The Leechlab is our name for the Vassar College facility on the third floor of Olmsted hall. Here we keep our leeches (Hirudo medicinalis) and study their neurobiology. The lab is home to a number of students who do independent research (178, 298, 399, 303, URSI) on a variety of topics including such things as cell growth in culture, feeding behavior, immunocytochemistry, basic neurobiology, etc. The Leechlab is equipped with a complete electrophysiology setup including an Axopatch 1D patch clamp amplifier and Pclamp 6.0.3 software driving a DigiData 1200 interface, as well as a WPI Series 7000 intracellular recording system with capabilities for iontophoresis. We also have a Sutter P-97 pipette puller.
The Leechlab is also the breeding and proving ground for experiments and demonstrations which become incorporated in classes such as Evolution of Animal Structure and Neurobiology. Our leeches see a variety of duties, including being featured at presentations to prospective students and even to potential donors to the college.
Note: click on any of the links below to see the figures they refer to.
Beginning this summer (1997) we have been looking at the properties of the photoreceptors of the leech. Here's what we've found so far...
Leech photoreceptors are most unusual in a number of aspects, in fact, possibly unique. The "eyes" of the leech are found as five pairs on segments II through VI. Each "eye" consists of a roughly cylindrical cup of pigment cells, is about 200 microns in diameter and 500 microns deep, contains anywhere from 60 to 100 photoreceptive cells, and is covered on the top by a modified patch of epidermis which forms a translucent "cornea". The eyes are set at varying degrees of perpendicularity to the surface, presumably to convey some directional information. The photoreceptive cells themselves are the unusual part of the eye. Each cell is about 50 microns in diameter and is connected to the central nervous system by an axon. The cells are rhabdomeric in origin as are all annelid photoreceptors; that is, they contain their photopigment in microvilli protruding from the surface of the cell, unlike the internal arrangement of vertebrate photoreceptors. Now for the unique part: each cell contains a large "vacuole" which occupies most of the interior of the cell. The actual cytoplasm is restricted to a thin (about 2 microns) cortex. The microvilli containing the photopigment are located on the interface between the cytoplasm and the vacuole, not between the cytoplasm and the extracellular fluid as is the common case. This provides a unique opportunity to study the phototransductive process, since one can gain access to the vacuole with a microelectrode and thus be recording from the extracellular fluid yet at the same time have that environment contained so that one can readily manipulate it. You see, while the vacuole is not truly a vacuole in the sense that it has some connection to the extracellular fluid, those connections are via highly morphologically specialized canals which markedly restrict diffusion. Thus one can record from the 'extracellular' side of the photoreceptors without facing the normal problem of ions diffusing away, or one can record from the cytoplasmic side (although this is a bit harder given the thinness of the cytoplasm). An additional feature is that the site of generation of action potentials is anatomically separated from the phototransductive apparatus.
I f one records from the cytoplasm, one can see a depolarizing response to light. The cells have a resting potential of around -40mV. If on the other hand you record from the vacuole the response is hyperpolarizing. Often one finds that the electrode starts in the cytoplasm but slips into the vacuole, which can be annoying but does show that the cytoplasmic and vacuolar responses are symmetrical responses. So far so good; these are the same kinds of responses seen by Lasansky and Fuortes in 1969. Fioravantini and Fuortes also studied the dynamics of the photoreceptor responses, and found that the responses increase in amplitude and speed as the light stimulus increases in intensity. We have seen this also for both the depolarizing and hyperpolarizing responses. Going a bit further, we have shown that both the dynamic range of both responses can be fit with a hyperbolic tangent function (depolarizing fit, hyperpolarizing fit) as described for vertebrate photoreceptors by Naka and Rushton. The dynamic range (from no response to saturation) is about one log unit in stimulus intensity (as opposed to about seven or eight log units for the human eye - though each separate receptor in the human eye has less of a range than that). We have also seen, as did Fioravantini and Fuortes, that as the stimulus intensity increases, the shape of the response changes dramatically and cannot be predicted by scaling up a unit response fitted to the low-intensity response, indicating that some new mechanism is coming into play. What that mechanism is remains to be understood.
Now for some really new stuff: to begin with, we've tracked the dark adaptation of the photoreceptor, that is, how quickly does it recover from a bleach. It doesn't take long! This can be seen from the raw responses or from a tracking of the response amplitude over time following the bleach. The recovery begins after about ten seconds and is complete about ten to fifteen seconds later. We'd love to know how the cell does that! We've also tracked the amplitude of the response to a constant-intensity stimulus in the presence of a constant background light. At the onset of the background light one gets a diminution of the response and a hyperpolarization of the cell, but this hyperpolarization diminishes spontaneously to a steady level, as does the amplitude of the response. This can be seen in the raw responses or the tracking of the response over time. How does the cell adapt to the light? Stay tuned for future developments from the Leechlab. Finally, we've looked at the wavelength or spectral sensitivity of the eye to try to get a handle on the specific photopigment underlying the phototransduction process. We've measured the response versus intensity at several wavelengths. Using these data, one can measure the action spectrum of the pigment by picking a criterion response (we used -10mV) and seeing how much intensity it takes at each test wavelength to obtain that criterion. The logic is that if it takes less intensity to obtain -10mV at say 550nm than it does at say 460nm, then the eye is more sensitive to 550nm than to 460. This produces a spectral sensitivity curve. The curves we've obtained reveal that the data are best fit by a nomogram derived for rhodopsin, but one whose wavelength of maximum sensitivity is set at 540nm rather than the usual 500-510nm. Why? Also, we consistently see a drop in sensitivity at around 500nm. Why? Is it because the eye shows some kind of spectrally specific diffraction due to the alignment of the microvilli (they are all parallel and about 80nm in width; also, when you look at the eye it looks blue so it probably is scattering blue light)? Stay tuned.
That's where we are at the moment. There's a lot to be learned from these photoreceptors yet. For example, what cellular mechanisms underlie the dark- and light-adaptations? Why does sensitivity drop at 500nm? Why don't the action potentials seem to correlate with the amplitude and duration of the graded responses? Are there OFF and ON type cells? How is the information coded by action potentials? What is the mechanism of phototransduction? And so on. This should keep us busy for some time to come!
