Introduction
Two exercises will be performed on the central nervous system of the medicinal leech, Hirudo medicinalis, to determine some physiological and morphological features of neurons.
The purpose of the first exercise is to record resting potentials and action potentials in a segmental ganglion. Differences in the size and shape of the action potentials will be used to distinguish between different neurons. Intracellular microelectrodes will be used to record from the neurons, as well as to pass current into them. The injected current will be used to measure the input resistance of the neurons and to trigger action potentials from the cells.
In the second exercise, dyes will be injected into identified neurons, which will be viewed under a fluorescence compound microscope to see the structural details of the neurons. These anatomical studies will be used to confirm the identification of the neurons and the location of their axons that had previously been deduced from the physiological studies. These studies will also show the extreme complexity of the branching pattern of these neurons, especially in the central part of the ganglion where synaptic contacts are made among neurons.
Anatomy of the Medicinal Leech
Leeches are annelids (segmented worms), closely related to earthworms. Unlike earthworms, leeches have a fixed number of segments (32) plus a non-segmental prostomium (Fig. 1A). The anteriormost four ("head") segments are fused, forming specialized structures including 5 pairs of eyes dorsally and a mouth surrounded by the anterior sucker. The posteriormost seven ("tail") segments are also fused, forming the large caudal sucker. Between fused head and tail segments lie 21 unfused midbody segments, designated in rostrocaudal order as M1 to M21. Along the entire length of the body, the skin is subdivided into circumferential rings, or annuli, of which there are 5 per segment in the middle of the medicinal leech. Leeches are hermaphrodites, each individual being both male and female. Segments M5 and M6 are specialized for male and female function, respectively.
Below the skin lie three layers of muscle fibers (Fig. 1B). The outer layer consists of circular muscle fibers and the inner layer of longitudinal muscle fibers. The intermediate layer is formed by two thin sheets of crossed oblique muscle fibers. The body cavity is traversed by a fourth set of fibers, the dorsoventral muscles, which insert into the dorsal body wall at one end and into the ventral body wall at the other. Contraction of each type of muscle works against the hydrostatic skeleton provided by the fluid-filled leech body tube to change the body shape: contraction of the circular fibers causes lengthening; contraction of the longitudinal fibers causes shortening; and contraction of the dorsoventral fibers causes flattening and lengthening. The effect of contraction of the oblique fibers depends upon which other types of fibers happen to be contracting. During longitudinal fiber contraction (i.e., in a shortened animal), oblique fiber contraction produces elongation; during circular fiber contraction (i.e. in a fully extended animal), oblique fiber contraction produces shortening; when no other fibers are contracted, contraction of the oblique fibers stiffens the body wall at an intermediate body length. A fifth set of muscles, the annulus erector muscles, are composed of short longitudinal fibers that traverse a single annulus just below the epidermis. Contraction of the erectors raises the annuli, forming a series of sharp ridges that make the epidermis resemble the surface of a washboard.
The Leech Central Nervous System
The leech nervous system reflects the segmental body plan (Fig. 1A); it consists of a ventral nerve cord of 32 segmentally iterated ganglia. The four most anterior ganglia are fused to form the anterior brain (or "head ganglion" consisting of a super- & subesophageal ganglion) and seven most posterior segmental ganglia are fused to form the posterior brain. The segmental ganglia are linked via an unpaired, median connective, called "Faivre's nerve", and two paired, lateral connectives (Fig. 1B). The connectives contain, in addition to interganglionic axons, several longitudinal muscle fibers, whose contraction or distension is coordinated with changes in body length caused by the body wall musculature.
Figure 1. Anatomy of the medicinal leech
1A. A. Diagram of the external body, with an outline showing the position of the central nervous system
1B. A cut-away diagram of a mid-body segment, showing the layers of muscles in the body wall and the major internal organs.
Figure 2. Leech segmental ganglion.
Each segmental ganglion contains about 200 bilateral pairs of neurons, as well as a few unpaired neurons (Fig. 2). Their cell bodies form an outer cortex around the ventral and lateral aspects of the ganglion. The neurons are monopolar; their processes project initially into a central neuropil, where they make synaptic contacts. From there, the axons of some neurons project to other ganglia via the connective nerves. Sensory and motor neurons send processes to peripheral targets via segmental nerves, whose nerve roots emerge from the lateral edges of the ganglion. From either side of the typical midbody ganglion emerge four main segmental nerves: the anteroanterior (AA), the medioanterior (MA) the dorsoposterior (DP) and the posteroposterior (PP) nerve (Fig. 1B). In each ganglion, the neuronal cell bodies are distributed among six cell packets: a pair of anterolateral packets; a pair of posterolateral packets; and a pair of ventromedial packets. The latter pair lies anteriorly and posteriorly in all segmental ganglia. Each neuronal packet is enveloped by one giant glial cell.
The anatomy of the leech ganglion is sufficiently stereotyped, and its cell bodies are sufficiently accessible to electrophysiological and anatomical analysis that a substantial fraction of its neurons have been identified (Fig. 2). After characterizing a particular neuron by morphological and physiological criteria, homologous neurons can usually be found on the other side of that same ganglion, in other ganglia of that same specimen, in the ganglia of other specimens of the same species, and even in other leech species. It is likely that all neurons of the segmental ganglia are identifiable in this sense.
About one quarter of the neurons in the segmental ganglia have been identified according to various criteria, including function (Fig. 2). For instance, the identified sensory neurons include three types of mechanosensory cells, designated as T (for touch), P (for pressure) and N (for nociception; i.e., pain). Each of these neurons projects its axons from the ganglion to a particular territory of the segmental skin, where its endings form specialized mechanoreceptors that respond specifically to slight (T), moderate (P), or intense (N) deformation of the skin. In the typical midbody ganglion there are pairs of TV, TD, TL, PV, and PD cells, where the subscripts V, D and L designate that the ventral, dorsal or lateral skin, respectively, on the same side as the cell body is the principal territory of innervation. In addition, there are two pairs of N cells, each of which appears to innervate the entire hemilateral skin.
Dissecting the Medicinal Leech (this will be done for you, but please try an on-line simulation)
2. Using a pair of sharp-blunt scissors, make a medial, longitudinal incision through the dorsal body and dorsal gut walls of the leech. The incision should extend along the dorsal midline from the fused head to the fused tail segments.
4. Suction away the contents of the gut (a fire-polished Pasteur pipette works well) into a flask connected to a vacuum system. Replace any cloudy saline with chilled, fresh leech saline.
It is important to irrigate the preparation frequently (every 10 to 15 min.) with chilled saline to prevent dehydration and overheating of the ganglia.
5. Using an iridectomy scissors ("student grade" works fine), lift the ventral wall of the gut away from the underlying nerve cord and make a medial, longitudinal cut through this wall. This exposes the blood sinus (stocking) which surrounds the nerve cord.
6. Starting at posterior end of the midbody, open the stocking that surrounds the nerve tract. (A reasonable good pair of iridectomy scissors and Dumont #5 forceps are needed for this procedure.)
8. When the section of the nerve cord to be used has been cleared of the surrounding stocking, return to each ganglion and carefully cut away the connective tissue that lies under it.
9. Remove the connective tissue that surrounds the four segmental nerve roots that emerge laterally from each ganglion. Leave a small amount of stocking around the nerves near each ganglion. These tissues are difficult to remove cleanly without damaging the nerves, but they are useful in pinning out the isolated section of the ganglia preparation.
10. The dorsoposterior (DP) nerve will be used in some experiments for extracellular recording. Remove the connective tissue that surrounds the DP nerve root by:
12. Free up a connected pair of ganglia from the leech by cutting the connectives on either side of the connected pair. (Cut halfway between adjacent ganglia, so that the remaining ones can be used as another preparation.)
13. Transfer the pair of ganglia into a Sylgarded petri dish filled with cold leech saline by sucking them into a Pasteur pipette. (It is easy to lose the preparation at the surface of the saline if transfer by forceps is attempted.)
14. Pin the preparation to the Sylgard using the .002" tungsten wire pins. (Use a coarse Dumont #5 forceps for handling the pins or the tips will be bent backward and destroyed.) Push one pin into the connective tissue around each later pair of nerve roots, and one into each of the connectives.
15. Smash the
connectives with the fine forceps,
to destroy the muscles in the connectives.
Retrieve a leech ganglion in a Sylgard-filled Petri dish from the refrigerator. Set it up with light coming from the side so you can see the outlines of neurons and the glial packets in the ganglion. Which cells can you identify so far? Which way is anterior and ventral? Approximately how big are these neurons? Set up the Neuroprobe and an intracellular glass electrode filled and backfilled with 3 M KAc (potassium acetate). (Acetate ions not as mobile as chloride ions, but chloride can leak into the neurons from the electrode and dramatically change some of the inhibitory postsynaptic potentials [IPSP’s]. Acetate ions are large enough that, even if they leak into the neuron, they do not affect the IPSP’s. Why does size matter?)
Put the microelectrode into the holder, put a ground electrode into the bath, and measure the resistance of the microelectrode. Zero the Neuroprobe output, and make sure you can see the record from the Neuroprobe on the computer using the PowerLab template in Chart and make sure you know where 0 mV is on the screen so you can measure the resting potential. Make sure you Neuroprobe is set correctly.
2. Turn on the audio amplifier and set the volume to a low, but audible, level. Turn the volume as low as possible before removing the electrode from the bath solution and before buzzing the electrode; the noise can be very loud and the speaker can be damaged.
3. Lower the microelectrode by using the micromanipulator drive, until the cell dimples under the pressure of the electrode tip. The circumference of the cell also expands in response to pressure from the electrode. Make sure you are lightly touching the cell, with a small amount of compression so the electrode tip is bowed and bit, and the ganglion has an indentation in it.
4. Gently tap the base or the back end of the micromanipulator. This vibration should be sufficient to penetrate the cell. While tapping, watch the recording on the screen, not the cell. Be CERTAIN to stop tapping when the voltage drops to resting potential (negative 30 to 80 mV).
Successful impalement is seen when there is a sharp negative deflection of the recording, usually accompanied by action potentials and synaptic potentials. A good impalement can also be heard as a sharp click on the audio system. With practice, different cells can be recognized by the sound of their action potentials.
Later, print out recordings of spikes from you neuron.
5. If the first attempt is unsuccessful, advance the microelectrode
slightly and try again. If this does not succeed, reposition the
microelectrode
and try again. Each time the electrode is repositioned, the impedance
should
be measured; to make sure the tip is neither broken nor clogged.
If impedance is below 10 MOhms, get a new electrode. If above 200
MOhms, then trying clearing the clog by pressing the override button on
your amplifier several times. Then try impaling a cell again.
6. Try to impale one cell for each lab partner. For each of the cells that is impaled, note the following:
a. The value of the resting membrane potential and its stability. Measure the membrane potential as the voltage change when the cell is first impaled, as well as when you pull it out of the cell. These are not always the same. Why not? Use the larger value of the two as the membrane potential. Why?
b. Any impulse activity in the cell, and the amplitudes and time courses of these impulses.
c. Stimulate the cell to fire single action potentials to pulses of current that are 1-2 nA in amplitude. Turn the monitor on the Neuroprobe to display current while you inject positive current. If action potentials cannot be triggered using these strengths, check with the instructors before proceeding.
b. T1, T2, or T3 (TL, TV, or TD, respectively), in either of the anterolateral packets (Fig. 3). Short (~ 5 ms) after-potential. ~ –60mV RP. Appreciable accommodation. ~ 45-50 mV AP. AP duration shorter than P or N.
c. P1 or P2 (PD or PV, respectively), in either of the posterolateral packets (Fig. 3). Short (~5 ms) after-potential. ~ –50mV RP. Little accommodation. ~ 70-80 mV AP.
d. N1 or N2 in either of the
anterolateral
packets (Fig. 3). Long (~1 s) after-potential. ~ –50mV
RP. Strong accommodation. ~60-80 mV AP.
2. To be the envy of your friends and joy of
your
family, also identify the following cells:
b. AE (annulus erector), in the posterior ventromedial packet. These are motor neurons to the AE muscles. They are medium-sized cells at the posterior edge of the ganglion. The action potentials are similar to those in the AP neurons--the AE's were, in fact, the "posterior pagodas" until their function was discovered. Small AP < 20 mV.
3. To earn superstar status, record from the S
cell, in the anterior ventromedial packet. This cell is
unpaired,
unlike all the other cell types listed above, and it is much smaller
than
the other neurons listed. It is the only neuron in this packet, besides
the Rz cells, that has an action potential greater than 10 mV in
amplitude.
Leech CNS II: Identifying Neurons
Morphologically
Lucifer Yellow (LY)
This dye was created specifically as a bright and convenient marker to be injected into neurons (Stewart, 1978, 1981). It is negatively charged, so that it can be injected into cells by passing hyperpolarizing current; it is a small, highly mobile molecule, so that it diffuses readily to all parts of the cells; it is highly fluorescent, so that a small amount of it will produce a very bright signal; it has appropriate chemical groups on it so that aldehyde fixatives readily link it to intracellular proteins; and it is extremely non-toxic. For these reasons, LY has become a workhorse in identifying neurons. Routinely, LY-filled microelectrodes are used to record from neurons, then hyperpolarizing current is passed to mark the cell for later identification.
One major problem in using LY is its insolubility in solutions with a high concentration of Na+ or K+. Fortunately, it is soluble in concentrated solutions of Li+. Therefore, LY is typically made up as a highly concentrated solution (3-10% by weight) in distilled water, a small amount is injected into the tip of the electrode, and the rest of the shaft of the electrode is filled with a concentrated solution of LiCl for electrical recording. (LY is too expensive to use it to fill the whole electrode.) The electrode filled in this manner has an impedance that is 3-5 times greater than it would be if the electrode were filled with KCl or potassium acetate. Therefore, a good LY-filled electrode will have an impedance in the 60-120 megaohm range.
A second major problem with LY is inherent in the use of fluorescence: it fades with viewing. (The electrons of each fluorescent molecule absorb light at one wavelength, emit it at another wavelength, and go into a state where they cannot absorb any more light energy. This means that, at best, every light-absorbing molecule can give off just one photon.) When viewing a fluorescently labeled cell, therefore, the length of time the preparation is exposed to the bright UV light should be minimized. (Long-term storage should be in the dark, because there is enough UV in daylight or even room light to cause the dye to fade.) When drawings are done, for instance, the shutter between the UV light source and the preparation should be pulled whenever the preparation is not being viewed.
Rhodamine dextran
It is convenient to have a dye that emits a second color, to determine, for instance, where the processes of two different neurons lie relative to one another. A particularly good dye for this purpose is rhodamine, which emits a bright red fluorescence. If rhodamine is injected into a neuron, however, it leaks out quickly and cannot be fixed to the proteins in the cell. To make it useful, rhodamine can be attached covalently to another molecule that CAN be fixed to proteins, and is large enough to remain within the neuron. In this exercise, rhodamine bound to lysinated dextran is used. Dextran is a polymer of sugars, available in a variety of molecular weights. The dextran used in this exercise has a molecular weight of 3000. Lysine, an amino acid, has been conjugated to the dextran, so that the molecule can be fixed to intracellular proteins by exposing the filled neurons to paraformaldehyde.
I. Filling neurons with LY.
2. Record from a neuron, identify it by its electrical properties, be sure to have a good recording, the pass 10 or more nA hyperpolarizing pulses (900 msec at 1 Hz) or constantly (direct current, DC) into the cell for 5 minutes. Set up the stimulator by choosing Stimulator from the Setup menu. Set the duration to 900 ms. Make sure it is set to deliver pulses at 1 Hz, and that the pulse amplitude is -10 V. This -10V signals will be sent to the Neuroprobe current input, and the Neuroprobe will inject 1 nA of current per V applied. Note that the baseline voltage during stimulation is set to 0.5 V. This small positive current reverses ionic flow and helps prevent electrode clogging with the dye. Choose Stimulator Panel from the Setup menu so you can turn off stimulation when ready.
Have the microscope light turned off during this time.
3. Turn on the microscope light briefly to observe the cell. If it is no more yellow than any other neuron, buzz the electrode briefly with the capacity button and continue for 5 minutes more, at a higher intensity. Continue buzzing and increasing the intensity until the neuron is distinctly more yellow than any other cell, or until the cell is distinctly dead. This should occur after 15-30 minutes
4. When the cell looks yellow, discontinue
stimulation,
pull the electrode out of the cell and let the ganglion sit for another
15 minutes, to allow the dye to diffuse into all the processes. (If the
preparation sits for more than 60 minutes, there is a danger that the
dye
will start to leak out of the cell.)
Use the same techniques as was used for filling the neurons with Lucifer Yellow, except that 5 or more nA of POSITIVE current should be used. So set the pulse amplitude to +10V and the baseline to -0.5V.
III. Fixing and clearing the ganglion.
a. For immediate viewing.
Mount on a slide in 80% glycerol/20% buffered saline solution. View under the dissecting scope and when clear, transfer to 100% glycerol.
b. For better image quality and longer lasting color.
Pin the preparation to the bottom of a glass Petri dish (marked with an "F" for Formalin) dish with a Sylgard rubber bottom, by placing small pins in the cut ends of the connectives (only use pin forceps when handling pins). Put on rubber gloves. Under the hood, replace the saline with 4% paraformaldehyde fixative and let it sit for 15 minutes. Pour the fixative into the fixative waste bottle in the hood only. Next, dehydrate the preparation with an ascending EtOH series of 5 minutes each: 70%, 90%, 95% and 100%. These can be done outside the hood. Finish with one final dehydration of 100% EtOH for 5 minutes. Basically, remove one solution and add the next in the series. Please dispose of solutions in the designated waste containers. Now, back to the hood! Using your pin forceps, carefully remove the pins and transfer the preparation to a glass slide by grasping only the ends of the cut connectives. Not how stiff the tissue is. Add 2-3 drops of methyl salicylate on top of the preparation to clear it. After 5 minutes, remove the methyl salicylate with a Kimwipe and add 2-3 drops of glycerol to the preparation. Carefully place a cover slip on top of the preparation.
View it under the fluorescent microscope with your instructor and observe the morphologies of these neurons. Sketch their morphologies for your lab notebook, take photos, and consult the key to see if your morphological data correlate with your physiological data.
In addition to the questions found within the lab exercise, answer the following questions in your lab notebook. Please restate the question so that you will be able to place the answer in context when reviewing your lab notebook in the near future.
1. Which
neuron did you identify?
2. What were the physiological criteria you used
to identify the neuron?
3. Why do resting potentials, action potential
amplitude, AP duration, and after potential vary? Include printouts of
these data.
4. How might accommodation work? Describe a method based on ion channel
modifications.
5. What were the morphological criteria you used
to identify your neuron?
References
When done, clean your tools using the sonicator and the ethanol
rinse. Pat them dry with Kim-wipes and replace the covers your
removed
from the sharp-tipped tools.
MAKE SURE ALL OF YOUR TOOLS AND CABLES ETC. ARE IN THE CAGE.
Before leaving show your instructor that all of your data are saved in your named User folder and give the instructor you lab notebook. Also, turn off your PowerLab and Quit all programs (to do so you need to choose File, Quit for each program [leave the Launcher and Stickies open].
Make sure your understand the lab questions. Check with me before leaving.
Work the "Axon diameter" tutorial in the program NeuroLab of Neurons in Action. Find this file in you Lab 1 or 2 folder on the iMacs in lab. You can open it in Internet Explorer or in Netscape.
modified 10-7-07
