Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain.
BACKGROUND
5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain.


RESULTS
We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2) gene which selectively (97% co-localisation with TPH-2) target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of approximately 28000 molecules from varicosities and approximately 34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average approximately 800000 molecules.


CONCLUSIONS
For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this study open a host of new experimental opportunities and will greatly facilitate further studies of the central serotonergic system.
Benzekhroufa
Kheira
K
Department of Physiology and Pharmacology, University of Bristol, BS8 1TD, UK. Kheira.Benzekhroufa@bristol.ac.uk
Liu
Beihui
B
Tang
Feige
F
Teschemacher
Anja G
AG
Kasparov
Sergey
S
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D020013:Calcium Signaling; D005089:Exocytosis; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D011903:Raphe Nuclei / Q000378:metabolism*; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D014365:Tryptophan Hydroxylase / Q000235:genetics / Q000378:metabolism*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012701:Serotonin; D014365:Tryptophan Hydroxylase; C504569:tph2 protein, rat