ABSTRACT Suction capillary electrodes were employed to study the response of the osphradial nerve to electrical and chemical stimulation in the limpet, Colisella limatula. The osphradial nerve was filled with CoCl to identify individual cell bodies and axons along the posterior edge of the osphradium.) Chemo-sensitive paddle cilia on the surface of the osphradium were not abundant, if present at all, when observed under the scanning electron microscope. The organ proved to be sensitive to the application of IM NaCl, IM Licl, and 0.5M Na-Gluconate + ASW; it gave a heightened response both in frequency and amplitude of action potentials observed passing along the osphradial nerve (O.N.). A second effect was also observed: the organ was sensitive, on a smaller scale, to 0.6M Sucrose +ASW, and 0.5M Nacl + ASW. The application of these substances resulted in smaller increases in amplitude and frequency, but still visibly greater than that of natural sea water (NSW) which had no effect. Ineffective substances for osphradial stimulation were 0.5M N-Methyl-Glucamine-Cl, light, Pisaster ochraceus tube feet, dacron line, and distilled water. INTRODUCTION The function of the gastropod osphradium is, in general, poorly understood. Based on morphological studies, the osphradium in many prosobranchs has been identified as chemosensory (Haszprunar, 1985 A). The only physiological work has been carried out on the opistobranch. Aplysia, where recordings were made from central nervous system cell bodies which presumably were targets for axons coming from the osphradium via the O.N. In these studies osmotic, mechano, and chemo all proved successful stimulants (Stinnakre & Tauc. 1968) (Jahan-Parwar, et-al, 1969). A body of behavioral literature also exists and supports the idea that the osphradium in the predatory gastropod Biomphalcria glabrata serves as a food detector (Townsend. 1973). The osphradium of prosobranch limpets has not been previously investigated physiologically. It is unlikely that it would serve as a mechanoreceptor or sediment detector because limpets are found exclusively on rocks. It is also unlikely that it is a food detector in this non-carnivorous browsing gastropod. Therefore, the project attempted to detect an osmo, or chemo receptor in the limpet, Colisella limatula. These animals are subjected to osmotic stresses in their intertidal habitats. The approach taken was to record electrical activity in the osphradial nerve following stimulation of the organ's sensory epithelium. These experiments represent the first such successful recording from the O.N. of any gastropod. MATERIALS AND METHODS Specimins of Colisella limatula were collected from the Great Tide Pool in Pacific Grove, California. Limpets were maintained in holding tanks in running natural sea water at 16°0. Dissection Technique To expose the osphradial nerve, an animal was deshelled with a scalpel blade, placed in a slygard bottomed dish filled with sea water, and restrained using insect pins. An incision was made in the mantle tissue along the dorsal midline running posteriorly from the head of the organism. The two flaps of mantle were then gently pulled back and the connective tissue cut away from the mantle- exposing the osphradial nerve. The left osphradium was used in all experiments because it was more easily isolated and recorded from than was the right. All tissue posterior to the O.N. was removed to reduce the possibility of muscular contraction during stimulation. All dissections and experimentations were carried out in natural sea water. Experimental Set Up An electrical stimulating electrode was constructed of fine Pt wires embedded in silicon glue to expose only the ends of the wires. The electrode was placed on different areas on the left osphradium to map sensitivity and confirm that the recording electrode was properly positioned on the O.N. Rectangular pulses of 0.3 ms duration and 8-40 volts were employed. Recording suction electrodes made from glass hematocrit tubes were pulled on a micro-pipette puller and broken to a diameter approximately equal to that of the nerve (150 um). The recording electrode measured the voltage difference between two Ag:AgCl wires: one placed inside the syringe, and the other wrapped around the glass tip— careful insulation of this wire with Q-dope, except for the tip, was important to minimize the stimulus artifact. The recording electrode was filled with NSW. Recordings were amplified 1000 times by a Tektronix FM122, filtered at 1000Hz (high-pass) and 8Hz (low-pass), and displayed on a storage oscilloscope and Gould 220 chart recorder. Positioning of the electrode was on the osphradial nerve before the split leading to the left and right pleural ganglia (see Fig. 1). Slight suction was applied to the nerve until either spontaneous activity was observed or the nerve was sucked up into the electrode, forming an omega shape. The nerve was then slightly raised from the body to reduce the possibility of mechanical noise interferring with the recording of action potentials. In all cases, before testing for chemically stimulated nerve activity, positive responses were obtained with electrical stimulation of the osphradium or of the nerve itself. Chemical stimulation of the nerve originally consisted of introducing a hand micropipettor into the bath, slowly releasing 250 ul of solution onto the organ, and then removing the pipettor from the bath. To wash the organ with a new substance the process was repeated. It was felt that this method of application left too many uncontrolled variables and often generated unacceptable electrical and mechanical noise. The application method was then changed to a three barreled solution distributor mounted on a micromanipulator which could be turned on and off at will, allowing one solution to be dontindousky fidetng. Sach bagcel cioposasie pipettor tip) was fed from Intramedic Polyethylene tubing attached to an individually valved 60ml syringe. Flow from each of the barrels could be started or stopped with minimal mechanical or electrical disturbance, Flow rate was approximately one drop per second into a dish containing 50 ml of NSW. Three barrels for bathing were used so that one could be stopped and another started without stopping the general flow into the medium. During each experiment one barrel was filled with NSW. One barrel was filled with a solution known to elicit a positive response (e.g. 1H Nacl) as a check of organ viability. The third barrel was filled with an experimental solution to be tested and compared. Control solutions of NSW and ASW were allowed to flow on the organ for 2 to 4 minutes to ensure equiliberation. Experimental and the positive response check solutions flowed between 1 and 5 minutes. At the conclusion of each experiment the nerve was electrically stimulated to reconfirm that the nerve was still in the recording electrode. Experimental Solutions Hypertonic IM Nacl was used to increase Na, Cl, and osmolarity. O.SM Na-Cluconate i ASW was used to vary the Na while keeping Cl constant, while raising the osmolarity. O.5M N-Methyl-Clucamine-Cl 4 ASW was used to raise the number of Cl ions in the solution, keeping the Na normal, and raising the osmolarity. O.6ti Sucrose + ASW was used to raise the osmolarity while keeping the Na and Cl concentrations normal. A lM LiCl solution was prepared to test if the osphradium was a Na specific receptor, or sensitive to high osmolarity (see table 1 for actual concentrations). Scanning Electron Microscope Preparation specimens were prepared for scanning electron microscopy by fixation in a 12 glutaraldehyde, 12 para-formaldehyde, 0.22 Na Cacodylate, 857 sea water solution; dehydration was in acetone. Critical point drying with Co was employed to avoid the grape-to-raisin effect. CoCla Filling of the Nerve The preparation of the animal for the cobalt fill was done in a cold room (4'C) to keep the specimen fresh. The nerve was cut and sucked up into a capillary electrode filled with 350mM CoCla buffered to PI 7.0 with Tris. This solution was slightly hypotonic (830 mosmols). The electrode was attached to a 6 volt battery through a 10M ohm resistor, and a current passed between the electrode and an external Ag:AgCl wire. The preparation was allowed to sit for 9-18 hours. The electrode was then removed and the specimen was washed in NSW for four minutes. The cobalt was then precipitated with a 1-5% solution of ammonium sulfide, afterwhich, it was placed in 102 formalin + NSW overnight to fix. Dehydration was with ethanol. Specimens were mounted in Permount and viewed under a compound microscope, SULTS Anatomy of the Osphradium and Osphradial Nerve Filling nerves with CoCly allowed positive identification of the nerve that extends to the left osphradium, but no part of the actual osphradial organ was stained with cobalt. Individually stained cell bodies (glou dia. were found lying within the mantle tissue along the posterior edge of the organ (see Fig. 2). Single filled axons eminating from these cell bodies were seen transversing the mantle beneath the organ in the direction of the nerve trunk. Positive identification of filled cell bodies or axons in other areas (i.e. along the medial edge of the organ) was not made. Presumably the cell bodies which were stained are part of the osphradial ganglion often referred to in the literature, and axons of these cells pass out via the O.N. to the more centralized ganglia. The use of the scanning electron microscope to identify chemo-receptors yielded no definite results. The organ appears to be covered with fissures and cracks running throughout (Fig 34). Under greater magnification (Fig 3B) it is evident that the entire surface of the osphradium is composed of microvilli. Chemoreceptive paddle cilia were not abundant, if present at all, in the Colisella limatula osphradia examined. Electrical Stimulation of the Osphradium Osphradial nerve activity due to electrical stimulation of the osphradium was routinely observed. Electrically stimulated action potentials occurring with the lowest threshold, and presumably reflecting single unit activity (Fig 44) and spontaneously occurring action potentials (Fig 4B) were roughly similar in magnitude and time course. The lowest threshold responses could only be obtained from areas of the organ which were much more sensitive to electrical stimulation than others. These areas were the posterior and medial edges of the organ (Fig 2). These areas probably correspond to where the cell bodies of the O.N. axons lie. Stimulation of areas of the maatle adjacent to the organ did not lead to significan activity in the O.N. (Fig 5). Osphradial Nerve Activity Due to Stimulation by Hypertonic Nacl Activity in the O.N. was also routinely stimulated by application of 250ul of 1M Nacl. Figure 6 shows the result of applying 1M Nacl directly to the sensory epithelium of the left osphradium. Both frequency and amplitude increased in the spikes recorded from the O.N. (see Fig 7). It can be seen that the time course of spikes during 1M Nacl exposure (Fig 6) was very similar to those of the control solution in figure 4B. The increased spike amplitude and frequency were maintained until the osphradium was washed with NSW (in Fig 7 260sec). Once washed, frequency and amplitude soon returned to normal. Application of IM Nacl to the anterior and posterior ends of the mantle or foot of the limpet did not vield results. Similarly, application to the cephalic tenticles did not cause excitation of units in the O.N. Several problems with the hand pipettor method used to obtain the results in figure 7 stimulated development of the three barreled method of applying solutions (see methods). Mechanical and electrical noise produced by solution changes was thereby minimized. All of the remaining experiments to be described were carried out using this three barreled method. Figure 84 shows that turning off and on the sea water control flow caused little change in O.N. activity. The application of artificial sea water also caused no stimulation (not illus.). Bathing the organ in M NaCl yielded a characteristic effect upon every application. Both spike amplitude and frequency increased markedly in IM NaCl and returned to normal when NSH was reintroduced (Fig 80). Application of 1M Nacl, besides causing an increase in the frequency and amplitude of O.N. spikes, also apparently resulted in a heightened ammount of baseline noise (as in Fig. 8A & B). This enlargement was only seen when solutions led to an obvious increase in spikes of ) 2-3uv, and promptly dissapeared when the control solution (NSW) was reapplied and the spikes decreased in'frequency (Fig 8B & C). Increased baseline could be due to either a large increase in small spikes (2-3 uv, or coincidental amplification of background electrical noise, but there is no apparent reason for the latter possibility. Frequency of O.N. Discharge in Response to lM Nacl Figure 9 shows the frequency of spikes (in 2 sec bins), which are larger than 2uv, through time. Application of 1M Nacl solution causes a large increase in the frequency of such spikes. The time to reach peak effect and return to the control level, following readmission of NSW, is approximately four seconds. This trend of heightened frequency was typical for all experiments using 1M Nacl. Analysis of Spike Amplitude Distributions Because of the uncertain origin of the increased baseline noise accompanying the Nacl stimulated spikes of large amplitude and high frequency, the distribution of spike amplitudes was initially analyzed by measuring spikes protruding from the baseline noise, thus assuming the increase in the baseline was due to amplification of ambient electrical noise (Fig 7 inset). As discussed above, however, a more reasonable source of the apparent 'noise' is a large increase in small spike activity. Spike amplitudes were therefore measured from the middle of the baseline as indicated in figure 7 inset. Using the first method of calculation it can be seen (Fig 104) that the application of 1M Nacl to the osphradium causes a great increase in the number of mid-sized spikes 3-6uv, a smaller increase of larger spikes (buy). and apparent decrease in spikes (3uv. Due to the heightened baseline problem, however, measurments of spikes 43uv are not very reliable in IM Nacl. When the second method described above to measure spike amplitude is used, it basically ignores spikes (3uv. This analysis (Fig 10B) shows that the 1M Nacl distribution probably follows the NSW curve at the smaller amplitudes, but deviates sharply from it above 4uv. The primary effect of 1M Nacl can be described as a great increase in spikes Youv which are very rare in NSW. It is important to note that the qualitative effect of IM Nacl is not dependent on the method of analysis. The effect of 0.5M Nacl + ASW on the spike amplitude distribution is also plotted in figures 10 A & B. Using the first method of analysis, the effect is a marginal increase in the 3-6uv spikes (Fig 10A). Following the second method, however, a dramatic increase in the number of mid-sized spikes becomes evident. Few very large spikes appear in 0.5M Nacl + ASW. Again. 10 the qualitative effect of 0.5M Nacl + ASW is the same in figures 10 A & B. but for reasons discussed above, the picture given in figure 10B is taken to be the more realistic one. Analysis of all further amplitude distributions were carried out using the second method of spike measurment. All calculations were done from four second blocks occurring four seconds after changing solutions (to allow equiliberation, see figure 9). Numbers smaller than 2-3uv are too ambiguous to count, because they are lost in the heightened baseline noise accompaning increased activity. Experimental Variations of Na, Cl, and Osmolarity Because 1M Nacl and 0.5M Nacl + ASW both had definite effects on activity in the O.N., it was believed that some aspect associated with the increased NaCl content of these media evoked the response. Variations of concentrations of Na, Cl, and osmolarity were tested. O.5M Na-Gluconate + ASW (see table I) was used to double the concentration of Na, and to increase osmolarity, while keeping Cl at its normal level. O.5M N-Methyl-Glucamine-Cl + ASW was used to increase the concentration of Cl and osmolarity, while keeping Na normal. Finally, O.6M Sucrose + ASW was used to increase osmolarity alone, while keeping both Na and Cl concentrations normal. In three separate experiments the O.N. response to 0.5M Na-Gluconate + ASW applied to the osphradium was similar to that observed using IM Nacl- an increased ammount of larger spikes. One example is shown in figure 11. On the other hand, application of O.5M NMG-Cl + ASW yielded no definite effects on O.N. activity. Figure 12 shows an example, and similar results were obtained in two other experiments. The solution designed to produce a more specific osmotic stress (0.6M Sucrose + ASW) evoked a response similar to that caused by 0.5M Nacl + ASW, namely a large increase in the mid-sized spikes (Fig 13). Although the reaction to O.54 Na-Gluconate t ASW is not as profound as that in figure 11, there is still an increased number of the larger sized spikes, but this effect appears to be shared by 0.6M Sucrose t ASW. One other solution proved to be a potent stimulus for the osphradium, Im Licl elicited a response similar to that given by IM Nacl. There was a great increase in amplitude and frequency following application of this substance. Substances which proved to be ineffective stimulants were light. Pisaster ochraceous tube feet, dacron line (mechanical), and distilled water (applied by the 250ul pipettor method). DISCUSSION The findings with the scanning electron microscope that the osphradium of Colisella limatula does not show abundant sensory structures, in particular, paddle cilia, appears to be congruent with data on the osphradium of Patella and other prosobranch limpets. Possibly most sensory elements are only free nerve endings buried under the microvillar coat, and very few special sensory structures are visible protruding through this coat (Haszprunar, 1985 A). However, the complete lack of ciliated sensory elements noted in the present study must be questioned. This could possibly be attributed to preparative techniques and would have to be verified with additional observations of the osphradium under the scanning electron microscope. Transmission electron microscopy would be necessary to observe those areas beneath the microvilli-coated surface of the osphradium. Electrical stimuli proved very useful in confirming that the recording electrode made tight contact with the O.N.. Because the posterior edge and 12 medial corner of the osphradium were electrically sensitive it is likely that the cell bodies found in these areas, using cobalt back fill, were stimulating the O.N. to fire. These cell bodies possibly are constituents of the osphradial ganglion. Osphradial sensitivity to 1M Nacl, O.5M Nacl + ASW, O.SM Na-Gluconate. and not to O.5M N-Methyl-Glucamine-Cl suggests that it is a chemosensor for Na. However, because it is also sensitive to a high osmotic pressure (0.6M Sucrose + ASW), with no increase in Na content, it also suggests that there is a hyperosmotic receptor present in the osphradium. Why the organ does not respond to hypertonic NMG-Cl is mysterious. Osphradial stimulation by hypertonic media is a result similar to that observed in the opisthobranch Aplysia (Stinnakre, J & L Tauc, 1968). The organ in Colisella does not appear to respond to hypotonic sea water. It is believed that the Na is not directly acting on the neural processes near the osphradium increasing their excitability and causing the increased O.N. activity. The cell bodies in question lie embedded in mantle tissue, away from the surface of the organ. Excess flow from the solution distributor does not directly reach the O.N. after it exits from the mantle until long after the four seconds required for stimulation to start or reverse. The flow is blocked by mantle tissue and the solution dissipates away from the organ and nerve. Although additional controls for such concerns are desirable, it is felt that the responses to chemical stimulation are of fibers triggered by receptors in the organ proper. Two effects of chemical stimulation are described in this report. The first is an increase in both frequency and amplitude of the largest spikes Obuv). Solutions evoking this response are 1M Nacl, O.5M Na-Gluconate + ASW, and IM LiCl. The second effect is an increase in medium sized spikes (3-6uv) due to the application of O.5M Nacl + ASW or 0.6M Sucrose + ASW. I believe that one of two possibilities can account for these chemically stimulated affects on the osphradium. The different effects on spike amplitudes could be caused by different osphradial receptors stimulating larger O.N. axons to fire in response to the 1M Nacl, O.5M'Na-Gluconate + ASW, and 1M LiCl. This would result in larger action potentials than occur normally. Smaller axons stimulated by other receptors for O.5M NaCl + ASW or O.6M Sucrose + ASW would account for the effect of these solutions. Another explanation for the two distinct responses involves a heightened frequency of firing in one population of O.N. axons per unit sampling time. Even thouzh the spike amplitude of single axons active during stimulation does not increase, many additional axons may be activated thereby causing the increased signal. Because data was displayed on a chart recorder with limited frequency response it was not feasible to distinguish between these two possibilities. Very fast, continuous sampling using either an oscilloscope (recording on film) or a computerized data acquisition system would resolve this uncertainty. Further Cl, Na, and osmotic substitutes need to be tested to confirm the results reported here and increase understanding of mechanisms underlying these stimulants. Other intriguing substances such as food, gametes, body wastes, and particulate matter beg investigation to uncover possible influences on the osphradium, and thereby advance our knowledge of this cryptic organ. ACKNOWLEDGEMENTS I would like to thank everyone associated with the 175H program for making this experience one of the best to date. All of the professors which made this course possible I extend an extra allotment of appreciation; Chuck Baxter. Mark Dermy, and William Gilly. Thank you class for being with me 25 hours a day. And to Tom Hahn our fearless T.A. REFRENCES Haszprunar, G. 1985. The fine morphology of the osphradial sense organs of the mollusca. I. Gastropoda, Prosobranchia, Philosophical Transactions of the Royal Society of London 307, 457-496. Jahan-Parwar, B., M. Smith, R. VonBaumgarten. 1969. Activation of neurosecretory cells in Aplysia by osphradial stimulation. American Journal of Physiology 216, 1246-1256. Stirnakre, J. and L. Tauc. 1969. Central neuronal response to the activation of osmoreceptors in the osphradium of Aplysia. Journal of Experimental Biology 51, 347-361. lownsend, C.R. 1973. The role of the osphradium in chemoreception by the snail Biomphalaria Glabrata (say). Animal Behavior 21, 549-556. TABLE I. Artificial Sea Water- 44OnM Nacl, 15nM Cacl,, 50nM NgCl,, 1OnM KCI. 10m Hepes, 967m osmols, PH-8,0. O.5M Na-Gluconate + ASW- 1750m osmols, PH-8.1. N-Methyl-Glucamine-Cl + ASW-- 1850m osmols, PH-8 Titrated with 377 HCl 1M Naci-- 1850m osmols. 0.6M Sucrose + ASW— 1742m osmols. PH-8.0. 1M LiC1- 1900m osmols. Table I: Solutions used and their concentrations and osmolarities. FIGURE LEGENDS 1. Colisella limatula, diagram of dissection; osphradium, osphradial nerve, and location of recording electrode. 2. Osphradium as seen after CoCl, fill of the nerve trunk. Area where cell bodies are located'is most sensitive to electrical stimulation. 3A. View of left osphradium under scanning electron microscope magnified 850 times. Fissures are visible. 3B. Surface of the left osphradium magnified 17k times. Surface is composed of microvilli; cilia are not obvious, but possibly present. 4A. Action potentials as seen due to electrical stimulation. Voltages are between 7 and 10 volts for O.3ms. 4B. Spontaneous action potentials; these are of the same relative size and time-course as the electrically stimulated ones, 5. Electrical stimulation of mantle adjacent to the osphradium and corresponding lack of response. Osphradial nerve activity due to the application of 250ul of 1M NaCl. 7. Spike amplitude increase and decrease through time due to the application of 1M NaCl through a hand micropipettor. /(inset). Methods for measuring spike amplitude. Top half represents method that assumes varying widths in the baseline. Bottom half takes into account a constant baseline through time, 8A. Response of O.N. to the bathing of the osphradium with NSW. 8B. Response of O.N. to the changing of solutions from NSW to IM NaCl, Larger slow spikes are caused by bubbles and movement of the organism. 80. Decrease in spike amplitude due to the addition of NSW. 9. Frequency of spikes greater than 2uv above the baseline in response to the addition of NSW and 1M NaCl. Readings were taken in 2 second blocks. Time to reach full response is approximately 4 seconds after changing solutions. 10A. Number of spikes, occurring in four second blocks, vs. spike amplitude before adjusting for constant baseline (see text for details). 10B. Same as 10A after adjusting for a constant baseline. 11. Number of spikes vs. spike amplitude in response to the addition of 1M NaCl, and O.5M Na-Gluconate + ASW. 12. Number of spikes as a function of amplitude due to the application of O.5M NMG-CI +ASW, and O.5M NaCl + ASW. 13. Osphradial nerve response to the addition of O.6M Sucrose + ASW, and 0.5M Na-Gluconate. Spike number vs. spike amplitude. ( Osphradiug Osph Nerve Recording Electrode I 1 Scanning Electron Microscope A Ant Osphradium Ciliated Protozoa- „Fissure Microvilli —Paddle Cilium? — C — I —.—.— -— — — — — S O —— —-1 — — — — — — —.— . —- 9 ——— . — —.— — —- — —-- -.---. :- LECTRICAL Mantle stimulation. SOMV —-— — —.— * —-— — MS —— -— i —— —--- ---- —----- -- —.— — — E —- —...... —— -— —— 1 —---- -- i ——.— —.-- — --------. O L -: ——-— - - -- — - â L 10 — .. L o - + —— —.— 5 0 0 —.——— — ( - â . . -. ( — O O C 0 --— — O . Z - 0 O Z 1 — 1 — —.—.— S — 1 + S —1— Z — OO —— J —— O 038 2 ud Salas 0 — — - — ------- - - - - A NOT ADJUSTED 80 — — -- 2 — . — — ——40 — . -4 —1— 2 — — —— - — — 9 ——6 ME AMPLITUDE CIC V. — + R ADJUSTED — . â — —-- — —— .—80 c0 L O —— - -.- .. — ---------— —— 40 S 1 A ------.---—.— / - 4 . . .. 6 SPIKE AMPLITUDE CIO V.) NSW . .* — —--- 5M Naci & ASW *-- --.—-- ——--- - —. MNaci --- - —-——— — t — — —-- ——- O O L 0 u -- — — —.— (O — —— — ——— — — — 8 — SNIdS JO ON ---. —-— —— — Z C —— - — — 1 —:— — : — SANIdS JO ON — — — ( 1 ——.— e —- —-- 20 —---- C 0 —--—— 9 —- 0 — —— — 9 —— â — — --- — S — 10 — — — 4 8 SANIdS JO ON —