Blockade of retrograde transport of nerve growth factor (NGF) in a peripheral sensory nerve is known to induce transganglionic degenerative atrophy (TDA) of central sensory terminals in the upper dorsal horn of the related, ipsilateral segments(s) of the spinal cord. The ensuing temporary blockade of transmission of nociceptive impulses has been utilized in the therapy of intractable pain, using transcutaneous iontophoresis of the microtubule inhibitors vincristin and vinblastin, drugs which inhibit retrograde transport of NGF. Since microtubule inhibition might inhibit (at least theoretically) mitotic processes in general, we sought to find a drug which inhibits retrograde transport of NGF without microtubule inhibition. Vinpocetine, a derivate of vincamine, which does not interfere with microtubular function, was found to inhibit retrograde axoplasmic transport of NGF in peripheral sensory nerves, similarly to vincristin and vinblastin. Blockade of NGF transport is followed by transganglionic degenerative atrophy in the segmentally related, ipsilateral superficial spinal dorsal horn, characterized by depletion of the marker enzymes of nociception, fluoride resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP) from the Rolando substance and by decrease of the pain-related neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from lamina I-II-III. Based upon these findings, it has been suggested that vinpocetine may result in a locally restricted decrease of nociception. Herewith, the structural and behavioral effects of perineurally administered vinpocetine are discussed. Nociception, induced by intraplantar injection of formalin, was mitigated by perineural application of vinpocetine; also formalin-induced expression of c-fos in the ipsilateral, segmentally related superficial dorsal horn, was prevented by this treatment. Since vinpocetine is not a microtubule inhibitor, its mode of action is enigmatic. It is assumed that the effect of vinpocetine might be related to interaction with membrane-trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein, involved in retrograde axoplasmic transport of NGF, or to interaction with glial elements, recently reported to be involved in the modulation of pain in the spinal cord. Based on animal experiments it is assumed that the temporary, locally restricted decrease of nociception, induced by vinpocetine applied via transcutaneous iontophoresis, might open up new avenues in the clinical treatment of intractable pain.

Vinpocetine, a derivative of vincamine, widely used in the clinical pharmacotherapy of cerebral circulatory diseases, inhibits retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve, resulting in transganglionic degenerative atrophy (TDA) in the related ipsilateral superficial spinal dorsal horn, as shown in our previous publications. TDA induced by vinpocetine has been demonstrated to be followed by depletion of the marker enzyme fluoride-resistant acid phosphatase (FRAP) and its isoenzyme thiamine monophosphatase (TMP), and by the decrease in the pain-related neuropeptide substance P from laminae I–II–(III) from the segmentally related, ipsilateral substance of Rolando of the spinal cord. In the present paper, we report on the behavioral effects of perineurally administered vinpocetine. Nociception, induced by intraplantar injection of formalin, was mitigated by vinpocetine; increased expression of c-fos in the ipsilateral, segmentally related upper dorsal horn was also prevented. Since vinpocetine is not a microtubule inhibitor, and its chemical structure differs from that of vincristin and vinblastin (used formerly by us in the therapy of intractable, chronic neuropathic pain), its mode of action is enigmatic. We assume that the effect of vinpocetine in blocking retrograde axoplasmic transport of NGF might be related to its interaction with membrane trafficking proteins, such as signalling endosomes and the endocytosis-mediating “pincher” protein. Temporary, locally restricted decrease of nociception, induced by vinpocetine, might be useful in the clinical treatment of intractable, chronic neuropathic pain, since vinpocetine can successfully be applied by transcutaneous iontophoresis.

Vinpocetine, a derivate of vincamine, is widely used in the clinical pharmacotherapy of cerebral circulatory diseases. Herewith we report on a novel effect of vinpocetine: inhibition of retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve. Blockade of retrograde transport of NGF results in transganglionic degenerative atrophy (TDA) in the segmentally related ipsilateral superficial spinal dorsal horn, which is characterized by depletion of the marker enzymes fluoride-resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP). At the same time, pain-related neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), are depleted from lamina I–III from the segmentally related, ipsilateral Rolando substance of the spinal cord. On the basis of these experiments it is suggested that vinpocetine may result in a locally restricted decrease of nociception, that might be useful in clinical treatment of intractable pain. Pilot self-experiments support this assumption.

Parkinson’s disease (PD), a progressive neurodegenerative disorder, is characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC). Neurons in the SNPC are known to express tyrosine hydroxylase (TH); therefore, in a commonly used PD model, 6-hydroxydopamine (6-OHDA), a selective catecholamine neurotoxin, induces neuronal death in SNPC. We have shown with immunohistochemical techniques that kynurenine aminotransferase-I (KAT-I), the enzyme taking part in the formation of kynurenic acid (KYNA)—the only known endogenous selective NMDA receptor antagonist and a potent neuroprotective agent—is also expressed in the rat SNPC. We found that KAT-I and TH co-exist in the very same neurons of SNPC and that 6-OHDA injected into the lateral ventricle produced loss of the majority of nigral neurons. Densitometric analysis proved that, in consequence of 6-OHDA treatment, not only TH but also KAT-I immunoreactivity diminished considerably in the remaining SNPC neurons. Astrocytes in the substantia nigra were found to express KAT-I under normal conditions; the amount of this enzyme increased after administration of 6-OHDA, whereas microglial cells became KAT-I immunoreactive only after 6-OHDA treatment. Since intrinsic KYNA in SNPC neurons is perceptibly insufficient to protect them from the deleterious effect of 6-OHDA, it is hypothesized that biochemical approaches which increase KYNA content of the central nervous system might prevent the deleterious effect of 6-OHDA and, supposedly, also the neuronal degradation characterizing PD.

Large calyciform synapses in the rat reticular thalamic nucleus are characterized by the presence of gamma-aminobutyric acid. Presynaptic terminals are also loaded with calcium-binding proteins such as parvalbumin, calbindin, calretinin and calcineurin. The number of calyciform terminals containing gamma-aminobutyric acid and parvalbumin is 2005 in young adult rats; calbindin is present in 1500, calretinin in 850 and calcineurin in 560 calyciform terminals. Developmental studies revealed that gamma-aminobutyric acid and calcium-binding proteins are virtually absent from calyciform terminals at birth but their occurrence increased considerably during postnatal life, suggesting increasing regulation of presynaptic calcium signaling during postnatal life. It is concluded that synaptic activity of large calyciform gamma-aminobutyric acid-containing synapses of the reticular thalamic nucleus is mediated, regulated or accompanied by calcium ions.

In the reticular thalamic nucleus of the rat, nearly all neurons are parvalbumin-immunoreactive. We found that in addition, though superficially similar to large parvalbumin-immunoreactive neurons, also numerous peculiar parvalbumin-immunoreactive complexes are present in the reticular thalamic nucleus which are not identical with parvalbumin-immunoreactive perikarya, as shown by nuclear variation curves. Light and electron microscopic immunocytochemical studies revealed that these parvalbumin-immunoreactive complexes are brought about by parvalbumin-immunoreactive calyciform terminals which establish synapses with large, parvalbumin-immunonegative dendritic profiles. Transection of thalamo-reticular connections did not cause any alteration of calyciform terminals in the reticular thalamic nucleus. Nuclear counterstaining revealed that parvalbumin-immunoreactive calyciform terminals originated from local parvalbumin-immunoreactive interneuronal perikarya, which, depending of the length of the “neck” protruding from the perikaryon, establish somato-dendritic, axo-dendritic or dendro-dendritic synapses. Light and electron microscopic immunocytochemical investigations prove that the parvalbumin-immunoreactive calyciform complexes contain also GABA, that are likely to be inhibitory. In accordance with literature data, our results suggest that parvalbumin-immunoreactive GABAergic calyciform terminals in the reticular thalamic nucleus may be instrumental in intrinsic cell-to-cell communications and, as such, may be involved in synchronisation of thalamo-cortical oscillations, in the production of sleep spindles and in attentional processes.

Electrical stimulation of the reticular nucleus of the rat thalamus results in activation of c-fos immunoreactivity in nerve cells of the ipsilateral retrosplenial cortex. The c-fos immunoreactive neurons are mainly concentrated in lamina IV of the retrosplenial cortex. Conversely, electrical stimulation of the retrosplenial cortex induced c-fos immunoreactivity in the ipsilateral reticular nucleus of the thalamus. The results of the electrical stimulation suggest a direct synaptic connection between the cerebral cortex and the ipsilateral reticular thalamic nucleus. Simultaneous immunohistochemical staining proves that the majority of nerve cells and dendro-dendritic terminals in the reticular thalamic nucleus contain parvalbumine and, at the same time, also GABA. The role of GABA-ergic parvalbumine immunoreactive terminals in the reticular thalamic nucleus seems to be related to integration and processing of impulses and attentional gating, distinguishing between noxious and innocuous inputs.

Nitric oxide (NO) was recently proposed to be involved in the sleep-wake cycle and cortical spreading depression. As a structural correlate of these functions, we found that bNOS IR was expressed by three cell types in the prefrontal cortex, viz. bipolar, multipolar, and stellate cells. Dendrites of bipolar cells established bundles resulting in a columnoid organization; in addition, the monoclonal antibody mAb 35 which labels subunits α1, α3 and α5 of nAChR, also visualized apical axons proceeding alongside the columnoids. In contrast, α-bungarotoxin which labels the α7-subunit of nAChR, visualized only perikarya of interneurons from where the apical axons arose. In the prefrontal cortex of monkeys which were anesthetized for 6–24 hours, only traces of the columnoid organization were found, while perikaryal bNOS and nAChR were invariably expressed. It is suggested that interactions between NO and presynaptically released ACh might be involved in cortical functions such as the sleep/wake cycle.

Following transganglionic degenerative atrophy of primary afferent terminals induced by a crush-injury of the sciatic nerve, a regenerative process takes places in the upper dorsal horn of the lumbar spinal cord in the primate Macacus rhesus. Axonal growth cones are characterized by cisterns of axoplasmic reticulum; filopodia emanating from growth cones are electron-optically translucent sheet-like expansions, often containing growth-cone vesicles. Axoplasmic reticulum appears also in preterminal portions of regenerating axons. Dendritic growth cones contain a fine, filamentous matrix; electron-dense membrane specializations can be seen in well-defined areas of their surfaces. Immature synapses are formed between filopodia of axonal growth cones and dendritic growth cones. Electron-microscopic structures of this unique CNS regeneration are similar to those seen in the course of embryonic development of the spinal cord.

In the reticular nucleus of the rat thalamus, about 30% of the synapses are brought about by the perikarya of parvalbumin-immunopositive neurons, which establish somato-dendritic synapses with large dendrites of nerve cells of specific thalamic nuclei. Although the parvalbumin-immunopositive presynaptic structures bear resemblance to goblet-like or calyciform axonal endings, electron microscopic immunocytochemistry and in situ hybridization revealed that these structures are parts of the perikaryal cytoplasm studded with synaptic vesicles. In about 15% of the somato-dendritic synapses, axons are seen to be in synaptic contact with the parvalbumin-immunoreactive perikaryon. Double immunohistochemical staining revealed that the parvalbumin immunoreactive presynaptic perikarya and dendrites contained GABA. It is assumed that the peculiar somato-dendritic synaptic complexes subserve the goal of filtration of impulses arriving at the reticular nucleus from various thalamic nuclei, thus processing them for further sampling.

Development of glomerular synapses in the superficial dorsal horn has been studied in the embryonic macaque spinal cord using light and electron microscopic techniques including Golgi impregnation, 3H-thymidine radioautography and pre-embedding immunohistochemistry of substance P (SP), calcitonin gene related peptide (CGRP), calbindin D-28 K (CB) and parvalbumin (PV). The study revealed that substantia gelatinosa cells of the primate dorsal horn are generated last, but unlike in rodents, synaptogenesis in this region starts at early embryonic (E) stages of the 165-day long gestation. Already by E30, both Type 1 (light) and 2 (dark) dorsal root axons and their growth cones are identifiable within the oval bundle of His, before they form synaptic contact with their final target cells. Subsequently they invade the dorsal horn and enter the bisecting interfaces formed by orderly programmed cell death. Each type of scalloped (sinusoid) central primary afferent terminal (i.e. DSA, RSV and LDCV) have well defined pre- and post-synaptic specializations already by E40. Among the neuropeptides studied, SP appears first at E67 and CGRP at E70 in the lateral position but within a few days both of them are spread to the entire superficial dorsal horn. Both SP and CGRP are present in the thin dorsal root axons and their growth cones, giving rise to scalloped and simple axon terminals. PV is transiently present in the entire length of the thick dorsal root afferents before becoming concentrated in the synaptic boutons. CB is displayed mainly in neurons of the lamina I and III. Dendrites of CB-immunoreactive cells establish synaptic connection with each type of dorsal root afferents, including glomerular synaptic complexes. These data reveal that the superficial dorsal horn in the primate spinal cord develops its characteristic synaptic complexes much earlier in gestation than in any other mammalian species studied. Furthermore, characteristic cytological features of the prospective glomerular complex emerge before establishment of the final synaptic contacts.

Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds.

Because of the anatomical characteristics of the rat skull, the Gasserian ganghon has been approached by the infiaorbital route. Thiamine monophosphatase (TI") cytochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry of the Gasseriz ganghon, the supratentorial cerebral dura mater and the caudal trigeminal nucleus has been studied with light microscopic methods, 14 to 365 days after d a t e r a l treatment of the

Early development of the spinal cord was studied in macaque monkey embryos, using light- and electron microscopy, Golgi impregnation and [3H]thymidine radioautography. All neurons engaged in the first reflex arc are generated before E27 in the 165 day gestation period. The earliest-generated cells differentiate into motoneurons while the commissural and association neurons are generated later. Synaptogenesis starts at E27 in the basal plate, and 2 days later in the alar plate. The first synapses appear as symmetrical junctions situated on the neuronal perikarya and proximal dendrites. Closure of the first spinal reflex arc is established within 2 days and follows an antidromic pattern as related to the physiological spread of nerve impulses: synapses on motoneuronal somata and primary dendrites in the basal plate appear first and are followed by synapses in the alar plate, between dorsal root axon collaterals and somata of borderline (commissural/association) neurons. Commissural axons grow towards the floor plate, cross the midline and proceed caudo-rostrally, while association fibers remain ipsilateral. The first wave of apoptosis (programmed cell death) thins out dense populations of nerve and glial cells by E30. Some of the early-generated borderine cells that form commissural and association interneurons, seem to play the role of transient target cells and die once the definitive axonal pathways are established. Since transient cells form numerous synapses, deprivation from the afferent impulses is not a likely cause of their elimination. The present results indicate that the initial developmental events, including formation of the first reflex arc in the primate spinal cord, occur considerably earlier in respect to birth than in other mammals, but that the schedule of cellular events and cellular mechanisms seem to be the same.

Transection of the sciatic nerve in Rhesus monkeys and the consequent transganglionic degenerative atrophy (TDA) of central terminals of primary afferents result in transneuronal degeneration of substantia gelatinosa (SG) cells. Severe degeneration is characterized by an increased electron density of the nucleus and by conspicuous shrinkage of the cytoplasm, mitochondrial swelling, dilation of cisterns of the rough-surfaced endoplasmic reticulum, accumulation of free ribosomes and an electron-dense material in the cytoplasm. In the mild form, dilation of cisternal elements of the endoplasmic reticulum, swollen mitochondria and accumulation of free ribosomes takes place. About 10% of SG cells in segment L5 undergo the severe form whereas the rest shows signs of the mild form. Cytoplasmic alterations that occur during transneuronal degeneration seem to start at the level of subsurface cisterns. Dendrites and axons of transneuronally degenerating SG cells also show a conspicuous electron density. By analyzing the synaptic relationships of such “darkened” dendrites, connections in the upper dorsal horn can be deciphered. Modular units of the primary nociceptive analyzer that evaluate noxious and innocuous inputs on the basis of thin versus thick (AδC/Aβ) afferent activity and subjecting them to descending control appear to be recruited from structurally dispersed elements of synaptic glomeruli. These are arranged alongside dendritic processes of large antenna cells which relay impulses to projection cells of the spinothalamic tract.

The effect of sciatic nerve transection on its centrally located terminals in the spinal cord was analyzed by electron microscopy in adult rhesus monkeys one and three months following lesion. Although the peripheral and intermediate portions of the dorsal roots, where the axons are enveloped by Schwann cells were normal, their central portion and their terminals in the substantia gelatinosa were remarkably altered. Transganglionic degenerative atrophy (TDA) is characterized by three distinct types of electronmicroscopic alterations. The first type exhibits a conspicuous electron density of the terminal and pre-terminal axoplasm. Importantly, shrinkage replaces fragmentation and glial engulfement of the terminal seen in the course of Wallerian degeneration. The second type is characterized by the disappearance of synaptic vesicles from the terminals. The third type of TDA consists of intricate labyrinthine structures, composed of flattened profiles of axonal, dendritic and glial elements. The complex and diverse cellular changes that occur in the upper dorsal horn following peripheral nerve injury may provide the structural basis of plasticity of the primary nociceptive system.