Motor responses of the urinary bladder and skeletal muscle in Botulinum intoxicated rats

1. Type A or type D botulinum toxin administered to rats did not produce a generalized paralysis of skeletal muscles at the time of ventilatory arrest. However, if survival was extended by artificial ventilation complete blockade of neuromuscular transmission developed 6.5 hr after 100 MLD of type D and 5 hr after 1000 MLD of type A toxin. The onset of paralysis of a muscle was shortened by repetitive stimulation of the motor nerves.2. There was no consistent blockade of parasympathetically innervated viscera in animals dying after type A toxin. Animals given type D toxin displayed mydriasis and urinary retention before death.3. Motor responses to electrical stimulation, of bladder preparations in vitro were more vulnerable to type D than to type A toxin. When somatic paralysis was complete in animals treated with type A or type D toxin the excised bladders produced pressure elevations 45 and 25%, respectively, of control preparations.4. During electrical stimulation of bladder preparations nearly paralysed by either toxin, the ACh release was significantly diminished from controls. In the rat bladder botulinum toxin specifically disrupted the liberation of mediator from post-ganglionic nerve endings.     

Adrenergic and Cholinergic Innervation of the Rat Urinary Bladder

The autonomic innervation of the rat urinary bladder was studied using histochemical methods and nerve stimulations. A sparse adrenergic innervation of the detrusor muscle was found. It was supposed to originate from long adrenergic neurones. The trigonum area had a rich supply of adrenergic fibres, probably derived from short adrenergic neurones. A uniformly rich supply of acetylcholine-esterase (AChE)-positive nerves was found in the whole bladder. Postganglionic sympathetic denervation caused no detectable change of adrenergic or AChE-positive nerves in the bladder, while parasympathetic decentralization or denervation produced a total disappearance of adrenergic fibres. The AChE-positive nerves were appreciably reduced in number after parasympathetic decentralization and not detectable after postganglionic denervation. Neither adrenergic nor AChE-positive ganglion cells could be demonstrated in the bladder wall. Electrical stimulation of the hypogastric nerves or the pelvic nerves distal to the pelvic ganglia elicited contraction of the detrusor muscle. The responses were not affected by hexamethonium, dihydroergotamine or propranolol but were slightly reduced by guanethidine, reduced to about 40% by atropine and potentiated by eserine. Stimulation of the pelvic nerve proximal to the pelvic ganglion was partially blocked by hexamethonium. It is concluded that the urinary bladder of the rat is supplied by postganglionic adrenergic fibres mainly via the pelvic nerves and only to a lesser extent via the hypogastric nerves. Probably cholinergic fibres pass to the bladder mainly via the pelvic nerves but also via the hypogastric nerves, having their cellbodies outside the bladder wall, partly proximal to the pelvic ganglia.     

Compensatory increase of responses to nerve stimulation of the partially denervated rat urinary bladder

The rat urinary bladder was deprived of half of its innervation by removing the pelvic ganglion on one side. The motor responses of such a partially denervated bladder to stimulation of the pelvic nerve on the other side were examined I week, 1 month and 2 months postoperatively. On all three occasions the increase in pressure of the operated bladder was larger than that of the control bladder; the enlargement was most marked 2 months after operation. The responses were further enlarged by esenne, and markedly reduced by atropine. The present results combined with those of previous investigations, showing a rapid recovery in the activity of the acetylcholine forming enzyme from a reduced level and a transient supersensitivity to chemical stimuli after unilateral removal of the pelvic ganglion. suggest that the enlarged responses to nerve stimulation 1 week postoperatively are mainly due to sensitization, while those observed at the later stages are due to collateral sprouting from the cholinergic nerve fibres of the intact pelvic nerve.     

Unilateral denervation of the rat urinary bladder and reinnervation: a predominance for ipsilateral changes

Unilateral removal of the pelvic ganglion affected the bladder-half on the denervated side more profoundly than that on the non-denervated side. At an early stage (3-7 days) the former was heavier, had a lower choline acetyltransferase activity, developed less tension to nerve stimulation and, in course of time, became more sensitized to methacholine in vitro than the latter. At a late stage (30-60 days) the nerve-evoked contractile responses on the denervated side had increased, which was almost wholly attributed to the atropine-sensitive part of the contraction, but the recovery was not complete. On the non-denervated side the nerve-evoked responses were not significantly changed. It was calculated that in the normally innervated bladders 25-30% of the cholinergic nerves of each half were of contralateral origin. The figure was lower (18%) for the nerves mediating the atropine-resistant response.     

Adrenergic and cholinergic innervation of the rag urinary bladder.

The autonimic innervation of the rat urinary bladder was studied using histochemical methods and nerve stimulations. A sparse adrenergic innervation of the detrusor muscle was found. It was supposed to originate from long adrenergic neurones. The trigonum area had a rich ssupply of adrenergic fibres, probably derived from short adrenergic neurones. A uniformly rich supply of acetylcholine-esterase (AChE)-positive nerves was found in the WHOLE BLADDER. Postganglionic sympathetic denervation caused no detectable change of adrenergic or AChE-positive nerves in the bladder, while parasympathetic decentralization or denervation produced a total disappearance of adrenergic fibres. The AChE-positive nerves were appreciably reduced in number after parasympathetic decentralization and not detectable after postganglionic denervation. Neither adrenergic nor AChE-positive ganglion cells could be demonstrated in the bladder wall. Electrical stimulation of the hypogastric nerves or the pelvic nerves distal to the pelvic ganglia elictied contraction of the detrusor muscle. The responses were not affected by hexamethonium, dihydroergotamine or proparanolol but were slightly reduced by guanethidine, reduced to about 40% by atropine and potentiated by eserine. Stimulation of the pelvic nerve proximal to the pelvic ganglion was partially blocked by hexamethonium. It si concluded that the urinary bladder of the rat is supplied by postganglionic adrenergic fibres mainly via the pelvic nerves and only to a lesser extent via the hypogastric nerves. Probably cholinergic fibres pass to the bladder mainly via the pelvic nerves but also via the hypogastric nerves, having their cellbodies outside the bladder wall, partly proximal to the pelvic ganglia.     

Neurophysiological evidence for and characterization of the post-ganglionic innervation of the adrenal gland in the rat

The aim of this study was to examine and characterize the post-ganglionic innervation of the adrenal gland, using a neurophysiological nerve recording technique. Adrenal multifibre nerve activity was recorded in chloralose-anaesthetized Wistar rats. To test for post-ganglionic nerve activity, trimethaphan, a ganglionic blocker, was given intravenously. About 60% of the adrenal nerve preparations tested responded with a marked decrease in nerve activity (to 52 +/- 11% of pre-trimethaphan activity, P less than 0.01), while other nerves responded with an increase in activity (to 152 +/- 29% of pre-trimethaphan activity, P less than 0.01). Based on these responses, the nerves were considered to contain predominantly post- or preganglionic fibres respectively, and the difference in response to an intravenous injection of trimethaphan between the two groups was significant (P less than 0.01). It was also demonstrated that the post-ganglionic adrenal nerve activity had a greater variability in firing pattern than preganglionic adrenal nerve activity. We also examined whether there was any cardiac rhythmicity in the investigated nerves. There was a weak cardiac rhythmicity in six out of 12 post-ganglionic adrenal nerves, but there was no cardiac rhythmicity in the remaining six post-ganglionic nerves, and we observed no cardiac rhythmicity in preganglionic nerves. In contrast, renal sympathetic nerves showed a profound cardiac rhythmicity. Our results might explain recent histological findings of a direct post-ganglionic innervation of the adrenal cortex. We speculate that this nerve population is involved in steroid synthesis indirectly via regulation of the cortical blood flow or directly via a direct innervation of parenchymal cells in the adrenal cortex.     

Adrenergic innervation and neurogenic response in large and small arteries and veins from the rat

A combined morphological and physiological analysis of the properties of the adrenergic vasoconstrictor innervation was carried out in the splanchnic vasculature in the rat. Three generations of vessels were studied: (1) the abdominal aorta and the caval vein; (2) the superior mesenteric artery and vein; (3) 200 microns branches of the superior mesenteric artery and their parallelling veins. The adrenergic innervation was visualized by the Hillarp-Falck fluorescence technique, and by the immunohistochemical localization of immunoreactivity to tyrosine hydroxylase and dopamine-beta-hydroxylase. Determination of responses to applied noradrenaline, to transmural nerve stimulation and to direct activation of the muscle was made on ring preparations mounted in a myograph. All vessels were found to be innervated at the adventitio-medial border by noradrenergic nerves with varying density; the small arteries had the highest nerve density, the abdominal aorta was least innervated. When related to the maximal isometric response to applied noradrenaline, the maximal neurogenic response parallelled the density of the adrenergic innervation. Variations in frequency for half-maximal response among the vessels could not, however, be ascribed to innervation properties only. The constriction under isotonic conditions amounted to 20% of the initial circumference in the aorta, and to 30% in the small arteries and veins. The relation between response to applied noradrenaline and to nerve stimulation was similar for isometric and isotonic responses. In the smaller veins, a beta-receptor-mediated decline in the response to applied noradrenaline was seen. This decline was much less pronounced in neurogenic responses. The results indicate a gradation from proximal to peripheral arteries towards denser innervation and greater neurogenic responses. On the venous side only minor differences were found.     

Nervous regulation of the tone of the retractor penis muscle in the goat

The nervous control of the retractor penis muscle (rp) was investigated in the anaesthetized goat. Also, isolated field stimulated strips of the muscle were studied. The noradrenaline (NA) and acetylcholine (ACh) content of the rp was determined, and histochemistry for adrenergic and acetylcholinesterase (AChE) positive nerves was performed. The muscle exhibited spontaneous activity that persisted after section of all nerves. There was, however, also a tendency of the activity to follow the general vasomotor tone, which disappeared after section of the sympathetic chains. The excitatory adrenergic nerves which innervate the muscle come from the sympathetic chains and run along the pudendal, the hypogastric and the pelvic nerves. The rp has a dense network of adrenergic fibres and is very sensitive to excitatory adrenergic stimulation. It has a fairly large NA content, which is higher in old goats (5.95 ± 0.42 μg g^-1) than in young goats (2.87 ± 0.78 μg g-¹). Inhibitory non-adrenergic non-cholinergic (NANC) innervation reaches it via the pelvic and the hypogastric nerves. The maximum inhibitory response is reached at low frequencies (2–4 Hz). Cholinergic prejunctional inhibition of the excitatory response to sympathetic chain stimulation was effected by simultaneous stimulation of the hypogastric nerves. In vitro experiments confirmed the presence of endogenous cholinergic muscarinic suppression of the excitatory adrenergic neurotransmission. Significant amounts of ACh (0.81 7 plusmn; 0.18 μg g^-1) are present in the muscle, and it contains strongly AChE positive nerve fibres and nerve cell bodies. It is concluded that the goat rp is innervated by sympathetic adrenergic excitatory nerves and parasympathetic NANC inhibitory nerves. It further has a direct sympathetic inhibitory NANC innervation, and an indirect inhibitory cholinergic innervation which at least in part is sympathetic.     

Effects of right and left vagal stimulation on left ventricular acetylcholine levels in the cat.

To test the effectiveness of, and the interactions between, right and left vagal stimulation on left ventricular acetylcholine (ACh) levels, we applied the dialysis technique to the heart of anaesthetized cats. Dialysis probes were implanted in the left ventricular myocardium and perfused with Krebs-Henseleit buffer containing eserine. Dialysate ACh content was measured as an index of ACh release from post-ganglionic vagal nerve terminals in the left ventricular myocardium. We electrically stimulated the right and left cervical vagal nerves separately or together and investigated the dialysate ACh response. In two different regions of the left ventricle, substantial dialysate ACh responses were observed by the stimulation (20 Hz) of both right and left cervical vagal nerves. At stimulation frequencies of both 10 and 20 Hz, the dialysate ACh response to the bilateral vagal stimulation was almost algebraically the calculated sum of the individual dialysate ACh responses to unilateral vagal stimulation. In conclusion, ACh levels in the left ventricle are affected by both right and left vagal nerves and show little evidence of interactions between right and left vagal nerves at the level of the cardiac ganglia.     

Involvement of opioid and nicotinic receptors in rectal and anal reflex inhibition of urinary bladder motility in cats

The present study was performed to investigate mechanisms involved in urinary bladder relaxation during reflex activation of the pelvic nerves in the cat. Electrical stimulation of the pelvic nerves produced a contraction of the urinary bladder (P less than 0.05) and colon (P less than 0.05). Reflex activation of the pelvic nerves by rectal distension or mechanical stimulation of the anus induced relaxation of the bladder (P less than 0.05), while a colonic contraction was elicited (P less than 0.05). Naloxone (1.5 mg kg-1, i.v.) abolished the reflex inhibition of bladder motility elicited by rectal distension or mechanical stimulation of the anus (P less than 0.05). However, the urinary bladder and colonic contraction produced by electrical stimulation of the pelvic nerves were not affected. Hexamethonium (10 mg kg-1, i.v.) or severing of the pelvic nerves completely abolished the responses of the urinary bladder and colon to electrical stimulation or reflex activation of the pelvic nerves. The results indicate that inhibitory reflexes from the rectum and anal canal to the urinary bladder are conveyed via efferents of the pelvic nerves, and involve both nicotinic and opioid receptor mechanisms.     

VIP antisera inhibit the relaxatory motor responses of the feline sphincter of Oddi and gall-bladder induced by VIP or vagal nerve stimulation

Regional administration of VIP elicited a dose-dependent relaxation of the feline sphincter of Oddi and gall-bladder. Relaxatory motor responses of these regions at efferent electrical stimulation of the vagal nerves were unmasked after atropine (resistant to propranolol but sensitive to hexamethonium). These findings in combination with the presence of a rich VIP-ergic innervation, including intrinsic VIP neurons, have made VIP a tentative post-ganglionic non-adrenergic, non-cholinergic neurotransmitter to these regions. The relaxatory motor responses elicited by VIP or vagal activation were selectively antagonized using regional administration of specific VIP antisera in support of this hypothesis.     

Partially overlapping territories of nerves to hindlimb foot skin demonstrated by plasma extravasation to antidromic C-fiber stimulation in the rat

The innervation territories of the cutaneous nerves to the hindfoot were examined in the rat. Plasma extravasation of Evans blue to antidromic stimulation of C-fibers in the saphenous, sural, peroneal and tibial nerves was the means to determine the innervation areas. A high degree of bilateral symmetry was found for the territory of each nerve in individual rats. Stimulation of pairs of nerves, one on each side, that innervate neighboring skin regions revealed the existence of overlapping territories for all pairs of nerves. The area of overlap was greatest on the toes and just proximal to the toes on the dorsum of the foot, whereas overlap on the volar aspect of the foot was minimal. The need to consider the existence of overlapping cutaneous innervation in normal animals in studies involving collateral sprouting after nerve injuries is discussed.     

Reflex activation of sympathetic pathways to vesical smooth muscle and parasympathetic ganglia by electrical stimulation of vesical afferents.

1. Activation of the sympathetic input to the urinary bladder by electrical stimulation of afferent fibres in the pelvic nerve evoked three responses: (1) an initial transient rise in intravesical pressure, (2) an inhibition of neurally evoked bladder contractions and (3) an inhibition of transmission in vesical parasympathetic ganglia. Similar responses were elicited by stimulation of the hypogastric nerve. 2. The reflex responses were observed in acute spinal preparations (T10-T12) and in cats with intact spinal cords, but were abolished by bilateral transection of the hypogastric nerves. 3. The inhibition of bladder contractions was antagonized by the administration of propranolol (200-400 mug, I.A.), a beta-adrenergic blocking agent. The inhibition of ganglionic transmission was antagonized by dihydroergotamine (30-75 mug, I.A.), an alpha-adrenergic blocking agent. The initial rise in intravesical pressure was not antagonized by either agent. 4. Electrical stimulation of other afferents (carotid sinus nerve and sciatic nerve) did not consistently elicit responses in the urinary bladder. However, stimulation of these afferents as well as pelvic nerve afferents evoked reflex firing in nerve filaments on the surface of the urinary bladder. The firing was abolished by transection of the ipsilateral hypogastric nerve. 5. It is concluded that stimulation of vesical afferents activates a spinal sympathetic reflex which results in closing of the internal urethral sphincter and a depression of bladder activity. The latter occurs by a direct depression of detrusor smooth muscle as well as a block of the neural input to the bladder. This vesico-sympathetic reflex represents a negative feed-back mechanism which may have an important role in the maintenance of urinary continence.     

The effect of increasing the innervation field sizes of nerves on their reflex response time in salamanders

1. A simple quantitative measure was sought which could describe the relationship between reflex coupling in the spinal cord of salamanders and the peripheral innervation fields of the nerves from which the reflexes were elicited.2. In decerebrate salamanders reflex responses were recorded between pairs of cut hind limb nerves. The latencies (S/R times) of these reflex responses were bilaterally symmetrical for a given pair of nerves and were shorter when the stimulated nerve of the pair had a large motor and sensory peripheral limb innervation field; this was especially obvious for reflexes between 15th and 17th segmental nerves.3. After cutting or crushing the 16th nerve in adult salamanders, the adjacent 15th and 17th nerves sprouted collaterally to innervate denervated skin and muscle. There was apparently complete recovery of normal tactile reflexes and walking movements within a month.4. The operation did not affect the reflex response (S/R) times for nerve combinations on the unoperated side, which were not significantly different from those of normal animals with similar sized peripheral nerve fields. The unoperated side therefore represented the preoperative condition.5. In animals where one or both the 15th and 17th nerves had increased its innervation field size, the S/R times between them were significantly shorter on the operated side when the nerve with the enlarged field was stimulated. The degree of shortening was greatest for nerves showing the largest increase in peripheral field area.6. The S/R times between the 15th and 17th nerves were similar to those measured in normal animals in which the peripheral fields were of similar size to the enlarged fields in the operated animals. In a few cases where the increase in field size was considerable, the S/R time between the 15th and 17th nerves became as short as that between the 15th and 16th nerves on the control side.7. After removal of the 15th nerve, the 14th nerve sprouted into the trunk skin and muscle previously innervated by the 15th nerve and the 16th nerve into denervated limb skin and muscle. In spite of the increased peripheral fields of both these nerves, there was no change in the S/R times between them, or between any other pair of limb nerves on the operated side.8. The decrease in the S/R times between the 15th and 17th nerves was only observed where the stimulated nerve had increased its peripheral limb innervation field. The possible causes and significance of this shortening reflex response times are discussed in the context of an apparently functionally appropriate adaptation in the spinal cord.     

Transient supersensitivity in the hypertrophied rat urinary bladder

The presence of a paraffin bolus intravesically for 1 or 4 weeks was accompanied by an approximate three-fold increase in bladder weight. A supersensitivity to methacholine was demonstrated in these hypertrophied bladders at 1 week but not at 4 weeks. At the earlier time of observation, the contractile responses in vitro to nerve stimulation were reduced, while at the later time of observation the responses were the same as those of controls; the atropine-sensitive fraction of the response was entirely responsible for the recovery. The concentration of choline acetyltransferase, indicating cholinergic nerve density and initially reduced by about 50%, rose in course of time. It is suggested that in the beginning of the experimental period, the muscle cells of the hypertrophied bladders in situ are exposed to 'subnormal' transmitter concentration. An extension of the field of innervation of the cholinergic nerves may explain the disappearance of the supersensitivity.     

Diabetes mellitus and responses of the urinary bladder to acetylcholine: an in vitro study

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenously-administered muscarinic agonists, especially acetylcholine (ACh). In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ACh (10(-8)-10 (-4) M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The magnitude and/or intensity of the diabetic bladder enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed.     

Respiratory responses to electrical stimulation of the cervical sympathetic nerves in decerebrate, unanaesthetized cats

1. The effects of respiration of electrical stimulation of the peripheral cut end of the cervical sympathetic nerve were studied in seventeen decerebrate, unanaesthetized cats.2. The tidal volume increased and the end-tidal CO(2) fell within 2-3 breaths after the onset of stimulation in fourteen cats.3. In three experiments tidal volume and respiratory rate fell during stimulation and increased above the control when the stimulus was interrupted.4. All respiratory responses to sympathetic stimulation were abolished after cutting the carotid sinus nerves and are attributed to activation of chemo- and baroreceptor afferents.5. With the carotid sinus nerves intact, respiratory responses could be eliminated completely only by cutting the post-ganglionic sympathetics that pass directly to the carotid body and the post-ganglionic sympathetics that join the glossopharyngeal nerves to course with the sinus nerves to the carotid bifurcation.6. Pentobarbitone (10 mg/kg) or chloralose (40 mg/kg) given intravenously depressed spontaneous ventilation and responses to sympathetic nerve stimulation.7. Stimulation changed blood pressure slightly in fourteen experiments; in ten it fell and in four it increased 10 mm Hg or less. After pentobarbitone or chloralose, stimulation elicited a pronounced pressor response.8. The results of the study indicate that activation of sympathetic pathways to the carotid body constitutes an effective stimulus to ventilation.     

Synaptic Connections of the Paracervical (Frankenhauser) Ganglion of the Rat Uterus Examined with the Electron Microscope after Division of the Sympathetic and Sacral Parasympathetic Nerves

The fine structure of the synapses in the paracervical (Frankenhauser) ganglion of the rat uterus was studied after sympathectomy and sacral parasympathectomy. Degenerative nerve endings to the ganglion cells were observed after division of both the sympathetic and parasympathetic nerves. No degeneration of the synapses to the chromaffin cells were seen. The results are interpreted as evidence of both sympathetic and parasympathetic innervation of the ganglion cells, which send their axons to the uterus, whereas the “chromaffin cells” are innervated by intraganglionic nerves.     

Follow-up study after replantation of the forearm and nerves resuture

OBJECTIVE: Clinical and electrophysiological evolution after total section of the forearm and nerves resuture. MATERIAL AND METHODS: A young boy aged 14 years with accidental amputation of the right forearm. The forearm was replanted within the first 6 hours after accident. Electromyography, nerve conduction, estimated number of the motor units, single fiber EMG and motor complex reflex responses were studied until 4 years after surgery. RESULTS: Functional recovery was reached in muscles innervated by median and ulnar nerves. After 4 years of evolution EMG showed signs of chronic neuropathy. Nerve conduction did not reach normal values. Single fiber EMG showed increased fiber density and jitter, and intermittent impulse blocking The estimated number of the motor units was severely reduced with high mean amplitude. Motor reflex responses were elicited by cutaneous stimulation consistent with axon reflexes or ephatic responses. CONCLUSIONS: Replanted limbs in selected cases and nerve's resuture may reach a functional recovery for daily activities.     

Atropine sensitivity of the rat urinary bladder during nerve degeneration.

The pressure response of the rat urinary bladder to electrical stimulation of the pelvic nerve at a low stimulation frequency was found to be almost completely atropine resistant. However, the response to stimulation of the degenerating pelvic nerve 20-30 h after section of the nerve postganglionically using maximal stimulation frequency was totally abolished by atropine or the parasympatholytic agent Hoechst 9980. The responses were not affected by hexamethonium or dihydroergotamine, but were potentiated by eserine. The non-adrenergic portion of the bladder response to stimulation of the degenerating hypogastric nerves was also abolished by atropine or Hoechst 9980. It is concluded that the transmitter activating the detrusor muscle at stimulation of the pelvic nerve or the non-adrenergic part of the hypogastric nerve probably is acetylcholine.