Paracrine neurotransmission in the CNS: involvement of 5-HT.

While GABA and glutamate have an established synaptic function in the CNS, recent evidence suggests 5-HT neurotransmission is predominantly paracrine. As the amino-acid neurotransmitters interact with receptors that produce effects rapidly, electrophysiological approaches can be used to assess the time delay between transmitter release and the postsynaptic response directly. However, this approach cannot be used for studies of 5-HT-mediated neurotransmission, because the majority of its receptors react more slowly, so anatomical and voltammetrical approaches have been used to provide insight into 5-HT-mediated events. These studies have revealed that extrasynaptic receptors and transporters for 5-HT exist, and that 5-HT escapes readily from the synaptic cleft. Attenuation of 5-HT binding by 5-HT-receptor antagonists and 5-HT-uptake inhibitors does not affect the synaptic efflux elicited by transient stimuli, although the effects of such drugs are apparent at later time points. Once it is extrasynaptic, 5-HT has a concentration that is similar to those estimated to be optimal for receptor and transporter activation, and it can diffuse a few micrometers until removed by its transporter. These properties of 5-HT raise the possibility that it can act on receptors that are distant from its release site and function as a paracrine transmitter.

Dissociation of dopamine release in the nucleus accumbens from intracranial self-stimulation.

Mesolimbic dopamine-releasing neurons appear to be important in the brain reward system. One behavioural paradigm that supports this hypothesis is intracranial self-stimulation (ICS), during which animals repeatedly press a lever to stimulate their own dopamine-releasing neurons electrically. Here we study dopamine release from dopamine terminals in the nucleus accumbens core and shell in the brain by using rapid-responding voltammetric microsensors during electrical stimulation of dopamine cell bodies in the ventral tegmental area/substantia nigra brain regions. In rats in which stimulating electrode placement failed to elicit dopamine release in the nucleus accumbens, ICS behaviour was not learned. In contrast, ICS was acquired when stimulus trains evoked extracellular dopamine in either the core or the shell of the nucleus accumbens. In animals that could learn ICS, experimenter-delivered stimulation always elicited dopamine release. In contrast, extracellular dopamine was rarely observed during ICS itself. Thus, although activation of mesolimbic dopamine-releasing neurons seems to be a necessary condition for ICS, evoked dopamine release is actually diminished during ICS. Dopamine may therefore be a neural substrate for novelty or reward expectation rather than reward itself.

Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission.

Whether neurotransmitters are restricted to the synaptic cleft (participating only in hard-wired neurotransmission) or diffuse to remote receptor sites (participating in what has been termed volume or paracrine transmission) depends on a number of factors. These include (1) the location of release sites with respect to the receptors, (2) the number of molecules released, (3) the diffusional rate away from the release site, determined by both the geometry near the release site as well as binding interactions, and (4) the removal of transmitter by the relevant transporter. Fast-scan cyclic voltammetry allows for the detection of extrasynaptic concentrations of many biogenic amines, permitting direct access to many of these parameters. In this study the hypothesis that 5-hydroxytryptamine (5-HT) transmission is primarily extrasynaptic in the substantia nigra reticulata, a terminal region with identified synaptic contacts, and the dorsal raphe nucleus, a somatodendritic region with rare synaptic incidence, was tested in brain slices prepared from the rat. Using carbon fiber microelectrodes, we found the concentration of 5-HT released per stimulus pulse in both regions to be identical when elicited by single pulse stimulations or trains at high frequency. 5-HT efflux elicited by a single stimulus pulse was unaffected by uptake inhibition or receptor antagonism. Thus, synaptic efflux is not restricted by binding to intrasynaptic receptors or transporters. The number of 5-HT molecules released per terminal was estimated in the substantia nigra reticulata and was considerably less than the number of 5-HT transporter and receptor sites, reinforcing the hypothesis that these sites are extrasynaptic. Furthermore, the detected extrasynaptic concentrations closely match the affinity for the predominant 5-HT receptor in each region. Although they do not disprove the existence of classical synaptic transmission, our results support the existence of paracrine neurotransmission in both serotonergic regions.

Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain.

Fast scan cyclic voltammetry with carbon fiber electrodes has been used to investigate the dynamics of the neurotransmitter 5-hydroxytryptamine (5-HT) in the extracellular fluid of two brain regions: the dorsal raphe and the substantia nigra reticulata. The method used previously was shown to be optimized to allow the time course of 5-HT concentration changes to be measured rapidly. Measurements were made in slices prepared from the brains of rats with the carbon fiber electrode inserted into the tissue and a bipolar stimulating electrode placed on the slice surface. Identification of 5-HT as the detected substance in both regions was based on voltammetric, anatomical, physiological, and pharmacological evidence. Autoradiography using [3H]paroxetine revealed highest 5-HT transporter binding densities in the regions in which voltammetric measurements were made. Evaluation of the pharmacological actions of tetrodotoxin and tetrabenazine, as well as the effects of calcium removal, suggested that 5-HT storage was vesicular and that the release process was exocytotic. The effects of fluoxetine (0.5 microM) were typical of a competitive uptake inhibitor, changing Km with little effect on Vmax. Release of 5-HT was found to be maximal with wide (2-ms) stimulus pulses in both regions, as expected for release from small unmyelinated processes, and to increase linearly with the number of pulses when high frequencies (100 Hz) were used. At lower frequencies, the concentration observed was a function of both release and uptake. Kinetic simulations of the data revealed that the major difference in 5-HT neurotransmission between the two regions was that release and uptake rates are twice as large in the dorsal raphe ([5-HT] per pulse = 100 +/- 20 nM, Vmax = 1,300 +/- 20 nM/s for dorsal raphe; [5-HT] per pulse = 55 +/- 7 nM, Vmax = 570 +/- 70 nM/s for substantia nigra reticulata). When normalized to tissue content, uptake rates in both regions were identical and similar to rates previously reported for dopamine in dopamine terminal regions. Nonetheless, compared with dopaminergic transmission in terminal regions such as the striatum, the absolute clearance rates in the substantia nigra reticulata and dorsal raphe were lower, resulting in a longer lifetime of 5-HT in the extracellular fluid and allowing long-range interactions.