- Email: [email protected]
Memory traces in spinal cord
207-214 45 Maruyama, K., Schiavi, S. C., Huse, W., Johnson, G. L. and Ruley, H. E. (1987) Oncogene 1,361-367 46 Borasio, G. D. etal. (1989) Neuron 2, 1087-1096 47 Lendahl, U., Zimmerman, L. and McKay, R. D. G. Cell (in
press) 48 Frederiksen, K. and McKay, R. (1988) J. Neurosci. 8, 1144-1151 49 Raft, M. C. (1989) Science 243, 1450-1455 50 Giotta, G. J., Heitzmann, J. and Cohn, M. (1980) Brain Res. 202, 445-458 51 Rosenberg, M. B. et al. (1988) Science 242, 1575-1578 52 Whittemore, S. R., Holets, V. R., Gonzalez-Carvajal, M. and
Levy, D. (1988) Soc. Neurosci. Abstr. 14, 586 53 Bj6rklund, A. et aL (1987) Trends Neurosci. 10, 509-516 54 Albritton, L. M., Tseng, L., Scadden, D. and Cunningham, J. M. (1989) Ceil 57, 659--666 55 Geller, A. I. and Breakefield, X. O. (1988) Science 241, 1667-1669 56 Joyner, A. L., Skarnes, W. C. and Rossant, J. (1989) Nature 338, 153-156 57 Zimmer, A. and Gruss, P. (1989) Nature 338, 150-153 58 Kuehn, M. R., Bradley, A., Robertson, E. J. and Evans, M. J. (1987) Nature 326, 295-298 59 Hooper, M., Hardy, K., Handyside, A., Hunter, S. and Monk, M. (1987) Nature 326, 292-295
Memory tracesin spinal cord J o n a t h a n R. W o l p a w
The complexity and inaccessibility of the vertebrate CNS impede the localization and description of memory traces and the definition of the processes that create them. Recent work has shown that the spinal stretch reflex (SSR), which is produced by a monosynaptic twoneuron pathway, can be operantly conditioned, and that memory traces responsible for this behavioral change reside in the spinal cord. The probable locations are the terminal of the Ia afferent neuron on the motoneuron and/or the motoneuron itself. Because it modifies a simple well-defined and accessible pathway, SSR conditioning may be a valuable experimental model .for studying vertebrate memory. One of the ultimate goals of neuroscience research is to understand human memory in its most impressive and sophisticated manifestations, such as the ability to recognize a face seen briefly long ago or the capacity to master a language or a musical instrument. Immediate goals are more modest - to understand a rat's avoidance of a platform where a shock was delivered yesterday, or the acquisition by a monkey of the ability to move a lever smoothly against an opposing force. The objectives of ultimate and immediate goals are the same - to describe the memory trace, the change in the nervous system that is responsible for a particular change in behavior, and to describe learning, the process that creates the trace. The prerequisite for each is also the same - a memory trace must be located before it can be described. However, most behaviors result from activity at multiple sites in the CNS, and these sites and their interactions are usually not fully defined. As a result, this basic requirement of localization is the main impediment to progress. In response to the problem, three strategies have developed.
a n d J o n a t h a n S. C a r p
relatively simple nervous systems of invertebrates 3-5. Studies of this type have elucidated the simplest forms of memory, habituation and sensitization, and have begun to provide insight into simple forms of associative conditioning. The third strategy seeks to study memory traces in the complex nervous system of vertebrates. The problem of localization is addressed by choosing relatively simple behaviors 3'6-8. These vertebrate models are potentially the most rewarding because they are most closely related to human memory. However, they have so far proved most difficult to develop and use due to incomplete pathway definition, limited accessibility and/or susceptibility to input from elsewhere. A vertebrate model based on an even simpler behavior might overcome these difficulties. The spinal stretch reflex (SSR) The simplest behavior of the vertebrate CNS is the initial response to sudden muscle stretch. It is called the tendon jerk, the M1 response, or, as in this article, the spinal stretch reflex (SSR) 9-12, and can be measured by its mechanical effect or by EMG. The SSR is produced by the two-neuron monosynaptic pathway shown in Fig. 1. This pathway
Fig. 1. Dinal cord
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Research strategies The first strategy circumvents the problem by using reduced preparations to study phenomena that might be similar to the memory traces that underlie behavioral changes in intact animals. During the past decade, this approach has focused on long-term potentiation in the hippocampus and elsewhere 1'2. The second strategy tries to minimize the problem of localization by studying memory traces in the TINS, Vol. 13, No. 4, 1990
Jonathan R. Wolpaw and Jonathan S. Carp are at the Wadsworth Center for Laboratoriesand Research,New York State Departmentof Health and State Universityof New York,Albany, NY 12201, USA.
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Two-neuron monosynaptic pathway of the spinal stretch reflex (SSR).It consistsof the la afferent neuron, its synapseon the :rmotoneuron, and the motoneuron itself. Sudden muscle stretch lengthens the musclespindle and excites the la afferent. The la afferent synapse excites the motoneuron and causesmuscle contraction.
Muscle
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The physiology and anatomy of this two-neuron pathway, and the characteristics of the behavior it mediates (i.e. the SSR) have been studied extensively since the time of Sherrington 1°' is. However, in spite of its unique simplicity and accessibility, the SSR pathway has attracted little interest as a model for the study of vertebrate memory. The spinal cord in general, and spinal reflexes in particular, are widely viewed as fixed and inflexible, responding in a stereotyped manner to inputs from the periphery or from supraspinal areas. This common perception is not correct. Spinal cord neurons and synapses, like those of cerebral cortex and other supraspinal structures, change in the course of development and in response to trauma 19'2°. This suggests that the spinal cord also changes with learning, i.e. that it can contain memory traces. Furthermore, the evidence suggests that memory traces form in a spinal cord pathway when the learning process involves prolonged change in the influences on the pathway.
A
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MODES: Control (all trials rewarded) Up (reward only if SSR > criterion) Down (reward only if SSR < criterion) Fig. 2. Experimental design. (A) Animal performing computer-controlled SSR conditioning task. The arm rests in a cast attached to a torque motor shaft. A rotary variable differential transformer below the motor monitors the elbow angle. Chronically implanted fine-wire electrodes monitor the biceps EMG. A light indicates the correct elbow angle. A solenoid-powered syringe derivers squirt of juice in reward. (B) SSR conditioning task. The animal maintains elbow angle and biceps EMG within preset ranges against a constant background extension torque from the motor. At the end of a randomly varying 1.2-1.8 s period, a brief pulse of additional extension torque extends the elbow and elicits the biceps SSR. In the control-mode, a reward squirt always occurs 0.2 s after the pulse. In the up- or down-mode, a reward squirt occurs only if the SSR (as measured by EMG) is above or below a criterion value. Animals average 3000-4000 trials per day. (Modified from Ref. 21 .) consists of the Ia afferent neuron, its excitatory synapse on the o~-motoneuron, and the motoneuron itself. The Ia afferent neuron innervates the muscle spindle and is excited by sudden muscle stretch. It in turn excites motoneurons that innervate the same muscle and its synergists, thereby causing muscle contraction, which opposes the sudden stretch*. While the SSR is wholly spinal, it can be affected by influence on the muscle spindle (exerted via fusimotor fibers13'14), on the Ia terminal (exerted via presynaptic inputs is' 16), and on the motoneuron (exerted via postsynaptic inputs17). * Other oligosynaptic pathways might contribute to the SSR; however, the data suggest that they do not have a significant role in the SSR conditioningdescribed here 21'28,31,32.
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The SSR can be operantly conditioned In the experiment shown in Fig. 2, monkeys are exposed to a conditioning task that requires prolonged change in neuronal activity influencing the SSR pathway, and thus is likely to produce a memory trace located in this pathway21. The animal maintains elbow angle and a specified level of ongoing biceps EMG against a constant extension torque. At an unpredictable time, a brief pulse of additional extension torque transiently extends the forearm and elicits the biceps SSR. Before the initiation of conditioning, the reward always follows the SSR ('control-mode'). During conditioning, the reward follows only if SSR size is above ('up-mode') or below ('down-mode') a criterion value. The crucial feature of this task is that muscle stretch occurs at an unpredictable time. Because the SSR is the earliest possible response, an animal can change SSR size only by maintaining a continual appropriate influence over the SSR pathway. By linking reward to SSR size, the up- or down-mode operantly conditions the animal to maintain such an influence. The expectation is that this continued influence produces a memory trace located in the SSR pathway, most probably at the Ia synapse on the motoneuron. By virtue of its location, this memory trace, and the process creating it, should be accessible to detailed physiological and anatomical study. Figure 3 shows what happens when an animal is switched from the control-mode to the up- or downmode. The size of the SSR changes appropriately in the ensuing days and weeks. On day 0, the SSR is nearly equal in the two animals. On day 80, the SSR of the up-mode animal is three times the size of the SSR of the down-mode animal. Furthermore, as Fig. 3 indicates, SSR conditioning changes the amplitude of the elbow extension caused by the torque pulse. As the SSR rises under the up-mode, it provides more opposition to the torque pulse and extension decreases. The opposite occurs under the down-mode. If the mode is switched (from up to down, or vice versa) the change in SSR size reverses in the same gradual fashion in which it first develops 22. If task performance is halted, change persists: gaps of several weeks in performance cause no loss of SSR decrease and only partial loss of SSR increase 23. TINS, Vol. 13, No. 4, 1990
Recent initial studies by Wolf, Segal and their colleagues24'~5 indicate that humans can also increase or decrease the size of the SSR when exposed to a comparable task. Humans and monkeys appear similar in both the time course and the magnitude of SSR change. Figure 4 combines data from many animals to define the development of SSR change 26. Within 6 h of the beginning of the up- or down-mode, SSR size increases (up-mode) or decreases (down-mode) by about 8%. This rapid initial jump, called phase I, is followed by very gradual change (1-2% per day) that continues for at least 40 days. This slow change is called phase II. Phase I change indicates that the animal quickly responds to the up- or down-mode with an appropriate alteration in influence on the SSR pathway. Phase II change suggests that the continuation of this influence over subsequent weeks gradually changes the Ia terminal or the motoneuron, and thereby further changes the SSR 26'27. However, the data leave open the possibility that the muscle spindle is the site of change or that change is only supraspinal and simply exerts steadily increasing influence over the SSR pathway. These issues are addressed by a modified form of the experiment shown in Fig. 2. The new design conditions a simplified form of the SSR, the H-reflex, in the triceps surae (posterior calf) muscles of the leg28. The H-reflex29 is elicited by direct stimulation of the Ia afferent fibers, so that the afferent volley eliciting the reflex does not depend on muscle spindle sensitivity. The absence of the spindle from the pathway does not prevent conditioning. Under the upmode, the size of the H-reflex gradually rises to about twice the initial size; under the down-mode it gradually falls to about two-thirds 2s. Little or no change occurs in the opposite (control) leg3°. These findings eliminate the muscle spindle as the site of change, and thus focus attention on the Ia terminal and the motoneuron. In addition, because this simplified design conditions the SSR pathway in the lumbosacral cord, it facilitates determination of whether the conditioning task does create a memory trace in the spinal cord*. Memory traces are present in spinal cord If the conditioning task does produce a memory trace in the spinal cord itself, then eviflence of the trace should remain even if the spinal cord is isolated from supraspinal influence by transection. This can be tested by anesthetizing animals in which the H-reflex of one leg has been increased (up-mode) or decreased (down-mode), transecting the spinal cord above the lumbosacral site of the reflex pathway, and measuring the reflexes on both sides under continued anesthesia for several days. The anesthetized transected animals display reflex asymmetries comparable to those seen in the awake behaving animals 31'32. These asymmetries are still present three days after transection. In up-mode animals, the reflex remains larger in the conditioned leg than in the control leg. In down-mode animals, it , The H-reflex conditioning task has the additional advantage of permitting conditioning of the SSR pathway in an unrestrained, fxeely moving animal41. TINS, Vol. 13, No. 4, 1990
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Fig. 3. Effects of up- and down-modes on SSR size and on movement. (A) Data from an animal exposed to the up-mode for 80 days. The five upper graphs show average daily SSR size for every 20th day beginning with the imposition of the up-mode. SSR size increases steadily without change in background EMG (represented here by the 12 ms before SSR onset). The single lower graph shows pulse-induced elbow extension before (solid line) and after (dashed line) up-mode exposure. Initial extension, the stimulus that elicits the SSR, is the same for both. Beginning at 35-40 ms, extension is less after upmode exposure than before, because the larger biceps SSR provides more opposition. The 20 ms delay between the SSR and its effect on movement is presumably due to the time needed for muscle contraction after excitation. (B) Analogous data from an animal exposed to the down-mode. A steady decrease in SSR size occurs without change in background EMG or in the initial pulse-induced extension. Beyond 35-40 ms, extension is greater after downmode exposure, because the smaller SSR gives less opposition. (Modified from Ref. 26.)
remains smaller in the conditioned leg than in the control leg. Figure 5 shows examples of these persistent asymmetries. Their existence indicates that, when monkeys modify the behavior mediated by the SSR pathway, a significant part of the memory trace responsible for the altered behavior resides in the spinal cord. Intended and compensatory plasticity Reflex asymmetry is not the only effect of conditioning that is apparent in the anesthetized transected animals. As Fig. 5 illustrates, reflexes are larger in the control legs of down-mode animals than in the control legs of up-mode animals. This difference is not present before anesthesia and transection; control-leg reflexes do not change over the weeks of 139
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Fig. 4. Two-phase course of 55R change. Average 55R size under the up- or down-mode for 18 mode exposures (11 up and seven down) in nine animals. Each value is 55R size (+_SEM)as a percentage of initial size. Values are shown for hours 0-6, 6-12 and 12-24 of the first day, for each subsequent day through 40 days, and for every two days from then on. The inset is a horizontally expanded view of the first week. The superimposed lines are computer-fits of the equation 'y = (a/ (b+(l/x))) + c'. (See Ref. 26 for details of calculations.) Each curve shows a nearly immediate (within 6 h) modeappropriate offset (i.e. phase I change), followed by slow change (i.e. phase II change) that continues indefinitely. (Modified from Ref. 26.)
conditioning, and are of comparable size in awake behaving up- and down-mode animals a°. Thus, anesthesia and transection reveal an effect of conditioning that is not apparent in the awake behaving animal. The origin of this unexpected change is obscure; nevertheless, its occurrence raises an important issue. Every learning process has a specific goal, an intended change in behavior. In the present context, the goal is defined by the experimenter, i.e. a larger or smaller SSR. The memory trace responsible for this behavioral change can be called 'intended plasticity'. Depending on its nature and location, this trace Fig. 5. Evidence for spinal cord memory traces. Average reflexes in conditioned and control legs of an up-mode animal and a down-mode animal, both under deep pentobarbital anesthesia, 2-3 days after thoracic spinal cord transection eliminates supraspinal influence. Conditioned-leg reflexes are shown by dashed lines, and control-leg reflexes by solid lines. Reflexes are elicited by supramaximal dorsal root stimulation and recorded from the nerve branches supplying the triceps surae. The horizontal bar is 1 ms and the vertical bar is 20% of the maximum possible response (i.e. the response to supramaximal ventral root stimulation 4 2"), Mode-appropriate reflex asymmetry is present in both animals: in the up-mode animal the reflex is bigger in the conditioned leg than in the control leg, and in the down-mode animal it is smaller in the conditioned leg than in the control leg. In addition, the control-leg reflex of the down-mode animal is bigger than the control-leg reflex of the up-mode animal Both these effects of conditioning were evident throughout the several days of post-transection study. (See Ref. 31 for full presentation.) 140
may have additional effects: it may interfere with other behaviors that involve some of the same neurons and synapses. Thus, it may provoke 'compensatory plasticity', modifications that allow the CNS to continue to perform these other behaviors properly. For example, suppose that up-conditioning causes greater transmitter release at the triceps surae Ia synapse. In addition to affecting the SSR, greater transmitter release would also affect other behaviors in which this synapse participates, such as walking or jumping. Compensatory plasticity would be needed to maintain proper performance (perhaps by increasing a specific inhibitory input to the triceps surae motoneuron). Compensatory plasticity may also accompany conditioning of the vestibulo-ocular reflex (VOR), the behavior that maintains a stable retinal image during head movement 7's'33. The sensitivity of floccular Purkinje cells to vestibular input appears to change in a direction opposite to that able to account for the change in VOR gain34. This apparently paradoxical change in floccular response to vestibular input may compensate for change in eye movement input to the flocculus, and thereby properly adjust the contribution of the cerebellum to eye movement 8. If compensatory plasticity does occur, it has two important implications for efforts to define the memory traces underlying specific changes in behavior. First, it indicates that change in even the simplest behavior involves CNS plasticity beyond that responsible for the intended behavioral change. Second, it emphasizes the extreme importance of simplicity in experimental models used to study memory traces. Without the relative simplicity of the SSR and VOR pathways, these putative compensatory changes would be difficult to detect and exceedingly difficult to analyse. R e l a t i o n s h i p s to other t y p e s of learning The memory traces created by SSR conditioning are probably not limited to studies specifically designed to produce them. Evidence is accumulating that SSR changes are common. In humans, gradual changes in the SSR accompany the acquisition of basic motor skills early in life35. They also appear to occur later in life during the learning of skills such as ballet a6. In a highly relevant study, Meyer-Lohmann et al. 37 trained monkeys to move a lever to a target, and randomly perturbed this movement with a brief torque pulse. Initially, the animal's response to perturbation was dominated by the later M2 component of the response to muscle stretch. However, Up-mode animal /\ / \ I I//~,\
Down-mode animal
\
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A B C over months of gaining, the M1 component (i.e. the SSR) gradually Presynaptic Whole Local motoneuron postsynaptic increased in size and M2 disappeared. In effect, the SSR gradually took over the task of opposing the perturbation. Gradual changes in the spinal cord and elsewhere in the CNS Phase probably underlie many learning I processes, and help explain why the mastery of many skills requires intense practice over long periods. The need for prolonged practice is not limited to sophisticated motor behaviors: non-motor skills also require it. For example, the acqui- Phase sition of skill in basic mathematical II operations such as multiplication is a gradual, labor-intensive process. The development of SSR change under the up- or down-mode (Fig. 4) has a course common to many Fig. 6. Possible locations of the spinal cord memory traces responsible for change in SSR size. In learning curves: improvement is each case, phase I change in influence on a site in the SSRpathway (indicated by arrows) eventually rapid at first and then continues at produces phase II change (i.e. the memory trace) at the site. (A) The most likely site is the la a gradually decreasing rate for a afferent terminal on the motoneuron. A memory trace here could be produced by prolonged long time. In the case of the SSR, change in presynaptic inhibition. (B) The memory trace could be a generalized change in the this form appears to be the result motoneuron that alters its response to any input. However, such a modification would have widespread effects on motoneuron function. (C) The trace could also be a very localized of a two-phase sequence: the rapid modification in the postsynaptic membrane, such as a change in receptor sensitivity or dendritic onset of appropriate influence on architecture. the SSR pathway causes phase I change, and the long-term continuation of this influence produces spinal modifications motoneuron itse#. Transmission through the Ia synapse is inhibited by that underlie slow phase II change (see below). Change in the VOR, another simple behavior, also presynaptic inputs from several supraspinal sites 1s'16. displays two distinct phases a8, but learning curves for Recent studies suggest that short-term changes in more complex behaviors typically do not. This dif- presynaptic inhibition have an important role in motor ference is not surprising. Changes in more complex behaviora9'4°. Long-term change in this inhibition behaviors probably involve appropriate adjustments in might modify the Ia terminal (Fig. 6A), perhaps many influences (i.e. many phase I changes), and affecting depolarization-induced calcium entry and these adjustments probably gradually modify many transmitter release, and thereby altering the size of sites in the CNS (i. e. many phase II changes). These the EPSP produced in the motoneuron. Motoneuron membrane properties, such as those multiple two-phase sequences presumably overlap and obscure each other, so that distinct phases are controlling resting potential or input resistance, help determine the response of the motoneuron to any not apparent. input, and might be modified (Fig. 6B) by prolonged changes in descending influence. However, the conMemory trace location(s) in the spinal cord* The most likely locations of the spinal cord memory straints imposed by the need to maintain constant traces created by SSR conditioning are shown in Fig. motoneuron tone during conditioning (Fig. 2 legend), 6. They are the sites in the SSR pathway that are and the great interference that such alterations would subject to influence from supraspinal structures, the exert on most behaviors involving the motoneuron, Ia afferent terminal on the motoneuron and the make generalized membrane modifications a more complicated and less probable explanation for SSR change than presynaptic (Fig. 6A) modification. A *The present discussion focuses on the possible locations of very localized postsynaptic modification (Fig. 6C), the memory trace responsible for the mode-appropriate perhaps in receptor sensitivity or dendritic architecreflex asymmetry. Discussion of the basis of the difference ture, could give greater specificity. However, pathin control-leg reflexes of anesthetized transected animals is ways that could produce such a selective modification comparable and appears elsewhere 31. are not yet defined. At present, a modification of the Ia terminal caused It is unlikely that the memory trace is limited to other by a prolonged change in presynaptic inhibition is spinal cord sites and simply influences the SSR pathway, probably the best candidate for the memory trace. because conditioned reflex changes are prominent even under deep pentobarbital anesthesia 31's2, which suppresses The significance of SSR conditioning as a model for neuronal and synaptic function and thereby tends to isolate studying memory is that the simplicity of the pathway neurons from one another. If a memory trace were not allows the search for the memory trace to focus on present at the Ia terminal or the motoneuron, its effect on this single synapse and its postsynaptic neuron, both reflex size in the pentobarbital-anesthetized animal would be of which are accessible physiologically and anaminimal. tomically. TINS, VoL 13, No. 4, 1990
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Acknowledgements Selected references We thankfiobertJ.
Brady,DavidO. Carpenter, ChongL. Lee, DavidL Martin, DennisJ. McFartand, Barry W. Peterson, RichardF. Seegal,N. TraverseSlaterand Elizabeth W. Wolpaw for criticalreviewof the manuscript.Our work wassupported in part by NIHgrant N522189, the United Cerebra/Palsy Researchand Educational Foundationand the SpinalCordResearch Foundation.
1 Teyler, T. J. and Discenna, P. (1984) Brain Res. 7, 15-28 2 Gustafsson, B. and Wigstrom, H. (1988) Trends Neurosci. 11, 156-162 3 Byrne, J. H. (1987) PhysioL Rev. 67, 329-439 4 Abrams, T. W. and Kandel, E. R. (1988) TrendsNeurosci. 11, 128-135 5 Crow, T. (1988) Trends Neurosci. 11, 136-142 6 Thompson, R. F. (1988) Trends Neurosci. 11, 152-155 7 Ito, M. (1982) Annu. Rev. Neurosci. 5, 275-296 8 Lisberger, S. G. (1988) Science 242, 728-735 9 Lee, R. G. and Tatton, W. G. (1975) Can. J. NeuroL Sci. 2, 285-293 10 Mat-thews, P. B. C. (1972) in Mammalian Muscle Receptors and Their Central Actions pp. 319-409, Williams and Wilkins 11 Henneman, E. and Mendell, L. M. (1981) in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 423-507, Williams and Wilkins 12 Magladery, J. W., Porter, W. E., Park, A. M. and Teasdall, R. D. (1951) Electrophysiological Studies of Nerve and Reflex Activity in Normal Man, IV: The Two-neuron Reflex and Identification of Certain Action Potentials from Spinal Roots and Cord pp. 499-519, Bull. John Hopkins Hospital 13 Harris, D. A. and Henneman, E. (1980) in Medical Physiology, VoL I (Mountcastle, V. B., ed.), pp. 703-717, Mosby 14 Mat-thews, P. B. C. (1981)in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 189-228, Williams and Wilkins 15 Baldissera, F., Hultborn, H. and Illert, M. (1981)in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 509-595, Williams and Wilkins 16 Burke, R. E. and Rudomin, P. (1977) in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I: Cellular Biology of Neurons) (Kandel, E. R., ed.), pp. 877-944, Williams and Wilkins 17 Capaday, C. and Stein, R. B. (1986) J. Neurosci. 6, 1308-1313 18 Brown, A. G. (1981) Organization in the Spinal Cord Springer-Verlag 19 Mendell, L. M. (1984) PhysioL Rev. 64, 260-324
20 Wolpaw, J. R. (1985) Cell. Molec. NeurobioL 5, 147-165 21 Wolpaw, J. R., Braitman, D. J. and Seegal, R. F. (1983) J. NeurophysioL 50, 1296-1311 22 Wolpaw, J. R. (1983) Brain Res. 278, 299-304 23 Wolpaw, J. R., O'Keefe, J. A., Noonan, P. A. and Sanders, M. G. (1986) J. NeurophysioL 55, 272-279 24 Evatt, M. L., Wolf, S. L. and Segal, R. L. (1989) Neurosci. Left. 105, 350-355 25 Segal, R. L. etal. (1989) Soc. Neurosci. Abstr. 15, 917 26 Wolpaw, J. R. and O'Keefe, J. A. (1984) J. Neurosci. 4, 2718-2724 27 Wolpaw, J. R., O'Keefe, J. A., Kieffer, V. A. and Sanders, M. G. (1985) Neurosci. Left. 54, 165-171 28 Wolpaw, J. R. (1987) J. NeurophysioL 57, 443-458 29 Brown, W. F. (1984) The Physiological and Technical Basis of Electromyography pp. 144-148 and 474-479, Butterworth 30 Wolpaw, J. R., Lee, C. L. and Calaitges, J. G. (1989) Exp. Brain Res. 75, 35-39 31 Wolpaw, J. R. and Lee, C. L. (1989) J. Neurophysiol. 61, 563-572 32 Wolpaw, J. R., Carp, J. S. and Lee, C. L. (1989) Neurosci. Lett. 103, 113-119 33 Berthoz, A, and Melvill Jones, G., eds (1985) Adaptive Mechanisms in Gaze Control Elsevier 34 Miles, F. A,, Fuller, J. H., Braitman, D. J. and Dow, B. M. (1980) J. Neurophysiol. 43, 1477-1495 35 Myklebust, B. M., Gottlieb, G. L. and Agarwal, G. C. (1986) Dev. Med. Child Neurol. 28, 440-449 36 Goode, D. J. and Van Hoeven, J. (1982) Arch. NeuroL 39, 323 37 Meyer-Lohmann, J., Christakos, C. N. and Wolf, H. (1986) Exp. Brain Res. 64, 393-399 38 Mandl, G., Melvill Jones, G. and Cynacler, M. (1981) Brain Res. 209, 35-45 39 Capaday, C. and Stein, R. B. (1987) J. Neurosci. Met& 21, 91-104 40 Capaday, C. and Stein, R. B. (1987)J. PhysioL (London)392, 513-522 41 Wolpaw, J. R. and Herchenroder, P. A. J. Neurosci. Meth. (in press) 42 Clamann, H. P., Gillies, J. D., Skinner, R. D. and Henneman, E, (1974) J. NeurophysioL 37, 1328-1337
Retinoic acid, a developmental signalling molecule Dennis Summerbell and Malcolm Maden DennisSummerbellis at the National Institute for Medical Research,The Ridgeway,MilI Hill, LondonNW71AA, UKandMalcolm Maden is at the Departmentof Anatomy, King's College,Strand, London WC2R2LS, UK.
Retinoic acid has been used as a tool both by embryologists studying the spatial organization of cells in the embryo and by molecular biologists studying the control of gene expression in the nucleus. Embryologists have shown that retinoic acid can modify the pattern of cell differentiation so as to duplicate complete parts of the embryo in a well-organized way; molecular biologists have shown that retinoic acid can act as the switch starting the sequence of differential gene expression that results in cell differentiation. In the past year these two approaches have converged so that there now seems a real possibility that we may soon for the first time understand how a particular vertebrate development system works. In 1969 Lewis Wolpert 1 published his influential 'theory of positional information'. This was a development of the 'gradient theory' of Child2 and of the 'field theory' of Huxley and de Beer 3. It proposed that cells within a developing system had their position specified with respect to one or more reference points or organizing regions. This positional information was then fixed in a more or less stable form as the cell's positional value. Each cell interpreted its positional value according to its genome and developmental
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history by differentiating in a particular way. The suggestion was that positional value was established by having a graded concentration profile of a morphogen across the embryonic field. Each cell, measuring the local concentration, was able to determine its position relative to the organizers. This theory revitalized interest in the search for biochemical signals and during the past ten years a number of potential morphogens have been identified in a variety of developmental systems, including the Coelenterates 4, the slime mold, Dictyostelium 5, the amphibian blastula6, and the Drosophila embryo 7. This review is concerned with the role of retinoic acid in vertebrate limb development and regeneration, and in the control of differentiation.
The chick limb bud During early development the fertilized egg is transformed into a multicellular layered structure (comprising ectoderm, mesoderm and endoderm) with a head end, a tail end and bilateral symmetry about the mid-line. This is sometimes known as the formation of the primary body plan8. At this stage we would describe the whole embryo as comprising a single embryonic field. This first field maps out the
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press) 48 Frederiksen, K. and McKay, R. (1988) J. Neurosci. 8, 1144-1151 49 Raft, M. C. (1989) Science 243, 1450-1455 50 Giotta, G. J., Heitzmann, J. and Cohn, M. (1980) Brain Res. 202, 445-458 51 Rosenberg, M. B. et al. (1988) Science 242, 1575-1578 52 Whittemore, S. R., Holets, V. R., Gonzalez-Carvajal, M. and
Levy, D. (1988) Soc. Neurosci. Abstr. 14, 586 53 Bj6rklund, A. et aL (1987) Trends Neurosci. 10, 509-516 54 Albritton, L. M., Tseng, L., Scadden, D. and Cunningham, J. M. (1989) Ceil 57, 659--666 55 Geller, A. I. and Breakefield, X. O. (1988) Science 241, 1667-1669 56 Joyner, A. L., Skarnes, W. C. and Rossant, J. (1989) Nature 338, 153-156 57 Zimmer, A. and Gruss, P. (1989) Nature 338, 150-153 58 Kuehn, M. R., Bradley, A., Robertson, E. J. and Evans, M. J. (1987) Nature 326, 295-298 59 Hooper, M., Hardy, K., Handyside, A., Hunter, S. and Monk, M. (1987) Nature 326, 292-295
Memory tracesin spinal cord J o n a t h a n R. W o l p a w
The complexity and inaccessibility of the vertebrate CNS impede the localization and description of memory traces and the definition of the processes that create them. Recent work has shown that the spinal stretch reflex (SSR), which is produced by a monosynaptic twoneuron pathway, can be operantly conditioned, and that memory traces responsible for this behavioral change reside in the spinal cord. The probable locations are the terminal of the Ia afferent neuron on the motoneuron and/or the motoneuron itself. Because it modifies a simple well-defined and accessible pathway, SSR conditioning may be a valuable experimental model .for studying vertebrate memory. One of the ultimate goals of neuroscience research is to understand human memory in its most impressive and sophisticated manifestations, such as the ability to recognize a face seen briefly long ago or the capacity to master a language or a musical instrument. Immediate goals are more modest - to understand a rat's avoidance of a platform where a shock was delivered yesterday, or the acquisition by a monkey of the ability to move a lever smoothly against an opposing force. The objectives of ultimate and immediate goals are the same - to describe the memory trace, the change in the nervous system that is responsible for a particular change in behavior, and to describe learning, the process that creates the trace. The prerequisite for each is also the same - a memory trace must be located before it can be described. However, most behaviors result from activity at multiple sites in the CNS, and these sites and their interactions are usually not fully defined. As a result, this basic requirement of localization is the main impediment to progress. In response to the problem, three strategies have developed.
a n d J o n a t h a n S. C a r p
relatively simple nervous systems of invertebrates 3-5. Studies of this type have elucidated the simplest forms of memory, habituation and sensitization, and have begun to provide insight into simple forms of associative conditioning. The third strategy seeks to study memory traces in the complex nervous system of vertebrates. The problem of localization is addressed by choosing relatively simple behaviors 3'6-8. These vertebrate models are potentially the most rewarding because they are most closely related to human memory. However, they have so far proved most difficult to develop and use due to incomplete pathway definition, limited accessibility and/or susceptibility to input from elsewhere. A vertebrate model based on an even simpler behavior might overcome these difficulties. The spinal stretch reflex (SSR) The simplest behavior of the vertebrate CNS is the initial response to sudden muscle stretch. It is called the tendon jerk, the M1 response, or, as in this article, the spinal stretch reflex (SSR) 9-12, and can be measured by its mechanical effect or by EMG. The SSR is produced by the two-neuron monosynaptic pathway shown in Fig. 1. This pathway
Fig. 1. Dinal cord
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Motoneuron
Research strategies The first strategy circumvents the problem by using reduced preparations to study phenomena that might be similar to the memory traces that underlie behavioral changes in intact animals. During the past decade, this approach has focused on long-term potentiation in the hippocampus and elsewhere 1'2. The second strategy tries to minimize the problem of localization by studying memory traces in the TINS, Vol. 13, No. 4, 1990
Jonathan R. Wolpaw and Jonathan S. Carp are at the Wadsworth Center for Laboratoriesand Research,New York State Departmentof Health and State Universityof New York,Albany, NY 12201, USA.
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Two-neuron monosynaptic pathway of the spinal stretch reflex (SSR).It consistsof the la afferent neuron, its synapseon the :rmotoneuron, and the motoneuron itself. Sudden muscle stretch lengthens the musclespindle and excites the la afferent. The la afferent synapse excites the motoneuron and causesmuscle contraction.
Muscle
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The physiology and anatomy of this two-neuron pathway, and the characteristics of the behavior it mediates (i.e. the SSR) have been studied extensively since the time of Sherrington 1°' is. However, in spite of its unique simplicity and accessibility, the SSR pathway has attracted little interest as a model for the study of vertebrate memory. The spinal cord in general, and spinal reflexes in particular, are widely viewed as fixed and inflexible, responding in a stereotyped manner to inputs from the periphery or from supraspinal areas. This common perception is not correct. Spinal cord neurons and synapses, like those of cerebral cortex and other supraspinal structures, change in the course of development and in response to trauma 19'2°. This suggests that the spinal cord also changes with learning, i.e. that it can contain memory traces. Furthermore, the evidence suggests that memory traces form in a spinal cord pathway when the learning process involves prolonged change in the influences on the pathway.
A
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MODES: Control (all trials rewarded) Up (reward only if SSR > criterion) Down (reward only if SSR < criterion) Fig. 2. Experimental design. (A) Animal performing computer-controlled SSR conditioning task. The arm rests in a cast attached to a torque motor shaft. A rotary variable differential transformer below the motor monitors the elbow angle. Chronically implanted fine-wire electrodes monitor the biceps EMG. A light indicates the correct elbow angle. A solenoid-powered syringe derivers squirt of juice in reward. (B) SSR conditioning task. The animal maintains elbow angle and biceps EMG within preset ranges against a constant background extension torque from the motor. At the end of a randomly varying 1.2-1.8 s period, a brief pulse of additional extension torque extends the elbow and elicits the biceps SSR. In the control-mode, a reward squirt always occurs 0.2 s after the pulse. In the up- or down-mode, a reward squirt occurs only if the SSR (as measured by EMG) is above or below a criterion value. Animals average 3000-4000 trials per day. (Modified from Ref. 21 .) consists of the Ia afferent neuron, its excitatory synapse on the o~-motoneuron, and the motoneuron itself. The Ia afferent neuron innervates the muscle spindle and is excited by sudden muscle stretch. It in turn excites motoneurons that innervate the same muscle and its synergists, thereby causing muscle contraction, which opposes the sudden stretch*. While the SSR is wholly spinal, it can be affected by influence on the muscle spindle (exerted via fusimotor fibers13'14), on the Ia terminal (exerted via presynaptic inputs is' 16), and on the motoneuron (exerted via postsynaptic inputs17). * Other oligosynaptic pathways might contribute to the SSR; however, the data suggest that they do not have a significant role in the SSR conditioningdescribed here 21'28,31,32.
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The SSR can be operantly conditioned In the experiment shown in Fig. 2, monkeys are exposed to a conditioning task that requires prolonged change in neuronal activity influencing the SSR pathway, and thus is likely to produce a memory trace located in this pathway21. The animal maintains elbow angle and a specified level of ongoing biceps EMG against a constant extension torque. At an unpredictable time, a brief pulse of additional extension torque transiently extends the forearm and elicits the biceps SSR. Before the initiation of conditioning, the reward always follows the SSR ('control-mode'). During conditioning, the reward follows only if SSR size is above ('up-mode') or below ('down-mode') a criterion value. The crucial feature of this task is that muscle stretch occurs at an unpredictable time. Because the SSR is the earliest possible response, an animal can change SSR size only by maintaining a continual appropriate influence over the SSR pathway. By linking reward to SSR size, the up- or down-mode operantly conditions the animal to maintain such an influence. The expectation is that this continued influence produces a memory trace located in the SSR pathway, most probably at the Ia synapse on the motoneuron. By virtue of its location, this memory trace, and the process creating it, should be accessible to detailed physiological and anatomical study. Figure 3 shows what happens when an animal is switched from the control-mode to the up- or downmode. The size of the SSR changes appropriately in the ensuing days and weeks. On day 0, the SSR is nearly equal in the two animals. On day 80, the SSR of the up-mode animal is three times the size of the SSR of the down-mode animal. Furthermore, as Fig. 3 indicates, SSR conditioning changes the amplitude of the elbow extension caused by the torque pulse. As the SSR rises under the up-mode, it provides more opposition to the torque pulse and extension decreases. The opposite occurs under the down-mode. If the mode is switched (from up to down, or vice versa) the change in SSR size reverses in the same gradual fashion in which it first develops 22. If task performance is halted, change persists: gaps of several weeks in performance cause no loss of SSR decrease and only partial loss of SSR increase 23. TINS, Vol. 13, No. 4, 1990
Recent initial studies by Wolf, Segal and their colleagues24'~5 indicate that humans can also increase or decrease the size of the SSR when exposed to a comparable task. Humans and monkeys appear similar in both the time course and the magnitude of SSR change. Figure 4 combines data from many animals to define the development of SSR change 26. Within 6 h of the beginning of the up- or down-mode, SSR size increases (up-mode) or decreases (down-mode) by about 8%. This rapid initial jump, called phase I, is followed by very gradual change (1-2% per day) that continues for at least 40 days. This slow change is called phase II. Phase I change indicates that the animal quickly responds to the up- or down-mode with an appropriate alteration in influence on the SSR pathway. Phase II change suggests that the continuation of this influence over subsequent weeks gradually changes the Ia terminal or the motoneuron, and thereby further changes the SSR 26'27. However, the data leave open the possibility that the muscle spindle is the site of change or that change is only supraspinal and simply exerts steadily increasing influence over the SSR pathway. These issues are addressed by a modified form of the experiment shown in Fig. 2. The new design conditions a simplified form of the SSR, the H-reflex, in the triceps surae (posterior calf) muscles of the leg28. The H-reflex29 is elicited by direct stimulation of the Ia afferent fibers, so that the afferent volley eliciting the reflex does not depend on muscle spindle sensitivity. The absence of the spindle from the pathway does not prevent conditioning. Under the upmode, the size of the H-reflex gradually rises to about twice the initial size; under the down-mode it gradually falls to about two-thirds 2s. Little or no change occurs in the opposite (control) leg3°. These findings eliminate the muscle spindle as the site of change, and thus focus attention on the Ia terminal and the motoneuron. In addition, because this simplified design conditions the SSR pathway in the lumbosacral cord, it facilitates determination of whether the conditioning task does create a memory trace in the spinal cord*. Memory traces are present in spinal cord If the conditioning task does produce a memory trace in the spinal cord itself, then eviflence of the trace should remain even if the spinal cord is isolated from supraspinal influence by transection. This can be tested by anesthetizing animals in which the H-reflex of one leg has been increased (up-mode) or decreased (down-mode), transecting the spinal cord above the lumbosacral site of the reflex pathway, and measuring the reflexes on both sides under continued anesthesia for several days. The anesthetized transected animals display reflex asymmetries comparable to those seen in the awake behaving animals 31'32. These asymmetries are still present three days after transection. In up-mode animals, the reflex remains larger in the conditioned leg than in the control leg. In down-mode animals, it , The H-reflex conditioning task has the additional advantage of permitting conditioning of the SSR pathway in an unrestrained, fxeely moving animal41. TINS, Vol. 13, No. 4, 1990
10
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Fig. 3. Effects of up- and down-modes on SSR size and on movement. (A) Data from an animal exposed to the up-mode for 80 days. The five upper graphs show average daily SSR size for every 20th day beginning with the imposition of the up-mode. SSR size increases steadily without change in background EMG (represented here by the 12 ms before SSR onset). The single lower graph shows pulse-induced elbow extension before (solid line) and after (dashed line) up-mode exposure. Initial extension, the stimulus that elicits the SSR, is the same for both. Beginning at 35-40 ms, extension is less after upmode exposure than before, because the larger biceps SSR provides more opposition. The 20 ms delay between the SSR and its effect on movement is presumably due to the time needed for muscle contraction after excitation. (B) Analogous data from an animal exposed to the down-mode. A steady decrease in SSR size occurs without change in background EMG or in the initial pulse-induced extension. Beyond 35-40 ms, extension is greater after downmode exposure, because the smaller SSR gives less opposition. (Modified from Ref. 26.)
remains smaller in the conditioned leg than in the control leg. Figure 5 shows examples of these persistent asymmetries. Their existence indicates that, when monkeys modify the behavior mediated by the SSR pathway, a significant part of the memory trace responsible for the altered behavior resides in the spinal cord. Intended and compensatory plasticity Reflex asymmetry is not the only effect of conditioning that is apparent in the anesthetized transected animals. As Fig. 5 illustrates, reflexes are larger in the control legs of down-mode animals than in the control legs of up-mode animals. This difference is not present before anesthesia and transection; control-leg reflexes do not change over the weeks of 139
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Fig. 4. Two-phase course of 55R change. Average 55R size under the up- or down-mode for 18 mode exposures (11 up and seven down) in nine animals. Each value is 55R size (+_SEM)as a percentage of initial size. Values are shown for hours 0-6, 6-12 and 12-24 of the first day, for each subsequent day through 40 days, and for every two days from then on. The inset is a horizontally expanded view of the first week. The superimposed lines are computer-fits of the equation 'y = (a/ (b+(l/x))) + c'. (See Ref. 26 for details of calculations.) Each curve shows a nearly immediate (within 6 h) modeappropriate offset (i.e. phase I change), followed by slow change (i.e. phase II change) that continues indefinitely. (Modified from Ref. 26.)
conditioning, and are of comparable size in awake behaving up- and down-mode animals a°. Thus, anesthesia and transection reveal an effect of conditioning that is not apparent in the awake behaving animal. The origin of this unexpected change is obscure; nevertheless, its occurrence raises an important issue. Every learning process has a specific goal, an intended change in behavior. In the present context, the goal is defined by the experimenter, i.e. a larger or smaller SSR. The memory trace responsible for this behavioral change can be called 'intended plasticity'. Depending on its nature and location, this trace Fig. 5. Evidence for spinal cord memory traces. Average reflexes in conditioned and control legs of an up-mode animal and a down-mode animal, both under deep pentobarbital anesthesia, 2-3 days after thoracic spinal cord transection eliminates supraspinal influence. Conditioned-leg reflexes are shown by dashed lines, and control-leg reflexes by solid lines. Reflexes are elicited by supramaximal dorsal root stimulation and recorded from the nerve branches supplying the triceps surae. The horizontal bar is 1 ms and the vertical bar is 20% of the maximum possible response (i.e. the response to supramaximal ventral root stimulation 4 2"), Mode-appropriate reflex asymmetry is present in both animals: in the up-mode animal the reflex is bigger in the conditioned leg than in the control leg, and in the down-mode animal it is smaller in the conditioned leg than in the control leg. In addition, the control-leg reflex of the down-mode animal is bigger than the control-leg reflex of the up-mode animal Both these effects of conditioning were evident throughout the several days of post-transection study. (See Ref. 31 for full presentation.) 140
may have additional effects: it may interfere with other behaviors that involve some of the same neurons and synapses. Thus, it may provoke 'compensatory plasticity', modifications that allow the CNS to continue to perform these other behaviors properly. For example, suppose that up-conditioning causes greater transmitter release at the triceps surae Ia synapse. In addition to affecting the SSR, greater transmitter release would also affect other behaviors in which this synapse participates, such as walking or jumping. Compensatory plasticity would be needed to maintain proper performance (perhaps by increasing a specific inhibitory input to the triceps surae motoneuron). Compensatory plasticity may also accompany conditioning of the vestibulo-ocular reflex (VOR), the behavior that maintains a stable retinal image during head movement 7's'33. The sensitivity of floccular Purkinje cells to vestibular input appears to change in a direction opposite to that able to account for the change in VOR gain34. This apparently paradoxical change in floccular response to vestibular input may compensate for change in eye movement input to the flocculus, and thereby properly adjust the contribution of the cerebellum to eye movement 8. If compensatory plasticity does occur, it has two important implications for efforts to define the memory traces underlying specific changes in behavior. First, it indicates that change in even the simplest behavior involves CNS plasticity beyond that responsible for the intended behavioral change. Second, it emphasizes the extreme importance of simplicity in experimental models used to study memory traces. Without the relative simplicity of the SSR and VOR pathways, these putative compensatory changes would be difficult to detect and exceedingly difficult to analyse. R e l a t i o n s h i p s to other t y p e s of learning The memory traces created by SSR conditioning are probably not limited to studies specifically designed to produce them. Evidence is accumulating that SSR changes are common. In humans, gradual changes in the SSR accompany the acquisition of basic motor skills early in life35. They also appear to occur later in life during the learning of skills such as ballet a6. In a highly relevant study, Meyer-Lohmann et al. 37 trained monkeys to move a lever to a target, and randomly perturbed this movement with a brief torque pulse. Initially, the animal's response to perturbation was dominated by the later M2 component of the response to muscle stretch. However, Up-mode animal /\ / \ I I//~,\
Down-mode animal
\
TINS, VoL 13, No. 4, 1990
A B C over months of gaining, the M1 component (i.e. the SSR) gradually Presynaptic Whole Local motoneuron postsynaptic increased in size and M2 disappeared. In effect, the SSR gradually took over the task of opposing the perturbation. Gradual changes in the spinal cord and elsewhere in the CNS Phase probably underlie many learning I processes, and help explain why the mastery of many skills requires intense practice over long periods. The need for prolonged practice is not limited to sophisticated motor behaviors: non-motor skills also require it. For example, the acqui- Phase sition of skill in basic mathematical II operations such as multiplication is a gradual, labor-intensive process. The development of SSR change under the up- or down-mode (Fig. 4) has a course common to many Fig. 6. Possible locations of the spinal cord memory traces responsible for change in SSR size. In learning curves: improvement is each case, phase I change in influence on a site in the SSRpathway (indicated by arrows) eventually rapid at first and then continues at produces phase II change (i.e. the memory trace) at the site. (A) The most likely site is the la a gradually decreasing rate for a afferent terminal on the motoneuron. A memory trace here could be produced by prolonged long time. In the case of the SSR, change in presynaptic inhibition. (B) The memory trace could be a generalized change in the this form appears to be the result motoneuron that alters its response to any input. However, such a modification would have widespread effects on motoneuron function. (C) The trace could also be a very localized of a two-phase sequence: the rapid modification in the postsynaptic membrane, such as a change in receptor sensitivity or dendritic onset of appropriate influence on architecture. the SSR pathway causes phase I change, and the long-term continuation of this influence produces spinal modifications motoneuron itse#. Transmission through the Ia synapse is inhibited by that underlie slow phase II change (see below). Change in the VOR, another simple behavior, also presynaptic inputs from several supraspinal sites 1s'16. displays two distinct phases a8, but learning curves for Recent studies suggest that short-term changes in more complex behaviors typically do not. This dif- presynaptic inhibition have an important role in motor ference is not surprising. Changes in more complex behaviora9'4°. Long-term change in this inhibition behaviors probably involve appropriate adjustments in might modify the Ia terminal (Fig. 6A), perhaps many influences (i.e. many phase I changes), and affecting depolarization-induced calcium entry and these adjustments probably gradually modify many transmitter release, and thereby altering the size of sites in the CNS (i. e. many phase II changes). These the EPSP produced in the motoneuron. Motoneuron membrane properties, such as those multiple two-phase sequences presumably overlap and obscure each other, so that distinct phases are controlling resting potential or input resistance, help determine the response of the motoneuron to any not apparent. input, and might be modified (Fig. 6B) by prolonged changes in descending influence. However, the conMemory trace location(s) in the spinal cord* The most likely locations of the spinal cord memory straints imposed by the need to maintain constant traces created by SSR conditioning are shown in Fig. motoneuron tone during conditioning (Fig. 2 legend), 6. They are the sites in the SSR pathway that are and the great interference that such alterations would subject to influence from supraspinal structures, the exert on most behaviors involving the motoneuron, Ia afferent terminal on the motoneuron and the make generalized membrane modifications a more complicated and less probable explanation for SSR change than presynaptic (Fig. 6A) modification. A *The present discussion focuses on the possible locations of very localized postsynaptic modification (Fig. 6C), the memory trace responsible for the mode-appropriate perhaps in receptor sensitivity or dendritic architecreflex asymmetry. Discussion of the basis of the difference ture, could give greater specificity. However, pathin control-leg reflexes of anesthetized transected animals is ways that could produce such a selective modification comparable and appears elsewhere 31. are not yet defined. At present, a modification of the Ia terminal caused It is unlikely that the memory trace is limited to other by a prolonged change in presynaptic inhibition is spinal cord sites and simply influences the SSR pathway, probably the best candidate for the memory trace. because conditioned reflex changes are prominent even under deep pentobarbital anesthesia 31's2, which suppresses The significance of SSR conditioning as a model for neuronal and synaptic function and thereby tends to isolate studying memory is that the simplicity of the pathway neurons from one another. If a memory trace were not allows the search for the memory trace to focus on present at the Ia terminal or the motoneuron, its effect on this single synapse and its postsynaptic neuron, both reflex size in the pentobarbital-anesthetized animal would be of which are accessible physiologically and anaminimal. tomically. TINS, VoL 13, No. 4, 1990
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Acknowledgements Selected references We thankfiobertJ.
Brady,DavidO. Carpenter, ChongL. Lee, DavidL Martin, DennisJ. McFartand, Barry W. Peterson, RichardF. Seegal,N. TraverseSlaterand Elizabeth W. Wolpaw for criticalreviewof the manuscript.Our work wassupported in part by NIHgrant N522189, the United Cerebra/Palsy Researchand Educational Foundationand the SpinalCordResearch Foundation.
1 Teyler, T. J. and Discenna, P. (1984) Brain Res. 7, 15-28 2 Gustafsson, B. and Wigstrom, H. (1988) Trends Neurosci. 11, 156-162 3 Byrne, J. H. (1987) PhysioL Rev. 67, 329-439 4 Abrams, T. W. and Kandel, E. R. (1988) TrendsNeurosci. 11, 128-135 5 Crow, T. (1988) Trends Neurosci. 11, 136-142 6 Thompson, R. F. (1988) Trends Neurosci. 11, 152-155 7 Ito, M. (1982) Annu. Rev. Neurosci. 5, 275-296 8 Lisberger, S. G. (1988) Science 242, 728-735 9 Lee, R. G. and Tatton, W. G. (1975) Can. J. NeuroL Sci. 2, 285-293 10 Mat-thews, P. B. C. (1972) in Mammalian Muscle Receptors and Their Central Actions pp. 319-409, Williams and Wilkins 11 Henneman, E. and Mendell, L. M. (1981) in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 423-507, Williams and Wilkins 12 Magladery, J. W., Porter, W. E., Park, A. M. and Teasdall, R. D. (1951) Electrophysiological Studies of Nerve and Reflex Activity in Normal Man, IV: The Two-neuron Reflex and Identification of Certain Action Potentials from Spinal Roots and Cord pp. 499-519, Bull. John Hopkins Hospital 13 Harris, D. A. and Henneman, E. (1980) in Medical Physiology, VoL I (Mountcastle, V. B., ed.), pp. 703-717, Mosby 14 Mat-thews, P. B. C. (1981)in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 189-228, Williams and Wilkins 15 Baldissera, F., Hultborn, H. and Illert, M. (1981)in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I1: Motor Control) (Brooks, V. B., ed.), pp. 509-595, Williams and Wilkins 16 Burke, R. E. and Rudomin, P. (1977) in Handbook of Physiology (Sect. 1: The Nervous System; Vol. I: Cellular Biology of Neurons) (Kandel, E. R., ed.), pp. 877-944, Williams and Wilkins 17 Capaday, C. and Stein, R. B. (1986) J. Neurosci. 6, 1308-1313 18 Brown, A. G. (1981) Organization in the Spinal Cord Springer-Verlag 19 Mendell, L. M. (1984) PhysioL Rev. 64, 260-324
20 Wolpaw, J. R. (1985) Cell. Molec. NeurobioL 5, 147-165 21 Wolpaw, J. R., Braitman, D. J. and Seegal, R. F. (1983) J. NeurophysioL 50, 1296-1311 22 Wolpaw, J. R. (1983) Brain Res. 278, 299-304 23 Wolpaw, J. R., O'Keefe, J. A., Noonan, P. A. and Sanders, M. G. (1986) J. NeurophysioL 55, 272-279 24 Evatt, M. L., Wolf, S. L. and Segal, R. L. (1989) Neurosci. Left. 105, 350-355 25 Segal, R. L. etal. (1989) Soc. Neurosci. Abstr. 15, 917 26 Wolpaw, J. R. and O'Keefe, J. A. (1984) J. Neurosci. 4, 2718-2724 27 Wolpaw, J. R., O'Keefe, J. A., Kieffer, V. A. and Sanders, M. G. (1985) Neurosci. Left. 54, 165-171 28 Wolpaw, J. R. (1987) J. NeurophysioL 57, 443-458 29 Brown, W. F. (1984) The Physiological and Technical Basis of Electromyography pp. 144-148 and 474-479, Butterworth 30 Wolpaw, J. R., Lee, C. L. and Calaitges, J. G. (1989) Exp. Brain Res. 75, 35-39 31 Wolpaw, J. R. and Lee, C. L. (1989) J. Neurophysiol. 61, 563-572 32 Wolpaw, J. R., Carp, J. S. and Lee, C. L. (1989) Neurosci. Lett. 103, 113-119 33 Berthoz, A, and Melvill Jones, G., eds (1985) Adaptive Mechanisms in Gaze Control Elsevier 34 Miles, F. A,, Fuller, J. H., Braitman, D. J. and Dow, B. M. (1980) J. Neurophysiol. 43, 1477-1495 35 Myklebust, B. M., Gottlieb, G. L. and Agarwal, G. C. (1986) Dev. Med. Child Neurol. 28, 440-449 36 Goode, D. J. and Van Hoeven, J. (1982) Arch. NeuroL 39, 323 37 Meyer-Lohmann, J., Christakos, C. N. and Wolf, H. (1986) Exp. Brain Res. 64, 393-399 38 Mandl, G., Melvill Jones, G. and Cynacler, M. (1981) Brain Res. 209, 35-45 39 Capaday, C. and Stein, R. B. (1987) J. Neurosci. Met& 21, 91-104 40 Capaday, C. and Stein, R. B. (1987)J. PhysioL (London)392, 513-522 41 Wolpaw, J. R. and Herchenroder, P. A. J. Neurosci. Meth. (in press) 42 Clamann, H. P., Gillies, J. D., Skinner, R. D. and Henneman, E, (1974) J. NeurophysioL 37, 1328-1337
Retinoic acid, a developmental signalling molecule Dennis Summerbell and Malcolm Maden DennisSummerbellis at the National Institute for Medical Research,The Ridgeway,MilI Hill, LondonNW71AA, UKandMalcolm Maden is at the Departmentof Anatomy, King's College,Strand, London WC2R2LS, UK.
Retinoic acid has been used as a tool both by embryologists studying the spatial organization of cells in the embryo and by molecular biologists studying the control of gene expression in the nucleus. Embryologists have shown that retinoic acid can modify the pattern of cell differentiation so as to duplicate complete parts of the embryo in a well-organized way; molecular biologists have shown that retinoic acid can act as the switch starting the sequence of differential gene expression that results in cell differentiation. In the past year these two approaches have converged so that there now seems a real possibility that we may soon for the first time understand how a particular vertebrate development system works. In 1969 Lewis Wolpert 1 published his influential 'theory of positional information'. This was a development of the 'gradient theory' of Child2 and of the 'field theory' of Huxley and de Beer 3. It proposed that cells within a developing system had their position specified with respect to one or more reference points or organizing regions. This positional information was then fixed in a more or less stable form as the cell's positional value. Each cell interpreted its positional value according to its genome and developmental
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history by differentiating in a particular way. The suggestion was that positional value was established by having a graded concentration profile of a morphogen across the embryonic field. Each cell, measuring the local concentration, was able to determine its position relative to the organizers. This theory revitalized interest in the search for biochemical signals and during the past ten years a number of potential morphogens have been identified in a variety of developmental systems, including the Coelenterates 4, the slime mold, Dictyostelium 5, the amphibian blastula6, and the Drosophila embryo 7. This review is concerned with the role of retinoic acid in vertebrate limb development and regeneration, and in the control of differentiation.
The chick limb bud During early development the fertilized egg is transformed into a multicellular layered structure (comprising ectoderm, mesoderm and endoderm) with a head end, a tail end and bilateral symmetry about the mid-line. This is sometimes known as the formation of the primary body plan8. At this stage we would describe the whole embryo as comprising a single embryonic field. This first field maps out the
© 1990,ElsevierSciencePublishersLtd,(UK) 0166 2236/90/$02.00 -
TINS, Vol. 13, No. 4, 1990