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Molecular Characterization of Mechanosensation in Mammalian Sensory Neurons
Introduction
The sensation of touch allows animals to explore their environment as well as to respond to painful stimuli.
Somatosensory neurons, those in the dorsal root and trigeminal ganglia, respond to both thermal and
mechanical stimuli. In culture, the cell bodies of these neurons replicate their in vivo properties; this
correlation has facilitated studies of their response properties to temperature. The mechanisms causing
thermal hypersensitivity following tissue damage have been elucidated (Chuang et al., 2001) but the
corresponding mechanisms sensitizing cells to mechanical stimuli are not well understood. Similarly, cell
surface receptors that detect thermal stimuli in somatosensory neurons have been cloned (Caterina et al.,
1997; McKemy et al., 2002), but the molecules responsible for mechanical sensitivity of somatosensory
neurons, particularly in the vertebrate system, remain largely unknown. I propose to ask whether thermal
and mechanical sensitization are mediated by the same extracellular agents and/or intracellular second
messengers. In addition, I propose to use properties unique to mechanically sensitive cells to identify
specific molecules that transduce mechanical stretch into electrochemical signals.
Specific Aims
1.
2.
Determine the molecular pathways by which neurochemical modulation lowers mechanical threshold
of somatosensory neurons
Identify the receptor(s) responsible for conferring mechanical sensitivity to somatosensory neurons
Aim 1: Determine the molecular pathways by which neurochemical modulation lowers mechanical
threshold of somatosensory neurons
Tissue injury and inflammation produce agents such as prostaglandins and nerve growth factor (NGF) that
cause a leftward shift in the stimulus-response curve, or sensitization, to thermal and mechanical stimuli
(Wall and Melzack, 1999). Recently, it has been shown that thermal sensitization is mediated by PLC
activation and consequent PIP2 hydrolysis, which releases the heat-activated channel TRPV1 from
inhibition (Chuang et al., 2001). It is not clear whether these same mechanisms also modulate the gain in
mechanosensitivity of these neurons. Experiments using dissociated somatosensory neurons have shown
that bathing neurons in prostaglandin E2 (PGE2) decreases the threshold for response to a local suction
application (Cho et al., 2002); however, it remains questionable if a local negative-pressure stimulus such
as patch suction mimics the whole-cell membrane tension resulting from touch.
Using an available stage-mounted system in which whole cells can be subjected to stretch/tension,
I propose to first examine effects of inflammatory agents on the electrophysiological response properties of
dissociated neurons subjected to stretch using whole-cell patch-clamping techniques. I hypothesize that
treatment with PGE2 or NGF will sensitize somatosensory neurons to stretch, in concordance with their
ability to sensitize these neurons to other types of mechanical stimluli. Similar experiments will be done
using protons and bradykinin, which are known to cause sensitization of the thermal receptor TRPV1
(Tominaga et al., 1998; Chuang et al., 2001).
This simplified primary culture system, instrumental in characterizing the responses of
somatosensory neurons to temperature, will allow further experiments to address intracellular mechanisms
of mechanical sensitization. If PGE2 and/or NGF can indeed modulate mechanosensory gain, I will go on
to ask what second messengers are required for the modulation. Using pharmacological inhibitors of PLC,
PKC, and other second messengers necessary for thermal sensitization, I can examine how these
compounds affect electrophysiological responses to stretch. If no change in mechanical threshold is seen
upon treatment with the above agents, I will conclude that modulation of thermal and mechanical gain in
somatosensory neurons is not mediated identically. Finally, if electrophysiological measurement during
cellular stretch proves to be technically difficult, I can instead examine cellular responses using ratiometric
calcium imaging with dyes like Fura-2.
Aim 2: Identify the receptor(s) responsible for conferring mechanical sensitivity to somatosensory
neurons
The XXX lab has used gene transfer and calcium imaging screens to successfully identify functional
cDNAs encoding hot and cold thermoreceptors from mammalian sensory ganglia (Caterina et al., 1997;
McKemy et al., 2002). Similarly, I propose to develop an imaging-based screening assay to identify a
mechanoreceptor from mammalian sensory neurons. This screen is based on the observation that lipophilic
dyes, such as FM1-43, enter cells through nonselective cation channels such as the hair cell’s channel (Gale
et al., 2001), ATP-gated P2X2 channels, and the capsaicin/heat-activated channel TRPV1 (Meyers et al.,
2001). The mechanically gated channel in sensory ganglia is also thought to be a nonselective cation
channel based on electrophysiological studies (Cho et al., 2002). Furthermore, mice injected with FM1-43
label mechanically sensitive neurons and affiliated cells (Meyers et al., 2001).
I hypothesize that mammalian sensory neurons containing mechanosensitive ion channels take up
FM1-43 into their cell bodies in response to physiological stretch. To test this, I will culture dissociated
somatosensory neurons and subject them to stretch in the presence of FM1-43. This should result in the
labeling of cells containing stretch-activated nonselective cation channels. If more neurons contain dye
after stretching than before stretching, I will conclude that stretch allowed FM1-43 to enter mechanically
sensitive cells. If no change is apparent, I will conclude that FM1-43 does not enter these neurons in
response to a stretch stimulus. This experiment will also be done with neurons isolated from mice deficient
in TRPV1, P2X2, and both channels to verify that a channel other than these is allowing stretch-dependent
FM1-43 uptake. Another important control for these experiments is to confirm that neurons taking up
FM1-43 by stretch also respond to stretch electrophysiologically or by intracellular calcium changes.
Finally, I will confirm that non-neuronal cells such as those in the human cell line HEK293 do not show
stretch-dependent dye uptake.
Provided that FM1-43 enters neurons in a stretch-dependent manner, an expression cloning
scheme emerges. I will transfect a cDNA library from sensory neurons into a stretch-insensitive cell line
and subject these cells to the stretch stimulus described above while in the presence of FM1-43. By
analyzing FM1-43 uptake, I can identify cDNAs which confer mechanical sensitivity to these cells.
Applying a range of tensions and examining which wells have increased fluorescence after stretch (using a
fluorimeter) may allow identification of channels gated at different mechanical thresholds.
In worms, extracellular molecules are hypothesized to be required for channel gating (Du et al.,
1996); the lack of these molecules in culture systems may prohibit cells from responding to stretch. To
circumvent this problem, I can grow cells in three-dimensional gels and thus provide the cells with an
environment more closely approximating their in vivo conditions.
Summary
My research will examine the mechanisms by which sensory neurons respond to touch and the way in
which sensitivity is achieved. My results should build upon the extensive knowledge already obtained
about the thermosensory properties of mammalian somatosensory neurons and thus achieve a more
complete picture of how these neurons allow mammals to sense environmental stimuli.
References
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) Nature 389, 816-24.
Cho H, Shin J, Shin CY, Lee S-Y, Oh U (2002) J Neurosci 22, 1238-47.
Chuang H-h, Prescott ED, Kong H, Shields S, Jordt S-E, Basbaum AI, Chao MV, Julius D (2001) Nature
411, 957-62.
Du H, Gu G, William CM, Chalfie M (1996) Neuron 16, 183-94.
Gale JE, Marcotti W, Kennedy HJ, Kros CJ, Richardson GP (2001) J. Neurosci 21, 7013-25.
McKemy DD, Neuhausser WM, Julius D (2002) Nature 416, 52-8.
Meyers JR, MacDonald RB, Duggan A, Lenzy D, Corwin JT, Corey DP (2001) Society for Neuroscience
Abstracts 27, 2168.
Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI,
Julius D (1998) Neuron 21, 531-43.
Wall PD, Melzack R. Textbook of Pain, 4th Ed. London: Churchill Livingstone, 1999.