Book of abstracts - British Neuroscience Association

29.14
Presynaptic modulation of nociceptive primary afferent terminals
by the endocannabinoids 2-AG in the outer dorsal horn of the rat.
Belle M, Maxwell D, Morris R
Department of Veterinary Preclinical Sciences, University of Liverpool,
Liverpool, L69 7ZJ, UK
Systemic administration of cannabinoid receptor agonists to rats
reduces allodynia and thermal hyperalgesia. CB1 receptor has been
localised in both LI /LII of the dorsal spinal cord, in regions that also
contain TRPV1 receptor. Several isoforms of diacylglycerol lipase
(DAG L) have been cloned and antibodies have been raised. In this
study DAG L has been localised, and the effects of cannabinoid
receptor activation tested on the rat outer dorsal horn.
Adult rats were terminally anaesthetised and perfuse-fixed.
Transverse sections from the spinal cord were subjected to
immunohistochemistry for DAG L, CGRP, SP, and IB4. Parasagittal
lumbar spinal cord slices were also prepared from neonatal rats and
whole-cell current-clamp recordings were made from LII(o) neurones
which were then morphological reconstructed.
Immunoreaction for DAG lipase (DAGL-ir) was seen in the primary
afferent terminals throughout LI and in LII(o) of the spinal cord. DAGLir
showed colocalisation with CGRP, SP and IB4. Immunoreaction for
DAG-L, CGRP and SP were significantly reduced, whilst IB4 staining
was almost absent in adults that were subcutaneously injected with
capsaicin as neonates (P0). In the spinal slice preparation capsaicin
significantly increased the frequency of EPSPs and action potentials.
CP55940 application significantly inhibited this capsaicin-evoked
excitation in a reversible manner.
This data reveals a mechanism by which pain sensation from thermal
and/or chemical sources can be modulated by presynaptic
cannabinoid receptors. The distribution of DAG L suggests that 2-AG
is produced locally in the terminals of primary afferents where it could
act on CB1 receptors to modulate synaptic transmission.
29.15
Differential control of NMDA receptors by calmodulin
Michiko Takahashi, Alasdair J. Gibb
Department of Pharmacology, University College London,, Gower Street,
London, WC1E 6BT
It has been previously shown that calmodulin reduces NMDA receptor
single channel open time and open probability (Ehlers et al., 1996; Rycroft
and Gibb, 2002). To investigate the influence of calmodulin on macroscopic
whole-cell NMDA currents, we have recorded NMDA responses with
intracellular calmodulin appliedvia the whole-cell patch pipette solution
(active concentration 1 µM) from medium spiny neurones of striatal slices
from humanely killed 7 day old rats. Application of the membranepermeable
calmodulin antagonist calmidazolium (20 µM) reduced the
whole-cell NMDA current from 350±39 pA to 250±43 pA; 62.8±10.5% of
control (mean±SE, n=9, P=0.01). Inclusion of calmodulin inhibitory peptide
(10µM) in the patch pipette in addition to calmodulin reduced the control
NMDA response to 262±67 pA (n=11) and further application of
calmidazolium (20 µM) reduced the size of the response to NMDA to
a lesser degree (81.8±9.1% of control, 206±60 pA, P=0.02). When
calmodulin was not added, calmidazolium had no significant effect. There
was no significant effect of calmidazolium on NMDA receptor single
channel currents recorded from outside-out patches. Inclusion of the
CaMKII inhibitor KN-93 (5 µM) in the patch pipette did not occlude, but
reduced the effect of calmidazolium. These results suggest that calmodulin
does not only modulate NMDARs by direct binding but also via CaMKII and
thus regulates NMDA receptor density.
Supported by the Wellcome Trust.
Ehlers et al., (1996). Cell. 84:745-755.
Rycroft and Gibb, (2002). J Neurosci. 22:8860-8868.
29.16
Investigation of NR2B and NR2D-containing NMDA receptors in
dopamine cells and GABAergic cells of rat substantia nigra
F. Suarez, D.T. Monaghan, D. Jane, S. Jones, A.J. Gibb
Department of Pharmacology, University College London, Gower
Street, London, WC1E 6BT. **Department of Physiology,
Development & Neuroscience, Anatomy Building, Downing Street,
Cambridge, CB2 3DY., *Department of Pharmacology & MRC Centre
for Synaptic Plasticity, University of Bristol.
NMDA receptors may contribute to excitotoxic neurodegeneration of
dopamine (DA) neurones in Parkinsonand#8217;s disease (PD). PD
patients and animal models of PD show a dramatic reduction in the
number of DA cells of the Substantia nigra pars compacta (SNc). In
parkinsonian models, the cells of the Substantia nigra pars reticulate
(SNr) neighbouring SNc cells, are much less affected. We have used
patch-clamp whole-cell recording methods to quantify the proportions
of NR2B and NR2D NMDA receptors in both cellular groups using 300
um coronal slices of substantia nigra from humanely killed 7 day old
rats.
Dopamine cells in SNc were identified by the presence of a
hyperpolarisation-activated slow inward current (Ih current) on
stepping the membrane potential from-60 mV to -120 mV. Responses
to 50 uM NMDA and 10 uM glycine in the presence of TTX (100 nM)
were recorded in control (SNc; -2352 ± 343 pA, SNr; -978 ±
113 pA) and following 5 min in presence of the NR2B selective
antagonist, ifenprodil (10 uM) (SNc; -1233 ± 252 pA, SNr; -534 ± 132
pA, n=10) or in control (SNc; -2127 ± 386 pA, SNr; -916 ± 133 pA) and
following 5 min in the presence of the partially NR2D selective
antagonist, UBP141 (3 uM) (Morley et al., 2005) (SNc; -1612 ± 127
pA, SNr; -653 ± 110 pA, n=9). These results suggest that SNc and
SNr cells both express a mixed population of NR2B and NR2D type
NMDA receptors.
29.17
Magnesium block of NMDA receptors in dopaminergic neurons of
neonatal rat substantia nigra pars compacta
Huang Z , Gibb A
Department of Pharmacology, University College London,, London WC1E
6BT, U.K.
In SNc, NMDA receptors may be triheteromers of NR1, NR2B and NR2D
subunits (Jones and Gibb, 2005). We have used patch-clamp whole-cell
recording to quantify magnesium block of NMDA receptors in 300 um
coronal slices of SNc from humanely killed 7 day old rats. NMDA currents
at -60mV, evoked by 0.1mM NMDA and 0.01mM glycine in the presence of
100nM of TTX were blocked by 59±3.0% (n=9) and 88.2±1.8% (n=9) in
0.1mM and 1.0mM magnesium respectively. Voltage ramps
(-100mV to +40mV) were used to quantify the voltage-dependence,
and#948;, and equilibrium constant, Kb (0mV), of magnesium block.
Magnesium IC50 values at -100mV, -80mV and -60mV were 5.98uM,
11.9uM and 55uM, respectively (n=9), similar to NR2A- and NR2Bcontaining
receptors. The residual NMDA current in 10 µM ifenprodil had a
lower magnesium sensitivity (IC50 = 25.1uM at -100mV, 73.2uM at -80mV
and 229uM at -60mV); similar to NR2C- and NR2D-containing receptors
(Kuner and Schoepfer, 1995; Qian et al., 2005). To analyse the data we
developed two new models that include trapping block of NMDA channels,
magnesium potentiation on the extracellular site of NR2B receptors and
proton block. The results suggest that ifenprodil-sensitive (NR2B) and lowmagnesium
sensitivity NR2D subunits are expressed by dopaminergic
neurons in SNc.
Supported by the Wellcome Trust and the BBSRC. Z.H. is funded by an
Overseas
Research Studentship and UCL Old Students Association
Trust Scholarship.
Supported by the Wellcome Trust and the BBSRC.
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