Low doses of urethane effectively inhibit spinal seizures evoked by sudden cooling of toad isolated spinal cord.

The effect of low doses of urethane on three phases of spinal seizures evoked by sudden cooling (SSSC) of toad isolated spinal cord was studied. In control toads, SSSC began with a latency of 91 +/- 3 sec (mean +/- S.E.M.) exhibiting brief tremors, followed by clonic muscle contractions and finally reaching a tonic contraction (tonic phase). The latency of onset of seizures was significantly enhanced. The tonic phase was markedly abolished in toads pretreated intralymphatically with 0.15 g/kg of urethane. Tremors were the only phase observed in 55% of toads that received doses of 0.2 g/kg, and a total blockade of seizures was seen after doses of 0.25 g/kg of urethane in 50% of the preparations. A possible depressant effect of urethane on transmission mediated by excitatory amino acids is suggested.     

Attenuation of reflex pressor and ventilatory responses to static contraction by an NK-1 receptor antagonist.

The chemical messengers released onto second-order dorsal horn neurons from the spinal terminals of contraction-activated group III and IV muscle afferents have not been identified. One candidate is the tachykinin substance P. Related to substance P are two other tachykinins, neurokinin A (NKA) and neurokinin B (NKB), which, like substance P, have been isolated in the dorsal horn of the spinal cord and have receptors there. Whether NKA or NKB plays a transmitter/modulator role in the spinal processing of the exercise pressor reflex is unknown. Therefore, we tested the following hypotheses. After the intrathecal injection of a highly selective NK-1 (substance P) receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Likewise, after the intrathecal injection either of an NK-2 (NKA) receptor antagonist or an NK-3 (NKB) receptor antagonist onto the lumbrosacral spinal cord, the reflex pressor and ventilatory responses to static contraction will be attenuated. We found that, 10 min after the intrathecal injection of 100 micrograms of the NK-1 receptor antagonist, the pressor and ventilatory responses to contraction were significantly (P < 0.05) attenuated. Mean arterial pressure was attenuated by 13 +/- 3 mmHg (48%) and minute volume of ventilation by 120 +/- 38 ml/min (34%). The cardiovascular and ventilatory responses to contraction before either 100 micrograms of the NK-2 receptor antagonist or 100 micrograms of the NK-3 receptor antagonist were not different (P > 0.05) from those after the NK-2 or the NK-3 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartate effect on spontaneous activity of 16–20-day-old chick embryo spinal cord

The effects of N-methyl-D-aspartate (NMDA) and the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (2-APV), on spontaneous activity of dorsal and ventral roots (DR and VR, respectively) generated by isolated spinal cord from 16–20-day-old chick embryo were studied. This activity was synchronous oscillations of electrotonic potentials in DR and VR. There was no impulse activity in the VR. When NMDA was applied at 2–25 µM, the amplitudes of the oscillations increased, the impulse activities in VR and DR developed, and the tonic component of electrotonic potentials appeared. At 20 µM, 2-APV decreased both, the spontaneous and NMDA induced activity. After sectioning of the spinal cord, the neuronal network of the isolated dorsal arm conserved the capacity to generate spontaneous activity in the DR which increased after NMDA application. There was no rhythm in the ventral part of the spinal cord. The localization of the NMDA-sensitive neuronal network, generator of the rhythmic (motor) activity, in the spinal cord is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 205–213, March–April, 1991.     

REGIONAL CHANGES IN CEREBRAL GABA CONCENTRATION AND CONVULSIONS PRODUCED BY d AND BY l-ALLYLGLYCINE

Abstract— The convulsant action of allylglycine (2-amino-4-pentenoic acid) is due to the metabolic conversion of allylglycine to 2-keto-4-pentenoic acid, a more potent glutamic acid decarboxylase inhibitor and more potent convulsant than the parent compound. We report regional changes in cerebral GABA concentration in rats after administration of d - and l -allylglycine. d -Allylglycine (3.75 mmol/kg) induced convulsions in 95–115 min, characterised by repeated clonic limb movements and rapid rotation around the head to tail axis. GABA concentrations were only reduced in cerebellum and ponsmedulla during the pre and post-convulsive periods. The localised reduction of GABA concentration is consistent with the enzymic conversion of d -allylglycine to 2-keto-4-pentenoic acid catalysed by cerebral d -amino acid oxidase, an enzyme known to be localised to the hind brain and spinal cord. l -allylglycine (1.2mmol/kg i.p.) induced convulsions in 65 -90 min, characterised by violent running followed by tonic flexion and extension. During the pre-convulsive period, GABA concentrations were reduced in all brain areas studied except the globus pallidus and ventral midbrain. The widespread decreases in GABA concentration suggest that the enzyme(s) which catalyse the conversion of l -allylglycine to 2-keto-4-pentenoic acid are widely distributed within the brain.     

Antinociceptive effects in mice after intrathecal injection of a substance P receptor antagonist, Spantide: lack of 'neurotoxic' action

The present investigation was undertaken to determine the antinociceptive potency and possible neurotoxic effects of a substance P (SP) receptor antagonist, [D-Arg,D-Trp,Leu]SP (Spantide), after intrathecal injection in mice. After the nociceptive tests had been carried out, the animals were sacrificed and the spinal cords were investigated for histopathological changes, since such have been reported previously to occur in rats. It was found that the reaction latency in the tail-flick test increased in the dose range 0-10 micrograms. The effect was maximal at 10 and 45 min after 10 micrograms Spantide, and somewhat lower when 5 micrograms was used. None of the animals showed the complete motor impairment reported previously to occur after intrathecal administration in rats. In some of the mice we observed a slight rigidity in the hind-legs. At histopathological examination, it was found that Spantide produced no histological changes indicative of 'neurotoxic' effects. In agreement with this, the immunohistochemical evaluation, using calcitonin gene-related peptide (CGRP) as a marker for motoneurons and central branches of primary sensory neurons, did not provide evidence that the intrathecal injection of 10 micrograms Spantide produced any effects when compared to vehicle-injected animals. In conclusion, the present results demonstrate an antinociceptive effect of Spantide when injected intrathecally in mice, and that this occurred without any signs of toxic reactions in spinal cord as previously has been reported for the rat.     

TRIM59 interacts with ECSIT and negatively regulates NF-κB and IRF-3/7-mediated signal pathways

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.     

Blockade effects of (Nphe1)Nociceptin(1-13)-NH(2) on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.     

Distribution of label after intrathecal administration of 125I-substance P in the rat

Despite the widespread use of the intrathecal route for the administration of neuroactive agents, little is known about the penetration of these agents into the spinal cord. In the present study, 125I-substance P was injected via a spinal catheter to the thoracic or sacro-coccygeal spinal cord in the rat (350-400 g) anesthetized with urethane (2.5 g/kg). Spinal cords were removed rapidly at 1 or 10 min after injection and immediately frozen in CCl2F2. Frozen sections, 20 micron thick, were cut and mounted for autoradiography. Autoradiographs of transverse sections demonstrated that the label penetrated 700 to 1800 micron from the surface of the spinal cord at both levels. In longitudinal sections, this penetration extended about 0.5 cm rostrally and caudally from the site of injection. Serial autoradiographs of transverse sections showed a similar penetration rostro-caudally. In addition, venous blood samples were taken at 1, 6, 11 and 16 min after injection of the labelled peptide. Quantification of the radioactivity in the samples revealed that 0.8 to 3.5% of the total CPM injected had passed into the general circulation at these times. These data indicate that after intrathecal administration of radiolabelled substance P, the label penetrates into the grey matter of the spinal cord to presumed sites of action. They also suggest that the rostro-caudal extent of penetration is more localized than suggested from earlier studies which looked only at levels of radioactivity in pieces of whole spinal cord. Finally, our study has indicated that passage of label into the circulation is negligible at least for substance P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of Calcitonin Gene-Related Peptide in Rat and Rabbit Spinal Cords: Effect of Intrathecal 5,7-Dihydroxytryptamine

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0 calcitonin gene-related peptide28-37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP-LI levels were 15-50-fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP-LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP-LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7-DHT, which caused 85-91% depletion of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7-DHT produced a threefold increase in CGRP-LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin-induced denervation of bulbospinal raphe neuronal input.     

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.     

Effects of WAY100635, a selective 5-HT1A-receptor antagonist on the micturition-reflex pathway in the rat

5-Hydroxytryptamine (5-HT) receptors in the central nervous system have been implicated in the control of micturition. The present study was undertaken to evaluate the effects of a selective 5-HT1A-receptor antagonist [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100635)] on the micturition-reflex pathway in urethane-anesthetized female Wistar rats. Rhythmic isovolumetric bladder contractions evoked by bladder distension were abolished by 0.3- to 3-mg/kg iv or 30- to 100-microg intrathecal (it) administration of WAY100635 in a dose-dependent manner for periods of 3-15 min. Intrathecal injection of WAY100635 was effective only if injected at the L6-S1 spinal cord level, but not at the thoracic or cervical cord levels. WAY100635 (30-100 microg it) significantly reduced the amplitude of bladder contractions evoked by electrical stimulation of the pontine micturition center. However, the field potentials in the rostral pons evoked by electrical stimulation of pelvic nerve were not affected by intrathecal or intravenous injection of WAY100635. These results suggest that 5-HT1A receptors at the L6-S1 level of the spinal cord have an important role in the tonic control of the descending limb of the micturition-reflex pathway in the rat.     

Effect of interleukin-1β on spinal cord nociceptive transmission of normal and monoarthritic rats after disruption of glial function

Introduction  

Cytokines produced by spinal cord glia after peripheral injuries have a relevant role in the maintenance of pain states. Thus, while IL-1β is overexpressed in the spinal cords of animals submitted to experimental arthritis and other chronic pain models, intrathecal administration of IL-1β to healthy animals induces hyperalgesia and allodynia and enhances wind-up activity in dorsal horn neurons.     

An interaction of opioids and galanin in dorsal horn of the spinal cord in mononeuropathic rats

The present study was performed in rats with experimentally induced mononeuropathy after common sciatic nerve ligation. The hind-paw withdrawal latencies to thermal and mechanical stimulation were increased significantly after intrathecal injection of 3 nmol of galanin. The increased hind-paw response latencies induced by galanin were attenuated by following intrathecal injection of 22 nmol, but not 11 or 2.75 nmol of the opioid receptor antagonist naloxone. Further, the increased hind-paw response latencies induced by galanin were prevented by following intrathecal injection of 10 nmol of mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA), but not by 10 nmol of delta-opioid receptor antagonist, natrindole or 10 nmol of kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI). Intrathecal 10 nmol of beta-FNA alone had no significant effects on the hind-paw withdrawal responses. These results demonstrate the existence of a specific interaction between galanin and opioids in the transmission of presumed nociceptive information in the spinal cord of mononeuropathic rats. This interaction involves the activation of mu-opioid receptor.     

Biosynthesis of 3-methoxycarbonylpropylglucosinolate in an Erysimum species

Incorporation of DL-2-aminohexanedioic acid, DL-2-amino-5-methoxycarbonyl-pentanoic acid and DL-methionine into 3-methoxycarbonylpropylglucosinolate have been demonstrated using an Erystmum species. The data support the following sequence of biosynthetic reactions: 2-aminohexanedioic acid is methylated by methionine; the resulting 2-amino-5-methoxycarbonyl-pentanoic acid is then converted into the glucosinolate. 2-Amino-5-methoxycarbonyl-pentanoic acid has been tentatively identified as a natural product in the plant.     

Intrathecal PACAP-38 causes increases in sympathetic nerve activity and heart rate but not blood pressure in the spontaneously hypertensive rat

The rostral ventrolateral medulla contains presympathetic neurons that project monosynaptically to sympathetic preganglionic neurons (SPN) in the spinal cord and are essential for the tonic and reflex control of the cardiovascular system. SPN directly innervate the adrenal medulla and, via postganglionic axons, affect the heart, kidneys, and blood vessels to alter sympathetic outflow and hence blood pressure. Over 80% of bulbospinal, catecholaminergic (C1) neurons contain pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA. Activation of PACAP receptors with intrathecal infusion of PACAP-38 causes a robust, prolonged elevation in sympathetic tone. Given that a common feature of most forms of hypertension is elevated sympathetic tone, this study aimed to determine in the spontaneously hypertensive rat (SHR) and the Wistar Kyoto rat (normotensive control) 1) the proportion of C1 neurons containing PACAP mRNA and 2) responsiveness to intrathecal PACAP-38. We further investigated whether intrathecal infusion of the PACAP antagonist, PACAP(6-38), reduces the hypertension in the SHR. The principal findings are that 1) the proportion of PACAP mRNA-containing C1 neurons is not different between normotensive and hypertensive rats, 2) intrathecal PACAP-38 causes a strain-dependent, sustained sympathoexcitation and tachycardia with variable effects on mean arterial pressure in normotensive and hypertensive rats, and 3) PACAP(6-38) effectively attenuated the effects of intrathecal PACAP-38, but had no effect alone, on any baseline variables. This finding indicates that PACAP-38 is not tonically released in the spinal cord of rats. A role for PACAP in hypertension in conscious rats remains to be determined.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Inhibition of electroshock-induced seizures by cholecystokinin-related peptides in mice

The effects of several doses of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS), and two CCK-related peptide analogues Ac-Thr5-caerulein, and nonsulphated Ac-Thr5-caerulein were investigated on electroshock-(ES)-induced seizures after intraperitoneal administration in mice. As parameters, the duration of the tonic and clonic phase of the fit, and those of postictal coma and behavioural depression were measured. CCK-8-SE decreased the duration of the clonic phase; its highest dose, 3.2 mumol/kg, shortened the coma. CCK-8-NS antagonized only slightly the clonic phase of seizure. Ac-Thr5-caerulein did not influence ES-induced seizures in any dose, only increased the duration of behavioural depression. Similarly to CCK-8-NS, the nonsulphated form of Ac-Thr5-caerulein inhibited selectively the clonic phase of seizures. The reference drugs, diazepam and phenobarbital, antagonized dose-dependently and most effectively the tonic phase of ES-induced seizures, but in much higher doses than did the CCK-related peptides. Besides, diazepam increased and phenobarbital decreased the duration of postictal coma. The results showed that the tested CCK-related peptides inhibit prevalently the clonic phase of ES-induced seizures after peripheral administration.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

Na, K-ATP-ase and acetylcholinesterase activity of the membrane structures of the rat brain and spinal cord during the seizure process]

The activity of ATP-ase and acetylcholinesterase (AChE) in crude mitochondrial fraction (CMF) and microsomal fraction of rat brain cortex and the spinal cord was studied in clonic seizures evoked by electroshock and 5 min after them. Inhibition of the Na, K-ATP-ase activity of the CMF of the brain at the clonic phase of convulsions and an increase in the activity of this enzyme in all the fractions of the tissues under study at the postconvulsive period were revealed. The activity of Ca-ATP-ase in the CMF of the brain increased during the convulsions and decreased at the postconfulsive period. The activity of Mg-ATP-ase remained unchanged. The AChE activity, as a rule increased during the convulsions, and grew even more during the postconvulsive period; the spinal cord tissue displayed a reduction of the activation effect. A possibility of structural reconstructions in the excitable neuron membranes during the convulsive activity is discussed.