Pharmacology of the vestibular hair cell-afferent fiber synapse in the frog

The isolated, intact, membranous labyrinth of the frog (Rana temporaria) has been investigated electrophysiologically in vitro to determine the nature of the transmitter substance at the synapse between the vestibular hair cells and afferent fibers. Spontaneous synaptic activity can be monitored with intra-axonal recordings from the afferents. Increased K+ in the bath results in an increase in frequency of presynaptic release, as indicated by an increased frequency of spontaneous synaptic potentials. Adding Mg2+ and lowering Ca2+ results in a decrease in synaptic potential frequency (often to zero) with no change in their mean amplitude, indicating pre-synaptic blockade. Extracellular recordings from individual vestibular afferents indicate that bath-applied glutamate and related acidic amino acids consistently increase the firing rates of these afferents in a dose-dependent manner with no evidence of desensitization. In the presence of presynaptic blockade (high Mg2+/low Ca2+), bath application of glutamate and its agonists results in a reversible depolarization of vestibular afferents, suggesting a postsynaptic action of these substances. 2-Amino-5-phosphonovaleric acid, kynurenic acid, and other acidic amino acid antagonists reversibly decrease the amplitudes of spontaneously occurring synaptic potentials without affecting their frequency, indicating subsynaptic blockade. These antagonists also block the postsynaptic depolarizations due to glutamate and its agonists. GABA and its agonists and antagonists have no consistent effect upon afferent activity. These findings suggest that glutamate, aspartate, or a related compound is the transmitter at this synapse. However, the antagonists used, or the receptors themselves, are not selective enough to discriminate adequately between the agonists. Therefore, which of these glutamate agonists are actually involved in synaptic transmission remains to be determined.     

Presynaptic inhibition of identified wind-sensitive afferents in the cercal system of the locust

The paired cerci located at the tip of the locust abdomen bear a large number of wind-sensitive filiform hairs, each of which sends an axon via the cercal nerve into the terminal ganglion of the CNS. The filiform afferents fire bursts of action potentials when their hairs are displaced by wind or mechanical stimuli. Filiform axon terminals in the CNS are depolarized concomitantly with the discharge of another type of unit (a primary afferent-depolarizing, or PAD, unit) recorded in the cercal nerve. The instantaneous spike frequency of PAD unit discharges matches the evoked depolarization very closely, and during such depolarizations spike amplitudes in the filiform afferent terminals are reduced by up to 55%. Depolarizing current pulses injected into the axonal terminals of an identified filiform afferent evoke spikes that are blocked by the PAD unit, probably via an intercalated interneuron. The PAD unit makes a monosynaptic connection with only one of the 4 giant interneurons (GIN 2) on each side of the terminal ganglion, and indirect connections with 2 others. Depolarizing current pulses injected into the neuropilar segments of GINs evoke fewer spikes when the PAD unit is active, consistent with the PAD unit's mediation of conductance changes in postsynaptic cells. Iontophoretic injection of Lucifer yellow shows the PAD unit to be an afferent with axon terminals overlapping those of filiform afferents and posteriorly directed branches of interneurons such as GIN 2 in the CNS. Passive movements of a cercus, monitored with a position transducer, show that the PAD unit fires discrete bursts during cercal displacement. The PAD unit most probably has its soma and dendrites in tissue spanning the cercal base. By responding to cercal movements sufficient to also activate filiform hairs, and by mediating conductance changes in both the presynaptic terminals of filiform afferents and the postsynaptic membranes of interneurons, the PAD unit desensitizes a pathway to movement-generated afferent input, and ensures that the locust remains sensitive to external wind stimuli.     

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.     

The development of the dorsal root potential and the responsiveness of primary afferent fibers to gamma-aminobutyric acid in the spinal cord of rat fetuses

The development of the dorsal root potential (DRP) and the responsiveness of primary afferent fibers to gamma-aminobutyric acid (GABA) were investigated in the isolated spinal cord of rat fetuses. At embryonic day 15.5, stimulation of the lumbar dorsal root was first effective in eliciting the DRP, which was not inhibited by bicuculline. A bicuculline-sensitive component of the DRP appeared at embryonic day 17.5. GABA (10 microM to 1 mM) caused a dose-dependent depolarization of the primary afferent fibers from embryonic day 13.5. The amplitude of the depolarization gradually increased with age until embryonic day 17.5 and was maintained thereafter. If the bicuculline-sensitive DRP solely reflects GABAergic activity, it is suggested that GABAergic activity develops at embryonic day 17.5 and the development of the responsiveness of primary afferent fibers to GABA precedes the functional onset of GABAergic neurons.     

Phase-frequency characteristics of afferent activity and depolarization of primary afferents during passive displacement of the hindlimb of the cat

Frequency response characteristics of afferent activity and primary afferent depolarization (PAD) evoked by this activity were investigated in decerebrated cats during passive movement of hindlimb in ankle joint. At the frequencies of 0.2-1.7 Hz the lead of the level of depolarization was determined by the value of phase changes of afferent activity. Above 1.7 Hz a phase lag of the level of depolarization was observed. This lag was caused by the dynamic properties of PAD generation system.     

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.     

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 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.     

Persistence of PAD and presynaptic inhibition of muscle spindle afferents after peripheral nerve crush

Two to twelve weeks after crushing a muscle nerve, still before the damaged afferents reinnervate the muscle receptors, conditioning stimulation of group I fibers from flexor muscles depolarizes the damaged afferents [M. Enriquez, I. Jimenez, P. Rudomin, Changes in PAD patterns of group I muscle afferents after a peripheral nerve crush. Exp. Brain Res., 107 (1996), 405-420]. It is not known, however, if this primary afferent depolarization (PAD) is indeed related to presynaptic inhibition. We now show in the cat that 2-12 weeks after crushing the medial gastrocnemius nerve (MG), conditioning stimulation of group I fibers from flexors increases the excitability of the intraspinal terminals of both the intact lateral gastrocnemius plus soleus (LGS) and of the previously damaged MG fibers ending in the motor pool, because of PAD. The PAD is associated with the depression of the pre- and postsynaptic components of the extracellular field potentials (EFPs) evoked in the motor pool by stimulation of either the intact LGS or of the previously damaged MG nerves. These observations indicate, in contrast to what has been reported for crushed cutaneous afferents [K.W. Horch, J.W. Lisney, Changes in primary afferent depolarization of sensory neurones during peripheral nerve regeneration in the cat, J. Physiol., 313 (1981), 287-299], that shortly after damaging their peripheral axons, the synaptic efficacy of group I spindle afferents remains under central control. Presynaptic inhibitory mechanisms could be utilized to adjust the central actions of muscle afferents not fully recovered from peripheral lesions.     

Depolarization of primary afferents evoked by cyclically modulated natural activity of proprioceptors of the hindlimb of the cat

The primary afferent depolarization (PAD) evoked during passive sinusoidal movements of a hindlimb in the ankle joint was investigated in decerebrated cats. The frequency of movements varied within 0.14-5.0 Hz, the amplitude of the joint angle with respect to the axis of the tibia changed from 90 degrees to 130 degrees. The dorsal root potential (DRP) negativity increased both during flexion and during extension of the joint. The amplitude of the evoked DRPs was about 50-100 mV. A strong negative correlation was observed between the latency and rise time of the DRP and the frequency of the joint angle changes. During flexion the latency changed from 650 ms at 0.14-0.16 Hz frequency to 100-110 ms at 2.0 Hz and higher frequencies; during extension at the same frequencies the latency changed from 300 ms to 80-85 ms. The latency and rise time became minimal at 2.0 Hz frequency and practically did not change during the further increase of the oscillation frequency. The cord dorsum potential (CDP) evoked by the cutaneous nerve stimulation was recorded in parallel with the DRP. Periodical changes of the N-component of the CDP were in the opposite phase to changes of the DRP. Mechanisms of the observed changes of the PAD and functional significance of these changes during rhythmical motor acts are discussed.     

Properties of functionally identified nociceptive and nonnociceptive facial primary afferents and presynaptic excitability changes induced in their brain stem endings by raphe and orofacial stimuli in cats

The activity of 221 single primary afferent units was recorded extracellularly in the trigeminal (V) ganglion of chloralose-anaesthetized cats to examine the receptive field properties of nonnociceptive and nociceptive cutaneous afferents and the effect of conditioning stimulation of the raphe system and orofacial afferents on the antidromic excitability of their brain stem endings in V subnucleus caudalis. In addition to slowly adapting and rapidly adapting low-threshold mechanosensitive afferents, we functionally identified three classes of cutaneous nociceptive afferents: these included A-delta high-threshold mechanoreceptive afferents (A-delta HTMs), C-fiber high-threshold mechanoreceptive afferents (C-HTMs), and C-polymodal nociceptive afferents (CPNs). Most of the CPNs could be activated by light tactile stimuli as well as by heavy pressure and pinch and noxious radiant heat applied to their mechanoreceptive field which usually involved a localized spot (approximately 1 mm in diameter) of skin. In contrast, the C-HTMs and A-delta HTMs could not be activated by radiant heat stimuli although some did show sensitization which was also a feature of the CPNs; they did respond to noxious mechanical stimulation of a localized area of skin. We noted that orofacial conditioning stimulation could produce an increase in antidromic excitability which was considered a reflection of primary afferent depolarization (PAD) in both nociceptive and nonnociceptive afferents innervating the cat's facial skin; nonnoxious mechanical stimuli and electrical stimuli were particularly effective in the low-threshold mechanosensitive afferents and noxious mechanical and high-intensity electrical stimuli were especially effective in the cutaneous nociceptive afferents. Raphe conditioning stimulation also was very effective in inducing PAD in these nociceptive afferents; however, the raphe conditioning effects were not limited to these nociceptive afferents since PAD was also frequently demonstrated in the low-threshold mechanosensitive afferents.     

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.     

Modification of primary afferent depolarization in cat group Ia afferents following high frequency intra-axonal tetanization of individual afferents

Intra-axonal tetanization of a single, functionally-identified group Ia afferent from the triceps surae muscle in the anesthetized cat produces marked enhancement and slowing of the primary afferent depolarization (PAD) generated in the Ia afferent by volleys in flexor muscle group Ia afferents, plus a pronounced transmembrane hyperpolarizing undershoot (HPU) which disappears more rapidly than the enhanced PAD. These alterations are qualitatively and quantitatively similar to those found after conditioning tetani are applied to the whole muscle nerve. The occurrence of these PAD changes after intra-axonal tetanization of a single group Ia afferent appears to rule out the participation of non-specific alterations in extracellular ionic concentrations or activation of polysynaptic pathways in their genesis.     

Activity-Dependent Changes in Rat Ventral Horn Neurons in vitro; Summation of Prolonged Afferent Evoked Postsynaptic Depolarizations Produce a d-2-Amino-5-Phosphonovaleric Acid Sensitive Windup

The synaptic responses of lumbar ventral horn neurons including identified flexor motoneurons, to graded stimulation of peripheral nerves have been recorded in vitro in the young rat spinal cord-hindlimb preparation. Single shock stimulation of low threshold myelinated afferents evoked short latency (< 20 ms) short duration (< 1.0 s, 391 +/- 42 ms n=43 SEM) compositive mono- and polysynaptic potentials. Recruitment of both thinly myelinated (A delta) and unmyelinated (C) afferent fibres elicited a prolonged postsynaptic depolarization (> 1 s) in all cells. In the majority of cells (67.4%), this depolarization exceeded 4.0 s in duration (8.01 +/- 0.4 s, n=26, maximum 14 s). In the remainder, shorter responses were evoked (< 3.0 s, mean=1.74 +/- 0.4 s, n=18). In those cells where the postsynaptic response to a single A delta or C fibre strength stimulus exceeded 4 s, low frequency (0.5 - 1.0 Hz) repetitive stimulation resulted in a temporal summation of the postsynaptic depolarizations, which generated a cumulatively increasing depolarization. This incrementing depolarization was sufficient in 33% of the cells to produce a progressive increase in spike discharge (windup). On cessation of the train of stimuli the depolarization decayed slowly (65 +/- 27 s). The N-methyl d-aspartic acid (NMDA) receptor antagonist d-2-amino-5-phosphonovaleric acid (d-APV) reduced the duration and amplitude of the prolonged postsynaptic depolarizations elicited by a single shock stimulation of small diameter afferents by 57% and 50% respectively. A smaller effect was produced on the low threshold afferent evoked early excitatory postsynaptic potentials (EPSP) (3% decrease in amplitude and 24% decrease in duration). In the presence of d-APV the cumulatively incrementing depolarization produced by repetitive stimulation was substantially reduced and windup failed to occur. Activity-dependent amplifications of primary afferent evoked responses in spinal neurons therefore involves a temporal summation of d-APV sensitive prolonged postsynaptic depolarizations.     

Specific and potassium components in the depolarization of the la afferents in the spinal cord of the cat

In the cat spinal cord, primary afferent depolarization (PAD) of group Ia fibers of extensor muscles is produced by high-frequency stimulation (100 Hz) of group I muscle flexor afferents without significant increases in extracellular potassium. On the other hand, the PAD produced by stimulation of mixed and pure cutaneous nerves correlates well with increases in potassium ions. We conclude that the PAD produced by group I muscle afferents results from the activation of specific pathways making axo-axonic synapses with the Ia fiber terminals. The PAD of Ia fibers resulting from activation of cutaneous nerves involves instead unspecific accumulation of potassium ions.     

Specific and potassium components in the depolarization of the Ia afferents in the spinal cord of the cat

In the cat spinal cord, primary afferent depolarization (PAD) of group Ia fibers of extensor muscles is produced by high-frequency stimulation (100 Hz) of group I muscle flexor afferents without significant increases in extracellular potassium. On the other hand, the PAD produced by stimulation of mixed and pure cutaneous nerves correlates well with increases in potassium ions. We conclude that the PAD produced by group I muscle afferents results from the activation of specific pathways making axo-axonic synapses with the Ia fiber terminals. The PAD of Ia fibers resulting from activation of cutaneous nerves involves instead unspecific accumulation of potassium ions.     

Phase-dependent modulation of dorsal root potentials evoked by peripheral nerve stimulation during fictive locomotion in the cat

To help elucidate the role of presynaptic mechanisms in the control of locomotor movements, the transmission of PAD pathways was investigated by recording dorsal root potentials (DRPs) evoked by electrical stimulation of cutaneous and muscle nerves of both hindlimbs at various phases of the fictive step cycle. Fictive locomotion occurred spontaneously in decorticate cats or by stimulating the mesencephalic locomotor region (MLR) as well as in low spinal cats injected with nialamide and L-DOPA. Evoked DRPs were superimposed on a fluctuating DRP accompanying the fictive locomotor rhythm (locomotor DRP) which typically consisted of two peaks of depolarization per cycle, the largest peak occurring during the flexor phase. The amplitude of evoked DRPs was substantially modulated throughout the locomotor cycle and followed a similar modulation pattern for all stimulated nerves whether ipsilateral (i-) or contralateral (co-). The amplitude of evoked DRPs decreased at the beginning of the flexor phase, dropped to a minimum later in the flexor phase and then increased during the extensor phase where it became maximum. Results were comparable in decorticate and spinal preparations and for L6 and L7 rootlets with cutaneous and muscle nerve stimulation. It is noteworthy that the modulation pattern for a given rootlet was similar for i- and co- stimulation, even though the bilateral locomotor DRPs fluctuate out-of-phase with each other, subjecting the stimulated fibres to opposite presynaptic polarization changes. This suggests that the modulation may depend more on the presynaptic mechanisms of the receiving fibres than on those of the stimulated fibres. These results demonstrate that the transmission in spinal pathways involved in primary afferent depolarization (PAD) is phasically modulated by the activity in the spinal locomotor network. It is further suggested that the presynaptic inhibition associated with PAD evoked by movement-related sensory feedback during real locomotion could be modulated in a similar way.     

Effect of chronic undernourishment on the cord dorsum potentials and the primary afferent depolarization evoked by cutaneous nerves in the rat spinal cord

The effect of chronic undernourishment on the cord dorsum potentials (CDPs) and the dorsal root potential (DRP), closely related to primary afferent depolarization (PAD) and presynaptic inhibition in the spinal cord of the rat, was analyzed in this study. Single electrical pulses applied to the sural nerve (SU) of control (n=14) and chronically undernourished (n=16) Wistar rats produced CDPs, which are composed of four components: afferent volley (AV), two negative components (N(1) and N(2)), and one positive component (P wave) and negative DRPs recorded in a small rootlet of the L6 segment of the rat. The CDPs of the control and undernourished rats with AV components of comparable amplitude (U(AV)/C(AV)=0.96), showed N(1) components of similar amplitude (U(N1)/C(N1)=0.94), but smaller P wave (U(PW)/C(PW)=0.23). A comparable reduction in the amplitude of the DRPs was obtained in the undernourished rats (U(DRP)/C(DRP)=0.36). When normalized as a function of the body mass of the animals, the CDPs and DRPs produced in undernourished rats were of significantly smaller normalized amplitude than those evoked in the control. According to these results, it is suggested that chronic undernourishment induce a depressive effect on the mechanisms generating the P wave component in the CDP and the DRPs either by decreasing the sensory input and/or the excitability of the dorsal horn neurones involved in the generation of PAD and presynaptic inhibition in the spinal cord of the rat.     

Changes in presynaptic processes during tonic subthreshold activation of the spinal center of scratching movements in the cat

In immobilized intercollicularly decerebrated cats tonic underthreshold activation of the spinal scratching generator (after application of tubocurarine or bicuculline on C1-C2 segments) is accompanied by an increase in primary different terminal depolarization, decrease in N1-component of cord dorsum potential evoked by stimulation of cutaneous afferents, decrease in the amplitude of DRP and early polysynaptic responses of motoneurons evoked by stimulation of cutaneous and muscle afferents; a respective rise and reduction in activity of intermediate nucleus interneurons which are mono- and di (oligo)-synaptically connected with afferent terminals. Spinalization of animal led to reverse changes. Injection of DOPA into spinal animals allowed comparing changes in the state of lumbar segmental apparatus during tonic underthreshold activation of spinal scratching and locomotor generators.     

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.