Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.     

Tetanus toxin effect on the metabolism and release of acetylcholine in rat central nervous system

The effect of tetanus toxin in doses of 30 mcg/kg on the content, synthesis and release of acetylcholine, and on the activity of choline acetylase and acetylcholine esterase in the central nervous system of the rat was studied. The investigations were carried out after the appearance of tetanus. We found that the tetanus toxin: a) caused no changes in the acetylcholine content in the cerebral cortex and brain stem, and also in the cervical and lumbar parts of the spinal cord; b) stimulated acetylcholine synthesis in the brain stem and in the cervical and lumbar parts of the spinal cord but not in the cerebral cortex; c) activated choline acetylase; d) had no effect on acetylcholine esterase activity; e) released acetylcholine from the neurons in the brain stem and spinal cord. The release could not be inhibited by low concentration of potassium ions in the medium or increased with electrical stimulation.     

Morphological and histochemical characteristics of the reaction of the body to Cl. botulinum toxin administration. III. The cellular reaction of the diaphragmatic nerve nucleus to the administration of Cl. botulinum type B toxin]

On the appearance in the animals (guinea pigs) of paralysis of the limbs and myasthenia after the administration of Cl. botulinum, type B, toxin, there was seen a considerable vascular hyperemia of the spinal cord, and in the neurons of the phrenic nerve nucleus there developed dystrophic-necrotic processes coursing with a marked swelling, hyperchromasia and tigrolysis. As revealed histochemically, at this stage of the botulin intoxication the neurons of the phrenic nerve nucleus displayed metabolic changes expressed in the altered activity of succinic dehydrogenase, acid phosphatase and cholinesterase.     

Endocytosis and retrograde axonal traffic in motor neurons

Spinal cord motor neurons control voluntary movement by relaying messages that arrive from upper brain centres to the innervated muscles. Despite the importance of motor neurons in human health and disease, the precise control of their membrane dynamics and its effect on motor neuron homoeostasis and survival are poorly understood. In particular, the molecular basis of the co-ordination of specific endocytic events with the axonal retrograde transport pathway is largely unknown. To study these important vesicular trafficking events, we pioneered the use of atoxic fragments of tetanus and botulinum neurotoxins to follow endocytosis and retrograde axonal transport in motor neurons. These neurotoxins bind specifically to pre-synaptic nerve terminals, where they are internalized. Whereas botulinum neurotoxins remain at the neuromuscular junction, tetanus toxin is retrogradely transported along the axon to the cell body, where it is released into the intersynaptic space and is internalized by adjacent inhibitory interneurons. The high neurospecificity and the differential intracellular sorting make tetanus and botulinum neurotoxins ideal tools to study neuronal physiology. In the present review, we discuss recent developments in our understanding of the internalization and trafficking of these molecules in spinal cord motor neurons. Furthermore, we describe the development of a reliable transfection method for motor neurons based on microinjection, which will be extremely useful for dissecting further the molecular basis of membrane dynamics and axonal transport in these cells.     

Effect of tetanus toxin on the content of glycine, gamma-aminobutyric acid, glutamate, glutamine and aspartate in the rat spinal cord

Tetanus toxin injected intramuscularly induced no significant changes in the levels of glycine, GABA, glutamate, glutamine or aspartate in extracts of spinal cord from rats killed at timed intervals during the development of local and generalized tetanus. The amino acid contents in the hemisegment (longitudinal one-half) of the spinal cord (L₂-L₆) on the injected side (left gastrocnemius muscle) did not differ significantly from the contents in the hemisegment of the spinal cord on the non-injected side. Nor were there any consistent changes in the contents of the amino acids in either hemisegment of the spinal cord as the tetanic symptoms became progressively more severe. Hence, the amino acid pool in the spinal cord was relatively stable despite the metabolic changes known to occur in tetanus. Our observations are consistent with the view of Johnston , De Groat and CURTIS (1969) who suggested that if glycine were indeed a spinal inhibitory neurotransmitter released by interneurons affected by tetanus toxin, the toxin should interfere with the release of the amino acid rather than deplete the transmitter stores.     

Stereotaxic Injection of Tetanus Toxin in Rat Central Nervous System Causes Alteration in Normal Levels of Monoamines

A single intraventricular injection of tetanus toxin produced a time-dependent elevation of serotonin levels in brain and spinal cord of adult rats. This tetanus toxin-induced increase was produced in areas of high density of serotonergic innervation, such as the hypothalamus, hippocampus, and spinal cord. Little or no effect was found in the thalamus, cerebellum, and frontal cortex, areas that are poorly innervated by serotonergic terminals. The responses of catecholamines (no change in dopamine level and generalized decrease in norepinephrine) pointed to a specific action of tetanus toxin on the serotonergic system. Stereotaxic injections of tetanus toxin in dorsal or magnus raphe nuclei did not have an evident effect on biogenic amine levels in the brain and spinal cord, respectively. Because direct stereotaxic injections of the toxin in the hypothalamus or hippocampus produced significant serotonin increases in both areas, it is proposed that tetanus toxin interacts with presynaptic targets to produce serotonin accumulation; this is probably due in part to an activation of tryptophan 5-hydroxylase.     

Effect of ICV pertussis toxin and N-ethylmaleimide on the levels of substance P and serotonin in brain and spinal cord of the rat.

The effects of single intracerebroventricular (icv) injections of either 0.5 microgram pertussis toxin or 5 micrograms N-ethylmaleimide (NEM) on the levels of immunoreactive substance P (ir-SP) and serotonin (5-HT) in the brain and spinal cord of rats have been assessed. At two and six days after pertussis toxin injection, the levels of ir-SP appeared significantly diminished in the spinal cord (about 34%). This reduction was even greater at two days after NEM injection (43%). These two agents did not alter the ir-SP of the midbrain and thalamus, whereas NEM increased the neuropeptide content in the pons-medulla. On the other hand, the thalamic content of serotonin was reduced two days after pertussis toxin (32%) or NEM (20%) injection. The indoleamine levels of the spinal cord were reduced by these treatments (20%), while in the midbrain a slight decrease could be observed. These findings suggest that pertussis toxin and NEM produce these effects by acting upon a common neural substrate.     

Asymmetric distribution of peptide regulators of muscle tonus and substance P in the spinal cord of rats with unilateral hyperactivity of the lumbar enlargement neurons

The injection of tetanus toxin in m. gastrocnemius of the left or right hind limb of rats evokes ipsilateral hyperactivity of lumbar neurons in the spinal cord. In this case the lumbar enlargement extract after its intracisternal injection to healthy animals increases the duration of hind limb passive extension on the side where the donor neurons are hyperactive. The extract of the spinal cord of healthy rats was ineffective. Proteolysis of the extract with pronase or co-injection of opiate antagonist--naloxone--completely eliminated the lateralized changes in the muscular tone of the recipient. Substances that cause the unilateral changes in the muscular tone of the recipient are believed to be peptides. They are assumed to be involved in the functioning of endogenous opioid system. The level of substance P in the donor spinal cord was elevated bilaterally, but was higher in the hyperactive half of the spinal cord.     

Manifestation of the neurotoxic effect of the organochlorine pesticide hexachlorobutadiene in the postnatal period of ontogeny in the rat

Daily peroral administration of chlororganic pesticide hexachlorobutadiene in doses 8.1 mg/kg (1/20 LD50) to pregnant rats results in certain ultrastructural changes of neurocytes and myelin fibers of the spinal cord both in the animals and their offspring (newborns and 1-2-month-old rats). By means of electron paramagnetic resonance (EPR) method, changes in intensity of the EPR-signals of free radicals in the spinal cord, ceruloplasmin of blood serum have been revealed in the experimental pregnant animals, as well as in 1-month-old rats (in the latter--in the brain, too). Gas-liquid chromatography reveals the preparation contents in the adrenals, heart, brain and spinal cord, in the uterus of the pregnant animals, as well as in corresponding organs of their offspring. Certain retardation in growth and decrease in body mass are noted in the offspring.     

Changes in the protein composition of the synaptic structures of the brain of rats in tetanus intoxication]

As shown by the method of electrophoresis on polyacrylamide gel, the number of proteins with low electrophoretic mobility proved to be increased in the triton extract fractions of synaptic structures isolated from spinal cord of rats with local tetanus; no changes in the protein spectrum were revealed in the dodecyl-sulphate extract. In vitro tetanus toxin stimulated the lysin-H3 incorporation into the total proteins of synaptosomes of rat brain cortex.     

Botulinum toxin's axonal transport from periphery to the spinal cord

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Effects of a Dendroaspis neurotoxin on synaptic transmission in the spinal cord and the neuromuscular junction of the frog

A neurotoxin from the venom of Dendroaspis jamesoni was tested at the neuromuscular junction and at a cholinergic pathway in the isolated spinal cord of the frog. The toxin reduced the amplitude and time constant of decay of miniature endplate currents in the presence of prostigmine, indicating a curare-like action. In the spinal cord it selectively blocked transmission in the cholinergic pathway and increased spontaneous activity. Partial protection against toxin action in the spinal cord was provided by atropine or carbachol. The results suggest that the toxin acts on cholinergic receptors at both sites and also provide further evidence that the pharmacology of the two sites is different.     

Botulinum toxin type A normalizes alterations in urothelial ATP and NO release induced by chronic spinal cord injury

The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.     

Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

Susceptibility of chickens to avian encephalomyelitis virus. IV. Behavior of the virus in laying hens.

Some laying hens 6 months of age were inoculated subcutaneously or orally with a chick embryo--adapted strain of avian encephalomyelitis virus and examined for propagation of the virus in the body. When inoculated subcutaneously, the virus appeared in liver, spleen, ovarian follicle, and muscle at the site of inoculation 1 day, in kidney and lumbar part of the spinal cord 3 days, in the pancreas 5 days, in heart, duodenum, and cervical part of the spinal cord 7 days, and in the brain 11 days after inoculation. After its appearance, it increased gradually in amount in liver, spleen, pancreas, muscle at the site of inoculation, and cervical and lumbar parts of the spinal cord, but remained at a low level in any other organ. When examined 14 days after inoculation and later, it was distributed mainly in the central nervous system. It was detected from 12 of 16 organs examined. The highest virus level in each organ was 10(2.6)/0.1 g in pancreas and lumbar part of the spinal cord, which were followed by muscle at the site of inoculation (10(2.0)/0.1 g), spleen (10(1.8)/0.1 g), cervical part of the spinal cord, heart, and liver in the order listed. When inoculated orally, the virus was found sporadically in spleen, pancreas, kidney, cecum, ovarian follicle, and lumbar part of the spinal cord. The virus level was low in these organs, of which pancreas, kidney, and lumbar part of the spinal cord showed the highest virus level, or 10(1.3)/0.1 g.     

Tetanus toxin fragment C binds to a protein present in neuronal cell lines and motoneurons

Tetanus Toxin Fragment C Binds to a Protein Present in Neuronal Cell Lines and Motoneurons Tetanus neurotoxin is one of the most powerful protein toxins known, acting in vivo at femtomolar doses. Two main factors determine its high potency: a protease activity restricted to a single intracellular substrate and its absolute neurospecificity. Whereas the enzymatic properties of tetanus toxin have been thoroughly defined, the nature of its neuronal receptor(s) and their involvement in the intracellular trafficking of tetanus toxin are poorly understood. Using binding and crosslinking experiments, we report here on the characterisation of an N-glycosylated 15-kDa interacting protein, which behaves as an integral membrane protein. This putative receptor specifically interacts with the binding domain (fragment C) of tetanus toxin and not with several related botulinum neurotoxins in spinal cord motoneurons and neuronal-like cell lines. Sialic acid-specific lectins antagonise the binding of tetanus toxin to the cell surface and to the 15-kDa protein, supporting the central role of sialic acid residues in the recognition process. Altogether, these results indicate the existence of a neuronal protein receptor for tetanus toxin whose identification is likely to constitute a key step in the analysis of the molecular machinery involved in the toxin internalisation and retrograde transport.     

Brain and spinal cord neuropeptides in adjuvant induced arthritis in rats

The concentrations of brain and spinal cord beta-endorphin, met-enkephalin, dynorphin and substance P were measured in rats bearing the Freund adjuvant induced arthritis. Beta-endorphin brain concentrations decreased gradually in time with a nadir on day twenty-one, when arthritis was at its maximum, and were back to normal by day thirty-five, when arthritis was no more evident. Met-enkephalin concentrations increased in brain areas and in the lumbar spinal cord and returned to normal with the same time pattern, while dynorphin and substance P concentrations did not change. These data indicate that peripheral lesions can induce important changes in brain concentrations of some opioid peptides involved in the modulation of pain.     

Expression and characterisation of the heavy chain of tetanus toxin: reconstitution of the fully-recombinant dichain protein in active form.

Tetanus toxin, composed of a disulphide-linked heavy (HC) and light (LC) chain, preferentially blocks the release of inhibitory neurotransmitters in the spinal cord by Zn2+-dependent proteolytic cleavage of synaptobrevin. This intoxication involves binding via HC to ecto-acceptors on peripheral nerve endings, followed by internalisation and retrograde transportation to its prime site of action in central neurons. To facilitate exploitation of the toxin's unique activities, HC was expressed at a high level in Escherichia coli as a fusion with maltose binding protein; after cleavage by thrombin, free HC was isolated and its identity confirmed by Western blotting and N-terminal microsequencing. The expressed and native HC gave very similar circular dichroism spectra, excluding any gross differences in their folded structures. Recombinant HC antagonised the neuromuscular paralysing activity of the native toxin, by competing for binding to neuronal ecto-acceptors. The HC was reconstituted with bacterially-expressed LC to create disulphide-bridged dichain toxin that blocked neuromuscular transmission. The fully-recombinant toxin produced spastic paralysis in mice characteristic of the blockade of central inhibitory synapses, revealing that it undergoes axonal transport to the spinal cord, like the native toxin but with a reduced efficacy. This first report of the large-scale production of recombinant tetanus toxin in active form should facilitate studies on the use of engineered innocuous forms of the toxin as neuronal transport vehicles.