MOLECULAR AND SYNAPTIC MECHANISMS
Mechanism of the medium-duration afterhyperpolarization
in rat serotonergic neurons
Philippe Alix,
1
Kumar Venkatesan,
1
Jacqueline Scuv
ee-Moreau,
1
Laurent Massotte,
1
Mai-Linh Nguyen Trung,
1
Charlotte A. Cornil
2
and Vincent Seutin
1
1
Neurophysiology Unit, GIGA Neurosciences, University of Lie `ge, Lie ` ge, Belgium
2
Behavioral Neuroendocrinology Unit, GIGA Neurosciences, University of Li ege, Li ege, Belgium
Keywords: apamin, bicuculline methiodide, SK channels, voltage-dependent Ca
2+
channels
Abstract
Most serotonergic neurons display a prominent medium-duration afterhyperpolarization (mAHP), which is mediated by small-
conductance Ca
2+
-activated K
+
(SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade
increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of
Ca
2+
which activates the mAHP channels in serotonergic neurons. In voltage-clamp experiments, an outward current was
recorded at À60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or
(-)-bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca
2+
channel blocker Co
2+
and was partially blocked by both x-conotoxin and mibefradil, which are blockers of N-type and T-type Ca
2+
channels, respec-
tively. Neither blockers of other voltage-gated Ca
2+
channels nor DBHQ, an inhibitor of Ca
2+
-induced Ca
2+
release, had any effect
on the SK current. In current-clamp experiments, mAHPs following action potentials were only blocked by x-conotoxin and were
unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced
to fire in an in vivo-like pacemaker rate, only x-conotoxin was able to increase their firing rate (by ~30%), an effect identical to
the one previously reported for apamin. Our results demonstrate that N-type Ca
2+
channels are the only source of Ca
2+
which
activates the SK channels underlying the mAHP. T-type Ca
2+
channels may also activate SK channels under different circum-
stances.
Introduction
The dorsal raphe nucleus (DRN) is a heterogeneous brainstem struc-
ture located in the midbrain and pons. It is implicated in various
physiological functions such as affect, memory and learning
(Michelsen et al., 2008) and its dysfunction may be involved in the
pathophysiology of major depression (Michelsen et al., 2007), anxi-
ety (Snyder, 2002) and possibly Alzheimer’s disease (Michelsen
et al., 2008; Simic et al., 2009). The DRN can be divided into five
subregions: the interfascicular, ventral (or ventromedial), ventrolat-
eral (or lateral), dorsal and caudal regions (Michelsen et al., 2008).
The vast majority of neurons within the ventromedial nucleus are
serotonergic and have two key electrophysiological characteristics: a
long-duration action potential with a shoulder on its repolarizing
phase (Beck et al., 2004) and a prominent medium-duration afterhy-
perpolarization (mAHP) which is blocked by apamin and is there-
fore due to the opening of small-conductance Ca
2+
-activated (SK or
KCa2.x) channels (Scuvee-Moreau et al., 2004). This mAHP may
be very important functionally, because local blockade of SK
channels in vivo induces irregular firing and sometimes bursting in
these neurons (Rouchet et al., 2008). Bursts mostly consist of dou-
blets of closely spaced action potentials (mean interspike interval,
7.7 ms; Hajos et al., 1995).This firing pattern, which is observed nat-
urally in a subpopulation of identified serotonergic neurons, is known
to increase terminal release of serotonin (Gartside et al., 2000).
Two of the three types of SK (or K
Ca
2.x) subunits have been
identified in the rat DRN: SK3 (K
Ca
2.3) > SK2 (K
Ca
2.2) (Stocker &
Pedarzani, 2000). In general, functional SK channels are homomeric
or heteromeric complexes of four a pore-forming subunits which
constitutively bind a calmodulin molecule at their C-terminus. The
exact stoichiometry of the subunits within the DRN is unknown. In
order to address this issue, the inhibitory potency of apamin and
tamapin (Pedarzani et al., 2002) was quantified in the present study,
as both peptides are known to preferentially block SK2 homomers.
SK channels quickly open when Ca
2+
binds to the four calmodulins
(Allen et al., 2007). Ca
2+
has a high affinity (EC
50
~ 300 nM) and
opens SK channels with a high cooperativity (Hill coefficient ~4;
Kohler et al., 1996).
Because modulation of the mAHP produces changes in the firing
pattern of DRN serotonergic neurons in vivo, the main aim of this
work was to study the physiological process involved in its genera-
tion. More specifically, we sought to isolate the SK current in DRN
neurons and to determine the source of Ca
2+
which activates their
Correspondence: Prof. Vincent Seutin, University of Li ege, Laboratory of Pharmacology
and GIGA Neurosciences, 1, avenue de l’H^ opital (B36), B-4000 Sart Tilman/Li ege 1,
Belgium.
E-mail: V.Seutin@ulg.ac.be
Received 16 July 2013, revised 27 September 2013, accepted 4 October 2013
© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, Vol. 39, pp. 186–196, 2014 doi:10.1111/ejn.12408
European Journal of Neuroscience