Pharmacological characterization of 5-HT
1B
receptor-mediated
inhibition of local excitatory synaptic transmission in the CA1
region of rat hippocampus
1
Boris Mlinar,
1
Chiara Falsini & *
,1
Renato Corradetti
1
Dipartimento di Farmacologia Preclinica e Clinica ‘Mario Aiazzi-Mancini’, Universita` di Firenze, V.le G. Pieraccini 6, 50139
Firenze, Italy
1 In the hippocampus, axon collaterals of CA1 pyramidal cells project locally onto neighbouring
CA1 pyramidal cells and interneurones, forming a local excitatory network which, in disinhibited
conditions, feeds polysynaptic epscs (poly-epscs). 5-hydroxytryptamine (5-HT) has been shown to
inhibit poly-epscs through activation of a presynaptic receptor. The aim of the present work was the
pharmacological characterization of the 5-HT receptor involved in this 5-HT action.
2 Poly-epscs, evoked by electrical stimulation of the stratum radiatum and recorded in whole-cell
voltage-clamp from CA1 pyramidal neurones, were studied in mini-slices of the CA1 region under
pharmacological block of GABA
A
, GABA
B
, and 5-HT
1A
receptors.
3 The 5-HT
1B
receptor selective agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrro-
lo[3,2-b]pyridin-5-one dihydrochloride (CP 93129) inhibited poly-epscs (EC
50
=55 nM), an e€ect
mimicked by the 5-HT
1B
ligands 5-carboxamidotryptamine (5-CT; EC
50
=14 nM) and methylergo-
metrine (EC
50
=78 nM), but not by 1-(3-chlorophenyl)piperazine dihydrochloride (mCPP; 10 mM) or
7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline dimaleate (CGS 12066B;
10 mM).
4 The e€ects of CP 93129 and 5-CT were blocked by the selective 5-HT
1B
receptor antagonist 3-[3-
(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR 55562;
K
B
&100 nM) and by cyanopindolol (K
B
=6 nM); methiothepin (10 mM) and dihydroergotamine
(1 mM). For both GR 55562 and methiothepin, application times of at least two hours were required
in order to achieve their full antagonistic e€ects.
5 Our results demonstrate that 5-HT
1B
receptors are responsible for the presynaptic inhibition of
neurotransmission at CA1/CA1 local excitatory synapses exerted by 5-HT.
British Journal of Pharmacology (2003) 138, 71 – 80. doi:10.1038/sj.bjp.0705026
Keywords: Electrophysiology; hippocampus; CA1; epsc; 5-HT; 5-HT
1B
receptor; presynaptic inhibition; CP 93129; GR
55562; CGS 12066B
Abbreviations: 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, (+)-8-hydroxy-2-dipropylaminotetralin hydrobromide; a.p.,
Action potential; CGP 35348, P-[3-aminopropyl]-P-diethoxymethylphosphinic acid; CGP 55845A, 3-N[1-(S)-
(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-phosphinic acid; CGS 12066B, 7-trifluoromethyl-
4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline dimaleate; CP 93129, 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-
pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride; GR 55562, 3-[3-(dimethylamino)propyl]-4-hydroxy-
N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride; mCPP, 1-(3-chlorophenyl)piperazine dihydrochloride; R
IN
,
Input resistance; r.m.p., Resting membrane potential; R
S
, Series resistance; WAY 100635, N-(2-(-4(2-
methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide
Introduction
The CA1 region of the hippocampus receives dense
innervation originating from the raphe nuclei (Segal, 1975)
and the released 5-hydroxytryptamine (5-HT) exerts multiple
actions on CA1 neurones, acting through a number of
distinct receptor types located at pre- and/or postsynaptic
levels (for review, see Hoyer et al. (1994) and Barnes & Sharp
(1999)). The resulting changes in synaptic transmission
produced by endogenous 5-HT are a likely consequence of
simultaneous activation of more than one 5-HT receptor.
Direct postsynaptic actions exerted by 5-HT through
changes in somatodendritic excitability on CA1 neurones
are fairly well known. In CA1 pyramidal cells, the 5-HT
1A
receptor-mediated increase and the 5-HT
4
receptor-mediated
block in K
+
conductances produce inhibitory and excitatory
e€ects, respectively (Andrade & Nicoll, 1987; Colino &
Halliwell, 1987). Furthermore, CA1 interneurones may be
excited by activation of their somatodendritic 5-HT
2
(Shen &
Andrade, 1998) and 5-HT
3
(Ropert & Guy, 1991) receptors.
Possible presynaptic modulation of CA1 neurotransmission
by 5-HT remains still largely unexplored, in spite of evidence
that 5-HT inhibits CA1 synaptic transmission at concentrations
lower than those required for eliciting changes in cell
excitability. In particular, stimulation of the principal excita-
British Journal of Pharmacology (2003) 138, 71 – 80 ª 2003 Nature Publishing Group All rights reserved 0007 – 1188/03 $25.00
www.nature.com/bjp
*Author for correspondence at: Dipartimento di Farmacologia
Preclinica e Clinica ‘Mario Aiazzi-Mancini’, Universita
`
di Firenze,
Viale G. Pieraccini 6, 50139 Firenze, Italy; E-mail: corradet@unifi.it

tory input to the CA1 region, Scha€er collaterals (CA3/CA1),
evokes polysynaptic responses in the CA1 region, which are
potently inhibited by 5-HT (Segal, 1990; Mlinar et al., 2001).
Both inhibitory and excitatory polysynaptic responses in
the CA1 region are generated by the activation of a local
excitatory network formed by collaterals of axonal branches
of CA1 pyramidal neurones which projects to basal dendrites
of other CA1 pyramidal cells as well as of CA1 interneurones
(Knowles & Schwartzkroin, 1981; Freund & Buzsaki, 1996).
These local excitatory recurrent connections between pyr-
amidal neurones have been electrophysiologically character-
ized (Christian & Dudek, 1988; Thomson & Radpour, 1991;
Deuchars & Thomson, 1996) and local polysynaptic
responses have been elicited following blockade of A
1
adenosine receptors (Klishin et al., 1995) and GABA
A
receptors (Crepel et al., 1997; Mlinar et al., 2001). This local
CA1/CA1 network appears to be implicated in physiological
CA1 signal processing (Thomson & Radpour, 1991; Radpour
& Thomson, 1991).
We have recently demonstrated (Mlinar et al., 2001) that 5-
HT decreases the polysynaptic excitatory and inhibitory
responses by presynaptic modulation of local excitatory
connections formed by axon collaterals of CA1 pyramidal
cells. In that study, an unexpected lack of sensitivity to
several broad spectrum 5-HT receptor antagonists, together
with the ine€ectiveness of some agonists (i.e.: CGS 12066B),
suggested unconventional pharmacological properties of the
5-HT receptor involved in the presynaptic inhibition of CA1
axon collateral transmission.
From the pharmacological profile of the e€ective agonists, a
putative receptor, named provisionally 5-HT
1G
, with nM
anity for 5-HT, 5-CT, and 5-MeOT and insensitive to several
antagonists (Castro et al., 1997) could not be dismissed.
Furthermore, an ‘atypical’ 5-HT
1A
receptor showing high-
anity for 5-CT and 8-OH-DPAT, while relatively insensitive
to antagonists has been described in the CA1 region of the rat
hippocampus (Waeber & Moskowitz, 1995). Finally, the
anatomical consideration that 5-HT
1B
were likely to be
expressed on axons of pyramidal cells in CA1 (Bruinvels et
al., 1994; Boschert et al., 1994) prompted us to re-appraise the
issue regardless of the ine€ectiveness of the putative 5-HT
1B
receptor agonist CGS 12066B.
In addition to 8-OH-DPAT, to investigate the possibility of
the involvement of an atypical 5-HT
1A
receptor, we used a
wider range of 5-HT receptor ligands, including rodent 5-
HT
1B
receptor selective agonist CP 93129 (Macor et al., 1990)
or antagonists such as CR 55562 (Walsh et al., 1995;
Lamothe et al., 1997) and cyanopindolol (Maura et al.,
1987; Hoyer et al., 1994).
In our investigation, we used whole cell voltage-clamp
recording of the polysynaptic excitatory response evoked in
CA1 pyramidal cells by electrical stimulation of the stratum
radiatum of rat dorsal hippocampal slices under block of
GABA receptor-mediated responses and ablation of the CA3
region. The e€ects of 5-HT at the presynaptic level within the
CA1/CA1 local network may be dicult to study in isolation.
However, a favourable feature of the CA1 local network
arrangement and of the selective sensitivity of CA1/CA1
synapses to 5-HT is that the poly-epscs originating from the
temporal summation of CA1/CA1 excitatory connections can
be easily separated from the monosynaptic component of
epscs (mono-epsc) originating from CA3 a€erents. This
allows for quantitative measurement of the action of 5-HT
agonists on CA1 pyramidal cell axons.
Methods
Experiments were carried out as previously described (Mlinar
et al., 2001). All animal manipulations were performed
according to the European Community guidelines for animal
care (DL 116/92, application of the European Communities
Council Directive 86/609/EEC) and approved by the
Committee for Animal Care and Experimental Use of the
University of Florence.
Experimental procedure
Twenty- to 28-day old Wistar rats (Harlan Italy, Udine,
Italy) were anaesthetised with ether and decapitated with a
guillotine. The brain was quickly removed and cooled in
partially frozen oxygenated artificial cerebrospinal fluid
(aCFS) which consisted of (in mM): NaCl 126; KCl 1.5;
KH
2
PO
4
1.25; NaHCO
3
26; MgSO
4
1.5; CaCl
2
2; D-glu-
cose 10 and was bubbled with 95% O
2
/5% CO
2
gas mixture
(pH 7.4). Transverse dorsal hippocampus slices of 350 mm
nominal thickness were cut with a vibroslicer (VSL, WPI,
Sarasota, FL, U.S.A.), and transferred to an incubation
chamber containing aCFS at room temperature. After at least
1 h of incubation, one slice was transferred to a Petri dish,
where, under the magnifying glass, the CA1 region was
disconnected from the CA3 region by surgical cut. Following
this procedure, the slice was returned to the incubation
chamber and after at least one additional hour of recovery,
was transferred to a recording chamber (volume 0.7 ml).
Slices were maintained submerged with a U-shaped platinum
wire and were continuously superfused with oxygenated
aCFS at a rate of 2 ml·min
71
. All experiments were
performed at room temperature (21 – 248C).
In all experiments bath solutions contained: (7)-bicucul-
line methiodide or (7)-bicuculline methochloride (10 mM) to
block GABA
A
receptors, 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]
amino-2-(S)-hydroxypropyl-P-benzyl-phosphinic acid (CGP
55845A; 1.5 mM) to block GABA
B
receptors (Davies et al.,
1993), and N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-
(2-pyridinyl) cyclohexane carboxamide (WAY 100635;
10 nM), to block 5-HT
1A
receptors. In some early experi-
ments P-[3-aminopropyl]-P-diethoxymethylphosphinic acid
(CGP 35348, 300 mM) was used instead of CGP 55845A as
GABA
B
receptor antagonist. Epileptiform activity was not
observed under these experimental conditions.
In experiments where 5-HT
1B
antagonists were applied for
2 h or longer, slices were exposed to the antagonist for the
required time in an incubation chamber containing oxyge-
nated aCFS at room temperature. After this pre-incubation,
the slice was transferred to the recording chamber, in which
the antagonist was present throughout the experiment.
Experiments were made on an upright microscope
(Axioskop; Zeiss, Go
¨
ttingen, Germany) with a 640 water
immersion objective lens. Visual guidance was achieved by
infrared di€erential interference contrast videomicroscopy
(Stuart et al., 1993), utilizing a Newvicon camera (C2400-
07; Hamamatsu, Hamamatsu City, Japan). All acquisition
was done with the use of an Axopatch 200B amplifier (Axon
British Journal of Pharmacology vol 138 (1)
5-HT
1B
receptors inhibit synaptic transmissionB. Mlinar et al72