Examining the pharmacology of stretch activated ion channels on mechanosensory proprioceptor responses in crayfish, crab and Drosophila

1
Examining the pharmacology of stretch activated
ion channels on mechanosensory proprioceptor
responses in crayfish, crab and Drosophila
Simpson, L.C. 1, Malloy C.1, DMahmood, D. 1,
Dabbain, N. 1, Van Doorn, J.1, Uradu, H.S.1,
Spence, A.E. 1, Martha, S.R.2, Potter, S.J.1,
Mattingly, M.X. 1, Kington, P.D. 1, King, M. 1,
Ho, A. 1, Hickey, T.N. 1, Goleva, S.B. 1,
Chukwudolue, I.M. 1, Alvarez, B.A. 1, Cooper,
R.L. 1 Bio 446 Bio 650 1Dept. of Biology,
2College of Nursing, Univ. of Kentucky.
Summary
Crayfish - MRO
Crab - PD organ
Introduction
LEFT Overview of general dissection to isolate
abdomen. A, B, and C are the series of steps in
dissecting the crayfish.
Joint proprioceptive organ in a walking leg
Proprioceptors are a group of specialized
receptors which detect position and movement
(kinesthetic). They monitor joint position,
direction, speed, and muscle-length. Arthropods,
like vertebrates have articulated appendages. It
is, therefore, not surprising that the
proprioceptors described for vertebrates have
their counterparts in arthropod limbs and
joints. The physiology of mechanosensory
transduction is diverse in that there are many
types of receptors which transduce mechanical
forces into an electrical neural impulses across
taxa. Receptors that are sensitive to
mechanosensation are used to monitor both
external and internal forces and are essential in
transferring information that allow for
appropriate behavioral responses to stimuli and
body positioning. Invertebrates serve as models
in understanding the physiology of
mechanoreception due to the diversity of receptor
types and the relative ease with which one can
utilize these models in experimentation. The
family of stretch activated ion channels is broad
and is currently being characterized by
gene/protein sequences and pharmacological
profiles. Common types of mechanoreceptors are
those associated with chordotonal organs (COs),
which monitor joint movements within the limbs of
arthropods. The MRO in the crayfish abdomen is a
well-described model system but only preliminary
studies have been conducted for examining
pharmacology of these stretch activated
receptors. In addition, the pharmacology of the
COs in the limbs of crabs has not previously been
investigated. The crab CO preparation offers
unique properties as the sensory endings are
embedded in an elastic strand with cell bodies
and endings that are relatively exposed. The
crayfish MRO is more complex as the sensory
endings are embedded within muscle fibers. When
the muscle fibers are stretched the displacement
stretches the sensory ending and opens stretch
activated ion channels. There are two types of
sensory neurons, each associated to their own
distinct muscle fiber. One MRO is referred to as
the rapidly adapting receptor and the other the
slow adapting neuron (see Rydqvist et al., 2007
for a review). We examined the effect of
amiloride and ruthenium red in an attempt to
profile the pharmacology of these receptors. For
comparison, we also examine cuticular
mechanosensors in larval Drosophila known to be
sensitive to amiloride. Our goal is to enhance
understanding of the physiology of COs which can
serve as models for mechanosensation. The
laboratory exercises we used in this
neurophysiology class demonstrates the use of
these two model preparations to address authentic
scientific based questions in regards to the
topic of examining pharmacological agents known
in other models to block stretch activated ion
channels.

1. The stretch activated channels in these model
   proprioceptors are not sensitive to the common
   pharmacological agents for some types of stretch
   activated channels.
2. Amiloride (1 mM) and ruthenium red (30 uM) did
   not block the channels even after 1 hour of
   incubation for the crab preparation. The crayfish
   MRO was only examined for 15 minutes
3. Since some TRP channels (i.e.TRP4), mammalian
   Deg/ENaC channels and the stretch activated
   channels comprised of the Piezo protein are
   mechanosensitive which blocked by ruthenium red
   or amiloride, we assume these crustaceans stretch
   activated channels with proprioception do not
   fall into these classes.
4. In mammals, it is noted that ENaCs ASICs (acid
   sensing mechanosensory channels) are inhibited by
   amiloride. The mammalian muscle spindle
   proprioceptors are amiloride sensitive, but have
   yet to be fully classified pharmacologically or
   by protein structure (Bewick and Banks, 2015) .
5. As for mammals, the mechanosensory receptors in
   these crustacean proprioceptor preparations have
   not been fully identified to the coding genes or
   protein composition.
6. Screening various compounds known to work on
   other mechanosensory receptors helps in further
   identifying these particular receptors.
7. Future studies will be to increase or sample size
   of the MRO preparations and to try intracellular
   recordings for recording receptor potentials for
   assessing more subtle changes in the effects of
   various pharmacological agents.

BELOW The schematic of an abdominal segment
illustrates the muscle groups (A) and a stained
preparation with methylene blue (B) helps to
delineate the muscle groups in an intact
preparation. The outlined area in A is shown in B
with an enlarged view. In B, the DEL1 and 2
muscle groups are not cut away as shown in the
lower half of the schematic as shown in A. Nerve
is pulled into recording electrode.
SALINE
AMILORIDE 1 mM
RUTHENIUM RED 30 mM
½ sec
½ sec
½ sec
SALINE
1 sec
1 sec
1 sec
AMILORIDE 1 mM (after 5 min)
2 sec
2 sec
2 sec
SALINE wash
4 sec
4 sec
4 sec
RUTHENIUM RED 30 mM (after 10 min)
5 sec
5 sec
5 sec
 
Another set of 5 preparations were amiloride was
tried before ruthenium red
Response condition amiloride Normal Saline ruthenium red Normal saline
Activity Prep1
Prep 2
Prep 3
Prep 4
Prep 5
Methods
Response condition amiloride Normal Saline ruthenium red Normal saline
Activity Prep1
Prep 2
References
The dissection procedures for recording neural
activity from the crab PD organ and the crayfish
MRO are previously described in detail with text
and video format (Majeed et al., 2013
Leksrisawat et al., 2010). The respective
proprioceptive nerves are exposed and recordings
are made with extracellular suction electrodes.
The signals are amplified and recorded on a
computer. All data were recorded by a computer
via PowerLab/4s A/D converter (ADInstruments).
The salines used are the normal salines
described previously (Majeed et al., 2013
Leksrisawat et al., 2010). Amiloride was used at
a concentration of 1mM where as ruthenium red
was used at 30 mM. Initial nerve recordings were
made with moving the PD segment in crabs or the
joint associated for the MRO in crayfish with
normal saline, then exchanged to one containing a
compound of interest followed by exchange back to
normal saline to regain the initial
responsiveness.
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