Book of abstracts - British Neuroscience Association

28.17
Synergic effect of cannabinoids and terpenes from Cannabis
sativa against PTZ-induced seizures
Utan* A, Speroni* E, Grassi** G
*University of Bologna, Department of Pharmacology, Via Irnerio, 48,
40126 Bologna, Italy, , **Research Institute for Industrial Crops (ISCI),
Via Amendola, 82, 45100 - Rovigo, Italy
The psychoactive effects of Cannabis preparations have been
attributed largely to the presence of delta-9-tetrahydrocannabinol, but
the non-psychoactive plant cannabinoid cannabidiol is assumed to
contribute to the attenuation of its side effects. Terpenoids may also be
involved in the clinical effects of Cannabis; in fact, its essential oil
possesses complex pharmacological activity.
The aims of the present project were to assess the potential
anticonvulsant activity of the cannabinoid-free essential oil of Cannabis
sativa and of the single major terpenes present in its mixture, and to
compare three different ethanol crude extracts of hemp aerial parts,
harvested by genetically selected genotypes (one rich in THC, one
CBD-rich, and one devoid of cannabinoids). The pentylentetrazol
(PTZ)-induced seizure model was used, with animals treated i.p. with
the compounds 60 minutes prior to injections with PTZ (85 mg/kg i.p.).
Animals were observed in individual cages for 30 minutes and
behavioural responses recorded to evaluate onset time of generalized
convulsions (1st end-point) and lethality (2nd end-point). THC-rich and
CBD-rich extracts prolonged the latency to the 1st end-point ten-times,
whereas the cannabinoid-free extract, essential oil and terpenes did
not show any significant effect. THC and CBD extracts, essential oil,
and terpenes prolonged the latency to the 2nd end point (seven-times,
four-times, and five-times, respectively), whereas cannabinoids-free
extract did not show any significant effect.
These data suggest that Cannabis-based terpenoids may contribute to
its anti-seizure activity. The extract results indicate that cannabinoids
are an essential anti-convulsant component of the phytocomplex.
28.18
Binding characteristics of selective NPY Y2 Receptor Antagonists
BIIE0246 and JNJ-5207787
Nepomuceno D, Bonaventure P, Jablonowski J, Rudolph D, Chai W,
Motley T, Carruthers N, Lovenberg T
3210 Merryfield Row, San Diego, CA 92121, USA
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant
neuropeptide in the central nervous system. NPY peptides bind to and
activate five G protein-coupled receptors, Y1, Y2, Y4, Y5 and Y6. The NPY
Y2 receptor is an autoinhibitory pre-synaptic GPCR and is expressed in
brain areas that are implicated in the development of psychiatric and eating
disorders. The role of hypothalamic NPY Y2 receptors in bone formation
has also been suggested.
Recent studies have demonstrated unique irreversible binding kinetics of
NPY ligands to Y2 but not to Y1 and Y5 receptors (Dautzenberg, 2005).
BIIE0246 was shown to behave as a competitive antagonist when coapplied
with agonist. However, when pre-incubated with the Y2 receptor,
BIIE0246 behaved as an insurmountable antagonist. Here we confirm the
slow/irreversible binding of BIIE0246 in radioligand binding studies as well
as in functional experiments. Pre-incubation of BIIE0246 with theY2
receptor resulted in a >20-fold shift in the Ki from 15 nM (no preincubation)
to 0.5 nM (with preincubation). Our functional experiments also confirm the
insurmountable antagonism of BIIE0246 when applied before agonist
addition. In addition to BIIE0246, we demonstrate the same binding and
functional characteristic with JNJ-5207787. Pre-incubation of JNJ-5207787
with the Y2 receptor caused a >20 fold shift in the Ki from 210 nM to 10 nM
in radioligand binding studies and an insurmountable antagonist profile in
our functional assay.
These findings will be an important aspect to monitor in the future
development of Y2-specific small molecule antagonists.
28.19
In vitro and in vivo pharmacological characterization of sedativehypnotic
targets in Zebrafish.
Motley T, Renier C, Faraco J H, Bourgin P, Bonaventure P, Rosa F,
Mignot E
Johnson&Johnson Pharmaceutical Research & Development, L.L.C.,
San Diego, CA, USA;Department of Psychiatry and Behavioral
Sciences, Stanford University School of Medicine, Palo Alto, CA,
94304;Unité 368 INSERM, École Normale Supérieure, Paris,
France;Howard Hughes Medical Institute, Stanford, CA
The Zebrafish model is increasingly being used for pharmacological
and behavioral research. Here we describe investigations utilizing
zebrafish to examine the effects on locomotor activity of human
hypnotics. We have identified the corresponding genomic and receptor
binding targets for GABA-A, GABA-B and H1. We identified the
Zebrafish homologs of histamine receptor H1, GABA A (alpha subunit)
and GABA B (1 and 2) receptor genes through translating queries of
the Zebrafish Zv4 database using human receptor protein sequences
as probes. We studied both radioreceptor binding and behavioral
responses to compounds with known sedative hypnotic properties.
These compounds represented multiple pharmacological classes. An
automated system was used to quantify behavioral effects. Immersion
of 5-7 day old larvae in drug resulted in reduced mobility. In some
cases, the drug produced a complete state of unresponsive immobility
similar to anesthesia. These effects were dose-dependent and rapidly
reversible in water. As established in mammals, (R)-baclofen was
more active behaviorally and had higher affinity in binding studies
when compared to (S)-baclofen. Radioreceptor binding studies
revealed high affinity binding sites for known GABA-A, GABA-B and
histaminergic ligands. These results demonstrate conservation of
gene, protein and function for many established sedative hypnotic
pathways and the utility of the zebrafish as a relevant vertebrate
model for examining compounds targeting these genes of
pharmacological interest.
29.01
Role of the hydrophobic core in the activation of glycine receptors
Miller P S, Smart T G
Pharmacology Department, Medical Sciences Building, UCL, Gower Street,
London, WC1E 6BT
Glycine receptors (GlyR) are fast activating inhibitory neuronal receptors
that are highly expressed in the mammalian spinal cord and hindbrain.
Defects in the gene encoding GlyRa1 can result in Startle Syndrome, a
disorder where patients experience an exaggerated response to stimuli. It
is important therefore, to ascertain the molecular mechanism of operation
of GlyRs, not only because of their physiological relevance, but also
because of their homology to the g-aminobutyric acid (GABAAR), nicotinic
acetylcholine (nAChR) and serotonin (5-HT3) receptors, which all share a
conserved general topology. Part of this conserved topology includes the
hydrophobic core in the ligand-binding, extracellular domain (ECD). This
sits between the agonist binding loops and the gating domain which
connects the ECD to the transmembrane helices forming the channel. The
hydrophobic core therefore represents an ideal conduit between these two
locations. Here we identify a key set of residues which contribute to a
hydrophobic ring around the ECDs of the GlyR a1 pentamer, and which are
required for retaining the GlyR in its closed conformation -- removing them
results in the GlyR opening spontaneously. We propose that receptor
activation in response to glycine binding, induces a reorganisation, via its
binding loops, of the hydrophobic core through the residues identified here,
and this conformational change is communicated downstream to the Cys
loop gating domain, so allowing the channel to open in response to glycine
binding.
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