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body wall preparation
The Application of Irritant Chemicals Selectively to the Skin of the leech Gan~lion/Body Wall Preparation
KATHLEENM. WESTON, R. W. FOSTER,ANDA. H. WESTON
A technique is described for applying chemical irritants selectively to the skin of a superfused ganglion/body wall preparation of the horse leech, Haemopis sanguisuga. Details are given of the intracellular recording from sensory neurones of the effects of the irritant dibenzoxazepine and of mechanical stimulation. Dibenzoxazepine produced changes in the spontaneous firing pattern in the nociceptive cells of the leech but did not affect the response to mechanical stimulation of the skin in any type of sensory cell. It is possible that the system described could be used as a model of cutaneous excitation evoked by drugs or mechanical stimulation in higher animals. Key Words:
Leech; Haemopis
sanguisuga; Irritants
INTRODUCTION Parallels have been drawn between the afferent neurones identified in the leech and those innervating human skin (Nicholls and Van Essen, 1974). The central nervous system of the leech consists of a chain of discrete segmental ganglia enclosed within a blood sinus and, apart from the fused ganglia at the head and tail, all are virtually identical (Coggeshall and Fawcett, 19641. Apart from the suckers at the extremities, the animal is made up of identical segments, each segment consisting of several annuli supplied by one ganglion. A ganglion consists of a connective tissue sheath enclosing about 350 unipolar neurones that are segregated into discrete groups or packets, together with several supporting glial cells (Coggeshall and Fawcett, 1964). Adjacent ganglia are connected by bundles of unmyelinated nerve fibers, the connectives. An anterior and posterior root, also containing unmyelinated axons, connects each side of the ganglion with the body wall and underlying viscera. The essential features in the horse leech (Haemopis sanguisuga) are the same as those described in the medicinal leech (Hirudo medicinalis) (Nicholls and Van Essen, 1974). Of the 350 nerve cell bodies in any one ganglion, seven pairs of sensory cells have been identified, all responding to mechanical stimulation of the skin. On either side of the ganglion are three touch (T), two pressure (P) and two nociceptive (N) cells responding to light touch, pressure, and noxious mechanical stimuli, respecFrom the Department of Pharmacology, Materia Medica and Therapeutics, University of Manchester, U.K. Address reprint requests to R. W. Foster, Department of Pharmacology, Materia Medica and fherapeutics, University of Manchester, Manchester Ml3 YPT, U.K. Received September 30,1983; accepted April 1984.
285 Journal of Pharmacological
Methods
D 1984 Elsevier Science Publishing
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tively (Nicholls and Baylor, 1968). Each cell occupies a fixed position in the ganglion by which it can be identified. In addition, the electrical characteristics of each type of cell differ and provide a further aid to identification. The sensory cells of the leech can be driven electrically or by mechanical stimulation of the skin, and the resulting electrical activity can be observed by use of intracellular microelectrodes. The size and shape of the action potential, its duration, and the magnitude of the positive phase all aid in the identification of the cell impaled (Nicholls and Baylor, 1968). An additional guide can be obtained from the size of the stimulus that must be applied to the skin to stimulate each type of cell. T cells fire in response to light touch or even to eddies in the bathing fluid, whereas much larger stimuli are required to activate P and N cells (Nicholls and Baylor, 1968). Each of the sensory cells has a particular region of the skin from which it can be stimulated-its receptive field. The receptive fields in the medicinal leech, H. medicinalis, have been mapped and, apart from a slight overlap, have been found to be specific for each cell of any one type (Nicholls and Baylor, 1968). Conflicting reports have appeared in the literature on the effects of capsaicin and other irritants on the responses to mechanical stimulation in avariety of mammalian systems (Jancso, 1958; Jancso and jancso-Gabor, 1959; Porszasz and Jancso, 1959; Green and Tregear, 1964; Hayes and Tyers, 1980; Foster and Ramage, 1981). The method described was evolved for the selective application of irritant chemicals to the skin of the leech and the measurement of the effects of such applications on the responses of sensory cells to mechanical stimulation. METHODS The Dissection
of the Leech/Ganglion
Body Wall Preparation
Horse leeches that ranged in length from S-10 cm when fully extended were used. Before experiments, animals were maintained at 4°C in distilled water containing 4% leech Ringer solution. For dissection, a leech was fully extended and pinned through the head and tail, ventral side uppermost, onto a hard paraffin block. An incision was made down the ventral midline to reveal the ventral blood sinus. The edges of the skin were pinned back and a ganglion was selected. Ganglia in the anterior region that served the genitalia were avoided. The remainder of the dissection was carried out using a binocular microscope. An incision was made in the wall of the ventral blood sinus to reveal the ganglion beneath. The nerve roots on one side of the ganglion were exposed and a l-mm sliver of the medial body wall on that side was removed to expose the roots further. The nerve roots on the opposite side of the ganglion were exposed and severed as closely as possible to the body wall and the connectives were cut as closely as possible to the adjacent ganglia. The severed roots and connectives were then pulled free from the blood sinus. To isolate the segment of body wall served by the ganglion, two transverse cuts were made, several annuli apart, on the side of the animal with intact innervation. Starting from the ventral midline, these cuts continued around the body wall to the lateral line on the opposite side. A longitudinal cut was then made between the
irritants and Leech Skin Afferents two transverse cuts along the lateral line. The body wall was dissected carefully from the viscera, and the inside surface of the longitudinal muscle layer was cleared of loose tissue. Throughout the dissection, the preparation was pinned as appropriate with small stainless steel pins comprising l-cm lengths of 0.15-mm diameter wire (dental grade, K.C. Smith). The preparation was bathed in leech Ringer solution that was changed frequently and care was taken to avoid injuring the nerves or bursting the gut. The ganglion/body wall preparation was removed to a perspex bath containing leech Ringer solution. The flap of body wall extended from the ventral midline around to the lateral line of the opposite side and contained about 12 annuli. The flap was larger than the segment of body wall served by the ganglion. Further preparations could be obtained throughout the day from the same leech, which was maintained at 4°C between dissections. The base of the perspex bath (maximum volume = 5ml) was coated in a layer of Sylgard 184 resin (Dow Corning) that had been allowed to cure for 2 to 3 days at room temperature. The body wall was pinned to the Sylgard, skin side uppermost and moderately stretched, with small stainless steel pins. The ganglion was pulled clear of the body wall, stretching the roots moderately, and was pinned ventral side uppermost by means of small tungsten hoops over the connectives and severed roots. Care was taken to avoid twisting the intact roots. Pinning these roots was avoided, for it appeared to cause nerve damage and to overstretch the ganglion, making impalement difficult. Tungsten Hoops Tungsten wire (0.2 mm diameter, Goodfellow Metals) was positioned as the anode in a simple electrolysis bath containing 10% KOH solution. Current (12 V d.c. source) was passed through the bath until the diameter of the wire was reduced to approximately0.05 mm. Al-2-mm length of wire was then bent into a hoop. By holding the tips of the hoop in the electrolysis bath and passing current, they became pointed. Perfusion
System
All experiments were carried out at room temperature (17-24°C). Leech Ringer solution had the following composition (mM): Na+ 115 (plus approximately 8 from the buffer solution); K’ 4.0; Cazl I .8; Cl- 122.6; glucose IO; morpholinopropane sulphonic acid (MOPS, Calbiochemf 10; pH 7.4. The leech Ringer solution bathing the preparation in the perspex bath was continuously exchanged by perfusion (Figure 1) at a flow rate of 2-3 ml/min, and the fluid in the bath (approximately 1 ml, 1 mm deep) just covered the preparation. It was found that if the depth of immersion were any greater the image of the ganglion seen through the binocular microscope was distorted and impalement of nerve cells was difficult. intracellular
Recording
System
A single microelectrode, in conjunction with a high-impedence probe and preamplifier (Mentor N-950) was used to record transmembrane potential. The indifferent electrode was a sintered silver/silver chloride/platinum black disc (Mentor N-9501).
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Dreschel
bottlefv-l
FIGURE 1.
Perfusion system used in the present study.
The output from the preamplifier was displayed on an oscilloscope (Tektronix) and on a polygraph (Crass Instruments). The output from the polygraph was connected to one channel of a tape recorder (Racaf Store 4) and signals were stored on tape (3M 207) at 3.75 inches per second. Silent ceils were identified immediately after impalement by stimulating them via the recording microelectrode. The stimulus artefact, dependent on the resistance of the microelectrode plus bathing fluid, was first balanced out. To accomplish this, a Grass S48 stimulator was used to deliver a 0.5-V pulse to the Mentor N-950 preamplifier, which converted it to a I-nA pulse before delivery to the cell. This pulse was far too brief (1 ms) to charge the membrane capacitance and the effective resistance of the cell membrane plus cytoplasm was thus negligible. The pulse was used to trigger the oscilloscope sweep and by balancing the bridge of the Mentor N-950, the deflection on the oscilloscope trace could be nulled, indicating that the effect of the resistance of the electrode plus bathing fluid had been balanced out. After balancing, a 3O-ms 0.5-1.5-V pulse was delivered from a second stimulator (Grass S48) to the preamplifier, the conversion resulting in a I-3-nA pulse being delivered to the cell. This elicited the firing necessary to aid cell identification. Standing potentials developed between the indifferent electrode and the probe input as a result of electrochemical action were cancelled before impalement by means of the bucking voltage facility on the Mentor N-950. In order to reduce the attenuation of high-frequency components of the input signal, the capacitance compensation facility on the Mentor N-950 was used to optimize the frequency response of the system. Eiec~ro~es Self-filling glass microelectrodes (Clark Electromedicalf with a blank outer diameter of 1 mm were used. Filtered (Millipore) KCI solution (3 M) was used as the
irritants and leech Skin Afferents electrolyte. Electrodes were mounted flexibly on a siiverfsilver chforide wire. The electrode was positioned using a micromanipu~ator (Leitzf and a binocular microscope (Olympus) with zoom magnification up to 80X and a dark-field attachment (Kyowa) . Electrodes were pulled on a vertical electrode puller (Narishige PE-2). They were then bevelled on an electrode beveller (WPI Model 1200) at an angle of 25” in a slurry of 20% w/v polishing alumina (0.05 pm, Banner Scientific) in KCI solution (3 M) (Eisner, et al., 1979). The electrode was connected to an ohm-meter (WPI Model F29) that generated the square wave and gave a digital read-out for the measurement of electrode resistance during bevelling. Initially, the resistance of an electrode in the KCI slurry was > 15 MfZ. After bevelling, electrodes had resistances of 6-10 Ma, higher values being used in later work to improve the stability of recordings. Electrodes had tip potentials of less than I mV. Tip potential was measured in a sample of electrodes by noting the change in potential difference between the microelectrode and indifferent electrode when the tip of the microelectrode was removed. Selective Application
of Chemical
Irritants to Primary Afferent
Nerve Endings
A method of applying chemical irritants selectively to the skin surface without allowing contact with the nerve cell body or synapses was evolved. A ring of polythene with one perfectly smooth edge was made by cutting the wide end from a disposable pipette tip (Hughes and Hughes). The ring had an internal diameter of 0.7 cm and was 0.3 cm high. The smooth edge was piped with a thin layer of cyanoacrylic glue (Loctite Superglue31, and the ring was immediately glued on to blotted, pinned-out leech skin. Gentle pressure was applied downwards on the ring for about IO sec. The ganglion and underside of the body wall could be perfused with leech Ringer solution while a solution of the chemical irritant being tested could be introduced into the ring. The height af the ring was sufficient to ensure that no mixing of the two solutions occurred. The size of the ring was such that a large part of the receptive field innervated by each sensory cell was enclosed. The flap of body wall excised was made sufficiently large to ensure that the walls of the ring did not obscure any more of the receptive fields than necessary. A diagram of the preparation is shown in Figure 2. Care was needed to use only the rnjn~rnurn amount of glue necessary for adhesion, otherwise the excess covered either part of the skin inside the ring or the ganglion outside. If the edge of the ring was placed too near the ventral edge of the body wall, the surrounding meniscus obscured the ganglion and made impaiement of sensory cells almost impossible. Care had to be taken in positioning the ring to prevent this without covering any more of the ventral receptive field than necessary, In order to ensure that an experiment had been conducted with an adequately sealed ring, eosin dye solution was introduced into the ring at the end of each experiment. If any leakage occurred, the experimental results were discarded. Each ring was used once only.
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K. M. Weston et al. Solution
Dorsal
of irritant
midline
Ventral midline
Segmental ganglion
FIGURE 2. Arrangement for the selective application of chemical irritant solutions to the skin of the leech (not to scale). The shaded area shows the black (dorsal) skin and the unshaded area the yellow (ventral) skin.
Mechanical
Stimulation
of the Skin
Von Frey i-lairs A series of von Frey hairs was made consisting of varying lengths of stainless steel wire or nylon bristle attached to a wooden handle. The von Frey hairs were calibrated on an analytical balance and delivered stimuli between 0.1 and 200 mN. For delivery of a stimulus, the von Frey hair was lowered vertically on to the body wall of the preparation until the first visible bending of the hair occurred. The hair was maintained in this state for about 1 set, after which the stimulus was removed. Characteristics of Sensory Cells and Receptive Fields In a series of preliminary experiments, the characteristics of action potentials elicited in N, P, and T cells were observed. Cells were driven either by electrical stimulation using a 30 ms depolarizing pulse with an amplitude of l-3 nA or by mechanical stimulation of the skin using a series of von Frey hairs ranging from 0.1 to 200 mN. By use of this method, a limited study of the receptive fields innervated by each type of cell was performed. Effect of the Ring Glued to the Skin on Responses to mechanical Sti~ui~tion A ganglion/body wall preparation was perfused with modified leech Ringer solution. As many sensory cells as possible were impaled in sequence, allowing at least 5 min between impalements. Immediately after penetration and stabilization of the membrane potential, a response to mechanical stimulation was elicited using a von Frey hair (0.1 mN for T cells, 50 mN for P cells, and 100 mN for N cells). These values were selected from preliminary experiments. The electrical activity of each cell during stimulation was recorded. The perspex bath was then removed from the perfusion system and drained. The skin of the leech was blotted, and a ring was glued in position. The bath was then
Irritants and leech Skin Afferents
returned to the system and perfused with modified leech Ringer solution. Modified leech Ringer solution (0.1 ml) was placed inside the ring to moisten the skin. The sensory cells were impaled in sequence as before, and the responses to mechanical stimulation were again elicited. Effect of Dibenzoxazepine Mechanical Stimulation
Applied
Selectively to the Skin on the Responses to
The chemical irritant dibenzoxazepine (CR) was kindly supplied by the Chemical Defence Establishment, Porton Down. A ganglion/body wall preparation with the ring glued in position was perfused with leech Ringer solution. Leech Ringer solution (0.1 ml) was introduced into the ring to keep the skin moist. With a sensory cell impaled, the skin was stimulated with a von Frey hair (0.1 mN for T cells, 50 mN for P cells, or 100 mN for N cells) 1 min before addition of 0.1 ml CR 20 mM in 20% ethanol to the ring. Five minutes later, the mechanical stimulation was repeated. Concurrent control experiments were performed to test the effect of the ethanol vehicle alone. Statistical Methods For comparison of several variables arising in one experimental series, data were assessed using an analysis of variance (AV). Since measurement of the mean firing rate of N cells alone before and after application of CR did not fully summarize the changes seen, a more elaborate method of comparison was used. The firing patterns for 1 min immediately before the first and second mechanical stimulations of the skin were compared in each cell in test and control groups. Each interspike interval was measured, and a cumulative frequency distribution of the interspike interval (bins of 1 set) was constructed for each cell during each of the I-min intervals outlined above. The greatest degree of separation between the two distributions was measured, and the deviation was estimated using the Kolmogorov-Smirnov test for goodness of fit (KS-test) (Lindgren, 1960). The incidence of change in firing pattern in test and control groups was used to construct 2 x 2 contingency tables, and the significance of the difference between the two groups was estimated (Fisher’s exact method). RESULTS Characteristics
of Sensory Cells and their Receptive Fields
When the ventral surface of a segmental ganglion of H. sanguisuga was viewed through the microscope, the two Retzius cells that served as landmarks in the ganglion could easily be seen. With some preparations, it was difficult to distinguish the various sensory cells and adjustment of the illumination was found to be a critical factor. The positions of the sensory cells in H. sanguisuga were essentially the same as those described for H. medicinalis (Nicholls and Baylor, 1968). Two pairs of P and N cells were clearly recognizable. These cells were comparatively easy to impale although they often moved sideways under pressure of the electrode tip on the
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ganglion sheath. Penetration of T cells was more difficult to achieve because of their small size and only a few satisfactory impalements were obtained throughout the course of the study. The electrical characteristics of the sensory cells in H. sanguisuga were similar to those described for H. medicinalis (Nicholls and Baylor, 1968) and can be seen in Figure 3. In all cells, the downstroke of the action potential reached a more negative value than the resting membrane potential (a positive phase), which was greater in N and P cells than in T cells. T cells fired in bursts and were silent unless stimulated, while N cells invariably showed spontaneous activity and fired every few seconds. Such activity was seen with or without the ring in position. P cells could either be silent or show occasional spontaneous firing. A high-frequency burst of spontaneous activity sometimes occurred in N and P cells immediately on penetration by the microelectrode. A spontaneously active cell could be recognized by its action potentials; silent cells were identified by injecting a 30 ms, l-3 nA depolarizing current pulse to elicit firing. The receptive fields of P and N cells in H. sanguisuga, when mapped by mechanical stimulation, were similar to those reported for H. medicinalis (Nicholls and Baylor, 1968). The ventral (yellow) skin was innervated by the lateral cells, whereas the medial cells innervated the dorsal (black) skin. In the present study, fewer T cells than other types of sensory cells were impaled, and an extensive survery of receptive fields was not possible. However, the receptive fields of the T cells examined corresponded with the findings of Nicholls and Baylor (1968) in H. medicinalis.
T cells responded when a stimulus of 0.1 mN was applied to the skin. P cells responded to a stimulus as low as 1 mN, while a response in N cells could be elicited with a stimulus as low as 12.5 mN. N cells frequently continued firing after termination of a stimulus, particularly when the higher stimulus strengths were used;
N
.
FIGURE 3. Characteristic action potentials induced in N, P, and 1 cells by mechanical stimulation of the skin. b indicates zero potential.
Irritants and Leech Skin Afferents
FIGURE 4. Effect of applying CR (10 mM + ethanol 10%) to the skin of the leech on spontaneous activity in an N cell and its vehicle control. A. Resting spontaneous activity. B. Spontaneous activity 4 min after application of CR 10 mM + ethanol 10% to the skin. Two parts of a continuous recording. C. Resting spontaneous activity. D. Spontaneous activity 4 min after application of ethanol 10% to the skin. Two parts of a continuous recording. ä indicates zero potential.
such an effect was very occasionally seen in P cells. For this reason, in experiments employing CR, the response was measured as the number of action potentials occurring in the 10 set starting with application of the stimulus to the skin. Effect of Ring and Glue on Responses to Mechanical
Stimulation
In ganglion/body wall preparations with no ring attached, mechanical stimulation of the skin resulted in an observable reflex contraction of the body wall in a longitudinal direction, and the annuli became raised. This reflex could not be seen in the body wall enclosed within a ring, possibly because the ring acted as a splint. Responses to mechanical stimulation (0.1 mN for T cells, 50 mN for P cells, and 100 mN for N cells) were obtained before and after gluing a ring in position on the skin. There was no significant difference in either the rate of firing or the amplitude
293
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K. M. Weston et al. TABLE 1 Responses to Mechanical a Ring to the Skin Surface No.
OF ACTION
STIMULATION
POTENTIALS
CELL
BEFORE
T P N
16.8 2 2.4 10.3 t 1.8 2.2 2 0.4
of
DURING
c. 1 SEC)
(DURATION
(mean f
Stimulation of the Skin before and after Application
AMPLITUDE
OF ACTION
POTENTIAL
(mV)
(mean 2 SEMjb
SEM)” AFTER
BEFORE
16.0 t 2.3 11.5 -c 1.6 3.6 -t-1.2
48.8 2 6.3 93.8 rf-3.1 92.0 2 5.1
AFTER
36.3 k 4.3 87.5 2 7.2 87.0 k 6.6
n
4 4 5
=/J = 0.6 (AV); b p = 0.13 (AV)
of the action potential in any type of sensory cell when the responses before and after application of the ring were compared (Table 1). Effects of Dibenzoxazepine
on Electrical Activity in Sensory Neurones
In the presence of CR (IO mM + ethanol IO%), there was no significant difference in action potential size or the response to mechanical stimulation between test and
0'
2
4
6
8
10 Interspike
12 interval (~1
FIGURE 5. Cumulative frequency distribution for interspike interval showing effect of A. CR (10 mM + ethanol 10%) on spontaneous activity in an N cell that developed a “bursting” firing pattern after CR administration. B. A concurrent vehicle control (ethanol 10%). 0 Before CR or vehicle application; 0 After CR or vehicle application.
Irritants and Leech Skin Afferents
control (ethanol 10%) groups before and after drug or vehicle exposure in any type of cell (AV, p > 0.12; n = 5-9 P and N cells, 2-3 T cells). However, an incidental finding in this series was a change in the spontaneous activity of N cells some 2-3 min after application of CR (IO mM) to the skin. The single spike activity changed to a repetitive pattern or “bursting” with groups of high-frequency action potentials separated by periods of quiescence. In many cells, this change in pattern resulted in an overall increase in mean firing rate, but in others there was no increase since the interspike interval between bursts was longer than that in the resting state. The pattern was sustained until electrode withdrawal, and the second IOO-mN stimulus still evoked a response. The effects of CR (IO mM) and of the ethanol vehicle in N cells are shown in Figure 4. No significant change in the firing pattern occurred in any of the seven cells in the control group (KS-test, p > 0.051, but a significant change occurred in eight out of the nine cells exposed to CR (KS-test, p < 0.05). The high frequency of interspike intervals of O-l set indicated that in each cell the change was manifested as a bursting pattern. Typical cumulative curves for a cell under test where a bursting pattern developed and for a control cell where no significant change in firing pattern occurred are shown in Figure 5. Analysis of the incidence of change in firing pattern (i.e., eight out of nine test cells and zero out of seven control cells) showed that there was a significant difference between test and control groups (Fisher’s exact method, p = 0.001). DISCUSSION In the course of the present investigation into the effects of chemical irritants on the leech afferent nervous system, much preliminary work had to be carried out. No reports of the selective application of chemicals to the skin of the leech have appeared and problems were encountered in designing a method by which this could be achieved. The method finally adopted, gluing a polythene ring to the skin, was highly successful and although the seal was always tested at the end of the experiment, in fact no failures occurred. The ring itself did not affect the responses of sensory cells to mechanical stimulation of the skin. Capsaicin and other chemical irritants modify the responses to mechanical stimulation in other systems in different ways. Unchanged sensitivity, increased pressure thresholds and abolition of mechanically induced responses have all been reported (Jancso, “1958; Jancs6 and Jancso-Giber 1959; Porszlisz and jancsd, 1959; Green and Tregear, 1964; Hayes and Tyers, 1980; Foster and Ramage, 1981). For this reason, the effects of chemical irritation on the responses of the cells to mechanical stimulation were investigated during the present study. It was originally intended to obtain mechanical stimulation/effect curves before and after application of an irritant to the skin, and in pilot experiments several stimuiation intensities were therefore applied when impaling P and N cells (the use of von Frey hairs was too crude a method for producing stimulation/effect curves in T cells). This intention eventually proved to be too ambitious since the number of completely successful experiments was low, the electrode often becoming dislodged from the cell on application of a stimulus to the skin. In subsequent inves-
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tigations, therefore, only a single stimulus was applied before and after drug administration. The burst of action potentials seen immediately on impalement of N and P cells was most likely due to ionic changes occurring in the cell before sealing of the cell membrane around the electrode. Few authors have referred to the spontaneous activity in P and N cells, although Nicholls and Baylor (1968) make a passing reference to such activity in H. medicinalis, linking it with penetration of the cell body. The authors reported that the spontaneous activity disappeared eventually but no indication of the time-scale is given. In impalements in the present study that lasted approximately IO min, the spontaneous activity in P and N cells continued throughout the course of the impalement. it is possible that spontaneous firing in these cells is a physiological event and that its disappearance (Nicholls and Baylor, 1968) indicates a measure of damage or that a species’ difference exists. In the present investigation, it was unlikely to have been caused by the ring glued to the skin since spontaneous activity was observed in preparations with no ring attached. A simple behavioral experiment with live, intact leeches early in the study had shown that animals responded to concentrations of CR as low as 1 P,M with an avoidance reaction-a shortening reflex and writhing. A systematic preliminary study of the effects of CR applied to the skin on the sensory cells of the leech was therefore carried out. No consistent response was seen until a concentration of 10 mM was applied, when N cells responded. The high concentration of CR required to elicit a response in N cells does not correlate with its activity in other systems. CR (IO PM) was effective in altering the membrane properties of the giant amoeba (Foster, et al., 1981), polymodal nociceptors and warm thermoreceptors in the cat responded to a concentration of 500 ~_LM(Foster and Ramage, l981), while 1 PM and 100 FM produced blepharospasm in man and the guinea pig, respectively (Ballantyne and Swanston, 1974; Foster and Weston, unpublished observations). However, this lack of correlation may be a reflection of the barrier presented by the cuticle of the leech to drug penetration. The present study has demonstrated that chemicals can be selectively applied to known areas of skin without allowing contact with neighboring synapses or nerve cell bodies. Flexible mounting of microelectrodes allowed long-term intracellular recordings to be made in spite of considerable body-wall movement following tactile stimulation. The assessment of sensory neurone responses before and after skin exposure to chemical irritants and the effects of such irritants on responses to mechanical stimulation of the skin could be made with a single microelectrode impalement.
REFERENCES Ballantyne 6, Swanston DW (1974) The irritant effects of dilute solutions of dibenzoxazepine (CR) on the eye and tongue. Acta phar~aco~ toxicof 35:412-423. Coggeshall RE, Fawcett DW (1964) The fine struc-
ture of the central nervous system of the leech, Hirudo medicinalis. j Neurophysiol27:229-289. E’tsner DA,
Lederer WJ, Spindler AJ (1979) Thick slurry bevelling of microelectrodes. Proc Physiol Sot July 1979, unpublished.
Irritants and leech Skin Afferents Foster RW, Ramage AG (1981) The action of some chemical irritants on somatosensory receptors of the cat. Neuropharmaco/20:191-198. Foster RW, Weston AH, Weston KM (1981) Some effects of chemical irritants on the membrane of the giant amoeba. Br) Pharmaco/74:333-339. Green DM, Tregear RT (1964) The action of sensory irritants on the cat’s cornea.) Physio/175:37-38P. Hayes AC, Tyers MB (1980) Effects of capsaicin on nociceptive heat, pressure and chemical thresholds and on substance P levels in the rat. Brain Res 189:561-564. Jancso N (1958) Die Desensibilisierung des Organismus gegenuber reizenden und entzundungserreizenden chemischen Wirkungen. Arch Physiol Hung 12 (suppl):lS.
Jancs6 N, Jancso-Gabor A (1959) Dauerausschaltung der chemischen Schmerzempfindlichkeit durch Capsaicin. Arch exp Path Pharmak 236:142-145. Lindgren BW (1960) Statistical Theory. New York: Macmillan. Nicholls JC, Baylor DA (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. / Neurophysio/31:740-756. Nicholls JC, Van Essen D (1974) The nervous system of the leech. Scientific Am 230:38-48. Porszasz J, Jancso N (1959) Studies on the action potentials of sensory nerves in animals desensitised with capsaicine. Acta Physiol Hung 16:299-306.
297
KATHLEENM. WESTON, R. W. FOSTER,ANDA. H. WESTON
A technique is described for applying chemical irritants selectively to the skin of a superfused ganglion/body wall preparation of the horse leech, Haemopis sanguisuga. Details are given of the intracellular recording from sensory neurones of the effects of the irritant dibenzoxazepine and of mechanical stimulation. Dibenzoxazepine produced changes in the spontaneous firing pattern in the nociceptive cells of the leech but did not affect the response to mechanical stimulation of the skin in any type of sensory cell. It is possible that the system described could be used as a model of cutaneous excitation evoked by drugs or mechanical stimulation in higher animals. Key Words:
Leech; Haemopis
sanguisuga; Irritants
INTRODUCTION Parallels have been drawn between the afferent neurones identified in the leech and those innervating human skin (Nicholls and Van Essen, 1974). The central nervous system of the leech consists of a chain of discrete segmental ganglia enclosed within a blood sinus and, apart from the fused ganglia at the head and tail, all are virtually identical (Coggeshall and Fawcett, 19641. Apart from the suckers at the extremities, the animal is made up of identical segments, each segment consisting of several annuli supplied by one ganglion. A ganglion consists of a connective tissue sheath enclosing about 350 unipolar neurones that are segregated into discrete groups or packets, together with several supporting glial cells (Coggeshall and Fawcett, 1964). Adjacent ganglia are connected by bundles of unmyelinated nerve fibers, the connectives. An anterior and posterior root, also containing unmyelinated axons, connects each side of the ganglion with the body wall and underlying viscera. The essential features in the horse leech (Haemopis sanguisuga) are the same as those described in the medicinal leech (Hirudo medicinalis) (Nicholls and Van Essen, 1974). Of the 350 nerve cell bodies in any one ganglion, seven pairs of sensory cells have been identified, all responding to mechanical stimulation of the skin. On either side of the ganglion are three touch (T), two pressure (P) and two nociceptive (N) cells responding to light touch, pressure, and noxious mechanical stimuli, respecFrom the Department of Pharmacology, Materia Medica and Therapeutics, University of Manchester, U.K. Address reprint requests to R. W. Foster, Department of Pharmacology, Materia Medica and fherapeutics, University of Manchester, Manchester Ml3 YPT, U.K. Received September 30,1983; accepted April 1984.
285 Journal of Pharmacological
Methods
D 1984 Elsevier Science Publishing
12, 285-297
(19%)
Co., Inc., 52 Vanderbilt
0160-5402184603.00 Avenue, New York, NY 10017
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K. M. Weston et al.
tively (Nicholls and Baylor, 1968). Each cell occupies a fixed position in the ganglion by which it can be identified. In addition, the electrical characteristics of each type of cell differ and provide a further aid to identification. The sensory cells of the leech can be driven electrically or by mechanical stimulation of the skin, and the resulting electrical activity can be observed by use of intracellular microelectrodes. The size and shape of the action potential, its duration, and the magnitude of the positive phase all aid in the identification of the cell impaled (Nicholls and Baylor, 1968). An additional guide can be obtained from the size of the stimulus that must be applied to the skin to stimulate each type of cell. T cells fire in response to light touch or even to eddies in the bathing fluid, whereas much larger stimuli are required to activate P and N cells (Nicholls and Baylor, 1968). Each of the sensory cells has a particular region of the skin from which it can be stimulated-its receptive field. The receptive fields in the medicinal leech, H. medicinalis, have been mapped and, apart from a slight overlap, have been found to be specific for each cell of any one type (Nicholls and Baylor, 1968). Conflicting reports have appeared in the literature on the effects of capsaicin and other irritants on the responses to mechanical stimulation in avariety of mammalian systems (Jancso, 1958; Jancso and jancso-Gabor, 1959; Porszasz and Jancso, 1959; Green and Tregear, 1964; Hayes and Tyers, 1980; Foster and Ramage, 1981). The method described was evolved for the selective application of irritant chemicals to the skin of the leech and the measurement of the effects of such applications on the responses of sensory cells to mechanical stimulation. METHODS The Dissection
of the Leech/Ganglion
Body Wall Preparation
Horse leeches that ranged in length from S-10 cm when fully extended were used. Before experiments, animals were maintained at 4°C in distilled water containing 4% leech Ringer solution. For dissection, a leech was fully extended and pinned through the head and tail, ventral side uppermost, onto a hard paraffin block. An incision was made down the ventral midline to reveal the ventral blood sinus. The edges of the skin were pinned back and a ganglion was selected. Ganglia in the anterior region that served the genitalia were avoided. The remainder of the dissection was carried out using a binocular microscope. An incision was made in the wall of the ventral blood sinus to reveal the ganglion beneath. The nerve roots on one side of the ganglion were exposed and a l-mm sliver of the medial body wall on that side was removed to expose the roots further. The nerve roots on the opposite side of the ganglion were exposed and severed as closely as possible to the body wall and the connectives were cut as closely as possible to the adjacent ganglia. The severed roots and connectives were then pulled free from the blood sinus. To isolate the segment of body wall served by the ganglion, two transverse cuts were made, several annuli apart, on the side of the animal with intact innervation. Starting from the ventral midline, these cuts continued around the body wall to the lateral line on the opposite side. A longitudinal cut was then made between the
irritants and Leech Skin Afferents two transverse cuts along the lateral line. The body wall was dissected carefully from the viscera, and the inside surface of the longitudinal muscle layer was cleared of loose tissue. Throughout the dissection, the preparation was pinned as appropriate with small stainless steel pins comprising l-cm lengths of 0.15-mm diameter wire (dental grade, K.C. Smith). The preparation was bathed in leech Ringer solution that was changed frequently and care was taken to avoid injuring the nerves or bursting the gut. The ganglion/body wall preparation was removed to a perspex bath containing leech Ringer solution. The flap of body wall extended from the ventral midline around to the lateral line of the opposite side and contained about 12 annuli. The flap was larger than the segment of body wall served by the ganglion. Further preparations could be obtained throughout the day from the same leech, which was maintained at 4°C between dissections. The base of the perspex bath (maximum volume = 5ml) was coated in a layer of Sylgard 184 resin (Dow Corning) that had been allowed to cure for 2 to 3 days at room temperature. The body wall was pinned to the Sylgard, skin side uppermost and moderately stretched, with small stainless steel pins. The ganglion was pulled clear of the body wall, stretching the roots moderately, and was pinned ventral side uppermost by means of small tungsten hoops over the connectives and severed roots. Care was taken to avoid twisting the intact roots. Pinning these roots was avoided, for it appeared to cause nerve damage and to overstretch the ganglion, making impalement difficult. Tungsten Hoops Tungsten wire (0.2 mm diameter, Goodfellow Metals) was positioned as the anode in a simple electrolysis bath containing 10% KOH solution. Current (12 V d.c. source) was passed through the bath until the diameter of the wire was reduced to approximately0.05 mm. Al-2-mm length of wire was then bent into a hoop. By holding the tips of the hoop in the electrolysis bath and passing current, they became pointed. Perfusion
System
All experiments were carried out at room temperature (17-24°C). Leech Ringer solution had the following composition (mM): Na+ 115 (plus approximately 8 from the buffer solution); K’ 4.0; Cazl I .8; Cl- 122.6; glucose IO; morpholinopropane sulphonic acid (MOPS, Calbiochemf 10; pH 7.4. The leech Ringer solution bathing the preparation in the perspex bath was continuously exchanged by perfusion (Figure 1) at a flow rate of 2-3 ml/min, and the fluid in the bath (approximately 1 ml, 1 mm deep) just covered the preparation. It was found that if the depth of immersion were any greater the image of the ganglion seen through the binocular microscope was distorted and impalement of nerve cells was difficult. intracellular
Recording
System
A single microelectrode, in conjunction with a high-impedence probe and preamplifier (Mentor N-950) was used to record transmembrane potential. The indifferent electrode was a sintered silver/silver chloride/platinum black disc (Mentor N-9501).
287
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K. M. Weston et al.
Dreschel
bottlefv-l
FIGURE 1.
Perfusion system used in the present study.
The output from the preamplifier was displayed on an oscilloscope (Tektronix) and on a polygraph (Crass Instruments). The output from the polygraph was connected to one channel of a tape recorder (Racaf Store 4) and signals were stored on tape (3M 207) at 3.75 inches per second. Silent ceils were identified immediately after impalement by stimulating them via the recording microelectrode. The stimulus artefact, dependent on the resistance of the microelectrode plus bathing fluid, was first balanced out. To accomplish this, a Grass S48 stimulator was used to deliver a 0.5-V pulse to the Mentor N-950 preamplifier, which converted it to a I-nA pulse before delivery to the cell. This pulse was far too brief (1 ms) to charge the membrane capacitance and the effective resistance of the cell membrane plus cytoplasm was thus negligible. The pulse was used to trigger the oscilloscope sweep and by balancing the bridge of the Mentor N-950, the deflection on the oscilloscope trace could be nulled, indicating that the effect of the resistance of the electrode plus bathing fluid had been balanced out. After balancing, a 3O-ms 0.5-1.5-V pulse was delivered from a second stimulator (Grass S48) to the preamplifier, the conversion resulting in a I-3-nA pulse being delivered to the cell. This elicited the firing necessary to aid cell identification. Standing potentials developed between the indifferent electrode and the probe input as a result of electrochemical action were cancelled before impalement by means of the bucking voltage facility on the Mentor N-950. In order to reduce the attenuation of high-frequency components of the input signal, the capacitance compensation facility on the Mentor N-950 was used to optimize the frequency response of the system. Eiec~ro~es Self-filling glass microelectrodes (Clark Electromedicalf with a blank outer diameter of 1 mm were used. Filtered (Millipore) KCI solution (3 M) was used as the
irritants and leech Skin Afferents electrolyte. Electrodes were mounted flexibly on a siiverfsilver chforide wire. The electrode was positioned using a micromanipu~ator (Leitzf and a binocular microscope (Olympus) with zoom magnification up to 80X and a dark-field attachment (Kyowa) . Electrodes were pulled on a vertical electrode puller (Narishige PE-2). They were then bevelled on an electrode beveller (WPI Model 1200) at an angle of 25” in a slurry of 20% w/v polishing alumina (0.05 pm, Banner Scientific) in KCI solution (3 M) (Eisner, et al., 1979). The electrode was connected to an ohm-meter (WPI Model F29) that generated the square wave and gave a digital read-out for the measurement of electrode resistance during bevelling. Initially, the resistance of an electrode in the KCI slurry was > 15 MfZ. After bevelling, electrodes had resistances of 6-10 Ma, higher values being used in later work to improve the stability of recordings. Electrodes had tip potentials of less than I mV. Tip potential was measured in a sample of electrodes by noting the change in potential difference between the microelectrode and indifferent electrode when the tip of the microelectrode was removed. Selective Application
of Chemical
Irritants to Primary Afferent
Nerve Endings
A method of applying chemical irritants selectively to the skin surface without allowing contact with the nerve cell body or synapses was evolved. A ring of polythene with one perfectly smooth edge was made by cutting the wide end from a disposable pipette tip (Hughes and Hughes). The ring had an internal diameter of 0.7 cm and was 0.3 cm high. The smooth edge was piped with a thin layer of cyanoacrylic glue (Loctite Superglue31, and the ring was immediately glued on to blotted, pinned-out leech skin. Gentle pressure was applied downwards on the ring for about IO sec. The ganglion and underside of the body wall could be perfused with leech Ringer solution while a solution of the chemical irritant being tested could be introduced into the ring. The height af the ring was sufficient to ensure that no mixing of the two solutions occurred. The size of the ring was such that a large part of the receptive field innervated by each sensory cell was enclosed. The flap of body wall excised was made sufficiently large to ensure that the walls of the ring did not obscure any more of the receptive fields than necessary. A diagram of the preparation is shown in Figure 2. Care was needed to use only the rnjn~rnurn amount of glue necessary for adhesion, otherwise the excess covered either part of the skin inside the ring or the ganglion outside. If the edge of the ring was placed too near the ventral edge of the body wall, the surrounding meniscus obscured the ganglion and made impaiement of sensory cells almost impossible. Care had to be taken in positioning the ring to prevent this without covering any more of the ventral receptive field than necessary, In order to ensure that an experiment had been conducted with an adequately sealed ring, eosin dye solution was introduced into the ring at the end of each experiment. If any leakage occurred, the experimental results were discarded. Each ring was used once only.
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K. M. Weston et al. Solution
Dorsal
of irritant
midline
Ventral midline
Segmental ganglion
FIGURE 2. Arrangement for the selective application of chemical irritant solutions to the skin of the leech (not to scale). The shaded area shows the black (dorsal) skin and the unshaded area the yellow (ventral) skin.
Mechanical
Stimulation
of the Skin
Von Frey i-lairs A series of von Frey hairs was made consisting of varying lengths of stainless steel wire or nylon bristle attached to a wooden handle. The von Frey hairs were calibrated on an analytical balance and delivered stimuli between 0.1 and 200 mN. For delivery of a stimulus, the von Frey hair was lowered vertically on to the body wall of the preparation until the first visible bending of the hair occurred. The hair was maintained in this state for about 1 set, after which the stimulus was removed. Characteristics of Sensory Cells and Receptive Fields In a series of preliminary experiments, the characteristics of action potentials elicited in N, P, and T cells were observed. Cells were driven either by electrical stimulation using a 30 ms depolarizing pulse with an amplitude of l-3 nA or by mechanical stimulation of the skin using a series of von Frey hairs ranging from 0.1 to 200 mN. By use of this method, a limited study of the receptive fields innervated by each type of cell was performed. Effect of the Ring Glued to the Skin on Responses to mechanical Sti~ui~tion A ganglion/body wall preparation was perfused with modified leech Ringer solution. As many sensory cells as possible were impaled in sequence, allowing at least 5 min between impalements. Immediately after penetration and stabilization of the membrane potential, a response to mechanical stimulation was elicited using a von Frey hair (0.1 mN for T cells, 50 mN for P cells, and 100 mN for N cells). These values were selected from preliminary experiments. The electrical activity of each cell during stimulation was recorded. The perspex bath was then removed from the perfusion system and drained. The skin of the leech was blotted, and a ring was glued in position. The bath was then
Irritants and leech Skin Afferents
returned to the system and perfused with modified leech Ringer solution. Modified leech Ringer solution (0.1 ml) was placed inside the ring to moisten the skin. The sensory cells were impaled in sequence as before, and the responses to mechanical stimulation were again elicited. Effect of Dibenzoxazepine Mechanical Stimulation
Applied
Selectively to the Skin on the Responses to
The chemical irritant dibenzoxazepine (CR) was kindly supplied by the Chemical Defence Establishment, Porton Down. A ganglion/body wall preparation with the ring glued in position was perfused with leech Ringer solution. Leech Ringer solution (0.1 ml) was introduced into the ring to keep the skin moist. With a sensory cell impaled, the skin was stimulated with a von Frey hair (0.1 mN for T cells, 50 mN for P cells, or 100 mN for N cells) 1 min before addition of 0.1 ml CR 20 mM in 20% ethanol to the ring. Five minutes later, the mechanical stimulation was repeated. Concurrent control experiments were performed to test the effect of the ethanol vehicle alone. Statistical Methods For comparison of several variables arising in one experimental series, data were assessed using an analysis of variance (AV). Since measurement of the mean firing rate of N cells alone before and after application of CR did not fully summarize the changes seen, a more elaborate method of comparison was used. The firing patterns for 1 min immediately before the first and second mechanical stimulations of the skin were compared in each cell in test and control groups. Each interspike interval was measured, and a cumulative frequency distribution of the interspike interval (bins of 1 set) was constructed for each cell during each of the I-min intervals outlined above. The greatest degree of separation between the two distributions was measured, and the deviation was estimated using the Kolmogorov-Smirnov test for goodness of fit (KS-test) (Lindgren, 1960). The incidence of change in firing pattern in test and control groups was used to construct 2 x 2 contingency tables, and the significance of the difference between the two groups was estimated (Fisher’s exact method). RESULTS Characteristics
of Sensory Cells and their Receptive Fields
When the ventral surface of a segmental ganglion of H. sanguisuga was viewed through the microscope, the two Retzius cells that served as landmarks in the ganglion could easily be seen. With some preparations, it was difficult to distinguish the various sensory cells and adjustment of the illumination was found to be a critical factor. The positions of the sensory cells in H. sanguisuga were essentially the same as those described for H. medicinalis (Nicholls and Baylor, 1968). Two pairs of P and N cells were clearly recognizable. These cells were comparatively easy to impale although they often moved sideways under pressure of the electrode tip on the
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K. M. Weston et al.
ganglion sheath. Penetration of T cells was more difficult to achieve because of their small size and only a few satisfactory impalements were obtained throughout the course of the study. The electrical characteristics of the sensory cells in H. sanguisuga were similar to those described for H. medicinalis (Nicholls and Baylor, 1968) and can be seen in Figure 3. In all cells, the downstroke of the action potential reached a more negative value than the resting membrane potential (a positive phase), which was greater in N and P cells than in T cells. T cells fired in bursts and were silent unless stimulated, while N cells invariably showed spontaneous activity and fired every few seconds. Such activity was seen with or without the ring in position. P cells could either be silent or show occasional spontaneous firing. A high-frequency burst of spontaneous activity sometimes occurred in N and P cells immediately on penetration by the microelectrode. A spontaneously active cell could be recognized by its action potentials; silent cells were identified by injecting a 30 ms, l-3 nA depolarizing current pulse to elicit firing. The receptive fields of P and N cells in H. sanguisuga, when mapped by mechanical stimulation, were similar to those reported for H. medicinalis (Nicholls and Baylor, 1968). The ventral (yellow) skin was innervated by the lateral cells, whereas the medial cells innervated the dorsal (black) skin. In the present study, fewer T cells than other types of sensory cells were impaled, and an extensive survery of receptive fields was not possible. However, the receptive fields of the T cells examined corresponded with the findings of Nicholls and Baylor (1968) in H. medicinalis.
T cells responded when a stimulus of 0.1 mN was applied to the skin. P cells responded to a stimulus as low as 1 mN, while a response in N cells could be elicited with a stimulus as low as 12.5 mN. N cells frequently continued firing after termination of a stimulus, particularly when the higher stimulus strengths were used;
N
.
FIGURE 3. Characteristic action potentials induced in N, P, and 1 cells by mechanical stimulation of the skin. b indicates zero potential.
Irritants and Leech Skin Afferents
FIGURE 4. Effect of applying CR (10 mM + ethanol 10%) to the skin of the leech on spontaneous activity in an N cell and its vehicle control. A. Resting spontaneous activity. B. Spontaneous activity 4 min after application of CR 10 mM + ethanol 10% to the skin. Two parts of a continuous recording. C. Resting spontaneous activity. D. Spontaneous activity 4 min after application of ethanol 10% to the skin. Two parts of a continuous recording. ä indicates zero potential.
such an effect was very occasionally seen in P cells. For this reason, in experiments employing CR, the response was measured as the number of action potentials occurring in the 10 set starting with application of the stimulus to the skin. Effect of Ring and Glue on Responses to Mechanical
Stimulation
In ganglion/body wall preparations with no ring attached, mechanical stimulation of the skin resulted in an observable reflex contraction of the body wall in a longitudinal direction, and the annuli became raised. This reflex could not be seen in the body wall enclosed within a ring, possibly because the ring acted as a splint. Responses to mechanical stimulation (0.1 mN for T cells, 50 mN for P cells, and 100 mN for N cells) were obtained before and after gluing a ring in position on the skin. There was no significant difference in either the rate of firing or the amplitude
293
294
K. M. Weston et al. TABLE 1 Responses to Mechanical a Ring to the Skin Surface No.
OF ACTION
STIMULATION
POTENTIALS
CELL
BEFORE
T P N
16.8 2 2.4 10.3 t 1.8 2.2 2 0.4
of
DURING
c. 1 SEC)
(DURATION
(mean f
Stimulation of the Skin before and after Application
AMPLITUDE
OF ACTION
POTENTIAL
(mV)
(mean 2 SEMjb
SEM)” AFTER
BEFORE
16.0 t 2.3 11.5 -c 1.6 3.6 -t-1.2
48.8 2 6.3 93.8 rf-3.1 92.0 2 5.1
AFTER
36.3 k 4.3 87.5 2 7.2 87.0 k 6.6
n
4 4 5
=/J = 0.6 (AV); b p = 0.13 (AV)
of the action potential in any type of sensory cell when the responses before and after application of the ring were compared (Table 1). Effects of Dibenzoxazepine
on Electrical Activity in Sensory Neurones
In the presence of CR (IO mM + ethanol IO%), there was no significant difference in action potential size or the response to mechanical stimulation between test and
0'
2
4
6
8
10 Interspike
12 interval (~1
FIGURE 5. Cumulative frequency distribution for interspike interval showing effect of A. CR (10 mM + ethanol 10%) on spontaneous activity in an N cell that developed a “bursting” firing pattern after CR administration. B. A concurrent vehicle control (ethanol 10%). 0 Before CR or vehicle application; 0 After CR or vehicle application.
Irritants and Leech Skin Afferents
control (ethanol 10%) groups before and after drug or vehicle exposure in any type of cell (AV, p > 0.12; n = 5-9 P and N cells, 2-3 T cells). However, an incidental finding in this series was a change in the spontaneous activity of N cells some 2-3 min after application of CR (IO mM) to the skin. The single spike activity changed to a repetitive pattern or “bursting” with groups of high-frequency action potentials separated by periods of quiescence. In many cells, this change in pattern resulted in an overall increase in mean firing rate, but in others there was no increase since the interspike interval between bursts was longer than that in the resting state. The pattern was sustained until electrode withdrawal, and the second IOO-mN stimulus still evoked a response. The effects of CR (IO mM) and of the ethanol vehicle in N cells are shown in Figure 4. No significant change in the firing pattern occurred in any of the seven cells in the control group (KS-test, p > 0.051, but a significant change occurred in eight out of the nine cells exposed to CR (KS-test, p < 0.05). The high frequency of interspike intervals of O-l set indicated that in each cell the change was manifested as a bursting pattern. Typical cumulative curves for a cell under test where a bursting pattern developed and for a control cell where no significant change in firing pattern occurred are shown in Figure 5. Analysis of the incidence of change in firing pattern (i.e., eight out of nine test cells and zero out of seven control cells) showed that there was a significant difference between test and control groups (Fisher’s exact method, p = 0.001). DISCUSSION In the course of the present investigation into the effects of chemical irritants on the leech afferent nervous system, much preliminary work had to be carried out. No reports of the selective application of chemicals to the skin of the leech have appeared and problems were encountered in designing a method by which this could be achieved. The method finally adopted, gluing a polythene ring to the skin, was highly successful and although the seal was always tested at the end of the experiment, in fact no failures occurred. The ring itself did not affect the responses of sensory cells to mechanical stimulation of the skin. Capsaicin and other chemical irritants modify the responses to mechanical stimulation in other systems in different ways. Unchanged sensitivity, increased pressure thresholds and abolition of mechanically induced responses have all been reported (Jancso, “1958; Jancs6 and Jancso-Giber 1959; Porszlisz and jancsd, 1959; Green and Tregear, 1964; Hayes and Tyers, 1980; Foster and Ramage, 1981). For this reason, the effects of chemical irritation on the responses of the cells to mechanical stimulation were investigated during the present study. It was originally intended to obtain mechanical stimulation/effect curves before and after application of an irritant to the skin, and in pilot experiments several stimuiation intensities were therefore applied when impaling P and N cells (the use of von Frey hairs was too crude a method for producing stimulation/effect curves in T cells). This intention eventually proved to be too ambitious since the number of completely successful experiments was low, the electrode often becoming dislodged from the cell on application of a stimulus to the skin. In subsequent inves-
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K. M. Weston et al.
tigations, therefore, only a single stimulus was applied before and after drug administration. The burst of action potentials seen immediately on impalement of N and P cells was most likely due to ionic changes occurring in the cell before sealing of the cell membrane around the electrode. Few authors have referred to the spontaneous activity in P and N cells, although Nicholls and Baylor (1968) make a passing reference to such activity in H. medicinalis, linking it with penetration of the cell body. The authors reported that the spontaneous activity disappeared eventually but no indication of the time-scale is given. In impalements in the present study that lasted approximately IO min, the spontaneous activity in P and N cells continued throughout the course of the impalement. it is possible that spontaneous firing in these cells is a physiological event and that its disappearance (Nicholls and Baylor, 1968) indicates a measure of damage or that a species’ difference exists. In the present investigation, it was unlikely to have been caused by the ring glued to the skin since spontaneous activity was observed in preparations with no ring attached. A simple behavioral experiment with live, intact leeches early in the study had shown that animals responded to concentrations of CR as low as 1 P,M with an avoidance reaction-a shortening reflex and writhing. A systematic preliminary study of the effects of CR applied to the skin on the sensory cells of the leech was therefore carried out. No consistent response was seen until a concentration of 10 mM was applied, when N cells responded. The high concentration of CR required to elicit a response in N cells does not correlate with its activity in other systems. CR (IO PM) was effective in altering the membrane properties of the giant amoeba (Foster, et al., 1981), polymodal nociceptors and warm thermoreceptors in the cat responded to a concentration of 500 ~_LM(Foster and Ramage, l981), while 1 PM and 100 FM produced blepharospasm in man and the guinea pig, respectively (Ballantyne and Swanston, 1974; Foster and Weston, unpublished observations). However, this lack of correlation may be a reflection of the barrier presented by the cuticle of the leech to drug penetration. The present study has demonstrated that chemicals can be selectively applied to known areas of skin without allowing contact with neighboring synapses or nerve cell bodies. Flexible mounting of microelectrodes allowed long-term intracellular recordings to be made in spite of considerable body-wall movement following tactile stimulation. The assessment of sensory neurone responses before and after skin exposure to chemical irritants and the effects of such irritants on responses to mechanical stimulation of the skin could be made with a single microelectrode impalement.
REFERENCES Ballantyne 6, Swanston DW (1974) The irritant effects of dilute solutions of dibenzoxazepine (CR) on the eye and tongue. Acta phar~aco~ toxicof 35:412-423. Coggeshall RE, Fawcett DW (1964) The fine struc-
ture of the central nervous system of the leech, Hirudo medicinalis. j Neurophysiol27:229-289. E’tsner DA,
Lederer WJ, Spindler AJ (1979) Thick slurry bevelling of microelectrodes. Proc Physiol Sot July 1979, unpublished.
Irritants and leech Skin Afferents Foster RW, Ramage AG (1981) The action of some chemical irritants on somatosensory receptors of the cat. Neuropharmaco/20:191-198. Foster RW, Weston AH, Weston KM (1981) Some effects of chemical irritants on the membrane of the giant amoeba. Br) Pharmaco/74:333-339. Green DM, Tregear RT (1964) The action of sensory irritants on the cat’s cornea.) Physio/175:37-38P. Hayes AC, Tyers MB (1980) Effects of capsaicin on nociceptive heat, pressure and chemical thresholds and on substance P levels in the rat. Brain Res 189:561-564. Jancso N (1958) Die Desensibilisierung des Organismus gegenuber reizenden und entzundungserreizenden chemischen Wirkungen. Arch Physiol Hung 12 (suppl):lS.
Jancs6 N, Jancso-Gabor A (1959) Dauerausschaltung der chemischen Schmerzempfindlichkeit durch Capsaicin. Arch exp Path Pharmak 236:142-145. Lindgren BW (1960) Statistical Theory. New York: Macmillan. Nicholls JC, Baylor DA (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. / Neurophysio/31:740-756. Nicholls JC, Van Essen D (1974) The nervous system of the leech. Scientific Am 230:38-48. Porszasz J, Jancso N (1959) Studies on the action potentials of sensory nerves in animals desensitised with capsaicine. Acta Physiol Hung 16:299-306.
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