Terminals Of Primary Afferent Fibers Research Articles

GABA-B receptor-mediated spinal inhibition.

The intravenous administration to pentobarbitone-anaesthetized cats of the GABA-B antagonists CGP 46381 and CGP 35348 blocks the longer duration component of the inhibition of lumbar extensor monosynaptic reflexes by tetanic stimulation of low threshold flexor primary afferent fibres. GABA-B receptors thus appear to be associated with this inhibitory process, in addition to bicuculline-sensitive GABA-A receptors associated with shorter duration 'presynaptic' inhibition of reflexes and the depolarization of the terminals of primary afferent fibres.

Spinal 5-HT3 receptor-mediated antinociception: possible release of GABA

Although 5-HT is clearly involved in spinal analgesia, its mode of action remains obscure, perhaps because it has multiple and often opposing effects mediated by its multiple receptor subtypes. This investigation uses selective agonists and antagonists directed at the most recently defined class of 5-HT receptors (5-HT3 receptors) in behavioral and electrophysiological studies of nociception in the spinal cord of rodents. The results demonstrate uniformly inhibitory effects of a selective 5-HT3 agonist on responses to noxious stimuli. Intrathecally administered 2-methyl 5-HT produced dose-dependent antinociception in the tail-flick test and inhibited behaviors elicited by intrathecally administered agonists for excitatory amino acid and neurokinin receptors, namely NMDA and substance P (SP). All 20 dorsal horn neurons we examined, which projected to the brain and responded to both noxious stimuli and NMDA, were inhibited in a current-related manner by this 5-HT3 agonist applied iontophoretically. Both the behavioral and electrophysiological effects were blocked not only by the 5-HT3 antagonists zacopride and ICS 205-930, but also by antagonists to the inhibitory amino acid GABA. Therefore, 5-HT via an action at 5-HT3 receptors may evoked release of GABA, which may in turn inhibit nociceptive transmission at a site postsynaptic to terminals of primary afferent fibers. If the descending serotonergic analgesic system in humans operates similarly, understanding it may enable the development of new nonopioid, nonaddictive analgesics.

Increase of blood flow in skin and spinal cord following activation of small diameter primary afferents

Activation of unmyelinated primary afferents produces vasodilation and plasma extravasation in the skin. Here, using the laser Doppler technique to measure changes in blood flow and the Evans blue technique for quantification of plasma extravasation, we have asked whether the stimulation of C-fibre precipitates the same phenomena in the spinal cord. Our results show that there is an increase of blood flow, but no extravasation in the ipsilateral lumbar enlargement of the spinal cord following supramaximal electrical stimulation of the sciatic nerve. The blood flow increases were small and short-lived compared with those seen in skin, and could be completely explained by concomitant blood pressure changes. Hence, whilst the same substances are apparently released from the peripheral and central terminals of primary afferent fibres, their ability to produce vasodilatation and extravasation is absent or severely restricted in the spinal cord.

Nerve growth factor receptors in rat spinal cord: An autoradiographic and immunohistochemical study

The distribution of nerve growth factor receptors in the lumbar spinal cord of the rat was studied with autoradiographic and immunohistochemical techniques: [ 125I]nerve growth factor and specific monoclonal antibody (Mab 192) against nerve growth factor receptor were used to localize nerve growth factor binding sites. The distributions of nerve growth factor binding sites with highest density within the superficial layers (laminae I and II) of the dorsal gray matter were virtually identical as demonstrated by these two ligands; this suggests that Mab 192 can be used as a specific probe to identify nerve growth factor receptors in rat nervous system. Nerve growth factor receptor binding sites, as demonstrated by autoradiography, were also found in longitudinal bundles of fibers running dorsolaterally in the lateral funiculus. However, no immunoreactivity was detected in these areas by immunohistochemistry. No specific binding was found in the dorsal horn when [ 125I]nerve growth factor was co-injected with unlabeled nerve growth factor or after incubation with nonspecific monoclonal antibody. Dorsal root section produced a complete loss of nerve growth factor-specific labeling pattern throughout laminae I–II of the spinal cord. This suggests that nerve growth factor receptors are localized on the nerve terminals of primary afferent fibers which synapse in the region of the spinal cord. The presence of nerve growth factor binding sites in the dorsal horn of the spinal cord is consistent with the possibility that nerve growth factor, or a nerve growth factor-like substance, derived from the central nervous system, may have a role in trophic support of dorsal root ganglion neurons.

Mystacial vibrissae representation within the trigeminal sensory nuclei of the cat

Somatotopic arrangements of axon terminals of primary afferent fibers innervating follicles of the mystacial vibrissae were examined in the cat by the transganglionic horseradish peroxidase (HRP) method. Forty to 60 hours after injecting HRP into a single or a group of vibrissal follicles, transported HRP was visualized by the tetramethylbenzidine technique. HRP-labeled axon terminals were distributed in the ventral subnucleus of the principal sensory trigeminal nucleus (ventral Vp), in the oral and interpolar spinal trigeminal nuclei (Vo and Vi), and in the caudal spinal trigeminal nucleus (Vc) (layer I, deep part of layer II, layers III-V) with its spinal extension into the dorsal horn of the first cervical cord segment (rostral C1). In cross sections through the caudal parts of the ventral Vp, Vi, and layer IV of the Vc and rostral C1, a single mystacial vibrissa was represented in a one-to-one fashion by a patch of dense terminal arbors of primary afferent fibers. The more dorsally a horizontal row of the mystacial vibrissae was located, the more ventrally was it represented in the ventral Vp, the more ventrolaterally in the Vi, and the more ventrally in layer IV of the Vc and the rostral C1. In addition, the more anteriorly a vibrissa was located in a horizontal row of the mystacial vibrissae, the more medially was it represented in the ventral Vp, the more ventromedially in the Vi, and the more laterally in layer IV of the Vc and rostral C1; the most posteriorly located vibrissae in the horizontal rows of the mystacial vibrissae were represented along the lateral border of the ventral Vp and Vi, and most medially in layer IV of the Vc and rostral C1. Thus, the representation pattern in the ventral Vp was rotated clockwise at about 45 degrees angle in the Vi, and projected as a mirror image in layer IV of the Vc and rostral C1. It was also indicated that the anterior-posterior arrangement of the mystacial vibrissae was represented in a rostral-caudal organization within layer IV of the Vc and rostral C1. It was also indicated that the anterior-posterior arrangement of the mystacial vibrissae was represented in a rostral-caudal organization within layer IV of the Vc and rostral C1. Patchy patterns probably replicating the distribution of the vibrissae on the face of the cat were also revealed by the cytochrome oxidase histochemical staining in cross sections through the caudal parts of the ventral Vp, Vi, and layer IV of the Vc and rostral C1.

Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: A possible mechanism for neuropeptide release

It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70–130nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P < 0.01) on the ipsilateral side 1–24 h after a unilateral skin lesion in the vibrissae area. A second increase (P < 0.001) occurred 14–15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.

On the probable absence of GABA receptors on the terminations of motor axon collaterals in the cat spinal cord.

When administered microelectrophoretically, GABA and the GABA-mimetic piperidine-4-sulphonic acid (P4S) appear to have no direct hyperpolarizing or depolarizing effect on the terminations of motor axon collaterals excited electrically in the ventral horn of the lumbar spinal cord of the cat. This lack of effect on axon terminals of motoneurones, which contrasts with the bicuculline-sensitive depolarization by P4S of the spinal terminals of primary afferent fibres, is consistent with previous reports of the probable absence of pharmacologically detectable GABA receptors on the spinal terminals of other central excitatory neurones, namely those of the red and lateral vestibular nuclei.

Somatotopical arrangements of the axon terminals of primary afferent fibers innervating the mystacial vibrissae in the cat

Somatotopical arrangements of the axon terminals of primary afferent fibers innervating the mystacial vibrissae in the cat

Antispasticity drugs: mechanisms of action.

Several different drugs are now used, or are potentially useful, to treat patients with spasticity. Although these compounds vary in their actions on spinal neurons and reflex arcs, it is possible to formulate reasonable hypotheses regarding their modes of action. The benzodiazepines bind to specific benzodiazepine receptors linked to classic gamma-aminobutyric acid (GABA) receptors located on the terminals of primary afferent fibers. This binding results in an increased affinity of the GABA receptor for the amino acid, an augmented flux of chloride ions across the terminal membrane, and an increase in the amount of presynaptic inhibition. Baclofen activates GABAB receptors putatively located on the same terminals. Activation of these receptors retards the influx of calcium ions into the terminals, thereby reducing the evoked release of excitatory amino acids and possibly other transmitters. Progabide and its metabolites act on both classic and GABAB receptors. Glycine works on specific inhibitory receptors located on spinal interneurons and motoneurons. The phenothiazines act on the brainstem to alter the function of fusimotor fibers. Phenytoin and carbamazepine reduce the afferent output of muscle spindles. Dantrolene diminishes the activation of the contractile process in muscle fibers by reducing the release of calcium ions from the sarcoplasmic reticulum. This review summarizes the data supporting these concepts.

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord.

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.

Neuronal 5-HT receptors in the periphery

5-Hydroxytryptamine (5-HT) induces responses in neurones from all branches of the mammalian peripheral nervous system. Responses may be excitatory or inhibitory and are mediated through at least four distinct receptor sites. One receptor mediates excitation in motoneurones and preganglionic sympathetic neurones and can be designated a D (or possibly 5-HT 2) receptor since “classical” antagonists such as methysergide, metergoline or cinanserin are potent and selective antagonists at this site. A second receptor mediating neuronal excitation can be positively identified on the basis of susceptibility to blockade by small concentrations of 1αH,3α,5αH-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) and the weak or negligible affinity, relative to 5-HT, of certain agonists such as 5-methoxytryptamine. Such sites mediate depolarization of sympathetic and parasympathetic neurones and excitation of both the cell bodies and terminals of primary afferent fibres. A third receptor, mediating neuronal excitation, is the classical M-receptor of Gaddum and Picarelli, at this stage clearly identified only on postganglionic parasympathetic neurones of the guinea-pig myenteric plexus. These sites can be differentiated from other excitatory 5-HT receptors since MDL 72222 is neither potent nor selective as an antagonist and 5-methoxytryptamine approaches the potency of 5-HT as an agonist. (3α-Homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid is a potent, surmountable antagonist of 5-HT at the M-receptor of the ileum, but is non-selective. Neuronal inhibitory responses have been observed using electrophysiological techniques or by monitoring the decrease in depolarization-evoked release of transmitter in enteric, parasympathetic and sympathetic neurones. Largely negative results, using selective agonists and antagonists, allow the receptor(s) mediating inhibition to be clearly differentiated from the three neuronal excitatory receptors for 5-HT. Comparison of relative potencies of agonists suggests similarities with the 5-HT 1 recognition site of the central nervous system; no selective antagonist has yet emerged to permit their positive identification.

The effect of GABA on lumbar terminations of rubrospinal neurons in the cat spinal cord.

Although GABA and piperidine-4-sulphonic acid depolarize I a afferent terminations in the cat spinal cord by activation of bicuculline-sensitive GABA receptors, no evidence was obtained for a bicuculline-sensitive alteration by either gabamimetic of the electrical threshold of rubrospinal terminations in the spinal intermediate nucleus. The terminal axonal arborizations in the spinal cord of neurons in the red nucleus thus do not have GABA receptors similar to those on the cell bodies. The results are discussed in relation to the depolarizing action of GABA on some central neurons, and on neurons with peripheral cell bodies, and to probable differences in the intracellular chloride content of neurons having peripheral or central cell bodies, and thus of different embryological origin. A presynaptic depolarizing inhibitory process mediated by GABA appears to be confined to the terminals of primary afferent fibres in the mammalian central nervous system.

Intraneuronal substance P contributes to the severity of experimental arthritis.

There is evidence that substance P is a peptide neurotransmitter of some unmyelinated primary afferent nociceptors and that its release from the peripheral terminals of primary afferent fibers mediates neurogenic inflammation. The investigators examined whether substance P also contributes to the severity of adjuvant-induced arthritis, an inflammatory disease in rats. They found that, in the rat, joints that developed more severe arthritis (ankles) were more densely innervated by substance P-containing primary afferent neurons than were joints that developed less severe arthritis (knees). Infusion of substance P into the knee increased the severity of arthritis; injection of a substance P receptor antagonist did not. These results suggest a significant physiological difference between joints that develop mild and severe arthritis and indicate that release of intraneuronal substance P in joints contributes to the severity of the arthritis.

Is ATP a central synaptic mediator for certain primary afferent fibers from mammalian skin?

The possibility that ATP acts as a synaptic mediator at the central terminals of primary afferent fibers was examined by applying it iontophoretically to neurons of the outer layers of the cat spinal cord in vivo. ATP proved to be selectively excitatory for a limited subset of spinal neurons. Those units consistently excited by ATP iontophoresis with very small currents (2-15 nA) responded to gentle mechanical stimulation of the skin and usually evidenced excitatory input from unmyelinated primary afferent fibers. Most units excited by ATP were specifically mechanoreceptive; a few neurons receiving excitatory input from both low-threshold mechanoreceptors and nociceptors also responded to ATP. Selectively nocireceptive neurons were unresponsive. Generally, the mechanoreceptive neurons excited by ATP were located in the deeper substantia gelatinosa or in the immediately adjacent nucleus proprius of the dorsal horn. The results suggest the presence of a purinergic excitatory receptor on central neurons receiving excitatory projection from tactile mechanoreceptors with fine-diameter afferent fibers and are consistent with the possibility that an ATP-like agent may mediate central synaptic excitation for this set of sense organs.

Laminar-related projection of primary trigeminal fibers in the caudal medulla demonstrated by transganglionic transport of horseradish peroxidase

The mode of termination of primary afferent fibers within the cat trigeminal nucleus caudalis was investigated by means of the transganglionic transport of horseradish peroxidase (HRP). Several types of laminar-related labeling were observed, depending upon the survival time after HRP application. At the earliest survival time (28–34 h) the highest density of labeling was found in laminae I and II. At 2 and 3 days survival laminae III and IV were heavily labeled, in addition to laminae I and II where the amount of labeling was greatly increased in lamina I, but not in lamina II. At 5 days survival time an abrupt drop of labeling occurred in laminae I and II, while this pattern was not predominant in laminae III and IV. In lamina V the pattern of labeling was less intense and not changeable through all survival times observed. These findings indicating a differentiation of the primary afferent terminals have good correspondence with a functional specialization of neuronal locations since the functional properties of neurons vary according to their locations.

Catecholamine effects on frog dorsal root terminals

Dopamine, norepinephrine and epinephrine applied to the isolated superfused frog spinal cord had complex effects on the terminals of primary afferent fibers. The most cosistent finding was a slow hyperpolarization of terminals with lower concentrations (10 μM or lower), but depolarization either following or admixed with the hyperpolarization were seen. These were particularly prominent when the catecholamines were applied in high concentrations or for prolonged periods of time. A part of the response of afferent terminals appears to be indirect since the potential changes were reduced following exposure of the cord to tetrodotoxin, Mn 2+, or mephenesin. The hyperpolarizations were augmented by imipramine, a known inhibitor of catecholamine uptake. These observations are consistent with a role of catecholamines in the processing of sensory input in the spinal cord.

Distribution of terminals of primary afferent fibers connected polysynaptically with motoneurons in the frog spinal cord

Parallel intracellular recordings of potentials in primary afferent fibers (in the region of their entry into the spinal cord) and motoneurons were made in experiments on an isolated perfused preparation of frog spinal cord preserving its connections with hind limb nerves. It was shown by injection of horseradish peroxidase through a microelectrode inserted into the fiber that fast-conducting cutaneous, tendon, and muscular afferents connected polysynaptically with motoneurons reach only the upper or middle third of the dorsal horn. Terminal branches of these fibers are characterized by numerous short terminal twigs given off at short distances apart from larger collaterals. Terminal boutons and en passant contacts, stained with horseradish peroxidase, were found on bodies of interneurons. In some cases, trans-synaptic staining of interneurons was found to take place. It is suggested that peroxidase-labeled interneurons form axo-axonal synapses with primary afferents.

GABA ‘desensitization’ of frog primary afferent fibers

GABA (γ-aminobutyric acid) depolarizes the terminals of primary afferent fibers of the in vitro hemisected frog spinal cord. During sustained or repetitive exposure to GABA or to muscimol, the amplitude of the depolarization is characterized by a rapid and exponential decline to a steady plateau level (desensitization). Desensitization to muscimol was eliminated by removal of Ca 2+ and addition of Mn 2+ to the superfusate — a finding consistent with the presence of ‘receptor’ (‘true’) desensitization (i.e., receptor inactivation). GABA desensitization was significantly reduced by exposure of the cord to either low Na +, low temperature, ouabain, dinitrophenol, (±)-nipecotic acid, or cis-1,3-aminocyclohexanecar☐ylic acid. These treatments also significantly decreased the high affinity uptake of GABA when the latter process was studied by incubating frog spinal slices in Ringer's solution containing a low concentration of [ 3H]GABA. These results suggest that cellular transport processes can influence the form of GABA responses and indicate that neuronal removal of GABA is responsible in part for GABA desensitization.

Effects of intravenously administered enantiomers of baclofen on functionally identified units in lumbar dorsal horn of the spinal cat

The present study investigated the effects of intravenous administration of the d- and l-enantiomers of baclofen on extracellular single unit spikes recorded from the lumbar dorsal horn of the spinalized cat, either anaesthetized with chloralose or decerebrated. Both enantiomers had two types of depressant effect. One was a transient (complete within 2 min), non-specific depression of the discharge of synaptically-elicited responses and of the postsynaptic glutamate-evoked excitation; it is reasoned that this effect was due to a postsynaptic action of baclofen and its antagonism by bicuculline suggests it is via classical GABA receptors. The other type of depression was more prolonged, lasting usually for more than 1 hr. It was observed as a preferential blocking of spontaneous activity, as well as of evoked responses to noxious stimuli or innocuous thermal stimuli applied to the skin, with little or no effect on responses to hair movement or to the iontophoretic application of glutamate. In addition, baclofen preferentially reduced the late, higher threshold component of evoked responses of wide dynamic range neurones to electrical stimulation of the cutaneous receptive field. Thus, whether tested on naturally- or electrically-evoked responses, baclofen had a preferential effect on those mediated by small diameter afferents. Doses giving approximately equal effects were 0.1 mg/kg for l-baclofen and 10 mg/kg for d-baclofen. The results of the present study support recent evidence of a prolonged antinociceptive effect of baelofen, indicate that at least some of this effect is mediated by an action at the spinal level and support the possibility that the action is presynaptic, perhaps on the terminals of primary afferent fibres. In addition, a similar mechanism may account for the depression of spinal polysynaptic reflexes and thus, perhaps, additionally for some of the antispastic properties of baclofen.

Pharmacological studies on the prolonged depressant effects of baclofen on lumbar dorsal horn units in the cat

The pharmacological basis of the prolonged depressant effect of baclofen on small diameter afferent inputs to dorsal horn neurons was studied because of the possibility that this depression represents, at the cellular level, the mechanism of the analgesic effects of baclofen. When l-baclofen (0.1–3.0 mg/kg, i.v.) was given, the ensuing prolonged depression could not be mimicked or modified by the GABA-mimetic drug, muscimol (0.5–5.0 mg/kg, i.v.). Administration of the GABA antagonist, bicuculline (1–5 mg/kg, i.v.), partially reversed the depression induced by baclofen but even after large doses of bicuculline, subsequent administration of baclofen produced depression. The opiate antagonist naloxone (0.1–0.3 mg/kg, i.v.) and the glycine antagonist strychnine (0.1–0.2 mg/kg, i.v.) failed to block the prolonged depression induced by baclofen. These results suggest that baclofen is acting in the dorsal horn via a mechanism which excludes opiate receptors and glycine receptors and which only partially includes bicuculline-sensitive GABA receptors. The results are consistent with a possible action of baclofen on the recently proposed ‘novel GABA receptor’, or GABA B receptor, which is insensitive to bicuculline; if this indeed proves to be the case, then the present results suggest that at least in the spinal cord, these receptors are associated differentially with small diameter afferent fibres. However, regardless of the receptors involved, the most likely effect of baclofen is to reduce the amount of transmitter (or modulator) released from terminals of primary afferent fibres, and evidence is examined implicating glutamate and substance P. The transient, non-specific depressant effect (described in more detail in another publication) was prevented by bicuculline, and thus its effects are likely to be mediated by bicuculline-sensitive GABA receptors.