1. The effect of CO 2, 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; 1 College of Nursing, 2 Dept. of Biology, Univ. of Kentucky. Introduction 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). 100% CO 2 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 CO 2. 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 pH i a 20 mM propionic acid saline was prepared with the normal saline and adding propionic acid. This is estimated to reduce pH i to 5.0. Joint proprioceptive organ in a walking leg Crab - PD organ 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 CO 2 and low pH on cellular function, particularly neurons, are of interest to understand as most animals, have a narrow window of tolerance for CO 2 and pH intracellularly (pH i ). Acute high levels of CO 2 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 CO 2 levels in blood or hemolymph. Abnormal responsiveness to hypercapnia is commonly considered a mechanism of failure in sudden infant death syndrome (SIDS). The production and regulation of intracellular protons is mostly due to production of CO 2 from cellular metabolism. Metabolic activity as well as electric activity reduces pH i. Since CO 2 can rapidly transverse bi-lipid membranes the CO 2 equilibrium is driven by diffusion and compounds which are in chemical equilibrium (CO 2 +H 2 O ↔ H 2 CO 3 ↔ HCO 3 - +H +, Stone and Koopowitz, 1974). The action of CO 2 exposure is of interest not only for understanding the consequences of manipulating pH i 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 CO 2. In dogs, CO 2 has been used as an anesthesia (Eisele et al., 1967) and CO 2 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). The potential effects on limb proprioceptive neurons have not commonly addressed clinically in relation to hypercapnia and low pH i. 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. We examined the effect on neuronal function by raised CO 2 in saline, low pH in the saline as well as reduced pH i 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 CO 2 and low pH i on neuronal function. Methods Nerve A B Crayfish - MRO Overview of general dissection to isolate abdomen. A, B, and C are the series of steps in dissecting the crayfish. 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. Low pH i within the terminal and muscle still allows evoked release to occur. 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 + & Ca 2+ channels being activated. This would also be expected to occur when exposed to saline containing high CO 2. References Badre, N.H., Martin, M.E. and Cooper, R.L. (2005). The physiological and behavioral effects of carbon dioxide on Drosophila larvae. Comparative Biochemistry and Physiology A. 140:363-376. Bierbower, S.M. and Cooper, R.L. (2010) The effects of acute carbon dioxide on behavior and physiology in Procambarus clarkii. Journal of Experimental Zoology. Journal of Experimental Zoology 313A:484-497 Breton S. (2001) The cellular physiology of carbonic anhydrases. JOP. 2(4 Suppl):159-64. Review. Caldwell, P.C. (1954) An investigation of the intracellular pH of crab muscle fibres by means of micro-glass and micro-tungsten electrodes. J. Physiol. 126:169-180. Caldwell, P.C. (1956) Intracellualr pH. Int. Rev. Cytology 5:229-277. Caldwell, P.C. (1958) Studies on the internal pH of large muscle and nerve fibres. J. Physiol. 142:22-62. 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-crayfish- abdomen-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/http://www.jove.com/ video/51050/proprioception-tension-receptors-crab-limbs-student-laboratory Putnam, R.W., Filosa, J.A., and Ritucci, N.A. (2004) Cellular mechanisms involved in CO 2 and acid signaling in chemosensitive neurons. Am J Physiol Cell Physiol 287: C1493-C1526. 1. Saline2. CO 2 - Saline 3. Saline Wash 5 sec 4. pH=55. Saline Wash6. Propionic Acid 20 mM 7. Saline Wash 5 sec 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. Saline 90° to 180° in ½ sec Saline 90° to 180° in 1 sec Saline 90° to 180° in 2 sec Saline 90° to 180° in 4 sec Propionic Acid 20 mM 90° to 180° in multiple 1 sec movements Different preparation from above 1 sec from 90° to 180° 5. Saline Wash4. pH=53. Saline Wash2. CO 2 - Saline1. Saline 180° 90° Summary 1.These neurophysiology laboratory preparations are readily accessible to address the effects of raised CO 2 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 CO 2 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 pH i ) there was a decrease in activity for 3 of 4 preps. 3.Crayfish MRO prep was bathed in 100% bubbled CO 2 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 pH i ) 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 CO 2 '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 CO 2 exposure. 7.It would be interesting to note if chronic exposure to low levels of CO 2 can result in cellular responses to condition in becoming less responsive to CO 2 and reduced pH such as might occur with COPD for humans. Also, there may be differences in neuronal responses with CO 2 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 CO 2 /pH changes which may occur with intertidal zone or burrowing or hibernating animals. Response condition CO 2 Normal Saline Saline pH 5.0 Normal saline Propionic acid Activity Prep1 No change Prep 2No change Prep 3No change Prep 4No change Response condition CO 2 Normal Saline Saline pH 5.0 Normal saline Propionic acid Activity Prep1 No change Prep 2No change Prep 3No change Prep 4No change Prep 5No change 5 sec