Elsevier

Neuroscience

Volume 122, Issue 1, 20 November 2003, Pages 193-204
Neuroscience

Neurochemical and anatomical identification of fast- and slow-firing neurones in the rat dorsal raphe nucleus using juxtacellular labelling methods in vivo

https://doi.org/10.1016/S0306-4522(03)00518-9Get rights and content

Abstract

GABA neurones in the dorsal raphe nucleus (DRN) influence ascending 5-hydroxytryptamine (5-HT) neurones but are not physiologically or anatomically characterised. Here, in vivo juxtacellular labelling methods in urethane-anaesthetised rats were used to establish the neurochemical and morphological identity of a fast-firing population of DRN neurones, which recent data suggest may be GABAergic. Slow-firing, putative 5-HT DRN neurones were also identified for the first time using this approach. Fast-firing, DRN neurones were successfully labelled with neurobiotin (n=10) and the majority (n=8/10) were immunoreactive for the GABA synthetic enzyme glutamic acid decarboxylase. These neurones were located in the DRN (mainly lateral regions), and consistently fired spikes with short width (1.1±0.1 ms) and high frequency (12.1±2.0 Hz). In most cases spike trains were regular but displayed low frequency oscillations (1–2 Hz). These neurones were morphologically heterogeneous but commonly had branching axons with varicosities and dendrites that extended across DRN subregions and the midline. Slow-firing DRN neurones were also successfully labelled with neurobiotin (n=24). These neurones comprised a population of neurones immunopositive for 5-HT and/or tryptophan hydroxylase (n=12) that fired broad spikes (2.2±0.2 ms) with high regularity and low frequency (1.7±0.2 Hz). However, a slow-firing, less regular population of neurones immunonegative for 5-HT/tryptophan hydroxylase (n=12) was also apparent.

In summary, this study chemically identifies fast- and slow-firing neurones in the DRN and establishes for the first time that fast-firing DRN neurones are GABAergic. The electrophysiological and morphological properties of these neurones suggest a novel function involving co-ordination between GABA and 5-HT neurones dispersed across DRN subregions.

Section snippets

Animals

Adult Sprague–Dawley rats (270–330 g) were housed in a temperature-controlled environment on a 12-h light/dark cycle (lights on 07:00 a.m.), and had free access to food and water. All experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986) and Home Office guidelines. All experiments were designed to minimize animal suffering and the number of animals used.

Electrophysiology

Animals were anaesthetized with 1.25 g/kg i.p. urethane (plus 4% lignocaine injected at the cranial

Juxtacellular labelling of slow- and fast-firing DRN neurones

A total of 34 slow- or fast-firing DRN neurones (one neurone/rat) were successfully labelled with neurobiotin and included in the analysis. We excluded from the analysis a small number of cases that demonstrated dense labelling in one cell but light ‘halo’ labelling in surrounding cells. This occasional spreading of neurobiotin over short distances was often due to the use of low resistance electrodes (5–10 Mohm) or the ejection of current to ‘clear’ the electrode of debris.

Electrophysiology and neurochemistry

Twenty-four neurones

Discussion

DRN GABA neurones are thought to play a role in the regulation of the ascending 5-HT system, but advances in the understanding of these neurones are hampered by the lack of knowledge of their physiological and anatomical properties. The present study applied in vivo juxtacellular labelling (Pinault, 1996) to characterise a population of fast-firing DRN neurones of unknown chemical identity. Slow-firing 5-HT neurones were analysed for comparison. It was found that DRN neurones with narrow spikes

Acknowledgements

This work was supported by a Medical Research Council (U.K.) programme grant (G9102310, T.S.). The authors would like to thank Dr. Peter J. Magill for assistance with the juxtacellular procedure.

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