Release and Uptake Rates of 5‐Hydroxytryptamine in the Dorsal Raphe and Substantia Nigra Reticulata of the Rat Brain

Abstract

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 µM) 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.

Severe Serotonin Depletion after Conditional Deletion of the Vesicular Monoamine Transporter 2 Gene in Serotonin Neurons: Neural and Behavioral Consequences

“…Previous studies have shown that the majority of brain monoamines resides in the vesicular storage pools and that pharmacological inhibition of VMAT prevents vesicular release of 5-HT (Adell and Artigas, 1998;Bunin et al, 1998;O'Connor and Kruk, 1991). We confirm here that VMAT2 is the only vesicular transporter implicated in this effect in the CNS, as its genetic ablation in 5-HT neurons entirely reproduced the effects on 5-HT uptake of complete VMAT inhibition with reserpine.…”

“…Microdialysis (Adell and Artigas, 1998) and voltametry (Bunin et al, 1998; O'Connor and Kruk, 1991) experiments showed that tetrabenazine, a selective VMAT2 blocker, prevents the release of 5-HT in the raphe and in axon terminal fields, whereas VMAT2-KO mice showed no release of amines (Fon et al, 1997;Wang et al, 1997). VMAT2-KO mice do not survive beyond the first postnatal days Fon et al, 1997;Wang et al, 1997) preventing the long-term evaluation of the consequences of a lack of monoamine/5-HT release.…”

Histamine H3Receptors Inhibit Serotonin Release in Substantia Nigra Pars Reticulata

“…2 D) by the characteristic doublereduction current profile for 5-HT as noted previously in SNr (Davidson and Stamford, 1996;Cragg et al, 1997b;Iravani and Kruk, 1997;Bunin et al, 1998). Furthermore, it has been identified previously that the primary substance detected in rat SNr using this method is 5-HT and not DA (Cragg et al, 1997b;Iravani and Kruk, 1997;Bunin et al, 1998).…”

“…Bath application of the SERT inhibitor citalopram (75 nM) prolonged the extracellular lifetime of 5-HT and significantly enhanced (Fig. 3A) (M-WU; p Ͻ 0.001; n ϭ 28) as shown previously with SERT inhibitors in the SNr (Iravani and Kruk, 1997;Bunin et al, 1998). Conversely, application of the DAT inhibitor GBR 12909 (1 M) had no effect on the lifetime or peak concentration of the detected substance, 5-HT (Fig.…”

Detecting Subsecond Dopamine Release with Fast-Scan Cyclic Voltammetry in Vivo

“…One of the advantages of cyclic voltammetry is multiple point identification (each cyclic voltammogram contains ϳ1000 points), which allows numerous species with unique oxidation and reduction potentials to be detected and differentiated. Fast-scan cyclic voltammetry can be used to detect catecholamines, indolamines, neurotransmitter metabolites, ascorbic acid, oxygen, nitric oxide, and pH changes (32,(42)(43)(44). The compounds detected and the ability to resolve two compounds can depend on the applied waveform.…”