An investigation of NMDA receptor subunit pharmacology

Mallon, Andrew Peter (2004) An investigation of NMDA receptor subunit pharmacology. PhD thesis, University of Glasgow.

Abstract

N-Methyl-D-aspartate (NMDA) receptors are critically involved in
synaptic transmission, neural development and various forms of neuronal
plasticity including long-term potentiation (LTP) and long-term depression
(LTD). They are also involved in the production of neuronal damage
following excessive activation by glutamate released as a result of hypoxia
or ischaemia. Each heteromeric receptor includes one or two NRl
subunits, at least two of the four NR2A-D subunits and less usually the
NR3AJB subunits. This study demonstrates that the putative NR2B
subunit-containing NMDA receptor antagonist Ro 25-6981 potentiates the
effects ofNMDA on rat hippocampal slices. The NR2A subunit antagonist
PEAQX blocks the effects of NMDA alone and the potentiated response
following Ro 25-6981 application. Furthermore, Ro 25-6981 was not
neuroprotective as reported previously but unexpectedly precipitated
excitotoxicity. The potentiating effect of Ro 25-6981 required around 20
minutes to become apparent, took a further 30 minutes to reach its
maximum effect and was irreversible. It was not prevented by
staurosporine (a broad-spectrum protein kinase inhibitor), okadaic acid (a
potent inhibitor of the serine/threonine protein phosphatases types 1 and
2A) or anisomycin (a protein synthesis inhibitor). However, the
potentiation was prevented by cyclosporin A (an inhibitor of
Ca2+/calmodulin-dependent phosphatase 2B [calcineurin]). The results indicate that in an intact neuronal network, NR2B subunits tonically gate
NR2A subunit-containing receptor function by a negative coupling
mechanism involving ca1cineurin activation.
NMDA receptor-dependent LTP induced by high frequency stimulation
was prevented by PEAQX, an NR2A antagonist. Ro 25-6981 was unable
to prevent L TP induction but was associated with a marginal reduction in
the magnitude of LTP induced.
There is evidence for the binding of homoquinolinic acid to an NMDAinsensitive
novel binding site in the brain. This study investigated the
pharmacology of homoquinolinate on the evoked field excitatory synaptic
potential (fEPSP) recorded from the CAl area of rat hippocampal slices.
Two NMDA receptor agonists, quinolinic acid 150/lM and homoquinolinic
acid 2.5/lM, caused an approximately 50% inhibition of fEPSP slope.
Paired-pulse studies suggested there might be a presynaptic component to
this action that is independent of presynaptic adenosine Al receptor
activation. The broad-spectrum EAA antagonist kynurenic acid and the
NMDA receptor blockers 2-amino-5-phosphonopentanoic acid and
dizocilpine could prevent the inhibition of fEPSP slope. None of these
antagonists revealed any other NMDA-insensitive activity of
homoquinolinic acid. The use of 2-carboxy-3-carboxymethylquinoline
(CCMQ) to displace the reported NMDA-insensitive binding had no effect
on either baseline fEPSP slope or the depression caused by homoquinolinic acid. It was also apparent that responses to homoquinolinic acid were
blocked completely by the NR2A subunit-selective antagonist PEAQX, but
not by the NR2B subunit-selective blocker Ro 25-6981. It was concluded
that the novel binding site for homoquinolinic acid does not affect synaptic
potentials in the hippocampus and that homoquinolinic acid appears to be a
selective agonist at NMDA receptors that include the NR2A subunit.
Although the NR2B agonist site may be maximally activated under normal
conditions and therefore it is not possible to observe any additional effects
upon fEPSP slope.
This study next investigated the negative coupling between NR2B and
NR2A subunit-containing receptors, combining the NR2A1B subunit
selective agonist HQA with the NR2B and NR2A selective antagonists
Ro 25-6981 and PEAQX. The negative coupling observed previously with
applications of NMDA was also seen using HQA and QA. The
potentiation of responses to HQA by Ro 25-6981 application was also
associated with an enhancement of paired-pulse interactions. The
subsequent application of PEAQX was able to block both the depression of
fEPSP slope and the associated enhancement of paired-pulse interactions.
The presence of a presynaptic element during applications of HQA alone
and potentiated responses alike and the blockade of these effects by
PEAQX suggests the NR2A subunit-containing NMDA receptor is
responsible for the presynaptic effects acting either directly at presynaptic sites or indirectly at postsynaptic sites leading to the raising of a retrograde
signal. The NR2B subunit in both its activated and antagonised state was
associated with enhancements in paired-pulse interactions which suggest
that it is not able to modulate directly the presynaptic element. However,
whilst paired-pulse interactions are generally accepted to he presynaptic
phenomena, it does not follow that postsynaptic effects cannot influence
the appearance of changes in these interactions in field recordings. The
absence of any observable difference between HQA, QA and NMDA
results suggests that the NR2D subunit is not obviously involved in these
processes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
• Supervisor's Name: Stone, Prof. Trevor and Nimmo, Prof. Hugh
• Date of Award: 2004
• Depositing User: Mr Toby Hanning
• Unique ID: glathesis:2004-3127
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Jan 2012
• Last Modified: 10 Dec 2012 14:04
• URI: https://theses.gla.ac.uk/id/eprint/3127