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

Abstract

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.

Fig. 1.

5-HT maximal release as a function of current amplitude. Each data point was obtained with a 20-pulse (2 msec, biphasic pulses) stimulation at 100 Hz (n= 4 slices). Open circles, DR; filled circles, SNr.

Fig. 2.

Representative 5-HT concentration profiles elicited in the DR and the SNr when 20 stimulation pulses (350 μA) are applied at 10 Hz. The open squares indicate the beginning and end of the stimulation. The insets are the cyclic voltammograms obtained at the maximal 5-HT concentrations elicited: the solid line is that obtained in the slice; the broken line is that obtained during postcalibration with 2 μm 5-HT and is normalized in amplitude.

Fig. 3.

Comparison of one- and two-pulse stimulations in the DR. Bottom left panel, The 5-HT concentration profile obtained when a single stimulus pulse (1p) is applied.Bottom right panel, The 5-HT concentration profile obtained when two stimulus pulses (2p) are applied at 100 Hz.Open squares indicate the time of stimulation. Theinsets are the cyclic voltammograms obtained at the maximal evoked 5-HT concentration. Top panel, Pooled data (mean ± SEM; n = 4 slices) comparing the maximal 5-HT concentration elicited by a single stimulus pulse and two stimulus pulses (100 Hz).

Fig. 4.

Comparison of 1- and 20-pulse stimulations (350 μA, 100 Hz) in the SNr and DR. The maximal 5-HT concentration in the SNr example is 54 nm for the single pulse (1p) and 1100 nm for the 20-pulse (20p) stimulation; in the DR example, these values are 97 and 2000 nm, respectively. The arrowsindicate the onset of the electrical stimulation. Theinsets are the cyclic voltammograms obtained at the maximal 5-HT concentration that was evoked.

Fig. 5.

Effects of methiothepin (0.5 μm) and fluoxetine (0.5 μm) on the concentration profile obtained with a single stimulation pulse in the DR. The concentration profiles shown were obtained in a single location within the same brain slice; the insets are cyclic voltammograms obtained at the maximal concentration of 5-HT that was elicited. The mean maximal 5-HT concentration from similar experiments (n = 4 slices) revealed that the presence of these drugs did not alter release (value with drugs present was 91% ± 2 of control;p = 0.67; Student’s t test).

Fig. 6.

Absence of failures in successive one-pulse stimulations in the DR. Each plot is an individual response obtained in the DR during an experiment in which single pulses were applied 10 times (120 sec apart). The concentration changes resulting from the first stimulation of the series is shown in the top left panel (labeled 1); the results of the last stimulation of the series is shown in the bottom right panel.