Presynaptic inhibition of the monosynaptic reflex produced by injections of nicotine or eserine in the spinal cat

In completely deafferented cats with spinal cord transected and paralyzed with gallamine, the close arterial injection of nicotine (5 to 33 μg) into the spinal cord circulation or the intravenous injection of eserine (2 mg/kg) produced a transient increase in excitability of the central terminals of primary afferent fibers. Continuous d-c records from dorsal roots during nicotine injections indicated that a depolarization of the terminals was probably responsible for the observed excitability increase. Mecamylamine prevented both the depolarization and the increase in excitability of primary afferent central terminals whereas atropine and gallamine did not. The monosynaptic reflex was always depressed following injections of nicotine or eserine, but the excitability of the motorpool was found to be increased by these drugs. It was concluded that the monosynaptic reflex depression following injections of nicotine or eserine had a significant presynaptic inhibitory component as a consequence of a drug-induced depolarization of the central terminals of Group Ia afferent fibers.     

Recurrent potentials in human peripheral sensory nerve: possible evidence of primary afferent depolarization of the spinal cord.

To study slowly conducted components of the orthodromic compound sensory action potential (CSAP), the response evoked at the lateral malleolus in the sural nerve was recorded through near-nerve needles at two to four sites along the nerve at midcalf. When 500 to 2000 responses were averaged at high gain, components with latencies of 30 to 80 ms were often recorded. In contrast to the main component and late components with latencies of less than 15 to 20 ms, the latencies of these extremely late components diminished the closer to the spinal cord that they were recorded. This suggested that the components were conducted antidromically from proximal to distal. This assumption was supported by abolishing the components by local anesthesia of the nerve proximal to the recording electrodes. These antidromic potentials therefore appear to be due to recurrent discharges in the sural nerve. Recurrent discharges were recorded from 65% of 60 subjects (18 normal subjects and 42 patients with peripheral or central nervous system disorders). The latencies of the recurrent discharges allowed conduction to and back from the spinal cord. Although the origin of these potentials remains unknown, we suggest that they are due to dorsal root reflexes within the spinal cord. In this case, the responses may be a direct expression of primary afferent depolarization (PAD) seen in presynaptic inhibition, and may be of value in further studies on the physiology and pathophysiology of presynaptic inhibition of cutaneous fibers in man.     

Differential decerebrate control of depolarization in the central terminals of cutaneous afferents in the sacral cord

Presynaptic depolarization of cutaneous afferents has been investigated in the sacral cord of decerebrate cats before and after spinal cord transection. In the decerebrate state the central terminals of caudal femoral cutaneous nerve are depolarized by ipsilateral volleys entering the cord via sacral and lumbar dorsal roots. A significant increase of depolarization occurring after severing the cord indicates that there is tonic decerebrate inhibition of presynaptic depolarization in terminals of caudal femoral cutaneous nerve. In contrast to this finding, presynaptic depolarization evoked in the central terminals of the pudendal nerve by ipsilateral volleys entering the cord through sacral and lumbar dorsal roots is not subjected to decerebrate inhibitory control. It is suggested that differential inhibitory control of depolarization in the central terminals of cutaneous nerves in the sacral cord is related to the intraspinal course of their fibres, to differences in the receptor types involved, and to the location of their innervation fields. In more than half of the decerebrate preparations stimulation of the central terminals of cutaneous afferents through microelectrodes evokes antidromic spikes appearing simultaneously in ipsi- and contralateral nerves. The time course of bilateral excitability changes is similar on both sides of the cord. It is assumed that presynaptic effects are transmitted to the contralateral side by collaterals of ipsilateral cutaneous afferents.     

Electrophysiological changes in the fasciculus gracilis of the cat following chronic clioquinol administration

Clioquinol was administered to cats for more than 200 days, in order to investigate the neural mechanisms underlying the sensory disturbances of subacute myelo-opticoneuropathy (SMON). Electrophysiological examination, carried out under urethane-chloralose anesthesia, revealed that there were 3 major abnormalities in the surface potentials of the nucleus gracilis evoked by sural nerve stimulation, i.e., a reduction in the peak-to-peak amplitude, prolongation of the N wave, and a reduction in P wave amplitude. The reduction in P-wave amplitude suggested suppression of presynaptic inhibition. This was confirmed by excitability tests of the presynaptic terminals of sural nerve fibers within the nucleus gracilis. Recordings of orthodromic volleys in the fasciculus gracilis, elicited by sural nerve stimulation, showed an increase in temporal dispersion. Increased temporal dispersion was also evident from recordings of antidromic volleys in the sural nerve. Peripheral axons of primary sensory neurons in the sural nerve and their terminals within the spinal cord showed no significant functional abnormalities in the chronic clioquinol cat. It is suggested that primary axons in the fasciculus gracilis near the nucleus gracilis are affected by chronic clioquinol administration.     

Presynaptic modulation of synaptic effectiveness of afferent and ventrolateral tract fibers in the frog spinal cord

In the isolated frog spinal cord, antidromic stimulation of motor nerves produces intraspinal field potentials with a characteristic spatial distribution. When recording from the ventral horn, there is a short latency (1–2 msec) response corresponding to activity generated by antidromic activation of motoneuron cell bodies and proximal dendrites. In addition, in the dorsal horn, a delayed wave (12–13 msec latency) corresponding in time with the negative dorsal root potential is also recorded. This wave (VR-SFP) is positive at the dorsal surface of the cord and inverts to negativity at more ventral regions. The negative VR-SFP is maximum between 300–500 μm depth from the dorsal surface and decays with increasing depth towards the motor nucleus. Six days after chronic section of the dorsal roots L7 to L9 in one side of the spinal cord, stimulation of the motor nerves on the deafferented side produces only the early response attributable to antidromic activation of motoneurons. No distinctive VR-SFPs are recorded at any depth within the cord. These findings are consistent with the interpretation that afferent fiber terminals are the current generators of the VR-SFP. The presynaptic and postsynaptic focal potentials recorded in the motor nucleus after stimulation of the ventrolateral tract, as well as the corresponding synaptic potentials electrotonically recorded from the ventral roots, are not depressed during conditioning stimulations which produce primary afferent depolarization. This contrasts with the depression of the presynaptic and post-synaptic focal potentials and synaptic potentials produced by stimulation of sensory fibers. It is concluded that, unlike the afferent fiber terminals, the terminals of the ventrolateral tract are not subjected to a presynaptic modulation of the type involving primary afferent depolarization.     

Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses

Electrical stimulation of the frog sciatic nerve was found to produce rapid, transient swelling of the 8th and 9th dorsal root ganglia followed by prolonged swelling of the spinal cord. Swelling of the ganglion is analogous to the rapid mechanical change observed in invertebrate axons during excitation. The mechanical change observed in the spinal cord is probably related to prolonged depolarization of the primary afferent fibers near their terminals.     

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.     

GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord

Multiple, dorsal rhizotomies were performed unilaterally at lumbar levels L1–L4 in adult rats. Following 24–48 h degeneration periods and fixation by intracardiac perfusions, spinal cord were removed and transversely cut into 150 μm thick sections. These sections were incubated in immunocytochemical reagents for the peroxidase-labeling of glutamic acid decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA). The sections were then prepared for electron microscopic examination, while other sections were processed for light microscopic, GAD immunocytochemistry and for Fink-Heimer staining of degenerating axons and axon terminals.Thirty-six hours following dorsal rhizotomies, the sections that were prepared for the light microscopic study of terminal degeneration showed large numbers of degenerating profiles in the ipsilateral substantia gelatinosa while degenerating profiles were virtually absent contralaterally. In electron microscopic preparations, degenerating primary afferent terminals were commonly observed at the centers of rosettes where they formed synaptic contacts with other axon terminals and with surrounding dendrites. Several types of synaptic relationships were observed in the rosettes which involved both GAD-positive and degenerating primary afferent terminals. Such synaptic relationships included those in which: (a) a single GAD-positive terminal was presynaptic to the central, primary afferent terminal, (b) two different GAD-positive terminals formed synapses with opposite sides of the same central, primary afferent terminal and were also closely apposed to the surrounding dendrites of the rosette, and (c) a GAD-positive terminal was presynaptic to a primary afferent terminal and both types of terminals were presynaptic to the same dendrite of the rosette.The synaptic relationships described in this study are discussed with respect to their possible functional roles in such GABA-mediated phenomena as: (a) primary afferent depolarization, (b) the dorsal root reflex and (c) primary afferent hyperpolarization. Our observations support the concept that GABAergic axon terminals are involved in the synaptic circuits which produce presynaptic inhibition and presynaptic facilitation of the primary afferent input to the dorsal spinal cord. Collectively the observed synaptic relationships could provide a morphological substrate that is compatible with an inhibitory surround system in the substantia gelatinosa.     

Morphine acts via mu-opioid receptors to enhance spinal regeneration and synaptic reconstruction of primary afferent fibers injured by sciatic nerve crush

The present study investigated whether morphine can promote regeneration and synaptic reconstruction of the terminals of injured primary afferent fibers in lamina II of the spinal cord in rats following sciatic nerve injury. Fluoride-resistant acid phosphatase (FRAP)-positive terminals in lamina II of the L4 spinal segment after sciatic nerve injury were assessed after treatment with vehicle, morphine, and naloxone plus morphine. Under the electron microscope, types I and II complex terminals of unmyelinated afferent fibers from the dorsal root, simple terminals of interneuronal axons, and terminals of descending axons at lamina II of the L4 spinal segment were documented in the different groups after injury. FRAP-positive terminals in lamina II were depleted after sciatic nerve injury in the vehicle group. Treatment with morphine increased the numbers of FRAP-positive terminals, and this was prevented by naloxone. The present study demonstrates that morphine may promote the regeneration and synaptic reconstruction of the terminals of injured primary unmyelinated afferent fibers in lamina II of spinal cord, by a process mediated by mu-opioid receptors.     

Propofol enhances GABA(A) receptor-mediated presynaptic inhibition in human spinal cord

Although the function of somatodendritic GABAA receptors is augmented by propofol, it is not known whether presynaptic GABAA receptor function is similarly affected. In the present study, we examined the action of propofol on the second positive wave (P2 component) of segmental spinal cord evoked potentials (seg SCEPs), which is believed to reflect GABAA receptor-mediated presynaptic inhibition of primary afferent terminals and can be recorded from spinal epidural space in man. In all seven patients tested while undergoing scoliosis surgery, a clinical dose of propofol (1 mg//kg, i.v.) significantly augmented the P2 component of seg SCEPs evoked by ulner nerve stimulation. We conclude that propofol enhances GABAA receptor-mediated presynaptic inhibition at primary afferent terminals in human spinal cord.     

Origins of spontaneous and noxious stimuli-evoked miniature EPSCs in substantia gelatinosa

The origins of spontaneous and noxious stimuli-evoked glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in substantia gelatinosa (SG) neurons were investigated by using whole-cell voltage-clamp technique on adult rat spinal cord slice. The properties of mEPSCs of SG neurons from rats either neonatally capsaicin-treated or sciatic nerve ligated showed no difference from those of intact SG neurons, indicating independence of spontaneous mEPSCs on primary afferent fibers. In the presence of tetrodotoxin (TTX), capsaicin, which noxiously stimulated fine primary afferent fibers, caused increase of the mEPSCs frequency, but did not affect the amplitude profiles or mean amplitudes. TTX affected neither the spontaneous mEPSCs nor capsaicin-induced mEPSCs frequency increase. The results suggest that spontaneous mEPSCs in SG are mediated by presynaptic spontaneous glutamate release predominantly originating from interneuron terminals rather than from primary afferent terminals; under noxious stimulation, however, mEPSCs frequency increase is mediated by primary afferent excitation.     

Unmyelinated primary afferent fiber stimulation depletes dynorphin A (1-8) immunoreactivity in rat ventral horn.

The present study demonstrates many dynorphin (DYN)-immunoreactive fibers and presumed presynaptic terminals in rat lumbar ventral horn. The fibers and terminals seem to arise largely from DYN-containing intrinsic neurons in the dorsal horn. The majority of the presumed terminals closely surround a subpopulation of motoneurons that tend to be located in flexor motoneuron columns. Acute C fiber, but not A fiber, primary afferent stimulation depletes the ventral horn DYN immunostaining. We interpret these findings to indicate that the spinal DYN neurons are well positioned to serve both as modulators of nociceptive input and as interneurons in motor reflexes. We further hypothesize that the depletion of DYN-immunoreactivity that follows either acute C fiber stimulation or intense nociceptive stimuli may be the trigger for the upregulation in spinal cord DYN that occurs in models of chronic pain states.     

Mu and delta opioid receptor-like immunoreactivity in the cervical spinal cord of the rat after dorsal rhizotomy or neonatal capsaicin: an analysis of pre- and postsynaptic receptor distributions

Opioid compounds have powerful analgesic properties when administered to the spinal cord. These effects are exerted through mu and delta opioid receptors, and both pre- and postsynaptic mechanisms have been implicated. To specifically address the relative pre- and postsynaptic contribution to spinal opioid analgesia, we have quantitatively assessed the pre- vs. postsynaptic distribution of the mu-opioid (MOR-1, MOP₁) and delta-opioid receptors (DOR-1, DOP₁). We also examined the rostro-caudal arborization of MOR-1 and DOR-1 immunoreactive primary sensory neurons, using an isolated dorsal root preparation. These results were compared to those obtained by labeling for calcitonin gene-related peptide (CGRP), a neuropeptide whose expression in the spinal cord is restricted to the terminals of small diameter primary sensory neurons. We estimate that approximately one half of MOR-1 and two thirds of DOR-1 immunoreactivity in the cervical spinal cord is located on primary afferent fibers. These fibers have a broad rostro-caudal distribution, extending at least three segments rostral and caudal to their segment of entry. Regardless of marker used, the rostral projection was greatest, however, the distribution of CGRP-immunoreactive fibers differed somewhat in that they had a much smaller projection to the most caudal segments examined. Our results suggest that presynaptic delta opioid actions predominate, but that there are mixed pre- and postsynaptic inhibitory effects exerted by opioid analgesics that act at the spinal cord mu opioid receptor.     

The role of 5-HT, GABA and opioid peptides in presynaptic inhibition of tooth pulp input from the medial brainstem

In decerebrate or chloralose-anaesthetized cats electrical stimulation in the spinal trigeminal nucleus evoked antidromic responses in the mandibular canine tooth pulp. Conditioning stimulation in nucleus raphe magnus (NRM) and in the adjacent contralateral medullary reticular formation, nucleus reticularis gigantocellularis (NRGC) and nucleus reticularis magnocellularis (NRMC), produced a decrease in the threshold for the antidromic responses in a proportion of the tooth pulp inputs. This was interpreted as being due to depolarization of the tooth pulp afferent terminals, reflecting presynaptic inhibition. The primary afferent depolarization (PAD) of tooth pulp afferent terminals by NRM stimulation could be selectively blocked by bicuculline applied intravenously or by iontophoresis in the terminal region. Intravenous naloxone, cinanserin and methysergide had no effect on the PAD evoked from NRM, NRGC or NRMC. Thus NRM appears to exert presynaptic inhibitory control of A delta tooth pulp input to the spinal trigeminal nucleus via GABA-containing neurones.     

Inhibitory amino acid transmitters associated with axons in presynaptic apposition to cutaneous primary afferent axons in the cat spinal cord

The purpose of the present study was to characterize the transmitter content of structures in presynaptic apposition to the central terminals of cutaneous afferent fibers in the dorsal horn of the spinal cord. Axons in the Aalphabeta conduction velocity range were identified in adult cats, stained intra-axonally with horseradish peroxidase, and prepared for combined light and electron microscopy. In total, we labeled two slowly adapting (Type 1) axons, two hair-follicle afferents, and one rapidly adapting (Krause) afferent. Ninety-nine labeled boutons were examined through complete series of serial sections. Approximately 80% of boutons originating from rapidly adapting and hair-follicle afferents were postsynaptic to other axons, but only 50% of boutons from slowly adapting axons were associated with this type of arrangement. Postembedding immunogold reactions revealed that between 80% (for slowly adapting axons) and 100% (for rapidly adapting axons) of boutons presynaptic to primary afferents were immunoreactive for gamma-aminobutyric acid (GABA). The vast majority of these terminals (in excess of 80%) were also enriched with glycine. Therefore, presynaptic inhibition of these three functional classes of Aalphabeta cutaneous primary afferents is mediated principally by the subgroup of GABAergic interneuron that also contains glycine.     

Peripheral nerve injury induces reorganization of galanin-containing afferents in the superficial dorsal horn of monkey spinal cord

Peripheral nerve injury-induced structural and chemical modifications of the sensory circuits in the dorsal horn of the spinal cord contribute to the mechanism of neuropathic pain. In contrast to the topographic projection of primary afferents in laminae I-IV in the rat spinal cord, the primary afferents of Macaca mulatta monkeys almost exclusively project into laminae I-II of the spinal cord. After peripheral nerve injury, up-regulation of galanin has been found in sensory neurons in both monkey and rat dorsal root ganglia. However, the nerve injury-induced ultrastructural modification of galanin-containing afferents in the monkey spinal cord remains unknown. Using immunoelectron microscopy, we found that 3 weeks after unilateral sciatic nerve transection, the number of galanin-containing afferents was increased in ipsilateral lamina II of monkey spinal cord. Branching of these galanin-positive afferents was often observed. The afferent terminals contained a large number of synaptic vesicles, peptidergic vesicles and mitochondria, whereas the number of synapses was markedly reduced. Some of the afferents-enriched microtubules were often packed into bundles. Moreover, galanin-labeling could be associated with endosomal structures in many dendrites and axonal terminals of dorsal horn neurons. These results suggest that peripheral nerve injury induces an expansion of the central projection of galanin-containing afferents in lamina II of the monkey spinal cord, not only by increasing galanin levels in primary afferents but also by triggering afferent branching.     

Direct observations of synapses between GABA-immunoreactive boutons and muscle afferent terminals in lamina VI of the cat''s spinal cord

Single group Ia muscle afferent fibers in the lumbar spinal cord of the cat impaled with microelectrodes and labelled with horseradish peroxidase. Two collateral axons were prepared for combined light and electron microscopy. Arbors selected from lamina VI were processed by the postembedding immunogold technique with antiserum which specifically recognizes GABA in glutaraldehyde-fixed tissue. Twelve Ia boutons were examined through series of thin sections with the electron microscope and all of them were associated with presynaptic axon terminals which were positively labelled for GABA. Some Ia boutons received synaptic contacts from several GABAergic terminals. The present study establishes that a GABA-like substance is present in axon terminals presynaptic to Ia afferent boutons in lamina VI of the spinal cord. This evidence provides a morphological basis for presynaptic inhibition of Ia afferent input into lamina VI.     

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.     

Light microscopic and ultrastructural analysis of GABA-immunoreactive profiles in the monkey spinal cord

It is hypothesized that terminals containing gamma-aminobutyric acid (GABA) participate in presynaptic inhibition of primary afferents. To date, few convincing GABA-immunoreactive (GABA-IR) axo-axonic synapses have been demonstrated in support of this theory. The goal of this study is to document the relationship between GABA-IR profiles and central terminals in glomerular complexes in lumbar cord of the monkey (Macaca fascicularis). In addition, the relationship between GABA-IR profiles and other neural elements are analyzed in order to better understand the processing of sensory input in the spinal cord. GABA-IR cell bodies were present in Lissauer's tract (LT) and in all laminae in the spinal gray matter except lamina IX. GABA-IR fibers and terminals were heavily concentrated in LT; laminae I, II, and III; and present in moderate concentration in the deeper laminae of the dorsal horn, ventral horn (especially in association with presumed motor neurons), and lamina X. Electron microscopic analysis confined to LT and laminae I, II, and III demonstrated GABA-IR cell bodies, dendrites, and myelinated and unmyelinated fibers. GABA-IR cell bodies received sparse synaptic input, some of which was immunoreactive for GABA. The majority of the synaptic input to GABA-IR neurons occurred at the dendritic level. Furthermore, the presence of numerous vesicle-containing GABA-IR dendrites making synaptic interactions indicated that GABA-IR dendrites also provided a major site of output. Two consistent arrangements were observed in laminae I-III concerning vesicle-containing GABA-IR dendrites: 1) they were often postsynaptic to central terminals and 2) they participated in reciprocal synapses. The majority of GABA-IR axon terminals observed contained round clear vesicles and varying numbers of dense core vesicles. Only on rare occasions were GABA-IR terminals with flattened vesicles observed. GABA-IR terminals were not observed as presynaptic elements in axo-axonic synapses; however, on some occasions, GABA-IR profiles presumed to be axon terminals were observed postsynaptic to large glomerular type terminals. Our findings suggest that a frequent synaptic arrangement exists in which primary afferent terminals relay sensory information into a GABAergic system for further processing. Furthermore, GABA-IR dendrites appear to be the major source of input and output for this inhibitory system. The implications of this GABAergic neurocircuitry are discussed in relation to the processing of sensory input in the superficial dorsal horn and in terms of mechanisms of primary afferent depolarization (PAD).     

Ultrastructural study of glutamate- and GABA-immunoreactive terminals contacting the primary afferent fibers in frog spinal cord. A double postembedding immunocytochemical study

The in vitro HRP application to the dorsal root of the frog spinal cord produced an intensive staining of primary afferent fibers. A double postembedding GABA and glutamate immunocytochemical study revealed GABA- or glutamate-immunopositive presynaptic boutons establishing axo-axonic synapses onto HRP-stained primary afferent fibers in the spinal cord intermedial zone.