Joseph G. Kunkel
Biology Department
University of Massachusetts at Amherst
Sabbatical Report, Sept 1, 1993 - Aug. 31, 1994

Marine Biological Laboratory, Woods Hole, MA

Preparations for Sabbatical

Planning a sabbatical has never been easy. This one, my third, was no different. I had to put it off  for one year to allow my younger son to have a 'normal' Amherst HS Senior year and improve his own career in soccer, ice hockey and baseball. Furthermore, my wife, Gerda, exerted substantial pressure on me not to leave the country on sabbatical while her last issue was a Freshman at college. The reputation of student suicides at Cornell had done its job.

Happily, I received an offer to participate in the development of two new interesting instruments at the National Vibrating Probe Facility at Woods Hole. One was an aerial probe which could be used to examine electrical fields about an object in air. A prime objective for this instrument was to be able to remotely measure the electrical field associated with the early pattern development in the Drosophila egg.

The other instrument was a newly developed oscillating specific ion electrode that would be used to noninvasively measure specific ions entering and exiting cells. It was the successor to the vibrating voltage probe which could measure the electrical fields in aqueous media around cells. I had substantial experience with this later instrument, having one in my lab in Amherst. Since I enjoyed the Woods Hole area I jumped at the opportunity and, with the new director of the facility, Peter Smith, I submitted a NSF Grant proposal to fund my years stay and research. The NSF Grant was not funded. Looking at my potentially available sabbatical years ahead and making a few monetary calculations I decided to do the full year sabbatical despite going on half salary. Giving up a full year sabbatical away would seem like selling my birthright.

Finding a sabbatical couple to rent our house ended up being 'easy'. It was done almost exclusively over the Information Data Highway! A Math Professor from Molde Norway was coming to Amherst and needed a rental. Several email messages later we had a tenant. Finding a house in Woods Hole was also easy since my good colleague, Hans Laufer, at U.Conn. had already been on me to rent his place for the off-season. He however would be using the house himself for the '94 Summer Season. That was OK with me since I originally planned to stay from September 1, 1993 to June 15, 1994 to avoid the hectic summer season.

Mid-August we packed our son up and moved him to Ithica for his Freshman orientation. Back in Amherst I missed my son's strong back as we packed up our possessions to move to Woods Hole. From my lab I was also transporting my own vibrating voltage probe rig which, if all went well, was to be upgraded to a specific ion rig during my stay at the NVP Facility. I was also bringing Ted Sargent's Robinson Trap which I would be using to collect Noctuid moths nightly at the Cape. The trap would prove to be a very rewarding part of my experience during the sabbatical.

Sabbatical, Sept 1, 1993 - August 31, 1994.

The move went quite well and Gerda and I settled into 506 Woods Hole Road. Initially we took the 45 min roundabout walk past Nobska Light twice a day, once in the morning and again in the evening. Soon we felt we owned the route and the light house. In September the wild roses were still blooming as brilliant red rose hips gradually replaced large pink single flowers. The variegated sea grapes festooned our path and their approach to ripeness told us the growing season was at an end. We soon became familiar with the water birds: cormorants, mergansers, ospreys, sea ducks, rafts of eider ducks, loons. We started a sea water tank and using a seine Gerda and I collected a variety of sea life. We have continued the sea water tank in Amherst with several inhabitants that we have cared for over a year now. We also began exploring the other light houses and beaches of the Cape: Highland Light, Chatham Light, Three Sisters and Nauset Light, Race Point, Wood End and Long Point Lights. It is funny that in our 24 years here in Massachusetts we had never before appreciated the treasure of the Cape Cod National Seashore. Favorite restaurants soon were being visited regularly, even on our tight budget. If you are ever in Provincetown, P'town, you should surely experience the Portuguese cooking at Gloria's and the steamed clams at The Dunes.

Immediately on arriving in Woods Hole I set to work collecting moths with the Robinson Trap each evening. The catch was frozen each morning and sorted and mounted each evening providing over 1200 mounted and identified noctuid moths for my collaborative work with Ted Sargent. Ted visited us in Woods Hole twice during the year to discuss our progress and check out the MBL Facilities. Another Amherst citizen, Dan Bentil, a new U. Mass. Math Faculty member also visited us at Woods Hole and started discussions with me on the mathematical modeling of developmental pattern. This initial meeting led to substantial email during the sabbatical and now is developing into a useful collaboration.

Talking with Peter Smith, over beer at the Captain Kid, we decided that our lack of funding for my sabbatical was a blessing in disguise! It now allowed me to do anything I wanted and not have to be accountable to any granting agency. With this attitude in mind I plunged into the world of the specific ion electrode. After substantial frustration with the then crude friendliness of the software which ran the specific ion electrode I asked for the code, written by Wiel Kuhtreiber, to see if I could make it friendlier and allow a greater variety of measurements to be performed. Within the next few months I became the de facto expert on the software and had achieved enough that I was asked to give a progress report to the NVP Board of Directors at its Annual Spring Meeting. The following is an end-of-stay report that outlines the progress made by June of 1994.

Improvements in the Calibration of the Non-invasive Vibrating Specific Ion Electrode

J. G. Kunkel, U. Massachusetts at Amherst

Sabbatical Visit, Fall 93 to Summer '94:

During the past year at the NVP Facility, Joseph G. Kunkel, on sabbatical from the University of Massachusetts at Amherst, applied his experimental, mathematical and programming skills to improving the non-invasive vibrating specific ion electrode. The starting point was that described in a recent review of the technique (Smith et al., 1994). The authors issued several warnings about the inadequacy of the theory to explain all the observed data from artificial sources. In the process of trying to understand those cautions, discoveries were made by Kunkel which have virtually eliminated the need for those warnings. Each newly improved approach was quickly applied to Kunkel's favorite biological system, the cockroach vitellogenic ovariole which is a model system for the physiology of polarity development (Kunkel 1991; Anderson, Bowdan and Kunkel, 1994).

Kunkel's improvements achieved a substantially closer agreement of observed data with the theoretically expected ionic flux coming from an artificial source, especially at the most critical point, close to the source. The crux of the improvements depended on Kunkel's proposal of a hitherto unimagined parameter that was being ignored in the practical implementation of the theoretical equations. Prior calibration data were taken using the point-of-nearest-approach to the orifice of a capillary diffusional source as both the geometric origin, {X,Y,Z} = {0,0,0} and the point of origin of the infinite source of ions. All distances were measured from this point. The theory (Smith et al., 1994) in which [ion] = Cb + K/r implies that an infinite concentration source of ion exists at some radial distance, r, from the measurement point. Kunkel's parameter, which needs to be estimated for each artificial source, is given the name ro or distance to infinite source. Once this parameter was identified there were several other improvements in fit of the theoretical to the observed that followed. These subsequent improvements involved changes in the software to correct errors in the digital movements which resulted in substantial errors in long movements made in many small steps versus long movements made in one big step. In addition, small errors were being added to an accumulating innacuracy of location when the direction or angle of vibration of the probe was changed. Programming solutions to these errors were achieved which virtually eliminate the accumulation of location errors.

Analysis of ion probe data was slowed by numerous non-automated steps which produced data that could not easily be analyzed. In order to speed the collection of calibration as well as experimental data it was necessary to change the file structure of the collected data. Automated static-mV recording was accomplished through an existing channel of the A/D board. This was automatically saved with X, Y and Z location and X-,Y- and Z- uV-difference readings. Automated 3-D ion current measurements in a 3-D grid pattern algorithm was added to achieve the current software 3DVIS. All 3-D data output files are in a rectangular format so that they can be easily imported into spreadsheet programs. One practical result of attempting to improve these formatting aspects of the data collection was the generation of a large number of sets of calibration data. These sets of calibration data (Table I) will be useful in testing future mathematical approaches (maximum likelihood estimation) to computing the parameters of the flux equations.

In the meantime a MathCad spreadsheet was established which semi-automates the calibration data analysis. The spreadsheet estimates the concentration of the ion at radial distances from the infinite source; it predicts the theoretical uV and flux at those radial distances and provides a way of estimating unknown parameters in an iterative manner. Confidence intervals for uV and flux measurements allow an approach to estimating the resolution and sensitivity of the technique. While the ideal solution is not yet achieved, the described approach is an improvement over the status described earlier (Smith et al., 1994).

Kunkel JG. 1991. Models of pattern formation in insect oocytes. In Vivo 5: 43-456.

Anderson M, E Bowdan and JG Kunkel. (1994) Comparison of defolliculated oocytes and intact follicles of the cockroach using the vibrating probe to record steady currents. Developmental Biology 162:???-???.

Bowdan E. and JG Kunkel. (1994). Ionic components of dorsal and ventral currents in vitellogenic follicles of the cockroach, Blattella germanica. J. Insect Physiol. 40:323-331.

Smith PJS, RH Sanger and LF Jaffe. (1994) The vibrating Ca2+ electrode: A new technique for detecting plasma membrane regions of Ca2+ influx and eflux. Meth. Cell Biol. 40:115-134.

Abstracts of papers presented at meetings:

Kunkel JG, RH Sanger, and PJS Smith. 1994. Direct measurement of porter and pump mediated ion flow using a non-invasive vibrating ion-selective probe. J. Physiol.

Kunkel, J. G., A. M. Shipley, R. H. Sanger, and P. J. S. Smith. 1994. Non-invasive voltage probe (NVPi) for the measurement of steady ionic currents. In Vitro, Cell. Dev. Biol. 30A:100.

Other Sabbatical accomplishments

Progress in understanding the cockroach ovarian follicle, a personal passion over the last decade, followed quickly on the heals of each technical advance that I made in improving the software for the ion probe. I had come to the NVP Facility with the hope of testing the hypthesis that had been slowly developing from using the vibrating voltage probe. In that study which was just reaching print (Bowdan and Kunkel, 1994) we had used ion replacement to infer that there was a potassium flux eminating from the dorsal surface of the follicle and a chloride flux eminating from the ventral surface. Using the more direct specific ion electrodes I quicky was able to show that there was no outward chloride current! The dorsal outward current was potassium and the ventral inward current was also potassium. The ion replacement experiments and voltage probe had led us down a garden path. The strength of the new NVP technology could not have a stronger endorsement!

The making of specific ion microelectrodes is considered by some to be an art. I religiously followed all the prescriptions available from local sages. At Woods Hole there was also some mystery surrounding possible barometric conditions and perhaps the conjunction of the moon with Mars that might be considered in deciding if one should try to make electrodes on a given day. There was even an unsetling (to me) joke that during the early Spring everyone might just as well pack up and go fishing as try to make a microelectrode. While I did not want to offend the fishing gods, in the spirit of getting something done (it being early Spring) I decided to make a concerted attempt to understand the variables involved in the local electrode puller and silanization procedure for making microelectrodes.

To make a long story short, I was awarded the newly inaugurated (but none-the-less coveted) Tinkerbell Award by Peter Smith for my development of an apparent foolproof method of making excellent electrodes. One of my new electrodes, which under previous fabrication methods might last one to two hours tops, lasted for two days in a functional condition. The key to my improved procedure was particular care to cleanliness of the operators fingers and proper attention to the shaping and alignment of the puller fillament.

Summer of 1994

As summer approached neither Gerda nor I, nor Peter Smith, wanted to see the sabbatical end. Since I was the most experienced user (and author) of the new 3-D software running the ion probe rigs, Peter offered to support my stay at the lab over the '94 summer. In exchange for an MBL cottage, phone instalation, five round trips to Amherst and per diem expenses, I would stay and help summer visitors in the making of electrodes and running of the rigs. Gerda and I were both pleased. We set about trying to outwit the summer tourists by taking the back routes around the Cape to our favorite spots (despite the local lore of holing up at your local beach while the tourists crammed Route 6 and 6A, the main arteries of the Cape).

During the late spring and summer several investigators came to use the NVP Facility and along with the other staff I was happy to help them get the most out of their stay. Erwin Huebner, a UMass Ph.D. of Everet Anderson, came with his student, William Diehl, to study Rhodnius ovarioles. Nina Allen (wife of the late Robert Allen, renouned cell biologist) used my rig to study alfalfa root hairs and we demonstrated the highest resolution of a specific ion pattern so far achieved (we resolved a 4 µ structure at 2 µ resolution at the tip of a root hair). David Keefe M. D., a fertility doctor from Yale New Haven Hospital, brought 2-cell mouse embryos to the NVP facility. He used my rig to initiate studies aimed at quantifying the health of mammalian embryos in order to allow implanting 1 or 2 healthy embryos rather than the shotgun approach of implanting 5 embryos which in some instances result in dangerous multiple pregnancies. We were able to measure an outward calcium current from the mouse embryo, indicating a substantial energy expenditure over the period of observation. The strength of this current could be a measure of embryo health. These summer visitors allowed us to gauge the friendliness and usefullness of our new 3DVIS software.

At the end of the summer I was able to bring an upgraded NVPi rig back to the Amherst campus and installation of the upgraded equipment is now in progress. Peter Smith has provided the 3-D motion control system and 3-D stepper motors to me on loan. Al Shipley, the builder of the NVPi amplifier I was using at the Cape has donated a Model 300 Ion Probe unit to the University for my use in consideration for the help I provided in testing out his unit and providing the software development for his hardware. The intention is that I continue to improve the software and share it with the NVP facility.

Some lessons for the future:

Health Care. Since our normal healthcare provider, Kaiser, does not operate on the Cape we had to change from Kaiser Permanente to the best alternative John Hancock. While this was not a complete disaster, it presented a substantial irritant during our stay in Woods Hole that continued for four months after the sabbatical was over. A family with a weaker stomach for hassel might decide never to go on sabbatical again. A medical emergency occured in the late Fall '93. In helping a visitor to the NVP Facility get to his airport bus on a Sunday morning, I strained a muscle in my back. The severity of the strain did not surface until Monday morning when I collapsed on our kitchen floor in severe pain and unable to move a muscle without severe spasms. Gerda, not knowing what was ailing me called the local 911. The rescue team brought me to the hospital emergency room and I was treated. Followup was done at a local medical practice. The paperwork ramifications, including medical bills from four different entities (emergency ambulance service, hospital emergency room, emergency room doctor, follow up practice), that followed were impressive and depressive. Without Gerda's heroic patience and fortitude in calling all the parties involved and effectively becoming my health lawyer, I might have given up and paid the bills outright. In the end, almost grudgingly, we received all the benefits for which we were insured. Another instance, involving Gerda, will not be recounted. The tenuous relationship between insurer and health care provider results in a guessing game of which services are covered and which are not. The patient becomes a victim of the tension between the doctors and the insurance company.

Dental Care. The University in its wisdom decided to change our dental insurance in the middle of our sabbatical. Notification of this change reached us quite abruptly a few days before it went into effect. Our previous insurer, Delta, had a network on the Cape with which people at the MBL were familiar since the MBL used the Delta Plan. The new Dental Blue plan was difficult to get referals for, particularly just at the time we needed it in early 1994. Had we had more lead time we might have been able to find the 'preferred provider' at a dental clinic about a mile away from our Dentist-from-Hell. We ended up paying several hundreds of dollars to get our teeth cleaned while at the Cape because the Dentist refused to clean our teeth without a full examination and evaluation. We had requested a referral from our Amherst dentist but he said that he did not have any connections on the Cape. In addition our dental records and X-rays were lost in the mail between Amherst and Falmouth. This lack of communication skills among our medical services is sobering.

Clearly, better and more consistent health care benefits are critical to making sabbaticals easier and less traumatic to family life. Changing health care benefits are confusing enough when they ocuur while you are on home campus. Some effort must be made to keep sabbatical families well informed of health benefits particularly if changes are imminent.

Summing it all up:

I am convinced more than ever of the value of sabbaticals spent reaching out to new experiences and new places.

Last modified 6/12/97 by JGK