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Wiping reflexes and nerve impulse patterns evoked by electrical stimulation of the skin in frogs
Physiology & Behavior, Vol. 21, pp. 789-792. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
Wiping Reflexes and Nerve Impulse Patterns Evoked by Electrical Stimulation of the Skin in Frogs M. A. C O R N E R A N D R. E. B A K E R
Netherlands Institute for Brain Research, IJdijk 28, Amsterdam-O, The Netherlands (Received 14 April 1978) CORNER, M. A. AND R. E. BAKER. Wiping reflexes and nerve impulse patterns evoked by electrical stimulation of the skin in frogs. PHYSIOL. BEHAV. 21(5) 789-792, 1978.--Electric shocks applied to the skin evoke well aimed wiping reflexes in the frogs, Discoglossus pictus and Rana esculenta. In skin-grafted animals, moreover, wipes were misdirected from those areas which gave misdirected responses to tactile stimulation. Recordings from cutaneous sensory neurons revealed that only one action potential, with varying latency, was evoked per shock in any given unit. It is concluded that the capacity for sensory localization in anurans cannot depend upon specific patterns of afferent nerve impulses.
Wiping reflexes
Afferentnerve impulses
Frog development
THE FROG is a good preparation in which to study the ontogeny of sensory innervation patterns, and for many years has been exploited for this purpose (for review see [1, 13, 14]). There is a wide variety of appropriately directed behavioral responses to tactile stimulation of different parts of the body, which serve as a convenient monitor for the animal's ability to correctly localize the source of cutaneous sensations. Furthermore, surgical intervention is feasible at larval stages, enabling an experimental analysis to be made of the mechanisms of sensori-motor development. A frequently employed technique consists of rotating large patches of skin so that belly skin (white) differentiates on the frog's back, while back skin (pigmented) differentiates on the frog's belly. If this operation is made early enough, wiping reflexes develop which are misdirected in the sense that they are not aimed at the point of stimulation, but rather at the region from which the skin was taken. The assumption seems plausible that insight into the processes responsible for this phenomenon will be equally informative as regards normal development. The earliest mechanism proposed was that of Sperry and Miner [1, 13, 14], suggested in turn by a more general theory of functional neurogenesis which was first formulated by Paul Weiss [16]. This theory (end-organ specification of central nervous system connectivity) was based upon a considerable amount of data which seemed to eliminate any possibility that selectivity plays a role either in the outgrowth of nerve fibers to the periphery or in their preferences for subclasses of end-organs, (i.e., different muscles, types of skin, etc.). Misdirected reflexes can indeed be accounted for if sensory nerve fibers become fully specified only after making peripheral connections, with the central synapses then following suit. This hypothesis, as applied to normal development, has the attraction of a considerable saving in the amount of genetic information required to specify correct
peripheral innervation patterns [7]. Under the weight of recent evidence, however, the idea has become highly doubtful with respect to efferent nerve fibers [10], while our own findings on the organization of frog sensory ganglia have cast doubts about its correctness on the afferent side [2-5, 8, 9]. The idea that nerve fibers are actually highly specified prior to outgrowth, and can succeed in finding their normal end-organs even when these have been translocated early in development, has recently received serious consideration from several authors [2,13]. Such a mechanism would indeed account for the development of misdirected responses in skin-rotated animals but, in a series of experiments designed to test this possibility, no support for selective switching of peripheral afferent connections could be obtained [4, 5, 8]. We have therefore given some thought to still a third possible explanation for correct localization of tactile stimuli, one which does not require there to be much in the way of anatomical specificity at all. If belly and back skin receptors were able to reliably generate characteristic patterns of impulses in the nerves connected to them, and if these differences could be recognized by the spinal cord and channelled to the appropriate set of motor neurons, then no synaptic shifting need occur in order for wiping reflexes to become misdirected [13,14]. In one variant of this hypothesis, the temporal sequence of nerve impulses in a given set of input channels would provide the requisite information for making sensory discriminations. Alternatively, it could be the conduction velocity spectrum over all the channels which conveys this information to the central nervous system. It therefore seemed worthwhile to approach the question experimentally, even though no suggestion of differential discharge patterns among belly, flank and back neurons has been reported [1, 4, 5, 8, 9, 12, 13]. Our experimental approach here was to see if wiping reflexes could be elicited using precisely controlled stimulus parameters and, if so,
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/110789-04502.00/0
790
CORNER A N D B A K E R
what the corresponding impulse patterns looked like in dorsal and ventral cutaneous neurons. METHOD AND RESULTS
Behavioral Responses to Electrical Stimulation of the Skin Single shocks of brief duration (<1 msec) proved sufficient to evoke well organized behavioral responses. The shocks were delivered via a pair of concentric steel electrodes, with a drop of saline at the tip so as to avoid having to actually touch the electrode assembly to the skin (making contact with the drop in itself occasionally sufficed to elicit a wiping or other kind of reflex, but there was usually no noticeable reaction until a shock was delivered). The nature of the immediate behavioral response to a given stimulus was largely unpredictable---hopping, back-flattening or arching, side stepping and wiping were all observed---while the threshold fluctuated noticeably in the course of an experiment. The frogs differed greatly among themselves, moreover, in the ease of eliciting motor reactions in general and/or wiping reflexes in particular. (The latter responses were scored with respect both to the limb used and to the direction of the wipe, with each response being referred to the site of the stimulus which evoked it.) Wiping reflexes evoked from normal skin were always correctly aimed at the site of stimulation, and were identical to the patterns seen in the same frogs when applying tactile (mechanical) stimuli. Reflexes evoked from translocated skin, on the other hand, were misdirected from those areas which had given misdirected responses to mechanical stimuli. Thus, portions of the graft bordering on normal skin often gave rise to normal reflexes in the experimental animals (Fig. 1). These results therefore indicate that single shock electrical stimulation produces an identical directional response pattern, for misdirected as well as for normal wipes, to that seen when tactile stimuli are used.
FIG. 1. Composite plots of wiping reflexes evoked by electrical stimuli applied at the indicated points on the skin. A=normal forelimb response; &=misdirected forelimb response; (3=normal hindlimb response; O=misdirected hindlimb response. (Left) single-shock stimulation (N=8 frogs with belly skin grafts covering most of the back). The rostrally evoked normal hindlimb wipes were always done with the foot, the caudally evoked wipes with the thigh, as is also the case when using mechanical stimulation. (Right) highfrequency stimulation (N=3 frogs with unilateral belly skin grafts on the back). The rostrally evoked normal responses were obtained despite simultaneous light tactile stimulation applied caudally ([]); all other responses were obtained with simultaneous rostral tactile stimulation ([2]).
A supplementary series of experiments was conducted in order to test the possibility that positional information might be supplied by subliminal mechanical stimulation prior to delivery of the shock. Light touch was therefore applied at a given point on the body, while a stimulating electrode carrying 500 shocks per sec (in order to prevent interspike interval fluctuations during evoked bursts of neuronal fining; also see [ll]) was rapidly touched to a variety of other sites on the skin. With weak stimuli, only rarely did any kind of behavioral response occur upon application of the electrode, either at the beginning or at the end of each test session. Stronger currents, on the other hand, readily evoked all of the normal reactions to tactile stimuli, including wiping reflexes if the shock intensity was not too great. All wipes which followed immediately upon contact with the electrode were directed towards electrically, rather than towards mechanically stimulated areas of the skin (Fig. 1). The interpretation of the above findings, however, requires knowledge of the afferent neuronal impulse patterns elicited by electrical stimulation of, respectively, belly and back skin receptors.
Afferent Nerve Responses to Electrical Stimulation of the Skin The next step, then, was to examine the nerve response patterns evoked by shocks adequate to elicit a wiping reflex. F o r this, we used mature Rana esculenta in addition to D. pictus (after having established that well aimed responses are given to single shock stimulation of the skin in this species too). The methods used for recording unit action potentials from cutaneous mechanoceptive neurons in the thoracic spinal ganglia have been described previously [5, 8, 9]. Shocks were delivered to the skin via a constant current probe, in the same way as for studying the behavioral responses except that the electrodes were positioned using a micromanipulator. When a unit was located by tactile stimulation, the stimulating electrode was placed at the center of the receptive field (CRF) and spike responses were elicited at several shock intensities. The same procedure was then repeated with the electrode placed just inside the border of the units' CRF. The threhold response and the supra-liminal response having the shortest latency were both photographed from the face of a storage oscilloscope. Latency measurements (from shock onset to peak of the major deflection) were made from these photos to the nearest 0.1 msec. A shock delivered near the center of a CRF always evoked a single action potential if the current exceeded a critical intensity (which varied greatly from one neuron to the next; also [11,15]). The characteristic latency for each unit did not change with higher stimulus intensities. Near the borders of a CRF, on the other hand, action potentials often had a considerably longer latency (by as much as a factor 10) which declined as an inverse function of the applied current. The minimum latency was equal to that of spikes evoked centrally within the receptive field (Fig. 2). These values are thus assumed to represent nerve impulse conduction times for reaching the ganglion after leaving the skin. Brief trains of shocks drove the afferent responses in a strict repetitive fashion, with interspike intervals as short as 1-2 msec and without any afterdischarge upon termination of the stimulus (also [liD. DISCUSSION AND CONCLUSIONS It is striking that there was no intensity of electrical stimulation, despite the demonstrated efficacy for eliciting wiping
STIMULATION OF FROG SKIN
791
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•
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I
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I
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I
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0
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m sec
FIG. 2. Action potentials evoked in spinal ganglioncells by electrical stimulation of the skin in Rana escalenta. Left, uriit with a large receptive field, showing a long latency spike (+2 mV) followingliminal stimulation at the border (top), but a much shorter latency (5 msec) when using the stronger shocks (middle) or when stimulating centrally in the field (bottom). Right, minimumlatencies (msec) to the spinal ganglion of 47 back (O) and belly (©) neurons, classified according to receptive field size: + =small (<5 ram2); + + =medium sized; + + + =large (>20 ram2).
reflexes, which evoked more than one action potential per shock in any given neuron. It is inconceivable, therefore, that a temporal pattern of nerve impulses in the afferent fibers provides information enabling correct localization of the stimulus. It might be argued that the required positional information had already been transmitted during contact with the skin prior to shock delivery (this last merely serving in a nonspecific way to bring the excitation up to threshold). However, even the briefest touch is capable of triggering a wiping reflex and, since there are no after-discharges in frog cutaneous afferent fibers following the cessation of tactile stimulation [5, 6, 9, 12, 14], such stimuli can evoke only very short bursts of action potentials. During continuous stimulation, furthermore, impulse patterns have in our experience never appeared to be related to the p o s i t i o n of a unit's CRF, but rather to provide information about the rate of stimulus movement (i.e., skin deformation) and, to a more limited extent, about the pressure being applied (also [6,11]). Finally, the results of the present experiments employing high frequency shock trains to elicit wiping reflexes are incompatible with an impulse patterning mechanism for sensory localization, even if possible subliminal effects are taken into consideration. An alternative mechanism for "impulse patterning" as a source of positional information could be based upon the arrival times of impulses along s e v e r a l nerve fibers, even though none of the channels were transmitting more than a single impulse. In this view, belly and back skin would differ with respect to, e.g,, the proportion of slow to fast fibers activated by an adequate stimulus (with the spinal cord
channelling the different spatio-excitation patterns into the appropriate motor neuron pools). Such a mechanism has in fact been proposed to explain the occurrence of lid-closure (i.e., eye-retraction) reflexes in frogs following stimulation of a regenerating limb blastema [12,14]. In the present experiments, however, there was no indication of differences between the belly and the back neuron pools which might be utilized for the signalling of stimulus positions. (The overall latencies are longer for belly units, but this is to be expected on the basis of the greater distance which action potentials must travel from ventral skin to the dorsal root ganglion). Still more convincing is the large variation noted in conduction times (also see [6]), depending upon w h e r e in the CRF a stimulus is applied. This factor would introduce such a great uncertainty into the impulse spectrum generated by a given set of cutaneous CRF's that it is difficult to imagine how any useful information could be transmitted about the location of receptors giving rise to the nerve impulses. We conclude, therefore, that an explanation of the misdirected reflex phenomenon on the basis of specific afferent impulse patterns is highly unlikely, in view of the neuronal response patterns described in the present report. This conclusion, albeit negative, makes comprehensible the high degree of anatomical selectivity seen in the majority of cutaneous CRF's [5, 6, 8, 9, 13, 15], as well as the failure of misdirected relexes to appear ff skin rotations are performed after a critical stage of development [1, 2, 13, 14]. The available evidence thus points to an explanation for anuran cutaneous reflex specificity in terms of neuronal circuitry rather than of encoded sensory messages.
792
CORNER AND BAKER REFERENCES
1. Baker, R. E. Some comments on central and peripheral plastic changes in nerve connexions. In: Molecular and Functional Neurobiology, edited by W. H. Gispen. Amsterdam: Elsevier, 1976, pp. 47-86. 2. Baker, R. E. Synapse selectivity in somatic afferent systems. In: Maturation of the Nervous System, edited by M. A. Corner, R. E. Baker, N. E. van de Poll, D. F. Swaab and H. B. M. Uylings, Progr. Brain Res., Vol. 48. Amsterdam: Elsevier, 1978, pp. 77-98. 3. Baker, R. E. and M. A. Corner. Development of cutaneous afferent connections in frogs: an experimental analysis. Zodn. 6, in press. 4. Baker, R. E., M. A. Comer and W. A. M. Veltman. Cutaneous receptive field enlargement following skin-grafting in the frog, D. pictus. Brain Res. Bull. 2: 475-477, 1977. 5. Baker, R. E., M. A. Corner and W. A. M. Veltman. Topography of cutaneous mechanoceptive neurones in dorsal root ganglia of skin-grafted frogs. J. Physiol., Lond., in press. 6. Catton, W. T. Cutaneous mechanoreceptors. In: Frog Neurobiology-A Handbook, edited by R. Llinas and W. Precht. Berlin: Springer, 1976, pp. 629-642. 7. Changeux, J.-P. and K. Mikoshiba. Genetic and 'epigenetic' factors regulating synapse formation in the vertebrate cerebellum and neuromuscular junctions. In: Maturation of the Nervous System, Prog. Brain Res., Vol. 48. Amsterdam: Elsevier, 1978, pp. 43-66. 8. Comer, M. A., R. E. Baker and W. A. M. Veltman. Receptive fields of cutaneous mechanoceptive neurons in the frog, Discoglossus pictus, following skin transplantation at larval stages. Brain Res. Bull. 2: 393-395, 1977.
9. Corner, M. A., W. A. M. Veltman, R. E. Baker and J. van de Nes. Topography of cutaneous spinal ganglion cells in the frog (Rana esculenta). Brain Res., in press. 10. Fambrough, D. M. Specificity of nerve-muscle interactions. In: Neuronal Recognition, edited by S. H. Barondes. London: Chapman and Hall, 1976, pp. 25-68. 11. Holloway, J. A., C. F. Ramsundar and L. E. Wright. Excitability and functional organization of cutaneous tactile units of the bullfrog (R. catesbeiana). J. Neurosci. Res. 2: 261-270, 1976. 12. Kornacker, K. Some properties of the afferent pathway in the frog corneal reflex. Expl Neurol. 7: 224-239, 1963. 13. Mendel, L. and M. HoUyday. Spinal reflexes with altered periphery. In: Frog Neurobiology-A Handbook, edited by R. Llinas and W. Precht. Berlin: Springer, 1976, pp. 793-810. 14. Sz6kely, G. Problems of neuronal specificity in the development of some behavioral patterns in amphibia. In: Aspects of Neurogenesis, edited by G. Gottlieb. New York: Academic Press, 1974, pp. 115--150. 15. Verveen, A. A. Fields of touch receptors in frog skin. Expl Neurol. 8: 482-492, 1963. 16. Weiss, P. Neurobiology in statu nascendi. In: Perspectives in Brain Research, edited by M. A. Corner and D. F. Swaab. Progr. Brain Res., Vol. 45. Amsterdam: Elsevier, 1976, pp. 7-38.
Wiping Reflexes and Nerve Impulse Patterns Evoked by Electrical Stimulation of the Skin in Frogs M. A. C O R N E R A N D R. E. B A K E R
Netherlands Institute for Brain Research, IJdijk 28, Amsterdam-O, The Netherlands (Received 14 April 1978) CORNER, M. A. AND R. E. BAKER. Wiping reflexes and nerve impulse patterns evoked by electrical stimulation of the skin in frogs. PHYSIOL. BEHAV. 21(5) 789-792, 1978.--Electric shocks applied to the skin evoke well aimed wiping reflexes in the frogs, Discoglossus pictus and Rana esculenta. In skin-grafted animals, moreover, wipes were misdirected from those areas which gave misdirected responses to tactile stimulation. Recordings from cutaneous sensory neurons revealed that only one action potential, with varying latency, was evoked per shock in any given unit. It is concluded that the capacity for sensory localization in anurans cannot depend upon specific patterns of afferent nerve impulses.
Wiping reflexes
Afferentnerve impulses
Frog development
THE FROG is a good preparation in which to study the ontogeny of sensory innervation patterns, and for many years has been exploited for this purpose (for review see [1, 13, 14]). There is a wide variety of appropriately directed behavioral responses to tactile stimulation of different parts of the body, which serve as a convenient monitor for the animal's ability to correctly localize the source of cutaneous sensations. Furthermore, surgical intervention is feasible at larval stages, enabling an experimental analysis to be made of the mechanisms of sensori-motor development. A frequently employed technique consists of rotating large patches of skin so that belly skin (white) differentiates on the frog's back, while back skin (pigmented) differentiates on the frog's belly. If this operation is made early enough, wiping reflexes develop which are misdirected in the sense that they are not aimed at the point of stimulation, but rather at the region from which the skin was taken. The assumption seems plausible that insight into the processes responsible for this phenomenon will be equally informative as regards normal development. The earliest mechanism proposed was that of Sperry and Miner [1, 13, 14], suggested in turn by a more general theory of functional neurogenesis which was first formulated by Paul Weiss [16]. This theory (end-organ specification of central nervous system connectivity) was based upon a considerable amount of data which seemed to eliminate any possibility that selectivity plays a role either in the outgrowth of nerve fibers to the periphery or in their preferences for subclasses of end-organs, (i.e., different muscles, types of skin, etc.). Misdirected reflexes can indeed be accounted for if sensory nerve fibers become fully specified only after making peripheral connections, with the central synapses then following suit. This hypothesis, as applied to normal development, has the attraction of a considerable saving in the amount of genetic information required to specify correct
peripheral innervation patterns [7]. Under the weight of recent evidence, however, the idea has become highly doubtful with respect to efferent nerve fibers [10], while our own findings on the organization of frog sensory ganglia have cast doubts about its correctness on the afferent side [2-5, 8, 9]. The idea that nerve fibers are actually highly specified prior to outgrowth, and can succeed in finding their normal end-organs even when these have been translocated early in development, has recently received serious consideration from several authors [2,13]. Such a mechanism would indeed account for the development of misdirected responses in skin-rotated animals but, in a series of experiments designed to test this possibility, no support for selective switching of peripheral afferent connections could be obtained [4, 5, 8]. We have therefore given some thought to still a third possible explanation for correct localization of tactile stimuli, one which does not require there to be much in the way of anatomical specificity at all. If belly and back skin receptors were able to reliably generate characteristic patterns of impulses in the nerves connected to them, and if these differences could be recognized by the spinal cord and channelled to the appropriate set of motor neurons, then no synaptic shifting need occur in order for wiping reflexes to become misdirected [13,14]. In one variant of this hypothesis, the temporal sequence of nerve impulses in a given set of input channels would provide the requisite information for making sensory discriminations. Alternatively, it could be the conduction velocity spectrum over all the channels which conveys this information to the central nervous system. It therefore seemed worthwhile to approach the question experimentally, even though no suggestion of differential discharge patterns among belly, flank and back neurons has been reported [1, 4, 5, 8, 9, 12, 13]. Our experimental approach here was to see if wiping reflexes could be elicited using precisely controlled stimulus parameters and, if so,
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/110789-04502.00/0
790
CORNER A N D B A K E R
what the corresponding impulse patterns looked like in dorsal and ventral cutaneous neurons. METHOD AND RESULTS
Behavioral Responses to Electrical Stimulation of the Skin Single shocks of brief duration (<1 msec) proved sufficient to evoke well organized behavioral responses. The shocks were delivered via a pair of concentric steel electrodes, with a drop of saline at the tip so as to avoid having to actually touch the electrode assembly to the skin (making contact with the drop in itself occasionally sufficed to elicit a wiping or other kind of reflex, but there was usually no noticeable reaction until a shock was delivered). The nature of the immediate behavioral response to a given stimulus was largely unpredictable---hopping, back-flattening or arching, side stepping and wiping were all observed---while the threshold fluctuated noticeably in the course of an experiment. The frogs differed greatly among themselves, moreover, in the ease of eliciting motor reactions in general and/or wiping reflexes in particular. (The latter responses were scored with respect both to the limb used and to the direction of the wipe, with each response being referred to the site of the stimulus which evoked it.) Wiping reflexes evoked from normal skin were always correctly aimed at the site of stimulation, and were identical to the patterns seen in the same frogs when applying tactile (mechanical) stimuli. Reflexes evoked from translocated skin, on the other hand, were misdirected from those areas which had given misdirected responses to mechanical stimuli. Thus, portions of the graft bordering on normal skin often gave rise to normal reflexes in the experimental animals (Fig. 1). These results therefore indicate that single shock electrical stimulation produces an identical directional response pattern, for misdirected as well as for normal wipes, to that seen when tactile stimuli are used.
FIG. 1. Composite plots of wiping reflexes evoked by electrical stimuli applied at the indicated points on the skin. A=normal forelimb response; &=misdirected forelimb response; (3=normal hindlimb response; O=misdirected hindlimb response. (Left) single-shock stimulation (N=8 frogs with belly skin grafts covering most of the back). The rostrally evoked normal hindlimb wipes were always done with the foot, the caudally evoked wipes with the thigh, as is also the case when using mechanical stimulation. (Right) highfrequency stimulation (N=3 frogs with unilateral belly skin grafts on the back). The rostrally evoked normal responses were obtained despite simultaneous light tactile stimulation applied caudally ([]); all other responses were obtained with simultaneous rostral tactile stimulation ([2]).
A supplementary series of experiments was conducted in order to test the possibility that positional information might be supplied by subliminal mechanical stimulation prior to delivery of the shock. Light touch was therefore applied at a given point on the body, while a stimulating electrode carrying 500 shocks per sec (in order to prevent interspike interval fluctuations during evoked bursts of neuronal fining; also see [ll]) was rapidly touched to a variety of other sites on the skin. With weak stimuli, only rarely did any kind of behavioral response occur upon application of the electrode, either at the beginning or at the end of each test session. Stronger currents, on the other hand, readily evoked all of the normal reactions to tactile stimuli, including wiping reflexes if the shock intensity was not too great. All wipes which followed immediately upon contact with the electrode were directed towards electrically, rather than towards mechanically stimulated areas of the skin (Fig. 1). The interpretation of the above findings, however, requires knowledge of the afferent neuronal impulse patterns elicited by electrical stimulation of, respectively, belly and back skin receptors.
Afferent Nerve Responses to Electrical Stimulation of the Skin The next step, then, was to examine the nerve response patterns evoked by shocks adequate to elicit a wiping reflex. F o r this, we used mature Rana esculenta in addition to D. pictus (after having established that well aimed responses are given to single shock stimulation of the skin in this species too). The methods used for recording unit action potentials from cutaneous mechanoceptive neurons in the thoracic spinal ganglia have been described previously [5, 8, 9]. Shocks were delivered to the skin via a constant current probe, in the same way as for studying the behavioral responses except that the electrodes were positioned using a micromanipulator. When a unit was located by tactile stimulation, the stimulating electrode was placed at the center of the receptive field (CRF) and spike responses were elicited at several shock intensities. The same procedure was then repeated with the electrode placed just inside the border of the units' CRF. The threhold response and the supra-liminal response having the shortest latency were both photographed from the face of a storage oscilloscope. Latency measurements (from shock onset to peak of the major deflection) were made from these photos to the nearest 0.1 msec. A shock delivered near the center of a CRF always evoked a single action potential if the current exceeded a critical intensity (which varied greatly from one neuron to the next; also [11,15]). The characteristic latency for each unit did not change with higher stimulus intensities. Near the borders of a CRF, on the other hand, action potentials often had a considerably longer latency (by as much as a factor 10) which declined as an inverse function of the applied current. The minimum latency was equal to that of spikes evoked centrally within the receptive field (Fig. 2). These values are thus assumed to represent nerve impulse conduction times for reaching the ganglion after leaving the skin. Brief trains of shocks drove the afferent responses in a strict repetitive fashion, with interspike intervals as short as 1-2 msec and without any afterdischarge upon termination of the stimulus (also [liD. DISCUSSION AND CONCLUSIONS It is striking that there was no intensity of electrical stimulation, despite the demonstrated efficacy for eliciting wiping
STIMULATION OF FROG SKIN
791
+++
•
•
•
I
-t"-t"
I
•
O0
I
+
..
0000
0
I
0
I
OI
1
I
•
•
O0
I
o
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I
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0
I
o
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I
'o
I
o
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,5
m sec
FIG. 2. Action potentials evoked in spinal ganglioncells by electrical stimulation of the skin in Rana escalenta. Left, uriit with a large receptive field, showing a long latency spike (+2 mV) followingliminal stimulation at the border (top), but a much shorter latency (5 msec) when using the stronger shocks (middle) or when stimulating centrally in the field (bottom). Right, minimumlatencies (msec) to the spinal ganglion of 47 back (O) and belly (©) neurons, classified according to receptive field size: + =small (<5 ram2); + + =medium sized; + + + =large (>20 ram2).
reflexes, which evoked more than one action potential per shock in any given neuron. It is inconceivable, therefore, that a temporal pattern of nerve impulses in the afferent fibers provides information enabling correct localization of the stimulus. It might be argued that the required positional information had already been transmitted during contact with the skin prior to shock delivery (this last merely serving in a nonspecific way to bring the excitation up to threshold). However, even the briefest touch is capable of triggering a wiping reflex and, since there are no after-discharges in frog cutaneous afferent fibers following the cessation of tactile stimulation [5, 6, 9, 12, 14], such stimuli can evoke only very short bursts of action potentials. During continuous stimulation, furthermore, impulse patterns have in our experience never appeared to be related to the p o s i t i o n of a unit's CRF, but rather to provide information about the rate of stimulus movement (i.e., skin deformation) and, to a more limited extent, about the pressure being applied (also [6,11]). Finally, the results of the present experiments employing high frequency shock trains to elicit wiping reflexes are incompatible with an impulse patterning mechanism for sensory localization, even if possible subliminal effects are taken into consideration. An alternative mechanism for "impulse patterning" as a source of positional information could be based upon the arrival times of impulses along s e v e r a l nerve fibers, even though none of the channels were transmitting more than a single impulse. In this view, belly and back skin would differ with respect to, e.g,, the proportion of slow to fast fibers activated by an adequate stimulus (with the spinal cord
channelling the different spatio-excitation patterns into the appropriate motor neuron pools). Such a mechanism has in fact been proposed to explain the occurrence of lid-closure (i.e., eye-retraction) reflexes in frogs following stimulation of a regenerating limb blastema [12,14]. In the present experiments, however, there was no indication of differences between the belly and the back neuron pools which might be utilized for the signalling of stimulus positions. (The overall latencies are longer for belly units, but this is to be expected on the basis of the greater distance which action potentials must travel from ventral skin to the dorsal root ganglion). Still more convincing is the large variation noted in conduction times (also see [6]), depending upon w h e r e in the CRF a stimulus is applied. This factor would introduce such a great uncertainty into the impulse spectrum generated by a given set of cutaneous CRF's that it is difficult to imagine how any useful information could be transmitted about the location of receptors giving rise to the nerve impulses. We conclude, therefore, that an explanation of the misdirected reflex phenomenon on the basis of specific afferent impulse patterns is highly unlikely, in view of the neuronal response patterns described in the present report. This conclusion, albeit negative, makes comprehensible the high degree of anatomical selectivity seen in the majority of cutaneous CRF's [5, 6, 8, 9, 13, 15], as well as the failure of misdirected relexes to appear ff skin rotations are performed after a critical stage of development [1, 2, 13, 14]. The available evidence thus points to an explanation for anuran cutaneous reflex specificity in terms of neuronal circuitry rather than of encoded sensory messages.
792
CORNER AND BAKER REFERENCES
1. Baker, R. E. Some comments on central and peripheral plastic changes in nerve connexions. In: Molecular and Functional Neurobiology, edited by W. H. Gispen. Amsterdam: Elsevier, 1976, pp. 47-86. 2. Baker, R. E. Synapse selectivity in somatic afferent systems. In: Maturation of the Nervous System, edited by M. A. Corner, R. E. Baker, N. E. van de Poll, D. F. Swaab and H. B. M. Uylings, Progr. Brain Res., Vol. 48. Amsterdam: Elsevier, 1978, pp. 77-98. 3. Baker, R. E. and M. A. Corner. Development of cutaneous afferent connections in frogs: an experimental analysis. Zodn. 6, in press. 4. Baker, R. E., M. A. Comer and W. A. M. Veltman. Cutaneous receptive field enlargement following skin-grafting in the frog, D. pictus. Brain Res. Bull. 2: 475-477, 1977. 5. Baker, R. E., M. A. Corner and W. A. M. Veltman. Topography of cutaneous mechanoceptive neurones in dorsal root ganglia of skin-grafted frogs. J. Physiol., Lond., in press. 6. Catton, W. T. Cutaneous mechanoreceptors. In: Frog Neurobiology-A Handbook, edited by R. Llinas and W. Precht. Berlin: Springer, 1976, pp. 629-642. 7. Changeux, J.-P. and K. Mikoshiba. Genetic and 'epigenetic' factors regulating synapse formation in the vertebrate cerebellum and neuromuscular junctions. In: Maturation of the Nervous System, Prog. Brain Res., Vol. 48. Amsterdam: Elsevier, 1978, pp. 43-66. 8. Comer, M. A., R. E. Baker and W. A. M. Veltman. Receptive fields of cutaneous mechanoceptive neurons in the frog, Discoglossus pictus, following skin transplantation at larval stages. Brain Res. Bull. 2: 393-395, 1977.
9. Corner, M. A., W. A. M. Veltman, R. E. Baker and J. van de Nes. Topography of cutaneous spinal ganglion cells in the frog (Rana esculenta). Brain Res., in press. 10. Fambrough, D. M. Specificity of nerve-muscle interactions. In: Neuronal Recognition, edited by S. H. Barondes. London: Chapman and Hall, 1976, pp. 25-68. 11. Holloway, J. A., C. F. Ramsundar and L. E. Wright. Excitability and functional organization of cutaneous tactile units of the bullfrog (R. catesbeiana). J. Neurosci. Res. 2: 261-270, 1976. 12. Kornacker, K. Some properties of the afferent pathway in the frog corneal reflex. Expl Neurol. 7: 224-239, 1963. 13. Mendel, L. and M. HoUyday. Spinal reflexes with altered periphery. In: Frog Neurobiology-A Handbook, edited by R. Llinas and W. Precht. Berlin: Springer, 1976, pp. 793-810. 14. Sz6kely, G. Problems of neuronal specificity in the development of some behavioral patterns in amphibia. In: Aspects of Neurogenesis, edited by G. Gottlieb. New York: Academic Press, 1974, pp. 115--150. 15. Verveen, A. A. Fields of touch receptors in frog skin. Expl Neurol. 8: 482-492, 1963. 16. Weiss, P. Neurobiology in statu nascendi. In: Perspectives in Brain Research, edited by M. A. Corner and D. F. Swaab. Progr. Brain Res., Vol. 45. Amsterdam: Elsevier, 1976, pp. 7-38.