This thesis examines aspects of sympathetic neuroeffector transmission in the rat and guinea-pig. The potential changes evoked in the smooth muscle of the rat tail artery, splenic arterioles and splenic capsule following transmural nerve stimulation are described in relation to the function of the effector tissue and the properties of the postganglionic neurones.
1. The neural control of vascular smooth muscle is reviewed. The relationship between membrane potential changes evoked by neurally released transmitter and contraction are discussed.
2. Intracellular recordings from the smooth muscle of isolated segments of rat tail artery at various ages between 45 and 150 days were made in order to relate the electrophysiological events and responses to perivascular nerve stimulation at different levels along the artery to differences in vessel structure during growth of the animals. Perivascular nerve stimulation evoked excitatory junction potentials (EJPs) lasting around 1 s, and neurogenic-alpha depolarizations (NADs) which lasted around 1 min. Both responses progressively decreased in amplitude along the length of the artery. NADs were abolished by blockade of ot2 adrenoceptors with phentolamine (1 |iM) or idazoxan (0.1 |iM). In addition to EJPs and NADs, slow depolarizing potentials (SDPs) could be evoked by transmural stimulation. SDPs were slighdy longer in time course that EJPs and could only be evoked by large stimulation voltages relative to those required to activate perivascular nerves. SDPs were not altered by removal of calcium or addition of tetrodotoxin (0.1 jiM) indicating that they arise independently of the innervation. As the strength of transmural stimulation was progressively increased, SDPs and EJPs were evoked simultaneously resulting in a depolarization of complex time course. During growth, the amplitudes of the EJP and NAD, recorded from cells deep in the media, declined significantly with age, the former to a greater extent than the latter. This decrease occurred during a period in which the density of the perivascular nerve plexus, as determined by catecholamine fluorescence, was increasing. The possibility that the decline in the amplitude of EJPs and NADs could be explained by changes in the electrical properties of the media related to hypertrophy, hyperplasia or changes in the electrical coupling was examined. The increase in wall thickness occurred without evidence of hyperplasia. EJPs recorded in the outermost smooth muscle cells of the media were relatively large in the older animals suggesting that the junctional current close to the neuromuscular junctions was larger in the older animals but was markedly dissipated in the radial direction throughout the media. This is consistent with a reduced intracellular resistance to current flow in the hypertrophied smooth muscle. The changes in electrical properties of this effector tissue occurred after an age at which rats are generally assumed to be mature. The findings reveal that changes in the mechanisms of sympathetic neurovascular transmission continue into early adult life.
3. The effects of the putative P2x-purinoceptor antagonist suramin were tested on EJPs and NADs, and depolarizations evoked by exogenous noradrenaline and ATP. EJPs were abolished in the presence of suramin (1 mM). NADs were unaffected by suramin indicating that release of noradrenaline was unaffected. The time constant of the decay phase of the EJP was unaffected during the onset of blockade by suramin. This suggests that suramin does not block the EJP by changing the resting conductance of the membrane. The depolarization evoked by exogenous ATP (10-100 pM) was inhibited but not abolished in the presence of suramin (1 mM) whereas depolarizations induced by exogenous noradrenaline (1-2 pM) were not inhibited. The data indicate that suramin acts postjunctionally to abolish EJPs by interfering with either a junctional receptor or its associated ligand gated channel.
4. Current knowledge of the regeneration of peripheral axons following nerve injury is reviewed, with emphasis on the reinnervation of vascular targets by sympathetic axons.
5. Sympathetic neurotransmission in the rat tail artery was studied at various times between 7 and 128 days after freeze lesions of all four collector nerve trunks, near the base of the tail at 21 days of age. The most proximal regions of tail artery (down to 35-40 mm from the lesion site) were not denervated Reinnervation was assessed by the reestablishment of a perivascular nerve plexus, visualized by catecholamine fluorescence, and by the reappearance of EJPs and NADs. Reinnervation was rapid within a few centimetres of the lesion site, where the maximum regeneration was around 1.5 mm/day. The time at which EJPs were first recorded in previously denervated sections of artery closely parallelled the return of patches of perivascular plexus. The earliest recorded EJPs were small in amplitude and showed marked facilitation. These were followed by NADs which were large relative to the EJP. At later times (57-128 days post-operatively) at distances 40-70 mm distal to the lesion site EJPs returned to control amplitudes but NADs were significantly larger than those evoked in control animals. However, the perivascular plexus returned to only 80% of control density. In contrast, more distal portions of the artery were very poorly reinnervated even in the oldest animals studied. These results reveal that postganglionic sympathetic axons are able to reestablish functional contacts with vascular smooth muscle over short distances but that reinnervation of more distal targets is slow and/or may fail to occur. The postjunctional response to release of transmitter is modified and may remain modified for long periods of time. 6. Intracellular recordings were made from the smooth muscle of arterioles (25-150 Jim diameter) and the capsule of the spleen of guinea-pigs and rats. The innervation of the spleens of both species was studied using catecholamine fluorescence and immunohistochemical techniques. Catecholamine-containing axons which were also immunoreactive for neuropeptide Y (NPY) were associated with the smooth muscle cells of arterioles, capsule and trabeculae and amongst the peri-arteriolar lymphatic sheath (PALS). EJPs but no NADs could be evoked in smooth muscle cells of the splenic arterioles. EJPs were not observed in the smooth muscle of the splenic capsule. However, trains of stimuli (> 10 Hz, 1 s duration) evoked a biphasic NAD with an initial component lasting around 10 s which was abolished by the a\-adrenoceptor antagonist prazosin (1 p.M) followed by a second component which lasted about 1 min and was inhibited by the <X2-adrenoceptor antagonist idazoxan (1 (iM). In some cells a small faster depolarization lasting around 1 s persisted after a-adrenoceptor blockade. The slow component of the NAD was identical in time course and idazoxan sensitivity to the NAD in evoked in the rat tail artery suggesting that similar mechanisms underly the potential change in these two muscles. The data indicate that sympathetic neuroeffector transmission from noradrenergic axons containing NPY to smooth muscle of splenic arterioles and capsule occur by markedly different mechanisms. Sympathetic transmission in the splenic capsule is similar to that seen in many veins.
7. Sympathetic postganglionic neurones can be classified as tonic, phasic or long afterhyperpolarizing (LAH) on the basis of action potential discharge during maintained depolarizing current, and the duration of the afterhyperpolarization following an action potential. The electrical properties of antidromically identified splenic neurones within the coeliac ganglion of the guinea-pig were studied with intracellular microelectrodes. The location of splenic neurones within the ganglion was determined using the retrograde tracing molecule Fluorogold. Most labelled neurones (80%) were in the proximal lobe of the left coeliac ganglion. Twenty one splenic neurones were identified. In 94% of neurones one suprathreshold synaptic potential as well as several subthreshold excitatory synaptic potentials were evoked by stimulation of the left splanchnic nerve. Fourteen neurones fired phasically at the beginning of a depolarizing current step and were classified as phasic/LAH. Ten of these could be clearly classified as LAH neurones. One neurone had a tonic pattern of action potential discharge but was different to non-splenic tonic neurones in all other characteristics. These results suggest that most splenic neurones have similar electrical properties despite differences in their effectors. As the majority of axons within the spleen are associated with arterial smooth muscle, the data suggest that some LAH neurones are vasoconstrictor.
8. Two calcium activated potassium conductances (gKCai and gKCa2> underlie the afterhyperpolarization which follows the action potential in sympathetic postganglionic neurones. These were investigated by measuring the tail currents (in single electrode voltage clamp) following a depolarizing voltage step (50 ms duration) which reached threshold for an "action current". The tail current in tonic and phasic neurones (gKCaj) peaked at the end of the voltage step and decayed with a time constant of 100-130 ms. LAH neurones had a biphasic tail current In addition to gKCaj, a second slower current was evoked. This current (gKCa2) peaked around 800 ms after the action current and decayed with a time constant of around 1.5 s. gKCal was inhibited by the bee venom derivative apamin (50-100 nM) consistent with the involvement of the SK class of Ca^+ activated K+ channel. gKCa2 was resistant to apamin but inhibited by ryanodine, a plant alkaloid which has been shown to block release of Ca?+ from internal stores. This suggests that gKCa2 results from Ca^+ influx during the action potential which triggers release of Ca^+ from internal stores which activates a different class of K+ channel. Noradrenaline (100 |iM) abolished gKCa2 selectively although the physiological significance of this is unclear.
9. Sympathetic nerve stimulation evokes several potential changes in smooth muscle. Each smooth muscle varies in the potential changes which can be evoked by transmural stimulation. These potential changes may be altered either during growth and maturation or as a result of a disruption of the innervation. The type of potential change is independent of the neurochemistry and the electrical properties of the sympathetic postganglionic neurones.