I. Introduction

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The release of a transmitter at a given synapse, whether in the central or peripheral nervous system, is far from being constant and subject to modulation by a variety of mechanisms. In contrast to the original view of one-way neurotransmission, where a chemical substance was suggested to be released from a neuronal site to act at the postsynaptic cell (e.g., Loewi, 1921), we have learned during the last four decades that neurotransmitters may act back onto the nerve endings from which they have been released. In addition, other transmitters, drugs, and hormones may also change the amount of neurotransmitter being released from neurons by acting directly onto the sites of transmitter release. Because transmitters are most commonly released from nerve endings, also called presynapses, this phenomenon is known as presynaptic modulation. An early example of presynaptic modulation of neurotransmission was provided by Riker and coworkers (1957), who reported stimulatory effects of quarternary ammonium compounds at the neuromuscular junction. These authors also appear to be the first to use the term "presynaptic receptor" for the site of action of these stimulatory agents. In the same year, a first example of inhibitory presynaptic modulation of neurotransmission was obtained in the spinal cord where -aminobutyric acid (GABA¹) was reported to diminish excitatory transmit-ter release from primary afferent nerve endings (Frank and Fuortes, 1957). Also at the same time, Brown and Gillespie (1957) found that the -adrenoceptor antagonist phenoxybenzamine raised sympathetic transmitter release. However, at that time, it was not recognized that this effect may be due to the blockade of the autoinhibitory feedback modulation via presynaptic -adrenoceptors, and the authors speculated that an inhibitory action on uptake mechanisms may underlie the effects observed. Since these early days of the investigation of presynaptic modulation, effects of either neurotransmitters or receptor agonists and antagonists on the amount of transmitter being released have been described for virtually each synapse investigated, and a number of excellent reviews have summarized these phenomena (e.g., Langer, 1977, 1997; Westfall, 1977; Starke, 1981; Starke et al., 1989; Vizi et al., 1991; Wu and Saggau, 1997; Miller, 1998). A large part of our current knowledge about the function of presynaptic receptors has been obtained with monoaminergic neurons. As a consequence, a considerable number of reviews have previously dealt with the receptor-dependent modulation of the neuronal release of catecholamines in general (e.g., Langer, 1974, 1981), and more specifically, with the presynaptic regulation of noradrenaline release (e.g., Starke, 1977, 1987; Fuder and Muscholl, 1995). In addition, there are excellent reviews on the pre- and postsynaptic modulation of adrenergic neurotransmission (Westfall, 1977; Vanhoutte et al., 1981) and neurotransmitter release from sympathetic varicosities (Stjärne, 1989). However, sympathetic neurons do release more than just noradrenaline (see below); and the presynaptic modulation of, for instance, ATP release has also been reviewed recently (Starke et al., 1996; von Kügelgen, 1996).

In light of the plethora of reviews dealing with either presynaptic receptors or neuromodulation in a more general sense, it appears somewhat surprising that we were unable to identify a single review that selectively describes the functions of presynaptic receptors in the sympathetic nervous system. Nevertheless, most types of receptors mediating presynaptic modulation in sympathetic neurons have been covered by the expert reviews mentioned above. Therefore, this paper does not aim at listing each and every report that described some kind of presynaptic receptor on sympathetic neurons. We will rather try to highlight recent developments in the field of presynaptic modulation in sympathetic neurons as a model to study general mechanisms of presynaptic modulation. For a detailed summary of reports on presynaptic receptors of peripheral noradrenergic neurons, the reader is referred to excellent previous reviews by Westfall (1977), Vizi (1979), Langer (1981), Starke (1977 and 1987), and Fuder and Muscholl (1995).

Presynaptic receptors may be activated by either exogenously applied receptor agonists or by endogenously released neurotransmitters. In the latter case, transmitters activating the presynaptic receptors of a given nerve terminal may be released from either the very same nerve ending or from a different axon terminal. In the first situation, the receptor involved is a presynaptic autoreceptor, whereas in the second situation, the receptor is called a presynaptic heteroreceptor. Although the functions of presynaptic heteroreceptors were elucidated first, the detection of presynaptic autoreceptors turned out to be decisively important, in particular for the description of receptor subtypes: ₂-adrenoceptors and H₃ histamine receptors were discovered as presynaptic receptors involved in the autoinhibition of transmitter release (Langer, 1974; Arrang et al., 1983). Several reviews have covered exclusively presynaptic auto- (e.g., Starke, 1987, Starke et al., 1989) and heteroreceptors (Fuder and Muscholl, 1995), respectively. Here, we will consider both types of presynaptic receptors.

By definition, presynaptic receptors are those located at the presynapse, in other words, at the neuronal structures from which transmitters are being released and that, therefore, contain large clusters of neurotransmitter containing vesicles. Transmitter release occurs at specialized regions of the presynapse called "active zones," but the entire nerve ending is much larger than these restricted sites of transmitter release (Matthews, 1996). Is, thus, a presynaptic receptor only a binding site located at or close to the active zone? Is a receptor located somewhere else at the nerve terminal also a presynaptic one? And what about receptors located at the axon in close proximity to the nerve terminal (see below)? Finally, is a receptor detected within a synapse indeed a presynaptic one, or could it also be a postsynaptic one? Taken together, it appears somewhat difficult to define by just morphological means what may be a real presynaptic receptor. Therefore, various procedures have been used to identify by functional means whether a receptor is a presynaptic one. These experimental strategies include the following techniques: (i) the use of synaptosomes, which are isolated nerve terminals (Gray and Whittaker, 1962; Whittaker, 1993); (ii) the use of dissociated neurons in cell cultures where appropriate postsynaptic cells are lacking (see Boehm and Huck, 1997a, for a review on sympathetic neurons); (iii) the application of tetrodotoxin in various in vitro preparations, which blocks the propagation of action potentials along the axons down to nerve terminals (see Methodological Considerations); and (iv) the destruction of nerve terminals by lesioning of the corresponding neuron and the subsequent demonstration of a loss of function or loss of ligand binding.

Dealing with presynaptic receptors, one also has to take into consideration the term "preterminal," or presynaptic, receptors. As specified by the latter expression, this would designate a receptor that is located at regions of the axons that are close to the terminals but not at the terminals themselves. In analogy to what has been said above, a distinction between presynaptic and preterminal receptors can hardly be made by morphological means and must therefore rely again on functional criteria. Among the techniques mentioned above, the use of synaptosomes and the blockade of Na⁺-dependent action potentials, for instance, by tetrodotoxin should avoid the contribution of preterminal receptors to presynaptic modulatory effects.

On a physiological level, a presynaptic receptor is one that may, upon activation, modulate some function of the presynapse. Vesicle exocytosis and resulting transmitter release is only one task presynaptic nerve terminals have to deal with. Other functions of nerve terminals include, for instance, endocytosis, reuptake, and neurotransmitter synthesis and metabolism. Hence, any receptor that interferes with one of these presynaptic mechanisms may be called a presynaptic receptor. Nevertheless, in this text, we will only discuss receptors that somehow influence the amount of transmitter being released from sympathetic neurons. In this context, it should also be mentioned that transmitter release at nerve terminals may occur via at least two different mechanisms: vesicle exocytosis and reverse transport through the appropriate plasmalemmal transporter, which otherwise mediates neurotransmitter reuptake (Attwell et al., 1993). Here, we will mainly deal with receptors that regulate vesicular transmitter release, although in some cases a role of transporter-mediated release cannot be excluded.

When stating that the presynaptic modulation of transmitter release is the focus of attention of this review, one also has to consider the meaning of "release." In principle, release denotes any passage of transmitter across the membrane into the extracellular space. As stated above, release of transmitters may be vesicular or transporter-mediated. Irrespective of the underlying mechanisms, release may occur spontaneously or in a stimulation-dependent manner. Stimulation paradigms most commonly used to experimentally induce transmitter release include the application of electrical fields, of depolarizing K⁺ concentrations, or of drugs triggering depolarization, for instance, through an activation of Na⁺ channels or via a blockade of K⁺ channels. In the presence of extracellular Ca²⁺, these types of stimulation will cause exocytotic transmitter release. In addition, a multitude of drugs used in experiments to assess sympathetic transmitter release do "stimulate" release in that they raise the amount of transmitter remaining in the extracellular fluid. Nevertheless, such a phenomenon does not necessarily reflect the type of stimulation-dependent release mentioned above, because, in many instances, it remains unclear whether the increase in the liberation of transmitter is related to an increase in neuronal activity. Therefore, such observations are usually described as increases in spontaneous transmitter release, and the underlying mechanisms remain mostly unknown. Unless indicated otherwise, such changes in spontaneous transmitter release will be neglected here.

At the sympathoeffector junction, the axons of postganglionic sympathetic neurons do not form conventional synapses as can be found, for instance, in the central nervous system. The sympathetic axons rather possess specialized regions that contain vesicles and are known as varicosities or boutons. Although these boutons may have close contact with effector cells such as smooth muscle cells, typical active zones are most commonly lacking, and opposed postsynaptic densities have not been identified at all (Hirst et al., 1992). Because of the fact that sympathetic axons do not form "real" synapses, the term presynaptic receptor may appear inappropriate. For this reason, several authors prefer to call the receptors located at sympathetic axonal varicosities "prejunctional" receptors (Fuder and Muscholl, 1995). In this paper, however, we prefer to use the term presynaptic receptor, because we believe that pharmacological characteristics and signal transduction mechanisms of the receptors located at sympathetic varicosities are the same as those of receptors located at other presynapses.

	II. Anatomical and Functional Organization of the Sympathetic Nervous System

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The autonomic nervous system regulates the function of all innervated effector tissues in vertebrate organisms with one exception, the skeletal musculature. It, thus, forms the major efferent component of the peripheral nervous system. Three major components contribute to the autonomic nervous system: (i) the sympathetic division originating in the thoracolumbar region of the spinal cord, (ii) the parasympathetic division, which arises in craniosacral regions of the spinal cord, and (iii) the enteric nervous system, an independent nervous system entirely residing within the intestine. Whereas the sympathetic and parasympathetic pathways originate in the central nervous system with neuronal somata located in cranial nerve nuclei and in the intermediate zone of the spinal cord, the enteric system is controlled by connections from the sympathetic and parasympathetic divisions. The most diverse of the autonomic systems is the sympathetic division, which sends axons to all parts of the organism, whereas the parasympathetic nervous system supplies only a limited number of target organs. Each of the autonomic pathways is interrupted by ganglia that receive a cholinergic input via preganglionic neurons and contain the somata of postganglionic neurons, which give rise to axons. These postganglionic axons run in bundles into the effector organs, where single axons give rise to a considerable number of branches.

In sympathetic ganglia, preganglionic axons form synapses with the somatodendritic region of postganglionic neurons. One preganglionic axon may innervate a number of postganglionic neurons, and one postganglionic neuron may receive input from a number of preganglionic axons. Thus, at the level of sympathetic ganglia, one can find neuronal convergence as well as divergence, and it appears, therefore, difficult to define specific pathways leading from the spinal cord directly to the effector organs. Nevertheless, postganglionic sympathetic neurons can be characterized by functional means and categorized into at least three groups: phasic, tonic, and long-after-hyperpolarizing neurons. The electrophysiological characteristics of these divergent types of neurons are caused by the differential expression of various voltage-dependent K⁺ channels. Phasic neurons are characterized by the presence of M-type K⁺ channels, tonic neurons by small Ca²⁺-activated K⁺ channels, and long-after-hyperpolarizing neurons by the expression of small and long-lasting Ca²⁺-activated K⁺ channels. Phasic and long-after-hyperpolarizing neurons usually receive a strong preganglionic input and appear to function primarily as relay neurons. In contrast, tonic neurons receive weak efferent as well as afferent inputs and may, thus, integrate signals to mediate sympathetic reflexes. Phasic neurons predominate in paravertebral ganglia and exert in many cases vasoconstrictor function, whereas tonic neurons prevail in prevertebral ganglia and regulate primarily the contraction of visceral smooth muscle cells (Jänig and McLachlan, 1992).

The transmitter released from the preganglionic axon terminals is primarily acetylcholine (Feldberg and Gaddum, 1934), which depolarizes postsynaptic neurons most commonly in two phases: an early one in the millisecond range, mediated by an activation of nicotinic receptors, and a later one in the range of seconds, mediated by an activation of muscarinic receptors (Brown, 1983). In addition to acetylcholine, ATP is released within the ganglia (Vizi et al., 1997), but it has remained a matter of debate, whether ATP is really involved in ganglionic transmission; positive results have been obtained in enteric ganglia (Galligan and Bertrand, 1994), and negative results have been obtained in sympathetic ganglia (Inokuchi and McLachlan, 1995). In addition, several neuropeptides are present in autonomic ganglia, and it is mainly enkephalin, neurotensin, somatostatin, substance P, VIP, and CGRP that have been detected in sympathetic ganglia. These peptides are not involved, per se, in ganglionic transmission (Furness et al., 1992), but may exert modulatory effects: for instance, enkephalins are believed to dampen ganglionic transmission, whereas substance P, neurotensin, and VIP rather mediate excitatory actions (Benarroch, 1994).

In sympathetically innervated effector organs, there are numerous bundles containing one to 100 postganglionic axons, which are most commonly surrounded by Schwann cells. The axonal branches become varicose when they approach the cells that they innervate. Intervaricose axons are 0.1 to 0.4 µm in diameter and contain microtubules, whereas the varicosities themselves are larger and characterized by the presence of vesicles and mitochondria. Originally, axonal varicosities were believed not to form specialized synaptic contacts with their effector cells, but to release their transmitters at various distances from the postsynaptic target quasi-like local hormones. However, more recent evidence indicates that varicosities of sympathetic axons are in close contact with postsynaptic cells with a cleft of less than 100 nm and with only one sheet of basal lamina between pre- and postsynaptic structures. Nevertheless, typical presynaptic active zones and postsynaptic densities are most commonly lacking at these sympathoeffector junctions (Hirst et al., 1996). There are, however, indications from functional studies on cardiac pacemaker cells, which showed that added and neurally released transmitter, despite causing similar effects, activate different second messenger pathways (Hirst et al., 1996). This is thought to reflect the selective activation of postsynaptically clustered receptors coupled to one signaling pathway, on one hand, and activation of extrajunctional receptors linked to another signaling cascade, on the other hand.

Most varicosities of sympathetic axons contain noradrenaline and ATP as predominant neurotransmitters. Nevertheless, up to 15% of the neurons in paravertebral and about 1% of the neurons in prevertebral ganglia give rise to axons that use acetylcholine instead of noradrenaline as transmitter. The expression of either neurotransmitter phenotype is a multiple-step process. Although the precise underlying mechanisms remain to be elucidated, it is obvious that sympathetic neurons express cholinergic markers early during development. At later stages, the innervated target organ determines which of the two neurotransmitters is produced. Sweat glands release a cholinergic differentiation factor that leads to the expression of choline acetyl transferase and consequently to the biosynthesis of acetylcholine in sympathetic neurons (Ernsberger and Rohrer, 1999).

In addition to ATP, noradrenaline or acetylcholine, several neuropeptides are also stored in and released from postganglionic sympathetic axons. It should be noted, however, that the peptides and the amine transmitters are not stored within the same compartments: whereas amines are found preferentially in small (<70 nm) dense-cored vesicles, peptides are contained exclusively in large (>70 nm) vesicles, most commonly together with amines (De Potter et al., 1997). The type of peptide to be expressed in sympathetic neurons is also determined by the target organs: neurons innervating sweat glands contain VIP, CGRP, and/or substance P as cotransmitters to acetylcholine, whereas neurons that innervate, for instance, the heart, blood vessels, or the vas deferens store and release NPY, galanin, and/or somatostatin as cotransmitters to noradrenaline (Elfvin et al., 1993; Benarroch, 1994). Among these peptides, it is only NPY for which an unequivocal neurotransmitter role in sympathoeffector transmission has been established in various tissues and different species (Donoso et al., 1997; Franco-Cereceda and Liska, 1998; Kotecha, 1998; Hoyo et al., 2000). The expression of a certain neuropeptide phenotype is governed by the same mechanisms as the expression of the amine transmitters (Ernsberger and Rohrer, 1999).

The probability of transmitter release at central synapses is believed to be restricted in that only about one third of the action potentials invading the nerve terminals are able to cause significant vesicle exocytosis (Goda and Südhof, 1997). At sympathoeffector junctions, the probability of transmitter release appears to be even lower: individual varicosities respond to only 1 to 3% of the invading action potentials by releasing an amount of transmitter that is able to elicit postsynaptic responses. These postsynaptic responses, whether they are stimulation-dependent or spontaneously occurring, are all of similar size, which supports the view that sympathetic transmitter release is monoquantal due to the exocytosis of single vesicles. Intermittence of autonomic transmitter release was earlier suggested to be due to the failure of action potential propagation to surmount the abrupt impedance increase at the transition from the narrow diameter nonvaricose axon to the wider diameter varicosity. However, focal extracellular recording showed that action potentials are capable of invading sympathetic nerve terminals faithfully with every stimulus (Cunnane and Searl, 1994). Adjacent varicosities on the same nerve terminal were found to have different probabilities for transmitter release, which may be due to the observed nonuniformity in the Ca²⁺ influx between the varicosities (Bennett, 1998). In the course of repetitive stimulation, recruitment of previously silent varicosities occurs, so that the rate of arrival of action potentials in the secretory terminals determines the number of active varicosities (Cunnane and Searl, 1994). At the same time, trains of stimuli may activate more release sites on the same varicosity. Both effects may contribute to the facilitation observed in the autonomic system (Cunnane and Searl, 1994).

	III. Methodological Considerations

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Exogenous application of drugs that potentially modulate transmitter release at the sympathoeffector junctions may exert their actions at several levels of an organism. First, via several sites of action within the central nervous system, receptor agonists and antagonists can modulate neuronal activity in the autonomic nervous system and, thus, alter sympathetic transmitter release (Van Zwieten, 1996). Second, all agents can potentially influence ganglionic transmission, either via a presynaptic or a postsynaptic mechanism, which will finally cause a change in the amount of transmitter being released in the sympathetically innervated tissues. Third, the drugs may act directly on the postganglionic sympathetic axons to increase or decrease neuronal transmitter release. Hence, in vivo experiments will hardly reveal whether a drug acts specifically onto presynaptic receptors of sympathetic neurons. Therefore, it is primarily in vitro experiments that have been used to investigate this issue.

Mechanisms of transmitter release within autonomic ganglia are most commonly studied in in vitro preparations of isolated ganglia. There, acetylcholine, ATP, and some neuropeptides are released from the terminals of the preganglionic neurons. The two nonpeptide transmitters cause rapid ionic postsynaptic responses that are readily detectable by conventional electrophysiological recording techniques (Jänig and McLachlan, 1992; Galligan and Bertrand, 1994). Alternatively, the release of peptide and nonpeptide transmitters can be determined directly by various radiochemical, biochemical, or immunological techniques. In addition to the release from preganglionic axons, some exocytotic transmitter release within ganglia may also occur at the somatodendritic region of the postganglionic neurons (Zaidi and Matthews, 1997). In sensory neurons in primary cell culture, vesicle exocytosis has also been found to occur at neuronal somata (Huang and Neher, 1996). However, the majority of experiments that addressed the question as to whether sympathetic neurons do release their transmitters from dendrites or somata revealed that at least noradrenaline is released exclusively from the axons (Przywara et al., 1993a; Koh and Hille, 1997; Boehm, 1999).

The release of transmitters from postganglionic neurons onto effector cells is most commonly investigated in tissue preparations containing sympathetic axon terminals, such as different arteries or the vas deferens. Among the transmitters present in sympathetic varicosities, only ATP is able to elicit instantaneous ionic responses in the target cells. Hence, the determination of either evoked or spontaneously occurring excitatory junction potentials or excitatory junction currents in smooth muscle cells of arteries or vasa deferentia can only reflect the release of ATP (Cunnane and Searl, 1994). Noradrenaline may also cause an excitatory postsynaptic response which, however, does not occur spontaneously, requires strong neuronal stimulation, and is typically slower than the depolarization elicited by ATP (Sneddon, 2000). The response of the smooth musculature to sympathetic transmitter release can also be registered by measuring stimulation-dependent contractions, which also reveal a rapid, primarily ATP-mediated component, and a slow primarily noradrenaline-dependent component (von Kügelgen and Starke, 1991; Sneddon et al., 1996). As the postjunctional responses to noradrenaline appear to be delayed, real-time measurement of the release of endogenous noradrenaline has to rely on an alternative technique, the amperometric detection of the catecholamine via oxidizing carbon-fiber microelectrodes (Stjärne et al., 1994; Stjärne, 2000).

In contrast to the methods mentioned above that detect sympathetic transmitter release quasi "online," preparations of sympathetically innervated tissues are frequently incubated in or superfused with physiological buffer, which is collected thereafter and subsequently assayed for its transmitter content. Using this procedure, endogenous noradrenaline can be determined by high-performance liquid chromatography followed by electrochemical detection of the amine, and ATP can also be measured by high-performance liquid chromatography and fluorimetric detection (Sneddon, 2000). Alternatively, ATP can be quantified by the luciferin-luciferase assay (von Kügelgen et al., 1994a). However, the most widespread technique is the determination of the release of a radiotracer. Pieces of tissues containing the postganglionic sympathetic axons are incubated in radiolabeled noradrenaline which is taken up by the varicosities and, at least to some extent, incorporated into the vesicles present in these varicosities. After removal of radioactivity adherent to the tissue under investigation by some unspecific mechanism, the release of radioactivity is generally accepted to parallel the neuronal release of endogenous transmitter. However, the reliability of the release of a radiotracer as a marker for the release of endogenous noradrenaline has also been questioned (e.g., Bitran and Tapia, 1997).

When measuring transmitter release in tissue preparations containing sympathetic nerve terminals, several problems may be encountered. For instance, the released neurotransmitter may originate not only from the axonal endings but also from the surrounding tissue. This is only a minor problem if the release of noradrenaline is determined, because noradrenaline, in general, is not stored in target cells, such as, for instance, cardiomyocytes (Wakade et al., 1988). However, ATP may be released either from smooth muscle due to neurogenic contractions (Vizi and Sperlagh, 1999) or within the vasculature from endothelial cells (Shinozuka et al., 1994). Furthermore, electrically evoked noradrenaline release from tissue preparations containing sympathetic axon terminals is greatly enhanced in the presence of antagonists at presynaptic autoreceptors, ₂-adrenoceptors, and P2Y purinoceptors by as much as 100% or even more. This phenomenon is generally believed to reflect the interruption of the autoinhibitory feedback mediated by endogenous noradrenaline and ATP, respectively (e.g., von Kügelgen et al., 1993). This interpretation is supported by results obtained in experiments that applied electrical field stimulation using a single pulse only. Before the one pulse stimulation, there is no preexisting biophase concentration of endogenous agonist to be expected that could activate the autoreceptors. With the single pulse stimulation, antagonists at inhibitory autoreceptors accordingly failed to alter evoked noradrenaline release. However, when 10 or 100 pulses were applied instead of one, autoinhibition did develop, and the antagonists did augment stimulation-evoked release (Marshall, 1983). Hence, the investigation of pharmacological characteristics of release-modulating presynaptic receptors always has to take into account that an unknown concentration of endogenous agonist may be acting at presynaptic autoreceptors.

The problems mentioned above may be overcome by the use of primary cell cultures of dissociated postganglionic sympathetic neurons. Because these cell cultures are lacking target cells such as smooth muscle cells, transmitters are not released but from the neurons. Accordingly, cultures of sympathetic neurons have been employed successfully to directly demonstrate the sympathetic corelease of noradrenaline and ATP (von Kügelgen et al., 1994a). Moreover, in the monolayer cultures of dissociated neurons, autoinhibitory feedback modulation of transmitter release is either lacking or negligible, even though the presence of functional presynaptic autoreceptors can be demonstrated by the use of agonists (Boehm et al., 1991; Boehm and Huck, 1995). The most likely explanation for this lack of autoinhibition is the immediate dilution of the released neurotransmitter by the surrounding buffer to yield inactive concentrations only. Hence, in monolayer cell cultures, results obtained with exogenous agonists or antagonists at autoreceptors can be assumed to be hardly affected by the presence of endogenous agonists (see Boehm and Huck, 1997a, for a review).

The accumulation of a biophase concentration of endogenous noradrenaline in tissue preparations also prevented the detection of a feature of presynaptic ₂-autoreceptors that has finally been described in cultures of dissociated sympathetic neurons: the rapid, agonist-induced desensitization of release modulating ₂-adrenoceptors. This phenomenon has been investigated not only in cell cultures, but also in brain slices where no signs of agonist-induced desensitization could be detected, and it was the concentration of endogenously released noradrenaline in brain slices that was inferred as major reason for the failure of detecting desensitization (Boehm et al., 1995b).

Taken together, dissociated neurons appear to represent an ideal model to investigate receptors that modulate stimulation-evoked transmitter release, because (i) non-neural sources of endogenous modulators are absent and (ii) autoinhibitory feedback is virtually lacking. However, one also has to consider problems when using primary cultures of sympathetic neurons, because there are not only the axons and axon terminals, but the entire neurons. Dissociated postganglionic sympathetic neurons in cell culture develop only a small number of short dendrites (Furshpan et al., 1986; Lein et al., 1995) and a network of branching axons. The neuronal somata tend to form aggregates that are interconnected by axons that frequently occur in bundles. Within the regions of these aggregates of neuronal somata, numerous quasi synaptic contacts can be revealed by punctate staining with antibodies directed against synaptic vesicle proteins (Mochida et al., 1994; Zhou and Misler, 1995) or against syntaxin, a membrane protein participating in sympathetic transmitter release (Mochida et al., 1994). However, the branching axons with their presynaptic specializations are the sole sites of transmitter release as detected by amperometric recordings (Zhou and Misler, 1995) and by biochemical techniques (Przywara et al., 1993a). The neuronal somata, by contrast, hardly store and do not release noradrenaline (Przywara et al., 1993a; Boehm, 1999). It has also been shown in explant cultures of rat superior cervical ganglia that sprouting axons, growth cones, or axon terminals are the predominant sites of noradrenaline uptake and release (Vogel et al., 1972). Even though the neuronal cell bodies, per se, do not release transmitters, stimulatory or inhibitory effects at these somata and/or dendrites may contribute to the modulation of transmitter release from cultured neurons by receptor agonists and antagonists. Hence, one has to establish whether modulatory effects arise at presynaptic sites. This can be achieved by performing experiments in the presence of Na⁺ channel blockers, which prevent action potential propagation and, thereby, abolish signaling between neuronal somata and axonal varicosities (Allgaier and Meder, 1995; Kristufek et al., 1999a). One can alternatively generate cultures devoid of neuronal somata and dendrites by putting whole ganglia into culture dishes. When neurites have sprouted out of the ganglia and built up a network, the ganglia containing the cell bodies can be removed to leave only the neuronal processes behind. Immunocytochemical investigations have revealed that these neurites are only axons and not dendrites (Boehm, 1999). Drug effects on transmitter release observed in these "axonal" cultures cannot involve sites of action other than preterminal or presynaptic ones.

In vivo, postganglionic sympathetic neurons that innervate sweat glands utilize acetylcholine, and not noradrenaline, as the predominant neurotransmitter, and the development of a certain neurotransmitter phenotype is known to be target-dependent (see above). In vitro, a large number of environmental factors may cause dissociated sympathetic neurons to develop noradrenergic and/or cholinergic neurotransmitter traits (Landis, 1990). In vitro sympathetic neurons that are cholinergic form functional cholinergic synapses, either onto adjacent neurons (O'Lague et al., 1974) or onto themselves (so-called autapses; Furshpan et al., 1976). In addition, synaptic transmission between cultured sympathetic neurons may also occur via ATP (Evans et al., 1992). Because acetylcholine and ATP are known to activate sympathetic neurons via ligand-gated ion channels that mediate cationic currents, i.e., excitatory postsynaptic currents, with a resulting depolarization, i.e., an excitatory postsynaptic potential, release of these transmitters in cell culture can be determined by electrophysiological methods (Furshpan et al., 1976; Evans et al., 1992).

If sympathetic neurons are put in culture together with target cells, they form not only neuroneuronal synapses, but also neuro-effector contacts. One possible type of target cells are cardiomyocytes, which beat spontaneously in vitro. Stimulation of a neuron in synaptic contact with cardiomyocytes may then cause either acceleration of the spontaneous beating frequency of the cardiomyocyte, when the neuron is primarily adrenergic, and/or hyperpolarization and deceleration, when the neuron is primarily cholinergic (Furshpan et al., 1986). The cardiomyocytes mentioned above not only show spontaneous contractions, but also concomitant oscillations of the intracellular Ca²⁺ concentration. The frequency of these Ca²⁺ oscillations may also be regulated by transmitter release from cocultured sympathetic neurons, and stimulation-evoked release of acetylcholine and/or noradrenaline has been shown to alter this frequency (Toth et al., 1993).

The electrochemical, biochemical, and radiochemical methods that are frequently used to determine sympathetic transmitter release in innervated organs have also been applied to sympathetic neurons in cell culture. Oxidizing microelectrodes, for example, have been used successfully to investigate not only the secretion of catecholamines from chromaffin cells (Wightman et al., 1991), but also the release of noradrenaline from primary cultures of neurons from rat superior cervical ganglia. When carbon fiber microelectrodes were positioned in the clefts between neuronal somata, which harbor a large number of axonal varicosities, spikes that corresponded to the quantal packets of transmitter were observed either in the presence of depolarizing K⁺ concentrations or after exposure to -latrotoxin (Zhou and Misler, 1995). In addition, the release of endogenous noradrenaline was analyzed by reverse-phase high-performance liquid chromatography and electrochemical detection (May and Braas, 1995). Finally, release of ATP stored in sympathetic neurons in primary cell culture was quantified by the luciferin-luciferase technique by using commercially available bioluminescence kits (von Kügelgen et al., 1994a).

The method that has been employed most frequently with primary cultures of sympathetic neurons relies on the labeling of the endogenous transmitter pool by tritiated noradrenaline. The radioactivity released from cultures previously incubated in [³H]noradrenaline was found to consist primarily of authentic noradrenaline (Wakade and Wakade, 1988; Schwartz and Malik, 1993) and is, therefore, believed to reflect the release of endogenous transmitter. After labeling with [³H]noradrenaline, sympathetic neurons plated onto cover slips have been superfused with physiological buffers, and the superfusate was collected continuously to determine the time course of [³H] outflow (Boehm et al., 1991; Schwartz and Malik, 1991). Alternatively, neurons in culture dishes have been submerged in small amounts of balanced salt solutions which were then removed by pipetting to determine the radioactivity in the supernatant (Wakade and Wakade, 1988).

	IV. Ionotropic Receptors

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Neurons within sympathetic ganglia posses several types of ligand-gated ion channels, as evidenced by various techniques including reverse transcription of RNA and subsequent polymerase chain reaction, immunocytochemistry, autoradiography, and functional investigations such as electrophysiological recordings. Among the known ligand-gated cation channels, nicotinic acetylcholine receptors (for a review, see McGehee and Role, 1995), P2X receptors for extracellular ATP (Collo et al., 1996; Evans and Surprenant, 1996), and serotonin 5-HT₃ receptors (Yang et al., 1992; Rosenberg et al., 1997) have been detected on sympathetic neurons, and among the ligand-gated anion channels, GABA_A receptors (Adams and Brown, 1975; Ballanyi and Grafe, 1985; Amenta et al., 1992) and glycine receptors (Boehm et al., 1997). However, the detection of these receptor subtypes within the sympathetic ganglia or within primary cultures of isolated sympathetic neurons does not necessarily mean that these receptors are also present at the axon terminals of these neurons.

Among the neurotransmitters by which these receptors are activated, ATP and acetylcholine may be present in sympathetic neurons, and the appropriate receptors may, thus, be classified as autoreceptors. However, to our knowledge, presynaptic nicotinic autoreceptors have not been described on cholinergic sympathetic axon terminals, although such receptors have been investigated extensively at the neuromuscular junction (Wessler, 1992). Therefore, we will classify only the presynaptic P2X receptors as autoreceptors, whereas the nicotinic acetylcholine receptors will be dealt with as presynaptic heteroreceptors, because their function has been investigated on noradrenergic sympathetic axon terminals, which do not release acetylcholine.

A. Ionotropic Autoreceptors

1. P2X Nucleotide Receptors. Receptors for purines and pyrimidines comprise binding sites for either adenine nucleosides (P1 receptors) or adenine and uracil nucleotides (P2 receptors). Within the family of nucleotide receptors, there are ligand-gated ion channels named P2X and G protein-coupled receptors named P2Y. P2X receptors are activated by adenine nucleotides only, whereas some of the P2Y receptors are sensitive to uridine nucleotides. Currently, at least seven different P2X receptor subunits are known that may coassemble into functional heteromeric receptors (Ralevic and Burnstock, 1998).

B. Ionotropic Heteroreceptors

1. Nicotinic Acetylcholine Receptors. Nicotinic acetylcholine receptors are acetylcholine-gated cation channels composed of five subunits each having four membrane spanning domains. Currently, at least 16 different nicotinic receptor subunits are known, which are named by Greek letters (₁-₉, ₁-₄, , , and ). In neurons, -subunits either assemble to form functional homomers or coassemble with -subunits to build heteromeric receptors (Lukas et al., 1999).

2. -Aminobutyric Acid_A Receptors. GABA_A receptors are anion channels gated by -aminobutyric acid. In analogy to what has been stated about nicotinic receptors, GABA_A receptors are composed of five of a repertoire of at least 18 different subunits, which are named by Greek letters (₁-₆, ₁-₃, ₁-₃, ₁-₃, , , and ). Although functions and pharmacological characteristics of GABA_A receptors in the central nervous system are well known, their counterparts in the periphery are less well characterized (Barnard et al., 1998).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   -Aminobutyric acid stimulates [3H]noradrenaline release from primary cultures of dissociated rat superior cervical ganglia. The cultures were loaded with 0.05 µmol l1 [3H]noradrenaline at 36°C for 1 h. After labeling, the cultures were transferred to small chambers and superfused with a physiological buffer at 25°C at a superfusion rate of about 1.0 ml min1. Collection of 4-min superfusate fractions was started after a 60-min washout period. GABA was present as indicated by the black bar. In A, the buffer contained either no or 1 µM tetrodotoxin (TTX), to suppress action potential propagation. In B, the Cl concentration of the buffer was either 130 or 70 mM, and the omitted Cl was replaced by gluconate. This procedure shifts the Cl equilibrium by about 13 mV to more positive potentials and, thus, will facilitate Cl efflux. Both graphs show the time course of fractional tritium outflow per minute, n = 6 in each case. For experimental details, see e.g., Vartian et al. (2001).

3. Glycine Receptors. Glycine receptors are ligand-gated anion channels closely related to GABA_A receptors. They are composed of - and -subunits. Currently, at least four different -subunits (₁-₄) are known, and alternative splicing of these may contribute to additional heterogeneity. - and -subunits form heterooligomers with a stoichiometry of 3:2, but -subunits can also form homomeric receptors (Kuhse et al., 1995).

4. Serotonin 5-Hydroxytryptamine₃ Receptors. Within the huge family of serotonin receptors, the 5-HT₃ receptor is peculiar because it is a single ligand-gated cation channel among a large number of G protein-coupled receptors (Hoyer et al., 1994). Like nicotinic acetylcholine receptors, the 5-HT₃ receptor is composed of five subunits, but there are only two subunit plus one alternatively spliced variant that have been characterized by molecular means (Fletcher and Barnes, 1998; Davies et al., 1999).

	V. Metabotropic Receptors

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As mentioned above, postganglionic sympathetic axon terminalswith the exception of those innervating sweat glandsstore noradrenaline, ATP, and several peptides, such as NPY, somatostatin, galanin, and endorphins (Elfvin et al., 1993; Benarroch, 1994). However, from a functional point of view only ATP, noradrenaline, and NPY have been consistently shown to contribute to sympathoeffector transmission (Franco-Cereceda and Liska, 1998). Therefore, receptors for these transmitters will be considered autoreceptors, whereas the receptors for the other peptides mentioned above will be assumed to rather represent presynaptic heteroreceptors. In this context, it appears necessary to reconsider the term "autoreceptor": as stated above, this designates a receptor that is activated by a neurotransmitter, which is released from the very neuron on which the receptor is located. Hence, if ATP, noradrenaline, and NPY are released as cotransmitters from one neuron, each receptor on this neuron that is activated by one of these transmitters can be considered an autoreceptor. This also holds true, if the release of only one of the cotransmitters is being determined. In contrast to this interpretation, some authors categorize receptors for NPY regulating the release of noradrenaline from sympathetic neurons as heteroreceptors (Fuder and Muscholl, 1995). Because this review focuses on the presynaptic modulation of sympathetic transmitter release, all receptors activated by one of the sympathetic cotransmitters will be dealt with as autoreceptors.

As mentioned under Methodological Considerations (Section III.), in sympathetically innervated tissues, presynaptic autoreceptors mediate negative feedback modulation of transmitter release. This phenomenon is evidenced by a facilitatory effect of respective receptor antagonists either on stimulation-evoked transmitter release or on postsynaptic responses. Such results have been obtained with antagonists at ₂ and P2Y receptors, but not with a recently developed antagonist at NPY Y2 receptor subtypes (Smith-White et al., 2001). Nevertheless, this apparent lack of NPY-dependent autoinhibitory feedback modulation of sympathoeffector transmission does not necessarily imply that presynaptic Y2 receptors cannot function as autoreceptors either under adequate experimental conditions or in vivo.

In analogy to what has been said above, facilitatory presynaptic autoreceptors may mediate positive feedback modulation of transmitter release. In addition to -adrenoceptors, sympathetic axon terminals possess presynaptic -adrenoceptors, the activation of which causes facilitation of stimulated transmitter release. Nevertheless, antagonists at these -receptors do not alter stimulation-induced transmitter release or sympathoeffector transmission under conditions that otherwise permit noradrenaline-dependent autoinhibition (e.g., Brock et al., 1997). Thus, released noradrenaline apparently fails to activate presynaptic -receptors, and, therefore, these receptors are classified as heteroreceptors rather than autoreceptors.

Characteristics of presynaptic autoreceptors, although in a more general sense and not only in sympathetic neurons, have been reviewed in detail by Starke and coauthors (Starke, 1987; Starke et al., 1989), and those of presynaptic heteroreceptors by Fuder and Muscholl (1995). Therefore, we will summarize only results that have been obtained since the publication of these highly informative reviews, and the reader is referred to these papers for older references.

A. Metabotropic Autoreceptors

1. ₂-Adrenoceptors. For more than 50 years it has been known that adrenoceptors do not represent a homogenous group of binding sites but rather a family of receptor subtypes that were initially divided in - and -adrenoceptors (for a review, see Bylund et al., 1994). Some 20 years later, the description of presynaptic -autoreceptors led to the subdivision of these adrenoceptors into ₁- and ₂-adrenoceptors (Langer, 1974; Starke, 1977), and nowadays at least four different subtypes of ₂-adrenoceptors are known (see below).