Transient synapses in the embryonic primate spinal cord.

Electron microscopic and tritiated thymidine autoradiographic analysis of the embryonic spinal cord in the rhesus monkey reveals considerable rearrangement of cellular and synaptic relationships in the posterior (sensory) quadrant during early developmental stages. This remodeling involves the death of an entire population of neurons that received synapses from sensory afferent axons and the possible relocation of these afferents upon subsequently generated viable substantia gelatinosa neurons.

The kynurenate analog SZR-72 prevents the nitroglycerol-induced increase of c-fos immunoreactivity in the rat caudal trigeminal nucleus: comparative studies of the effects of SZR-72 and kynurenic acid.

Administration of nitroglycerol in a migraine model results in an increased number of c-fos-expressing secondary sensory neurons in the caudal trigeminal nucleus. Since synapses between first- and second-order trigeminal neurons are mediated by excitatory amino acids, NMDA receptors are inhibited by kynurenic acid, though this crosses the blood-brain barrier only poorly. Systemic treatment of rats with SZR-72, a newly synthetized kynurenic acid analog, diminished the nitroglycerol-induced increase of c-fos immunoreactivity in the brain stem highly significantly, while treatment with kynurenic acid resulted in a significantly smaller decrease, proving that SZR-72 is much more effective than kynurenic acid.

Primary afferent origin of substance P-containing axons in the superficial dorsal horn of the rat spinal cord: depletion, regeneration and replenishment of presumed nociceptive central terminals.

Substance P-like immunoreactivity (SPLI) was localized in the superficial spinal dorsal horn of the rat by means of light and electron microscopic immunocytochemical techniques. Serial immunocytochemical sections were subjected to densitometric measurements with an electronic Image Analyser, and with aid of a computer program, a two-dimensional reconstruction of the fine neuroanatomical structure of the SPLI-active regions of the lumbosacral upper superficial spinal dorsal horn was obtained. SPLI activity in the superficial dorsal horn outlines four well-marked and distinctly differing regions, called, in the mediolateral sequence, areas A, B, C, and D, plus Cajal's noyeau interstitiel ("lateral spinal nucleus" = "nucleus of the dorsolateral fascicle," L). Lumbosacral dorsal rhizotomy results in an almost complete depletion of SPLI from ipsilateral areas A, B, C, and D; it induces decreased SPLI in the area of the lateral spinal nucleus (L), ipsi- or contralaterally in an alternating fashion. Transection of the segmentally related, ipsilateral peripheral nerve induces a marked depletion of SPLI from areas A, B, and C but only a slight decrease in area D and virtually none in the area of L. Whereas a simple crush of the peripheral nerve (axocompression) induces only a slight depletion of SPLI, if any, semiautomatic densitometric analysis of serial immunocytochemical sections proves that a controlled crush injury (axocontusion) results in depletion of SPLI from the upper dorsal horn, similar to transection of the peripheral nerve. Following regeneration of the ipsilateral, segmentally related peripheral nerve, the original immunocytochemical structure of the superficial dorsal horn is re-established by SPLI-positive axonal sprouts originating from previously damaged dorsal root axons.

Ultrastructure of normal and degenerating glomerular terminals of dorsal root axons in the substantia gelatinosa of the rhesus monkey.

The fine structure of primary sensory terminals within glomerular complexes of lamina II of Rexed (substantia gelatinosa Rolandi) in the spinal cord was investigated in normal adult rhesus monkeys and in monkeys subjected to thoracic or lumbosacral dorsal root transection. Three types of "scalloped" primary sensory terminals were distinguished on the basis of their ultrastructural characteristics, size, and distribution of synaptic vesicle population: (1) dense sinusoid axon (DSA) terminals contain medium-sized (42--46 nm and 58--62 nm) and large (80 nm) clear synaptic vesicles; (2) large dense-core vesicles (LDCV) terminals are equipped with empty synaptic vesicles ranging from 30 to 106 nm, large, (80 nm) and very large, (100 nm) dense-core vesicles; and (3) regular synaptic vesicles (RSV) terminals contain a homogeneous population of 45--50 nm clear synaptic vesicles. Following transection of the dorsal roots, all three types of primary afferents degenerate and become engulfed and phagocytosed by glial cells. However, each type of terminal displays a different mode and tempo of degeneration as seen in monkeys sacrificed 36, 48, and 72 hours following rhizotomy. DSAs follow the osmiophilic degeneration pattern; LDCVs are characterized by a gradual increase in the number of "electron-dense bodies" and, less frequently, by a progressive osmiophilic process; RSVs exhibit signs of a filamentous degeneration, accompanied by clusters of synaptic vesicles. The three types of terminals are distributed in an overlapping but distinct pattern within the posterior horn. Thus DSAs are present in highest numbers in lamina II where they constitute the most frequent terminal type. LDCVs also occur in lamina II in its outer half but are most concentrated in lamina I. RSVs predominate in the deeper layers of the dorsal horn (lamina III) but are also found in the internal half of lamina II. On the basis of these ultrastructural data and a comparison with afferent profiles impregnated according to the Golgi method, it appears that DSAs and LDCVs correspond respectively to superficial and marginal collaterals of small, thin dorsal root fibers whereas RSVs represent terminals of deep collaterals from large, thick dorsal root axons.

Periterminal synaptology of dorsal root glomerular terminals in the substantia gelatinosa of the spinal cord in the rhesus monkey.

The synaptic glomerular complexes surrounding dorsal root terminals in the substantia gelatinosa were reconstructed from six sets of 50--140 gapless ultrathin serial sections prepared in the transverse plane of the spinal cord in adult rhesus monkeys. All three types of glomerular terminals described in the preceding paper (Knyihar-Csillik et al., '82) were identified: (1) DSA (endings of superficial collaterals); (2) LDCV (endings of marginal collaterals); and (3) RSV (endings of deep collaterals). Each type of terminal forms a glomerular complex which invariably includes presynaptic dendrites which are intercalated between primary terminals and the postsynaptic (conventional) dendrite. Since the latter also receives direct input from the primary sensory terminal the synaptic organization assumes triadic arrangements, suggesting that primary afferent impulses may be subjected to a postsynaptic modulation through inhibitory action of presynaptic dendrites. In glomeruli with DSA as the central element, several triadic systems are usually interrelated, possibly as a structural basis for prolonged retardation of impulses. Adjacent glomeruli, containing DSA and LDCV terminals, are coupled together by a series of triadic systems fed by DSA terminals enabling association between superficial and marginal collaterals. RSV terminals are presynaptic to somata and dendrites of substantia gelatinosa cells that presumably exert inhibition upon terminals of all three kinds of primary sensory collaterals. In addition RSV terminals are postsynaptic to numerous F boutons which presumably derive mainly from axons of substantia gelatinosa cells; similar F boutons impinge upon presynaptic and other dendrites surrounding DSA terminals. The complicated but orderly synaptic architecture of these types of primary afferents may be regarded as a structural basis for first-order analysis and modulation of the nociceptive information within the primate central nervous system.

Reversibility of microtubule inhibitor-induced transganglionic degenerative atrophy of central terminals of primary nociceptive neurons.

Microtubule inhibitor Vinca alkaloids applied around a peripheral nerve induce transganglionic degenerative atrophy of the central terminals of primary nociceptive neurons. This effect is reversible: 40-50 days later the original histochemical structure of the central terminals is restored. Restoration of fluoride-resistant acid phosphatase activity (the marker enzyme of primary nociceptive neurons) in the Rolando substance is due to regenerative sprouting of the formerly atrophied central terminals. Since peripherally-applied Vinca alkaloids induce transganglionic degenerative atrophy of the central terminals without inducing Wallerian degeneration of the peripheral nerve, and since this effect (virtually a synaptic uncoupling) is only temporary, this approach may be used in the treatment of otherwise intractable neuralgias without inducing irreparable alterations.

Decreased expression of kynurenine aminotransferase-I (KAT-I) in the substantia nigra of mice after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment.

Nerve cells in the substantia nigra pars compacta (SNPC) are known to express tyrosine hydroxylase (TH). By means of light and electron microscopical immunohistochemical techniques, we have shown that the dopaminergic neurons of SNPC express also kynurenine aminotransferase (KAT-I), the enzyme taking part in the formation of kynurenic acid, a neuroprotectant which is one of the endogeneous antagonists of N-methyl-d-aspartate receptors. It was also found that microglial cells and astrocytes express KAT-I. It has been shown that the highly selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), widely used as a model of Parkinson's disease (PD), affects not only TH of dopaminergic neurons in the SNPC but also their KAT-I immunoreactivity as well: MPTP treatment decreased the number and optical density of KAT-I immunoreactive SNPC neurons. Decrease of KAT-I after MPTP treatment has been proved also by Western blot analysis. MPTP also reduced KAT-I immunoreactivity of microglial cells, except for those involved in reactive gliosis, which were arranged in groups surrounding affected neurons of the SNPC; also the number of KAT-I immunoreactive (IR) astroglial cells was increased in SNPC. We conclude that MPTP treatment may have a dual effect: in addition to being deleterious for neurons expressing TH and KAT-I, it also affects glial cells which could exacerbate the neurodegenerative process characterizing PD.

Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons.

Thiamine monophosphatase (TMPase) has been selectively localized in small dorsal root ganglion cells and in their central and peripheral terminals. Light microscopic localization of TMPase, and its alterations due to transganglionic effects, are identical with those of fluoride-resistant acid phosphatase (FRAP), but are not contaminated by the ubiquitous lysosomal reaction inevitable in trivial acid phosphatase-stained sections. TMPase is inhibited by 0.1 mM NaF, which is slightly less than the concentration needed to inhibit FRAP (0.2-0.4 mM). It is assumed that TMPase and FRAP are identical enzymes. In the perikaryon of small dorsal root ganglion cells, TMPase is located in the cisterns of the endoplasmic reticulum and in the Golgi apparatus. The central terminals of these cells are scalloped (sinusoid) axon terminals, surrounded by membrane-bound TMPase activity. Central terminals outline substantia gelatinosa Rolandi throughout the spinal cord, as well as the analogous nucleus spinalis trigemini in the medulla. TMPase-active central terminals outline "faisceau de la corne postérieure" in the sacral cord, as well as Lissauer's tract in the thoracic, upper lumbar, and sacral segments, and the paratrigeminal nucleus and the terminal (sensory) nucleus of the ala cinerea in the brainstem. Peripheral terminals displaying TMPase activity are fine nerve plexuses of C fibers. The TMPase activity of the central terminals disappears after dorsal rhizotomy in the course of Wallerian degeneration, and is depleted in the course of transganglionic degenerative atrophy (after transection of the related peripheral sensory nerve). TMPase is an outstanding genuine marker for the study of transganglionic regulation in Muridae.

Distribution of calcitonin gene-related peptide in vertebrate neuromuscular junctions: relationship to the acetylcholine receptor.

Calcitonin gene-related peptide (CGRP), regarded by several authors to be involved in maintenance of the acetylcholine receptor, is present in the motor axons of various striated rat muscles. It is present, however, only in motor endplates of several selected striated muscles, where it is located in presynaptic axon terminals of neuromuscular junctions. No immunoreactivity could be seen within synaptic vesicles themselves. In the non-human primate Macaca fasciculata, neuromuscular junctions, including those in the diaphragm, display an intense CGRP reaction. The structure of the simian motor endplates is more elaborate than that of the rat. Amphibian motor nerve endings, both in tetanic and tonic muscles, display CGRP immunoreactivity. In tetanic muscles the CGRP reaction outlines "terminaisons en placque" (true motor end plates) and weakly reacting "terminaisons en grappe" (grape-like endings) in tonic muscles. On supramaximal stimulation of the motor nerve, CGRP is depleted from the affected neuromuscular junctions. Wallerian degeneration of the motor axon results in complete disappearance of CGRP. In most rat muscles in which motor endplates do not normally exhibit CGRP immunoreactivity, e.g., the diaphragm and buccinator muscles, the pre-terminal motor axons are CGRP-positive. After immobilization of such muscles by local bupivacaine injection to rats under brief chloral hydrate anesthesia, CGRP immunoreactivity of the neuromuscular junctions can be elicited because blockade of neuromuscular transmission results in accumulation of CGRP in the endplates. Even more striking is the appearance of CGRP immunoreactivity in normally non-reactive motor endplates during axon regeneration after an experimentally induced Wallerian degeneration of the motor axons. We conclude that CGRP is a regular, genotypically determined component of neuromuscular junctions, present either in a manifest or in a latent form. The latter can be elicited by various experimental approaches. The presence of CGRP in the motor endplate supports the theory that this peptide is instrumental in maintenance of the acetylcholine receptor.

Depletion of substance P, neurokinin A and calcitonin gene-related peptide from the contralateral and ipsilateral caudal trigeminal nucleus following unilateral electrical stimulation of the trigeminal ganglion; a possible neurophysiological and neuroanatomical link to generalized head pain.

Primary trigeminal neurons of the trigeminal ganglion (TG) innervate major parts of the face and head, including the dura. Electrical stimulation of the TG at specific parameters, can activate its nociceptive neurons and may serve as an experimental pain model. Markowitz [J. Neurosci. 7 (1987) 4129] reported that electrical stimulation of the trigeminal ganglion (TG) causes extravasation of plasma proteins from venules of the trigeminally innervated domain possibly due to the release of vasoactive substances. Neurogenic inflammation (vasodilatation, plasma protein extravasation, release of vasoactive peptides) in dura may serve as one of the possible pathomechanisms underlying vascular head pain [Moskowitz, Ann. Neurol. 16 (1984) 157]. We performed a unilateral electrical stimulation (7.5 Hz, 5 ms, 0.8-1.4 mA for 5 min) of the TG in rat, to induce a neurogenic inflammation in the peripheral trigeminal domain including the dura, looking for calcitonin gene related peptide (CGRP), substance P (SP) and neurokinin A (NKA) immunoreactivity (IR) in the caudal trigeminal nucleus (CTN) into which massive central trigeminal processes terminate. Here, we show patchy depletion(s) of CGRP-, SP- and NKA-IRs in the contralateral CTN of the rat in addition to their ipsilateral depletion. Such depletion is due to the release of these neuropeptides in the CTN leading to the activation of bilateral trigeminal nociceptive pathway. These data afford the possibility that under specific frequencies (which may roughly correlate to the intensity of the painful stimulus) and/or specific intensities (may correlate to specific areas of the peripheral trigeminal domain) of stimulation, activation of one side of the TG may activate bilateral trigeminal nociceptive pathway leading to the perception of an ill localized/generalized pain or headache rather than a unilateral one.

Functional immunohistochemistry of neuropeptides and nitric oxide synthase in the nerve fibers of the supratentorial dura mater in an experimental migraine model.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents.

Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater.

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF).

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.

Illusive transience of parvalbumin expression during embryonic development of the primate spinal cord.

Parvalbumin has been located by pre-embedding light- and electron microscopic immunohistochemical techniques in the spinal cords of monkey fetuses (Macaca fasciculata), ranging from E70 to E 123, and in young (P20) and young adult (3 years) Macaque monkeys. During the time window investigated, the main developmental events of parvalbumin-containing neural elements are that parvalbumin-positive dorsal root collaterals establish intercellular networks first around nerve cells of Clarke's nucleus, then in the motoneuron pool and finally in the upper dorsal horn. In each of these areas, location of the parvalbumin-positive network is gradually shifted from medial to lateral. Whenever an intercellular network is established, nerve cells innervated by parvalbumin-positive terminals of dorsal root collaterals start to express parvalbumin. Immunoreactivity of dorsal root axons is transient; it disappears first from the intercellular networks and, afterwards, also from the dorsal columns. However, the pericellular synaptic terminals and their post-synaptic nerve cells express parvalbumin into adulthood. It is concluded that some of the large (Type A) dorsal root ganglion cells are the first ones in the spinal reflex pathway to express parvalbumin, which is elicited and gradually increased in nerve cells synaptically innervated by parvalbumin-positive axon terminals. This seems to represent a specific case of activation (or desinhibiton) of the genome. Apparent "transience" of parvalbumin is due to the specific geometry of primary sensory neurons equipped with extremely long axonal processes, and the consequent specialities of axonal transport characteristics.

Lectin binding: a genuine marker for transganglionic regulation of human primary sensory neurons.

Medium-sized human dorsal root ganglion cells and their central terminals in the upper dorsal horn exert preferential affinity for Ulex europaeus I lectin. Lectin binding of glycoconjugates outlines lamina III (+IIi) and disappears after peripheral neurotomy, which renders it a useful marker to study transganglionic regulation in man.

Selective "labeling' by transsynaptic degeneration of substantia gelatinosal cells: an attempt to decipher intrinsic wiring in the Rolando substance of primates.

Transganglionic degenerative atrophy of primary sensory terminals in the head of the spinal dorsal horn that follows transection of the segmentally related peripheral sensory nerve, induces transsynaptic degeneration of substantia galatinosal (SG) cells, as was studied here in monkeys. On the basis of increased electron density as a selective "labelling', SG cells are shown to be postsynaptic both to thin (A delta and C) and to thick (A beta) afferents as well as to descending (or propriospinal) fibres, while their recurrent axon collaterals establish inhibitory synapses upon somata of neighbouring SG cells and upon A beta terminals. SG cells are envisaged as biasing elements with a contrast-enhancing function that, by means of inhibitory dendro-dendritic and axo-axonal synapses, realize a gating mechanism.

Alterations in glial fibrillary acidic protein immunoreactivity in the upper dorsal horn of the rat spinal cord in the course of transganglionic degenerative atrophy and regenerative proliferation.

Transection of a peripheral nerve induces marked increase in the glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral, segmentally related upper dorsal horn. Increase of GFAP immunoreaction is similar to, but not identical with, that observed after dorsal rhizotomy. If the peripheral nerve succeeds in regenerating, GFAP immunoreactivity in the upper dorsal horn returns to normal. It is concluded that the amount and distribution of GFAP is determined by transganglionic degenerative atrophy. Wallerian degeneration and regenerative proliferation of dorsal root axon terminals, respectively.

Treatment of chronic pain syndromes with iontophoresis of vinca alkaloids to the skin of patients.

Repeated iontophoretic administration of the microtubule inhibitors vinblastine or vincristine to the segmentally related dermatomes of patients suffering from postherpetic, trigeminal and other neuralgias permanently alleviates chronic, autochthonous pain. The beneficial effect of this therapy is probably due to transganglionic degenerative atrophy of primary central sensory terminals in the Rolando substance by blockade of retrograde axoplasmic transport in sensory nerves.

Vasoactive intestinal polypeptide in dorsal root terminals of the rat spinal cord is regulated by the axoplasmic transport in the peripheral nerve.

Vasoactive intestinal polypeptide (VIP) immunoreactivity in the upper spinal dorsal horn is markedly increased after transection, crush or vinblastine treatment of the ipsilateral, segmentally related peripheral nerve. After regeneration of the peripheral nerve, VIP disappears from the upper dorsal horn. Transection-induced VIP increase is abolished by rhizotomy. It is concluded that the expression of VIP is restricted by factor(s) carried by retrograde axoplasmic transport to dorsal root ganglion cells.

Effect of the pesticide Deltamethrin on the Mauthner cells of Lake Balaton fish.

Deltamethrin, a synthetic pesticide [(S)alpha-cyano-3-phenoxybenzyl-(1R)-cis-3-(2.2-dibromovinyl)-2,2-dim ethylcyclopropane-carboxylate] used for extermination of mosquitoes on the shores of lake Balaton, has been found to induce severe impairments of the nervous system of several Lake Balaton fish, such as carp (Cyprinus carpio), goldfish (Carassius auratus gibelis Bloch), eel (Anguilla anguilla) and wels (Silurus glanis). It has been shown that Deltamethrin, in a concentration of 1 microgram/liter in the aquarium water, inhibits acetylcholinesterase enzyme activity of the giant Mauthner's nerve cells as well as of the axon terminals synapsing with these cells. Even more importantly, however, Deltamethrin in a concentration of 10 micrograms per liter, induces blockade of the expression of choline acetyltransferase in the bulbous axon terminals synapsing with the lateral dendrites of the Mauthner cells. Since, under normal conditions, the function of the Mauthner cells is to co-ordinate the C-start reaction, by which fish rapidly leave sites of nociceptive stimulation, it stands for reason to assume that Deltamethrin intoxicated fish may be prone to become victims of various factors which endanger survival of the individual. During the last decade, waves of fish deaths were observed in Lake Balaton, which is the largest fresh-water lake in Europe. Fish death coincided with airborne mosquito-killing campaigns. Results of the enzyme- and immunohistochemical studies described in this paper, together with the deleterious effects of Deltamethrin to the enteric nervous system of fish which has been reported earlier (Lang et al., 1997) suggest that fish death might be caused by the indiscriminate use of Deltamethrin airborne spray in the mosquito-extermination campaigns.