From
molecular physiological techniques to behavioral
test
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The team uses all the facilities of the Institute
of Neurosciences of Montpellier (INM). This allows a multidisciplinary
approach to our research, including genome analysis (sequencing,
genotyping), routine molecular, cellular and biochemical techniques,
electrophysiology (patch-clamp) on isolated and ex-vivo organs
(cochlear explants and cochlear slices), neuroimaging (video-,
confocal, two-photon and electron microscopy). In addition,
the team develops specific techniques which meet the special
characteristic of the cochlea.
Patch-clamp and calcium imaging on cochlear slices
Patch-clamp recordings on auditory neurons are performed on
cochlear slices according to Jagger et al. (J. Neurosci. Methods
2000;104: 77-86). To determine if the results obtained from
isolated spiral ganglion neuron somata (Figure 1) may be extrapolated
to the events occurring at the sensory inner hair cell synapse,
we use retrograde labelling protocol with high-affinity calcium-sensitive
dyes in conjunction with two-photon imaging technique to monitor
[Ca2+]i change at the level of single postsynaptic afferent
boutons.
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Figure 1:
Patch-clamp recordings of auditory neuron somata from
cochlear slices. a-b: Cochlear slices from neonatal
rat. c: Optical observation of recording micropipette
positioned in the spiral ganglion area. Inset, intracellular
labelling of primary auditory neuron using the calcium
green dye d: patch- clamp traces illustrating the effects
of N-Methyl-D-Aspartate (NMDA) and glutamate (Glu) applications
on current responses in primary auditory neurons. |
Pharmacology and electrophysiology in vivo
In acute perilymphatic perfusions, drugs
are directly applied into the cochlea through a multibarrel
perfusion pipette (Figure 2). During the application of drug,
a variety of electrophysiological and otoacoustic responses
which represent the physiological activity of the cochlea
and the auditory nerve are monitored.
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Figure 2:
Intracochlear perfusion technique and electrophysiological
recordings in vivo. The electrode placed on
the round window allows to record cochlear potentials.
The compound action potential (CAP) reflects
the synchronous activity of the nerve fibers.
The cochlear microphonic (CM) and summating
potentials (SP) are related to the receptor
potentials of sensory hair cells. Single unit
recordings from the auditory nerve fibers are
used to study spontaneous activity, tuning curves
and sound driven discharge rate. The otoacoustic
acoustic emissions (DPOAEs) recorded in the
ear canal, provides a non-invasive tool for studying
the outer hair cell activity. (From Ruel
et al., Neuropharmacology 2000, 39:
1959-1973). |
Behavioural testing
It is well known that high doses of salicylate
induces tinnutus in animal and humans. To study the molecular
mechanisms of salicylate-induced tinnitus, we have developed
a behavioral paradigm to assess the occurrence of tinnitus
in rats (Guitton et al., J. Neurosci. 2003, 23: 3944-3952).
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Figure 3: Behavioral
paradigm to assess the occurrence of tinnitus in rats:
Animals were conditioned to perform a motor task (in
this case, to jump to a climbing pole) when exposed
to a 10 kHz tone. Two measurements were performed: the
number of correct responses to sound (score) and the
number of responses without sound (false positives).
Once conditioned, animals received daily intraperitoneal
injections of saline alone or containing 300 mg/kg of
sodium salicylate for 4 days. Injections were performed
two hours before behavioral measurements. Involvement
of cochlear NMDA receptors in behavioral responses (score
and false positive responses) was investigated by applying
drugs into the fluid of the cochlea via gelfoam placed
on the round window membrane of both ears (Courtesy
J. Ruel). |
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