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The effect of CO2, intracellular and extracellular pH on mechanosensory proprioceptor
responses in crayfish and crab
Martha, S.R. 1, Malloy, C. 2, DMahmood, D. 2, Dabbain, N. 2, Van Doorn, J. 2, Uradu, H.S. 2, Spence, A.E. 2, Simpson, L. C. 2, Potter, S.J. 2,
Mattingly, M.X. 2, Kington, P.D. 2, King, M. 2, Ho, A. 2, Hickey, T.N. 2, Goleva, S.B. 2, Chukwudolue, I.M. 2, Alvarez, B.A. 2, Cooper, R.L. 2
Bio 446 & Bio 650; 1College of Nursing, 2Dept. of Biology, Univ. of Kentucky.
Introduction
Proprioceptors are a group of specialized receptors,
which detect position and movement (kinesthetic). They
monitor joint position, direction, speed, muscle tension and
muscle-length. The effects of high CO2 and low pH on cellular
function, particularly neurons, are of interest to understand as
most animals, have a narrow window of tolerance for CO2 and
pH intracellularly (pHi). Acute high levels of CO2 can lead to
dysfunction and neuronal death. Slight acute elevations can
lead to altered mental status and cell responses even with
normal oxygenation in humans. Hypercapnia is elevated CO2
levels in blood or hemolymph. Abnormal responsiveness to
hypercapnia is commonly considered a mechanism of failure
in sudden infant death syndrome (SIDS).
Nerve
Saline
90° to 180° in ½ sec
2. CO2 - Saline
Saline
90° to
180°
in 2 sec
5 sec
5 sec
4. pH=5
The salines used are the normal salines described
previously (Majeed et al., 2013; Leksrisawat et al., 2010). 100%
CO2 was bubbled into the saline which was immediately
exposed to the preparations with a bath exchange. The saline
pH would decrease to pH 5.0 with bubbled CO2. Thus, normal
saline was reduced to pH 5.0 by addition of HCl and used as
exposure to the preparation for low extracellular pH. To reduce
pHi a 20 mM propionic acid saline was prepared with the
normal saline and adding propionic acid. This is estimated to
reduce pHi to 5.0.
These neurophysiology laboratory preparations are readily accessible to address the
effects of raised CO2 in saline, low extracellular pH saline and exposure of propionic
acid, which reduces intracellular and extracellular pH, and the consequences these
preps have on neuronal function.
2.
The PD organ of the blue crab was bathed in 100% bubbled CO2 saline, 3 of 4 preps
showed a decreased in activity. Changing to extracellular ph5 saline did not have an
effect but when bathed in propionic acid saline (reduces extracellular pH and pHi)
there was a decrease in activity for 3 of 4 preps.
3.
Crayfish MRO prep was bathed in 100% bubbled CO2 saline 3 of 5 preps showed a
decreased in activity. When changing to extracellular pH5 saline 2 of 5 preps showed
a decreased in activity and when bathed in propionic acid (reduces extracellular pH
and pHi) there was decreased in activity for 2 of 5 preps.
4.
Variability with these conditions in crayfish MRO preps may be due to their sensory
endings that are embedded to the muscle strand, while crab sensory endings are
attached directly to elastic strands. Small contractions in the muscle would allow for
differences in activity.
5.
To fully understand the mechanisms of CO2's action one needs to conduct
intracellular recordings to address changes in membrane potential in conjunction with
potential pH indicators which can be quantified.
6.
Preliminary evidence in neurons from rat spinal cord indicates propionic acid
application results in Ca2+ release from internal stores (ER). It would be interesting
to examine if this also occurs with high levels of CO2 exposure.
7.
It would be interesting to note if chronic exposure to low levels of CO2 can result in
cellular responses to condition in becoming less responsive to CO2 and reduced pH
such as might occur with COPD for humans. Also, there may be differences in
neuronal responses with CO2 and temperature which are thought to be potentially
related to occur with SIDS. On a comparative topic some animals may have better
abilities to allow changes or to homeostatic regulate fluctuation in CO2/pH changes
which may occur with intertidal zone or burrowing or hibernating animals.
3. Saline Wash
Saline
90° to 180° in
1 sec
5. Saline Wash
5 sec
6. Propionic Acid 20 mM
Saline
90° to 180°
in 4 sec
Propionic Acid 20 mM 90° to 180° in multiple 1 sec
movements
5 sec
5 sec
5 sec
5 sec
Different preparation from above
7. Saline Wash
1 sec from 90° to 180°
1. Saline
2. CO2 - Saline 3. Saline Wash
4. pH=5
Note: Even though the grid lines are of different
spacing all the traces are of 5 sec duration. Amplitudes
of the signals might not be reliable for quantitative
analysis due to variations in saline level on ground wire
and potentially the seal of recording electrode around
the nerve changing due to switching out the solutions.
Here we focus on the differences in frequency of
spikes.
5. Saline Wash
Methods
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 free nerve is shown floating over the
dissected abdomen (A). (C) Outlines the
nerve bundle and the plastic suction
electrode close by the nerve. The segmental
nerve is pulled into the suction electrode,
which is outlined in blue.
Overview of general
dissection to isolate
abdomen. A, B, and C are the
series of steps in dissecting
the crayfish.
1. Saline
1.
B
A
The action of CO2 exposure is of interest not only for
understanding the consequences of manipulating pHi within
cells but also for understanding the consequences with
exposure. In humans, chronic obstructive pulmonary disorder
(COPD) and apneas can lead to chronic conditions of lower
than normal blood pH due to reduce exhalation of CO2. In
dogs, CO2 has been used as an anesthesia (Eisele et al.,
1967) and CO2 was one of the first anesthetic agents for
humans by inducing one to pass out for a surgery and the
concept is still used today to reduce anxiety (i.e., rebreathing
bags).
We examined the effect on neuronal function by
raised CO2 in saline, low pH in the saline as well as reduced
pHi by exposure to propionic acid. 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 high CO2
and low pHi on neuronal function.
Summary
Joint proprioceptive organ in a walking leg
The production and regulation of intracellular protons
is mostly due to production of CO2 from cellular metabolism.
Metabolic activity as well as electric activity reduces pHi.
Since CO2 can rapidly transverse bi-lipid membranes the CO2
equilibrium is driven by diffusion and compounds which are in
chemical equilibrium (CO2+H2O ↔ H2CO3 ↔ HCO3- +H+,
Stone and Koopowitz, 1974).
The potential effects on limb proprioceptive neurons
have not commonly addressed clinically in relation to
hypercapnia and low pHi. We choose two invertebrate
crustacean models to address the effects of hypercapnia and
low pHi. The blue crab and crayfish are relatively easy to
obtain and are viable in minimal saline for several hours as
well as have readily accessible joint proprioceptors for
neurophysiological recordings.
Crayfish - MRO
Crab - PD organ
5 sec
Response
condition
Response
condition
Saline
pH 5.0
Normal
saline
Activity
Prep1
No change
No change
Prep 2
Prep 2
No change
No change
Prep 3
Prep 3
No change
No change
Prep 4
No change
No change
Prep 4
CO2
Normal
Saline
No change
Propionic
acid
No change
CO2
Normal
Saline
Activity
Prep1
Prep 5
No change
No change
Saline
pH 5.0
Normal
saline
No change
No change
No change
References
No change
No change
No change
No change
No change
Low pHi within the
terminal and muscle
still allows evoked
release to occur.
Propionic
acid
No change
No change
Experiments performed earlier with intracellular recording in the motor axon
to the opener muscle, in the walking leg of crayfish, revealed that the action potential
amplitude is greatly attenuated with exposure to propionic acid. Thus, the decrease
amplitude of “spikes” could occur and potentially decreased excitability due to fewer voltage
gated Na+ & Ca2+ channels being activated. This would also be expected to occur when
exposed to saline containing high CO2.
No change
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Leksrisawat, B., Cooper, A.S., Gilberts, A.B. and Cooper, R.L. (2010) Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in
Proprioception. Journal of Visualized Experiments (JoVE). Jove. 45: http://www.jove.com/video/2323/muscle-receptor-organs-crayfishabdomen- student-laboratory-exercise doi:10.3791/2323
Majeed, Z.R., Titlow, J., Hartman, H.B. and Cooper, R.L. (2013) Proprioception and tension receptors in crab limbs: Student laboratory exercises. Journal of
Visualized Experiments (JoVE). (80), e51050, doi:10.3791/51050 Professional movie and peer reviewed manuscript. http://www.jove.com/
video/51050/proprioception-tension-receptors-crab-limbs-student-laboratory
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Physiol 287: C1493-C1526.