Choline Acetyltransferase Activity in the Denervated Urinary Bladder of the Rat

After extirpation of the pelvic ganglia the choline acetyltransferase activity decreased markedly indicating that most of the postganglionic cholinergic neurones of the bladder take this route. A small decrease in the activity of this enzyme was found after section of the hypogastric nerves, showing that these nerves contribute to some extent to the cholinergic innervation. The residual enzyme activity found after a combination of the two surgical procedures suggests that neurones relay distal to the level of the section of the hypogastric nerves and pass outside the pelvic ganglia and (or) that neurones pass the pelvic ganglia and relay distal to them. Electrical stimulation of the hypogastric nerves after extirpation of the pelvic ganglia and the use of blocking drugs showed on the existence of cholinergic neurones passing outside the pelvic ganglia, some of them relaying distal to the point of stimulation.     

Recovery of choline acetyltransferase activity in the rat urinary bladder deprived of half of its innervation

In the urinary bladder of the rat, partially denervated by unilateral removal of the pelvic ganglion 3 days in advance, the activity of the acetylcholine-forming enzyme, choline acetyltransferase, measured by a radiolabelling method, was reduced to 58% of the control. A gain in enzyme activity of 28% was found to have occurred when the bladders were examined 25 days postoperatively; the main part of this increase took place during the period 3 to 6 days after the operation and beyond 25 days no further gain in enzyme activity was found. The present findings are compared with previous observations of a transient supersensitivity and an increased motor response to electrical stimulation of the intact pelvic nerve of such a partially denervated bladder.     

Functional organization of the sympathetic innervation supplying the hairless skin of the hindpaws in chronic spinal cats

1. Reflex activation of the sudomotor system supplying the cat's hindpaws to natural stimulation of skin has been investigated in chronic low thoracic spinal cats. The neuronal activation of the sweat glands was measured by recording the skin potentials from the surface of the hairless skin.
2. The sudomotor system was activated by mechanical and thermal noxious stimulation of skin and by stimuli which excited the Pacinian corpuscles in the paws. The reflexes were maximal some 50 to 75 days following spinalization.
3. Noxious stimulation of the skin was followed by an increase of the size and of the frequency of the resting activity in the sudomotor system. This increase of the resting activity lasted for 10 min or longer and was, in many preparations, especially pronounced 40 days or later after spinalization.
4. Neurophysiological investigations of postganglionic neurones in the medial plantar nerve in 10 chronic spinal cats (74 to 129 days after spinalization) revealed two populations of postganglionic neurones:type 1 neurones were excited by noxious cutaneous stimuli and by vibrational stimuli and classified as sudomotor neurones.Type 2 neurones were inhibited by noxious cutaneous stimuli applied to the ipsilateral hindfoot and mostly excited by noxious stimuli applied to the contralateral hindfoot and not affected by vibrational stimuli. These neurones were classified most likely as vasoconstrictor neurones.
5. The results argue that the chronic spinal cord contains reflex circuits relaying specific somatosympathetic reflexes to the sweat glands. Whether these spinal circuits are normally used in cats with intact CNS is an open question.

     

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.     

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.     

Enhancement of resting activity in postganglionic vasoconstrictor neurones following short-lasting repetitive activation of preganglionic axons

Resting activity in postganglionic neurones supplying the cat hindlimb was enhanced after activation of preganglionic axons in the lumbar sympathetic trunk with short trains of stimuli (50 stimuli at 25 Hz). The characteristics of this enhancement were as follows: 1) It has values of 120 to 600% and lasts for 4 to more than 40 min. 2) It can be elicited heterosynaptically by repetitive stimulation of the peripheral end of a cut white ramus. 3) It is dependent on the activation of thin, probably unmyelinated, preganglionic axons. 4) It is probably produced by non-nicotinic (muscarinic and non-cholinergic) synaptic mechanisms in the sympathetic chain ganglia. 5) Vasoconstrictor neurones can be activated in this way, but not sudomotor neurones. The results argue that nicotinic transmission of activity from pre- to postganglionic vasoconstrictor neurones can be modulated heterosynaptically by slow non-nicotinic synaptic processes.     

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.     

Reflex activation of postganglionic vasoconstrictor neurones supplying skeletal muscle by stimulation of arterial chemoreceptors via non-nicotinic synaptic mechanisms in sympathetic ganglia

Postganglionic sympathetic neurones supplying skeletal muscle and skin can be activated from the preganglionic site via cholinergic nicotinic, muscarinic and noncholinergic synaptic mechanisms. The experiments described in this paper were designed in order to show that postganglionic vasoconstrictor neurones supplying skeletal muscle can be activated by the naturally occurring discharge pattern in preganglionic axons when the nicotinic transmission is blocked. For this purpose, the activity was recorded simultaneously from postganglionic vasoconstrictor axons supplying skeletal muscle and vasoconstrictor axons supplying hairy skin. The preganglionic neurones were driven reflexly by stimulation of the arterial chemoreceptors. 1) During blockade of nicotinic transmission muscle vasoconstrictor neurones were activated via the CNS during stimulation of arterial chemoreceptors. This activation is either generated by muscarinic action of released acetylcholine or by a noncholinergic synaptic mechanism. 2) Postganglionic cutaneous vasoconstrictor neurones were inhibited during stimulation of arterial chemoreceptors. During blockade of cholinergic nicotinic transmission these neurones were not activated reflexly by stimulation of arterial chemoreceptors although they received inputs via cholinergic muscarinic and noncholinergic synaptic mechanisms. 3) The results illustrate that postganglionic vasoconstrictor neurones supplying skeletal muscle can not only be activated via non-nicotinic synaptic mechanisms through synchronous repetitive electrical stimulation of preganglionic axons but also by the discharge pattern produced in preganglionic neurones during stimulation of arterial chemoreceptors.     

Effect of pre- and postganglionic nerve divisions on normal postnatal and hydrocortisone-induced development of small intensely fluorescent cells in rat superior cervical ganglion

The left superior cervical ganglion of 3- or 23-day-old rats was subjected to pre- and/or postganglionic nerve division or sham operation, while the right ganglion was left intact. The animals were killed 20 or 60 days after the operation. Some animals were injected with 20 mg/kg hydrocortisone daily for 7 days and killed on the 8th day.Fluorescence microscopical examination revealed a normal postnatal increase in the number of small intensely fluorescent cells/ganglion after pre- or postganglionic nerve division, in spite of marked decreases in the volume of the operated ganglia. Combined pre- and postganglionic nerve division, which caused a dramatic loss of ganglion volume, entirely prevented the postnatal increase in the number of small intensely fluorescent cells. Hydrocortisone caused a large increase in the number of small intensely fluorescent cells both in intact and operated ganglia, including those in whom both pre- and postganglionic nerves had been divided.It is concluded that combined pre- and postganglionic denervation, in contrast to either operation alone, prevents the normal proliferation of the small intensely fluorescent cells possibly by causing an extensive loss of principal nerve cells which deprives the small intensely fluorescent cells of their normal contacts with the principal cells. Since the increase in the number of small intensely fluorescent cells due to hydrocortisone injections was not prevented by pre- and postganglionic denervation it must be due to a mechanism different from that responsible for the formation of small intensely fluorescent cells during normal postnatal development.     

Long-lasting discharge of postganglionic neurones to skin and muscle of the cat's hindlimb after repetitive activation of preganglionic axons in the lumbar sympathetic trunk

1. Postganglionic neurones to hairy skin and to skeletal muscle of the cat's hindlimb, probably vasoconstrictor in function, have been investigated for their response characteristics on repetitive stimulation of the preganglionic axons in the lumbar sympathetic trunk in chloralose anaesthetized cats.
2. Repetitive stimulation of the preganglionic axons with 50 stimuli at 25 to 50 Hz and stimulus strengths which excited all classes of preganglionic axons generated eartly short latency responses and late low frequency responses in postganglionic neurones. These late responses occurred predominantly in postganglionic neurones supplying skeletal muscle and in only 30% of postganglionic neurones supplying hairy skin. In most neurones the late discharges could only be elicited by trains of more than 5–10 preganglionic stimuli at frequencies of more than 5–10 Hz.
3. The threshold stimulus strength applied to the preganglionic axons to generate the late discharge was on average 4 times higher than for the early discharge. This finding implies that the late discharge requires the activation of thin preganglionic axons.
4. In most postganglionic neurones, the late discharges could be blocked by atropine, indicating that they were generated by muscarinic action of acetylcholine. The late discharges in a few cutaneous postganglionic neurones were unaffected by atropine. The early discharges were blocked by hexamethonium.
5. The results argue that the transmission of impulses from pre- to postganglionic neurones may be accomplished by two separate pathways acting via nicotinic and muscarinic receptors respectively.

     

Behaviour of the urethral striated sphincter and of the bladder in the chronic spinal cat

Nine cats were spinalized at the thoraco-lumbar junction (T12-L7) and the subsequent behaviour of the bladder and urethral striated sphincter was observed during periods of up to 27 days after spinalization by means of bladder manometry and of urethral electromyography. On the day following operation, the urethral sphincter responds to stimulation of its intact motor nerve, the pudic nerve by reflex (R) and direct (M) responses analogous to those of the intact animal anaesthetized with chloralose. The ratio R/M lies between 1 and 0.6 in the chronic spinal cat whereas it is generally less than 0.5 in the intact chloralose-anaesthetized cat. The tonic activity of the sphincter is weak or not present. The continence, however, is well maintained. The bladder activity appears only 4 to 8 days after spinalization. The bladder can thus void urine during brief contractions. These micturitions are always incomplete. The urethral reflex activity, either spontaneous or triggered by stimulation of the pudic nerve, may be inhibited, i: to a moderate degree by passive bladder distension; ii: almost completely by activation of vesicomotor neurones which provoke the bladder contraction. The first inhibition is seen the day after spinalization and is probably a protective reflex against vesical hypertension. The second inhibition develops progressively and in parallel to the functional recovery of vesical preganglionic neurones. It takes place on a background of antagnostic equilibrium of bladder and of urethral sphincter activities during micturition.     

Biphasic effects of substance P on respiratory activity and respiration-related neurones in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of substance P (SP) on respiratory activity in the brainstem-spinal cord preparation from neonatal rats (0-4 days old) were investigated. The respiratory activity was recorded from C4 ventral roots and intracellularly from three types of respiration-related neurones, i.e. pre-inspiratory (or biphasic E), three subtypes of inspiratory; expiratory and tonic neurones in the ventrolateral medulla (VLM). After the onset of SP bath application (10 nM-1 microM) a dose-dependent decline of burst rate (by 48%) occurred, followed by a weaker dose-dependent increase (by 17.5%) in burst rate. The biphasic effect of SP on inspiratory burst rate was associated with sustained membrane depolarization (in a range of 0.5-13 mV) of respiration-related and tonic neurones. There were no significant changes in membrane resistance in any type of neurones when SP was applied alone or when synaptic transmission was blocked with tetrodotoxin (TTX). The initial depolarization was associated with an increase in inspiratory drive potential (by 25%) as well as in bursting time (by 65%) and membrane excitability in inspiratory and pre-inspiratory neurones, which corresponded to the decrease in burst rate (C4 activity). The spiking frequency of expiratory and tonic neurones was also increased (by 36 and 48%). This activation was followed by restoration of the synaptic drive potential and bursting time in inspiratory and to a less extent in pre-inspiratory neurones, which corresponded to the increase in burst rate. The discharge frequency of expiratory and tonic neurones also decreased to control values. This phase followed the peak membrane depolarization. At the peak depolarization, SP reduced the amplitude of the action potential by 4-8% in all types of neurones. Our results suggest that SP exerts a general excitatory effect on respiration-related neurones and synaptic coupling within the respiratory network in the VLM. The transient changes in neuronal activity in the VLM may underlie the biphasic effect of SP in the brainstem respiration activity recorded in C4 roots. However, the biphasic effect of SP on inspiratory burst rate seems to be also defined by the balance in activity of other SP-sensitive systems and neurones in the respiratory network in the brainstem and spinal cord, which can modify the activity of medullary respiratory rhythm generator.     

The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission

The twitch response observed during low frequency electrical stimulation of postganglionic cholinergic neurones supplying the longitudinal smooth muscle of the guinea-pig ileum was markedly reduced by incubation with an homogeneous preparation of botulinum type A neurotoxin (4.3-8.6 nM). This intoxication of the autonomic cholinergic neurones was long-lasting, irreversible by washing, but readily reversed by 4-aminopyridine (50-1000 microM). The noradrenergic motor response of the rat anococcygeus following field stimulation was partially antagonised by the neurotoxin. The non-adrenergic inhibitory response of the guinea-pig taenia coli, elicited by field stimulation, was not antagonised by botulinum toxin, suggesting that a source of a non-adrenergic inhibitory transmitter exists, other than intramural cholinergic neurones. However, the neurogenic excitatory responses of the guinea-pig bladder, elicited by field stimulation in the presence of atropine and guanethidine, were virtually abolished by botulinum toxin. It is suggested that the parasympathetic neurones which supply the smooth muscle of the guinea-pig urinary bladder co-release acetylcholine and a non-cholinergic excitatory transmitter; ATP or polypeptides are possible candidates.     

Biphasic effects of substance P on respiratory activity and respiration‐related neurones in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of substance P (SP) on respiratory activity in the brainstem–spinal cord preparation from neonatal rats (0–4 days old) were investigated. The respiratory activity was recorded from C4 ventral roots and intracellularly from three types of respiration‐related neurones, i.e. pre‐inspiratory (or biphasic E), three subtypes of inspiratory; expiratory and tonic neurones in the ventrolateral medulla (VLM). After the onset of SP bath application (10 nM ^–1 μM ) a dose‐dependent decline of burst rate (by 48%) occurred, followed by a weaker dose‐dependent increase (by 17.5%) in burst rate. The biphasic effect of SP on inspiratory burst rate was associated with sustained membrane depolarization (in a range of 0.5–13 mV) of respiration‐related and tonic neurones. There were no significant changes in membrane resistance in any type of neurones when SP was applied alone or when synaptic transmission was blocked with tetrodotoxin (TTX). The initial depolarization was associated with an increase in inspiratory drive potential (by 25%) as well as in bursting time (by 65%) and membrane excitability in inspiratory and pre‐inspiratory neurones, which corresponded to the decrease in burst rate (C4 activity). The spiking frequency of expiratory and tonic neurones was also increased (by 36 and 48%). This activation was followed by restoration of the synaptic drive potential and bursting time in inspiratory and to a less extent in pre‐inspiratory neurones, which corresponded to the increase in burst rate. The discharge frequency of expiratory and tonic neurones also decreased to control values. This phase followed the peak membrane depolarization. At the peak depolarization, SP reduced the amplitude of the action potential by 4–8% in all types of neurones. Our results suggest that SP exerts a general excitatory effect on respiration‐related neurones and synaptic coupling within the respiratory network in the VLM. The transient changes in neuronal activity in the VLM may underlie the biphasic effect of SP in the brainstem respiration activity recorded in C4 roots. However, the biphasic effect of SP on inspiratory burst rate seems to be also defined by the balance in activity of other SP‐sensitive systems and neurones in the respiratory network in the brainstem and spinal cord, which can modify the activity of medullary respiratory rhythm generator.     

The Effect of Teeth Amputations on the Choline Aeetyltransferase Activity of Rat Submaxillary Glands

The choline aeetyltransferase activity in parasympathetically decentralized glands was unaffected by repeated teeth amputations over a period of 2 weeks, while after the same period of time the treatment caused the enzyme activity to increase in innervated glands. It appears that the enzyme in the postganglionic nerve is for its activity dependent on an intact connection with the central nervous system. The increase in enzyme activity is attributed to an enhanced reflex stimulation of the glands from pulpal receptors.     

Fall in choline acetyltransferase activity in the ventricles of the rat heart after treatment with a ganglion blocking drug

The choline acetyltransferase activity was found to be 27% lower in the ventricles of rats which had been given the ganglion blocking drug chlorisondamine than in those of untreated rats. Since the weight of the ventricles were also lower in the treated rats than in the untreated ones, it was wondered whether a change in the muscle mass as such affected the activity of the enzyme: isoprenaline treatment was found to cause the weight to increase markedly, while it left the enzyme activity unchanged. The decrease in enzyme activity observed after the prolonged ganglion blockade is interpreted as being the consequence of a reduced traffic of impulses along the postganglionic parasympathetic nerves of the ventricles. Thus, the results of the present investigation give further support for the idea of a vagal innervation of this part of the heart.     

Role of sympathetic innervation in the feline continence process under natural filling conditions.

1. The effect of the sympathetic innervation on the bladder detrusor muscle was assessed in pentobarbitone-anesthetized cats by measuring the changes in bladder wall tension that occurred during sympathetic ganglion blockade after filling the bladder at a natural rate. 2. At a point 60% of the way through the continence process, systemic sympathetic blockade was produced by intravenous trimethaphan, and selective blockade of postganglionic hypogastric nerve activity was produced by application of trimethaphan to the exposed inferior mesenteric ganglia. The level of blockade was monitored with an in-continuity hypogastric nerve recording. 3. During both systemic and selective ganglion blockade, there was an increase in baseline transmural bladder pressure and a decrease in the amplitude of nonmicturating contractions. 4. Although there was no change in the mean level of transmural bladder pressure during either systemic or selective blockade, there was a significant increase in the mean level of bladder wall mechanoreceptor discharge, suggesting that before the blockade sympathoinhibitory effects were greater than sympathoexcitatory effects. 5. Measurement of bladder wall mechanoreceptor discharge before and during ganglion blockade revealed a net sympathoinhibitory influence on the level of bladder wall tension under natural filling conditions. These results confirm that the detrusor muscle of the feline bladder is under both sympathoinhibitory and sympathoexcitatory influences for a significant portion of the continence process.     

Development of sympathetic ganglionic neurotransmission in the neonatal rat. Pre- and postganglionic nerve response to asphyxia and 2-deoxyglucose

To determine the time course of development of neurotransmission in the sympathetic ganglion of the rat, pre- and postganglionic activity was recorded from the cervical sympathetic trunk in anesthetized neonatal and mature preparations. Tonic activity and responses to two stimuli, cellular hypoglycemia induced by 2-deoxyglucose and asphyxia, which are known to evoke CNS-mediated sympathetic activation in mature rats were measured. In 2–11-day-old neonates, tonic preganglionic activity recorded from the cervical sympathetic nerve and responses to hypoglycemia and asphyxia were comparable to or greater than that in mature rats. In 17–19-day-old neonates these variables were elevated to twice the adult value. In contrast, tonic postganglionic activity recorded from the internal carotid nerve was barely detectable through 5 days of age and there was no response to hypoglycemia. During asphyxia, maximum postganglionic impulse frequency and total number of impulses discharged were 10–20% of the mature value through the 5th postnatal day and the duration of the postganglionic response was only 25% of the preganglionic response. Tonic postganglionic activity and responses to stimuli were equivalent to those in mature rats by the 10th postnatal day. The compound action potential evoked in the postganglionic axons by direct electrical stimulation was comparable in 4–5 and 10–13 day-old rats. It is concluded that functional ganglionic neurotransmission is established in the neonatal rat between the 5th and 10th postnatal day. The relation between biochemical changes associated with maturation of the postganglionic neuron, ganglionic synaptogenisis and neurotransmission is discussed.It is concluded that synaptogenisis and onset of neurotransmission are causally associated with development of CNS regulation of postganglionic activity and end organ response rather than with maturation of the postganglionic neuron and that cholinergic excitation of the postganglionic neuron adequate to evoke action potentials is not essential to initiate maturation of the neuron.     

Activation of postganglionic neurones via non-nicotinic synaptic mechanisms by stimulation of thin preganglionic axons

Activity in postganglionic neurones supplying skeletal muscle and hairy skin of the cat hindlimb was measured after single and repetitive stimulation of preganglionic axons in the lumbar sympathetic trunk at variable stimulation strengths. Simultaneously the compound action potentials were measured in one of the lumbar white rami L₂ or L₃. Early short latency (“nicotinic”) responses were obtained in the postganglionic neurones when preganglionic axons conducting at 8.9^±3.44 m/s (n=12) or less were stimulated. Late, long-latency non-nicotinic responses were elicited in the postganglionic neurones when preganglionic axons conducting at 1.52^±0.94 m/s (n=12) or less were stimulated.     

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia.

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), or the desensitising agonist, alpha,beta-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 microM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 microM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gamma S, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATP gamma S on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 microM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 microM).The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP.We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.