES IN THE GENERATION OF THE Two phenomena, which nevertheless have some link in common, for the paroxysmal waves occlude the evoked DRP. Such occlusion would appear as blockade, if DRPs were recorded by condenser-coupled amplifiers. In the presence of pentobarbital penicillin suppresses evoked DRPs, but under such circumstances seizure activity is not observed. Extracellular potassium activity within spinal gray matter transiently increases during seizure activity. Such increments of potassium activity are maximal in the ventral horns. This and several other observations suggest that in decapitate spinal cords systemically administered penicillin induces seizures which originate in the ventral gray matter. Accumulation of excess potassium may be the cause of paroxysmal depolarization of afferent nerve terminals. Excess potassium while not playing a principal role in initiating seizures, may influence the course of seizures by depolarizing afferent terminals. Such depolarization probably enhances tonic background release of transmitter substance, may modify the effect of synaptic input, and may favor synchronization of waves of neural excitability through extrasynaptic mechanisms.     

Control of Impulse Conduction in Long Range Branches of Afferents by Increases and Decreases of Primary Afferent Depolarization in the Rat

It has been shown previously that impulses in axons of the descending branches of myelinated afferents in rat dorsal columns may suffer a blockade of transmission along their course in the dorsal columns. This paper tests the effect of the mechanism of primary afferent depolarization on the orthodromic movement of impulses in descending dorsal column primary afferent axons originating in the L1 dorsal root. Orthodromic impulses were recorded in the L5 and 6 dorsal columns after stimulation of the L1 dorsal root. Twenty-seven out of 82 axons (33%) suffered a temporary transmission block if primary afferent depolarization had been induced by L5 stimulation before the L1 stimulus. The tendency to block peaked at 10–15 ms and persisted for up to 30–40 ms. The number of single unit orthodromic impulses originating from the L1 root and recorded during a search of the dorsal columns 15 mm caudal to L1 increased by a factor of 3.1 after the systemic administration of bicuculline (1 mg/kg). The number of single unit orthodromic impulses originating from the L1 root and recorded in axons descending in the dorsal columns 20 mm caudal to the root increased by a factor of 8.7 after the systemic administration of picrotoxin (5 mg/kg). It is concluded that the transmission of impulses in the long range caudally running axons from dorsal roots to dorsal columns may be blocked during primary afferent depolarization and that conduction may be restored by the administration of GABA antagonists.     

The influence of bemegride on frog spinal cord root potentials]

The effect of the convulsant bemegride (beta-ethyl-beta-methyl-glutharimide) on the electronic potentials in the frog spinal roots was investigated in situ. Intravenous injection of the bemegride subconvulsant doses (6.8 +/- 2.7 mg/kg) depressed rapidly the electrotonic dorsal root potentials (DRP) evoked by a stimulation of the adjacent dorsal root (DR) or the ventral root (VR). They were reduced by 55--67% 3-6 min after the bemegride injection. The effect of bemegride was reversible and the DRP amplitude restored its initial value within an hour. Ventral root potentials after bemegride injection revealed only greater amplitude fluctuations. A conclusion is made that bemegride is a selective and potent depressor of the primary afferent depolarization in the frog spinal cord.     

Spontaneous activity in the perinatal trigeminal nucleus of the rat

The role of electrical activity in establishing appropriate connections in the trigeminal pathway remains controversial. We report here a novel observation of spontaneous activity in the perinatal trigeminal sensory nucleus between embryonic day (E) 16 to postnatal day (P) 8. Most of these spontaneous bursts had the same polarity and were of comparable amplitude to the trigeminal nerve-evoked response. This synchronized activity was abolished by bicuculline or kynurenic acid. Recording from the teased trigeminal root during the spontaneous bursts also showed a corresponding afferent depolarization. We speculate that the GABAergic depolarization of pre- and postsynaptic cells may contribute to the generation of the spontaneous activity and its propagation throughout the trigeminal sensory pathway, even in the absence of activity initiated from peripheral sensory receptors.     

Some actions of catechol on synaptic transmission in the isolated spinal cord of the frog.

The isolated frog spinal cord was used to investigate the synaptic effects of the convulsant agent catechol. Addition of the compound to the superfusate consistently enhanced orthodromic reflex activity recorded from ventral roots and augmented primary afferent depolarization. Concomitantly catechol altered the polarization changes produced in ventral and dorsal roots by putative neurotransmitter amino acids when these compounds were applied in Mg2+-containing Ringer. Catechol reduced the hyperpolarizations induced in motoneurons by the neutral amino acids, GABA, beta-alanine, taurine and glycine, but did not affect the depolarizations produced by the dicarboxylic amino acids, L-glutamate and L-aspartate. In contrast, catechol increased the dorsal root depolarizations elicited by both neutral and dicarboxylic amino acids and also the depolarizations produced by elevated potassium concentrations. Catechol did not bring about significant changes in the passive electrical properties of motoneurons or dorsal root fibers. In addition, it did not alter either the high affinity uptake or the depolarization-evoked release of tritiated GABA, glycine, L-glutamate and L-aspartate. It appears that the postsynaptic actions of catechol explain its ability to enhance spinal reflexes.