Stimulusdassiflcationby ensemblesof climbingfiber receptivefields Lee T. R o b e r t s o n a n d G i n M c C o l l u m
Lee T. Robertsonis at OregonHealth SciencesUniversity, Deptof Anatomy, Schoolof Dentistry, 611 SW Campus Drive, Portland, OR 97201, USAand GinMcCollumisat RobertS. Dow NeurologicalSciences Institute, Good SamaritanHospital and MedicalCenter, 1120NW 20th Avenue, Portland, OR 97209, USA.
Although the local structure of the cerebellum is fairly uniform and its inputs are often widely shared, outputs .from different regions of the cerebellar cortex reach different parts of the cerebellar and vestibular nuclei, which can affect the rest of the nervous system in different ways. In this review, we explain how different ensembles of climbing fiber responses in the anterior lobe and paramedian lobule can be generated by a tactile stimulus to the distal hindpaw. Apart from differing in degree of activation, the cortical regions differ also in the detailed pattern of the activation transmitted. The anterior lobe can distinguish a greater divers@ of stimuli to various skin surfaces than can the paramedian lobule. This differential classification of particular stimulus arrays by the two cerebellar regions could produce distinct patterns of neuronal activity in various corticonuclear compartments. The nervous system achieves certain goals by correlating information from many receptors and by ordering the implementation of movement complexes. The cerebellum, as part of the motor system, receives input from receptors in a manner such that certain information can be used to enhance the smooth execution of particular movements. This article describes how the cerebellar cortex transforms tactile stimuli into spatial maps, in which the stimuli are encoded by activation of an ensemble of Purkinje cells - the output neurons of the cerebellar cortex. Although peripheral information is conveyed to the cerebellar cortex by the climbing and mossy fibers, this article focuses only on the climbing fiber system because its distinct postsynaptic excitatory influence on the Purkinje cell provides a unique opportunity to investigate a particular afferent system, and because climbing fiber input has the same effect on the Purkinje cell, regardless of the type of peripheral stimulation. Since the type of stimulation does not appear to be encoded, the location of the peripheral stimulus might be the significant element, which is revealed in its receptive field. We expand on the idea that ensembles of Purkinje cells process complex stimuli but that subsets of Purkinje cells operate differentially under varying stimulus conditions. Erickson 1 describes a nervous system in which ensembles of neurons are transformed at each synapse from peripheral sensory input to motor output. We describe one step in that sequence of transformations and two variations of the transformation (anterior lobe versus the paramedian lobule) within the cerebellar cortex. In this review, we recount briefly the different schemes to localize peripheral information in the cerebella cortex, describe the pattern of climbing fiber receptive field organization, compare the transformation of tactile stimuli in the anterior lobe versus the paramedian lobule and consider how tactile information might be processed in the cerebellum.
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Localization of peripheral information Three major concepts have been proposed for the organization of the body surface in the cerebellar cortex. The first concept of somatic representation was the 'distorted homunculus' scheme, which was originally proposed by Snider 2 (Fig. 1A). In this scheme, it is suggested that a point-to-point representation exists between the body surface and cerebellar cortex. By this scheme, the location of a tactile stimulus is specified according to a pixel strategy where the receptive fields are as small as the resolution required and are arranged with little overlap 3. Areas such as the distal extremities or the face presumably have small receptive fields and, therefore, occupy a large region of the cerebellar cortex. Areas such as the back have large receptive fields and are represented by relatively few Purkinje cells. The pixel strategy appears to occur also in the dorsal horn of the spinal cord 4 and the cerebral cortex S. Another organizational concept is based on anatomical and physiological evidence of a longitudinal arrangement of afferent and efferent cerebellar fibers, as well as various molecular correlates e-l°. Physiological studies that elicit climbing fiber responses by electrical stimulation of major peripheral nerves have identified eight longitudinal zones in the anterior lobe on the basis of latency, spinal pathway, and differences between ipsilateral and contralateral representations of the forelimb and hindlimb nerves (Fig. 1B) s'u-13. Some longitudinal zones are subdivided further, the divisions being based on the nerves represented and the origin of branching olivary axons 14'15. The concept of longitudinal organization suggests that stimulation of certain peripheral nerves will produce synchronous activity in longitudinal zones. Presumably, localization of tactile stimulation will be accomplished by activating a longitudinal zone or a portion of a zone that represents a dermatome supplied by a particular peripheral sensory nerve. However, some stimuli might produce a more complex pattern of activations than exciting simply a single zone, since a single climbing fiber can branch to contact many Purkinje cells that might be widely separated or in distinct zones or lobules ~6'17. The third organizational concept involves a patchlike arrangement where adjacent regions might contain representations of a noncontiguous body area and where patches of climbing fiber responses represent an assortment of receptive fields (Fig. 1C). The patch-like arrangement of the climbing fiber responses has been found consistently in studies employing natural tactile stimulation of the skin surface instead of electrical stimulation of nerves and using high resolution extracellular mapping procedures in cats anesthetized with sodium pentobarbital 18-21. Welker and associates 22 also describe a similar patch-like arrangement for the mossy fiber system.
© 1991,ElsevierSciencePublishersLtd,(UK) 0166-2236/91/$02.00
TINS, VOI. 14, NO. 6, 1991
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Fig. 1. Three organizational schemes of representation of the body are shown for Iobules III-VI of the anterior lobe. The boxed area on the diagram of the cerebellum of the cat (lower right) shows the region of Iobules IIl-Vl. (A) The body surface is represented as a continuous dermatomal sequence on the dorsal surface of the cerebellum. Each body area is represented in a specific cerebellar region. (Modified from Ref. 2 .) (B) Longitudinal zones (a, b, cl, c2, c3 and d) of the cat cerebellum are associated with short-latency afferent pathways of forelimb and hindlimb nerves. In this scheme, dermatomes of various sensory nerves would be represented in a certain longitudinal zone or zones. (Modified from Ref. 11 .) (C) Different areas of the skin surface are represented in a patch mosaic when tactile stimulation is used to elicit the responses. The parasagittal section [insert; the approximate location of which is indicated by the dashed line on the dorsal cerebellar surface in (C)] shows that the patches and overlapping patches extend into the deeper parts of the Iobules. Various body areas are represented in multiple patches, sometimes overlapping with other body areas. (Modified from Ref. 21.)
Within each patch there is a diversity of receptive fields that can include a mixture of large and small areas of skin, a blend of proximal and distal areas, or various combinations of glabrous and hairy surfaces. Each patch contains a unique combination of receptive fields, although some receptive fields are represented in multiple patches. The distribution and diversity of the climbing fiber receptive fields in various cortical regions suggest that most tactile stimuli would TINS, Vol. 14, No. 6, 1991
produce a complex pattern of cortical activity. For example, tactile stimulation of just the ventral (volar) surface of the forepaw would not be sufficient to drive all the climbing fiber responses of any longitudinal zone of the anterior lobe, but would activate a complex pattern of responses in several zones of the anterior lobe, as well as in various parts of the paramedian lobule, dorsal paraflocculus and posterior vel-mis21,23-25" 249
intermediate zone of lobules III-V of the anterior lobe and throughout • i/~ the caudal portion (sublobule C) of i the paramedian lobule. The all-ornothing properties of the climbing fiber response allowed us to define "., I' "-. I/ Ankle // the boundaries of the receptive \/x\ Dors // fields. Overlapping all the hindpaw receptive fields on a schematic / A " " ," drawing of the paw revealed that C "-,, / .'\ most receptive fields conform to the anatomical boundaries of the paw, and that most share common Tips/claws boundaries. Figure 2 shows four receptive fields (A-D) that are superimposed on the schematic drawing of the paw as projected onto a fiat surface (the dorsal, ventral, medial and lateral surfaces . . s ,' / on the same plane). The bound,i aries of several receptive fields L ',.,, / ~ntral Pad overlap. For example, the boundaries of receptive fields A, B and D I / B / Medial ~ Lateral share portions of the boundary of Fig. 2. Four examples of receptive fields of the hindpaw (A-D) are superimposed on a diagram of the central pad; receptive fields B the distal extremity as projected onto a plane, which allows all skin surfaces to be represented in the and D share a boundary with the same drawing, Most receptive fields share boundaries with other receptive fields and several medial-middle toe. Because most receptive fields are included within larger receptive fields. receptive fields share common boundaries with other receptive Overlapping receptive fields show fields, the paw can be described as being organized in asymmetrical regional distribution compartments of the skin surface. Compartments can To gain an understanding of regional variations in be defined as the smallest area of the skin that the the climbing fiber representations, we initially focused climbing fiber system can resolve 3, even though each on the receptive fields limited to the distal hindpaw a. compartment does not necessarily occur as a single High density recordings (with a 300-400 #m inter- receptive field. For the distal hindpaw, when 136 electrode penetration distance) were made in the receptive fields of the hindpaw are superimposed using the schematic drawing of the paw, then their common boundaries delineate 42 compartments, which are shown in the representation of the paw of Anterior Lobe Paramedian Lobule Figs 3-5. Most of the receptive fields are unions of two or more compartments. In Fig. 2, receptive field D is the union of the two middle toes, which also include the two toe pad compartments. Some receptive fields are also included completely in a larger receptive field. The toe pad of receptive field C is included in the receptive field of the two middle toes (D), which is included in the receptive field of the three lateral toes (B). Since stimulation of all parts of a receptive field can elicit a climbing fiber response, stimulation of the lateral-middle toe pad would also activate responses with receptive fields that include the lateral-middle toe pad. Inclusions provide an indication of the effectiveness Proportion ot C l i m b i n g Fil~er Responses of each area of the paw in activating a population of responses. A population of receptive fields can >0 >10 >20 >30 >40 >50 >60 preferentially include a particular compartment or set of neighboring compartments. Most hindpaw recepFig. :3. When 155 hindpaw receptive fields, obtained from climbing fiber tive fields of the paramedian lobule are concentrated responses in the rostral anterior lobe, and 198 hindpaw receptive fields from around the lateral-middle toe (Fig. 3). Thus, preferenthe paramedian Iobule were overlapped on the schematic drawing of the paw, tially stimulating the lateral-middle toe could synthe proportion of each compartment included in the total number of hindpaw receptive fields was revealed for each region. In the paramedian Iobule, the chronously activate a large subset of Purkinje cells in hindpaw receptive fields are centered around the three lateral toes, with the the paramedian lobule and a small set in the anterior highest proportion involving the lateral-middle toe. In the anterior lobe, the lobe, which contains a comparatively equal distrifields are concentrated around the three medial toes and involve compart- bution of compartments. In contrast, a stimulus ments of the lateral toe and the distal dorsal part of all toes. (See Fig. 2 for applied only to the medial toe could activate a rather details of the schematic representation of the paw.) small subset of responses in the paramedian lobule, A~
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Whereas the population of responses that could be activated in the anterior lobe would be similar in size to the population activated by stimulation of the lateral-middle toe. The two slightly separated stimuli could elicit strongly different balances of the activation of the paramedian lobule and anterior lobe.
Comparison of anterior lobe and paramedian lobule transformations of stimuli The transformation from a particular stimulus to a response ensemble in each region is characterized by its set of receptive fields. For this review, we assume that tactile stimulation of a skin area will synchronously activate all the climbing fiber responses with receptive fields that include the stimulated area. In actual fact, a more complex pattern is likely because of particular coupling mechanisms or the inhibition of inferior olivary neurons 26-~8, although the details of how these mechanisms influence afferent input are not yet understood. Because different sets of receptive fields exist in the anterior lobe and in the paramedian lobule, coincident tactile stimulation of various combinations of toes could result in different response ensembles activated in the two regions. The transformation of stimuli to the ventral surface of the hindpaw toes illustrates this point. For this illustration, we limited the receptive fields to the ventral surface of the four toes and did not consider the other compartments (such as pads, the area surrounding the pads, and toe tips). The first step in determining the transformation is to identify in each region the set of receptive fields that includes part or all of the ventral toe surfaces. In the anterior lobe, receptive fields exist for each of the four toes and for almost all neighboring combinations (left diagram in Fig. 4). The receptive fields are organized as an inverted pyramid - the four single Anterior Lobe
toes on top, then the three neighboring pairs (the lateral and lateral-middle, the two middle, and the medial-middle and medial toes), the two neighboring triples (lateral to medial-middle toes, medial to lateralmiddle toes) and, at the bottom, all four toes. This organization reflects an unbroken rule among receptive fields: whenever two or more toes on the same paw are involved in a receptive field, the toes are neighbors 29. In the paramedian lobule, receptive fields involving the medial toes are conspicuously absent, that is, no single medial or medial-middle toe, no medial pair, and no medial triple were identified (right diagram in Fig. 4). Stimulation of various combinations of the toes (a stimulus array) would produce different response ensembles in the two regions. We assume that the response to coincident stimulation of more than one toe is the union of responses to individual toe stimulation. For example, touching all four toes produces a response ensemble involving all the receptive fields that includes any of the toes. In the anterior lobe, touching all four toes would produce a response ensemble of all the possible receptive fields; in the paramedian lobule, the response ensemble would be only those receptive fields that include the lateral and the lateral-middle toes (Fig. 4). Because of its larger set of receptive fields, the anterior lobe can distinguish more stimulus combinations of the toes than can the paramedian lobule. Different stimulus arrays elicit different response patterns in the anterior lobe, but might elicit identical response patterns in the paramedian lobule. The possible combinations of stimuli to the ventral toes consist of 16 stimulus arrays. Each stimulus array elicits a specific response ensemble in the anterior lobe. In contrast, only seven different response ensembles are given for the 16 stimulus arrays in the paramedian lobule (boxes A-G in Fig. 5). Paramedian Lobule
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Fig. 4. A limited set of hindpa w receptive fields exempfifies differences between the distributions of receptive fields identified for the anterior lobe and the paramedian Iobule. The set is organized as an inverted pyramid: the top row is receptive fields of individual toe pads and the surroundinE hairy surface (the medial toe is on the left side whereas the lateral toe is on the right), the second row consists of pairs of toes, the third row shows three toes, and the bottom receptive field is of all four toes. Not encountered in the paramedian Iobule were any responses representing only the medial or medial-middle toes, the two medial toes, or the three medial toes (open circles). TINS, VoL 14, No. 6, 1991
251
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• _@@ (4iLl Fig. 5. With the inverted pyramid organization of the hindtoe receptive fields (shown in Fig. 4), various response ensembles could be elicited by stimulating (*) various combinations of toes. Each of the 16 different stimulus combinations of the toes could evoke a different response ensemble in the anterior lobe (AL), but the same stimulus combinations could evoke only seven different response ensembles (boxes A to G) in the paramedian Iobule (PML). For example, in box E, the four different stimulus combinations could evoke different response ensembles among the receptive fields of the anterior lobe but the same response ensemble in the paramedian Iobule, which are equivalent to each other. Thus, the hindtoe climbing fiber receptive fields of the anterior lobe can distinguish a greater diversity of stimulus combination than those of the paramedian Iobule.
Consider the stimulation of only the lateral toe (Fig. 5D). Four receptive field types (the lateral toe, the two lateral toes, the three lateral toes, and all four toes) include the lateral toe, so they would be activated by its stimulation in both the anterior lobe and paramedian lobule. The coincident stimulus combination of the medial toe with the lateral toe would activate the same four receptive fields that include the lateral toe, but the three other receptive fields that include the medial toe - the medial toe, the two medial toes, and the three medial toes - would be activated in the anterior lobe. Stimulation of either the lateral or the medial toe would activate the response with the receptive field of all four toes, but, in the paramedian 252
lobule, the response ensemble activated by the lateral toe would not be augmented by coincident stimulation of the medial toe. In the paramedian lobule, simultaneous stimulation of the medial and lateral toes is indistinguishable from stimulation of the lateral toe alone. These two stimulus arrays belong to a set that are equivalent with respect to the responses in the paramedian lobule, but each of the two stimuli elicits a distinct response ensemble in the anterior lobe. Within the seven response ensembles in the paramedian lobule, the different stimulus arrays are equivalent to each other. In addition to response ensembles for stimulation of the medial (Fig. 5C) and the lateral toes (Fig. 5D), the equivalent responses TINS, Vol. 14, No. 6, 1991
exist for stimulation of the medial-middle toe or the medial and medial-middle toes (Fig. 5A), for stimulation of lateral-middle toe alone or with combinations of the two medial toes (Fig. 5E), for stimulation of the two lateral toes with combinations of the two medial toes (Fig. 5F), or for stimulation of the lateral toe with the medial-middle toe or both medial toes (Fig. 5G).
Information processing in the cerebellum The balance of total climbing fiber activity between the anterior lobe and paramedian lobule can vary sharply depending on the stimulus location. There is also a difference in the representation of detail, with the paramedian lobule equating stimuli that are distinguished by the anterior lobe. Although these conclusions are based on assumptions that might be replaced in the future by more sophisticated information, knowing the features of the receptive fields is a valuable step towards understanding the transformation of neural activity carried out by the cerebellum. Ensemble activity, which we are currently studying, is expected to depend on the properties of individual olivary neurons, especially the long refractory period and the tendency to oscillate a°,al. These intrinsic electrophysiological properties of olivary neurons might influence how incoming stimuli are transformed into sets of climbing fiber responses. For example, the quick sequential stimulation of the lateral toe and then the lateral-middle toe would produce a different set of responses than the reverse stimulation sequence (stimulation of the lateral-middle toe followed by the lateral toe). Since the cells activated by the first stimulus are refractory when the second stimulus arrives, the set of responses for the second stimulus would not include responses with receptive fields that encompass the area of the first stimulus. The intrinsic electrical properties might also be modulated in ways not yet specified by the incoming stimuli. In such a case, repetition of a particular stimulus could produce a dynamic set of responses. The potential modulation of activity in cerebellar and vestibular nuclei by the cerebellar cortex also depends on the circuit properties, including the output from the local cerebella circuitry3z'a3, the interaction of various afferents within the nuclei 34, and the features of the corticonuclear connection 35. Since the olivo-corticonuclear projection displays, in general, a longitudinal organization 6'12'13'36, a different pattern of modulation will probably occur in the cerebellar nuclei if tactile stimuli elicit climbing fiber responses that are located within a limited region or longitudinal zone of the cerebellar cortex, as opposed to a pattern where responses are widely distributed, either rostrocaudaliy or mediolaterally. However, even within a longitudinal zone with common olivary input, the Purkinje cells do not necessarily project to the same part of the cerebellar nuclei 13,37. Further information is required before we can appreciate how the spatial characteristics of response ensembles at the level of the cerebellar cortex modulate the activity of cerebellar and vestibular nuclei. There is some evidence supporting the prediction that stimulation of different parts of the hindpaw will activate different ensembles of Purkinje cells, which will be reflected in the modulation of the nuclear activity. Eccles et al.3S observed that stimulation of TINS, Vol. 14, No. 6, 1991
various parts of the hindpaw produces variable patterns of activity in interpositus neurons. They reported, for example, that taps to the lateral hindtoe produced a response that was three times larger than the response to taps to the central pad or middle toe. The differential activation of response ensembles in the anterior lobe and paramedian lobule, and a concomitant differential modulation of the cerebellar nuclei, could contribute to postural adjustments in particular environmental conditions. For example, the postural reaction to perturbations during stance involves an increase in force of the lateral toes of each paw39. Such a change in force is likely to activate climbing fiber responses with receptive fields of the lateral toes, which would activate a higher proportion of responses in the paramedian lobule than in the anterior lobe. The response ensemble in the paramedian lobule would then modulate specific parts of the cerebellar nuclei. A change in direction of the perturbation would change the balance of activity between the various cerebellar nuclei, which could induce a prolonged postural change 4°. The postural change would complete the chain of transformations both in its detail and in its balance between cerebellar cortical regions.
Selected references 1 Erickson, R. P. (1984) Am. Sci. 72, 233-240 2 Snider, R. S. (1950)Arch. Neurol. Psychiatry 64, 196-219 3 McCollum, G. and Robertson, L. T. (1988) Neuroscience 27, 93-105 4 Brown, P. B. and Fuchs, J. L. (1975)J. Neurophysiol. 38, 1-9 5 Kaas, J. H., Nelson, R. J., Sur, M., Lin, C-S. and Merzenich, M. M. (1979) Science 204, 521-523 6 Voogd, J. and Bigar~, F. (1980) in The Inferior Olivary Nucleus: Anatomy and Physiology (Courville, J., de Montigny, C. and Lamarre, Y., eds), pp. 207-234, Raven Press 70scarsson, O. (1979) Trends Neurosci. 2, 143-145 80scarsson, O. (1980) in The Inferior Olivary Nucleus: Anatomy and Physiology (Courville, J., de Montigny, C. and Larnarre, Y., eds), pp. 278-289, Raven Press 9 Marani, E. and Voogd, J. (1977) J. Anat. 124, 335-345 10 Hawkes, R. and Leclerc, N. (1987)J. Comp. Neurol. 256, 29-41 11 Ekerot, C-F. and Larson, B. (1979) Exp. Brain Res. 36, 201-217 12 Trott, J. R. and Armstrong, D. M. (1987) Exp. Brain Res. 66, 318-338 13 Trott, J. R. and Armstrong, D. M. (1987) Exp. Brain Res. 68, 339-354 14 Andersson, G. and Oscarsson, O. (1978) Exp. Brain Res. 32, 565-579 15 Ekerot, C-F. and Larson, B. (1982) Exp. Brain Res. 48, 185-198 16 Armstrong, D. M., Harvey, R. J. and Schild, R. F. (1974) J. Comp. NeuroL 154, 287-302 17 Rosina, A. and Provini, L. (1987) J. Comp. Neurol. 256, 317-328 18 Eccles, J. C., Provini, L., Strata, P. and T~boF[kov~, H. (1968) Exp. Brain Res. 6, 195-215 19 Leicht, R., Rowe, M. J. and Schmidt, R. F. (1977) Exp. Brain Res. 27, 459-477 20 Miles, T. S. and Wiesendanger, M. (1975) J. Physiol. 245, 409-424 21 Robertson, L. T. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 281-320, Alan R. Liss 22 Welker, W. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 268-280, Alan R. Liss 23 Brons, J. F., Robertson, L. T. and Tong, G. (1990) Brain Res. 519, 243-248 24 Robertson, L. T. (1984) Soc. Neurosci. Abstr. 10, 538 25 Robertson, L. T. and Elias, S. A. (1988) Brain Res. 452, 97-104 253
Acknowledgements WethankDrCarol Pratt for the critical
review of the manuscript. This work was supported by NIH Grants NS18242 and N523209.
26 Llin&s, R., Baker, R. and Sotelo, C. (1974) J. Neurophysiol. 37, 560-571 27 Nelson, B. J. and Mugnaini, E. (1989) in The Ofivocerebellar System in Motor Control (Strata, P., ed.), pp. 86-107,
Springer-Verlag 28 de Zeeuw, C. I., Holstege, J. C., Ruigrok, T. J. H. and Voogd, J. (1989) in The Olivocerebellar System in Motor Control (Strata, P., ed.), pp. 111-116, Springer-Verlag 29 McCollum, G. and Robertson, L. T. (1987) Soc. Neurosci. Abstr. 13, 602 30 Bell, C. C. and Kawasaki, T. (1972) J. Neurophysiol. 35, 155--169 31 Llinb,s, R. and Yarom, Y. (1981) J. PhysioL 315, 569-584 32 Bishop, G. A., Blake, T. L. and O'Donoghue, D. L. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 29-56, Alan R. Liss
33 Ebner, T. J. and Bloedel, J. R. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 371-386, Alan R. Liss 34 Ito, M. (1984) The Cerebellum and Neural Control, Raven Press 35 Haines, D. E., Patrick, G. W. and Satrulee, P. (1982) in The Cerebellum - New Vistas (Palay, S. L. and Chan-Palay, V., eds), pp. 320-371, Springer-Verlag 36 Dietrichs, E. and Walberg, F. (1979) Anat. Embryol. 158, 13-39 37 Andersson, G. and Oscarsson, O. (1978) Exp. Brain Res. 32, 549-564 38 Eccles, J. C., Rosen, I., Scheid, P. and T~bo~ikov,~, H. (1974) J. NeurophysioL 37, 1438-1448 39 Macpherson, J. (1988)J. NeurophysioL 60, 204-217 40 Boylls, C. C. (1980)in Tutorials in Motor Behavior(Stelmach, G. E. and Requin, J., eds), pp, 83-94, Elsevier
Frombehaviorto molecules:an integratedapproachto the studyof neuropeptides N. T u b l i t z , D. B r i n k , K. S. B r o a d i e , P. K. Loi a n d A . W . S y l w e s t e r
Despite extensive information on many aspects of psptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory p@tides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes m target sensitivity and the distribution of the CAPcontaining neurons, including a set of peptidergic neurons that alter their transmitter pheno~pe postembryonically. Studies show that inositol 1,4,5USA. trisphosphate (IP3), linked to mtracellular Ca z+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.
N, Tublitz, D. Brink and P. K. toi are at the Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA, K. S. Broadieis at the Dept of Zoology, University of Cambridge, Cambridge CB23H, UK, and A. W. 5ylwester is at the Dept of Biology, University of Iowa, Iowa City, IA 52242,
It is now well documented that neuropeptides play a pivotal role in the control and proper expression of behavior in a wide range of organisms. Among the invertebrates, molting in insects is initiated by eclosion hormone 1, feeding in gastropod mollusks is regulated by bucculin 2, defensive postures in lobsters are triggered by proctolin 3, and egg hying in Aplysia is stimulated by egg laying hormone 4. In rats, drinking is activated by angiotensin II 5, maternal behavior is enhanced by oxytocin6, feeding is inhibited by bombesin and cholecystokinin 7'8, lordosis behavior is facilitated by LHRH 9"1°, and stereotyped grooming is induced by ACTH n. More complex, long-term behavioral changes, such as learning, memory, arousal, emotional states and schizophrenia, are also influenced by neuropeptides 12,13. Despite the extensive amount of literature on peptide-mediated behavior, there still remains a large gap in our knowledge of the connection between peptide effects on behavior and the cellular and molecular mechanisms underlying these responses. 254
For example, unequivocal identification of peptidergic neurons has been hampered in most cases by difficulties in satisfying many of the criteria for neurotransmitter identification that are routinely used to characterize non-peptidergic neurons. At present we have only a minimal understanding of the regulation of the activity of peptidergic neurons, and virtually nothing is known about the events surrounding the development and maturation of these unique neurons. Even in those areas where results have been forthcoming, e.g. the numerous reports on the sequences of peptide-coding genes 14, it has been difficult to apply this information to physiologically or behaviorally relevant issues. Because of these obstacles, it has become increasingly important to find experimental preparations in which this unique class of neural messenger can be investigated simultaneously at the behavioral, cellular and molecular levels. Such a preparation would accomplish three goals: (1) it would permit a complete examination of the functional relationship between peptides and behavior; (2) it would enable the identification of peptide-containing neurons and an analysis of their properties; and (3) it would allow the study of the molecular mechanisms that regulate peptide action at its targets. The tobacco hawkmoth, Manduca sexta, provides an excellent system for such an integrated analysis of neuropeptide function. This particular species has been the focus of intensive study over the past two decades, partly because of its large size, short life cycle (about six weeks), ease of growth in the laboratory, and an accessible CNS that contains relatively few neurons 15. As in other invertebrates, the CNS of M. sexta contains large, peptidergic neurons that are re-identifiable in different individuals and are amenable to a wide range of electrophysiological, biochemical, endocrinological, behavioral and molecular techniques. Their cell bodies are relatively large (30-50 ~tm in diameter) and frequently opalescent in situ, which greatly facilitates intracellular recordings and single cell dissections. Because many peptidergic neurons in M. sexta are neurosecretory in
© 1991,ElsevierSciencePublishersLtd,(UK) 0166- 2236/91/$0200
TINS, Vol. 14, No. 6, 1991
Lee T. Robertsonis at OregonHealth SciencesUniversity, Deptof Anatomy, Schoolof Dentistry, 611 SW Campus Drive, Portland, OR 97201, USAand GinMcCollumisat RobertS. Dow NeurologicalSciences Institute, Good SamaritanHospital and MedicalCenter, 1120NW 20th Avenue, Portland, OR 97209, USA.
Although the local structure of the cerebellum is fairly uniform and its inputs are often widely shared, outputs .from different regions of the cerebellar cortex reach different parts of the cerebellar and vestibular nuclei, which can affect the rest of the nervous system in different ways. In this review, we explain how different ensembles of climbing fiber responses in the anterior lobe and paramedian lobule can be generated by a tactile stimulus to the distal hindpaw. Apart from differing in degree of activation, the cortical regions differ also in the detailed pattern of the activation transmitted. The anterior lobe can distinguish a greater divers@ of stimuli to various skin surfaces than can the paramedian lobule. This differential classification of particular stimulus arrays by the two cerebellar regions could produce distinct patterns of neuronal activity in various corticonuclear compartments. The nervous system achieves certain goals by correlating information from many receptors and by ordering the implementation of movement complexes. The cerebellum, as part of the motor system, receives input from receptors in a manner such that certain information can be used to enhance the smooth execution of particular movements. This article describes how the cerebellar cortex transforms tactile stimuli into spatial maps, in which the stimuli are encoded by activation of an ensemble of Purkinje cells - the output neurons of the cerebellar cortex. Although peripheral information is conveyed to the cerebellar cortex by the climbing and mossy fibers, this article focuses only on the climbing fiber system because its distinct postsynaptic excitatory influence on the Purkinje cell provides a unique opportunity to investigate a particular afferent system, and because climbing fiber input has the same effect on the Purkinje cell, regardless of the type of peripheral stimulation. Since the type of stimulation does not appear to be encoded, the location of the peripheral stimulus might be the significant element, which is revealed in its receptive field. We expand on the idea that ensembles of Purkinje cells process complex stimuli but that subsets of Purkinje cells operate differentially under varying stimulus conditions. Erickson 1 describes a nervous system in which ensembles of neurons are transformed at each synapse from peripheral sensory input to motor output. We describe one step in that sequence of transformations and two variations of the transformation (anterior lobe versus the paramedian lobule) within the cerebellar cortex. In this review, we recount briefly the different schemes to localize peripheral information in the cerebella cortex, describe the pattern of climbing fiber receptive field organization, compare the transformation of tactile stimuli in the anterior lobe versus the paramedian lobule and consider how tactile information might be processed in the cerebellum.
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Localization of peripheral information Three major concepts have been proposed for the organization of the body surface in the cerebellar cortex. The first concept of somatic representation was the 'distorted homunculus' scheme, which was originally proposed by Snider 2 (Fig. 1A). In this scheme, it is suggested that a point-to-point representation exists between the body surface and cerebellar cortex. By this scheme, the location of a tactile stimulus is specified according to a pixel strategy where the receptive fields are as small as the resolution required and are arranged with little overlap 3. Areas such as the distal extremities or the face presumably have small receptive fields and, therefore, occupy a large region of the cerebellar cortex. Areas such as the back have large receptive fields and are represented by relatively few Purkinje cells. The pixel strategy appears to occur also in the dorsal horn of the spinal cord 4 and the cerebral cortex S. Another organizational concept is based on anatomical and physiological evidence of a longitudinal arrangement of afferent and efferent cerebellar fibers, as well as various molecular correlates e-l°. Physiological studies that elicit climbing fiber responses by electrical stimulation of major peripheral nerves have identified eight longitudinal zones in the anterior lobe on the basis of latency, spinal pathway, and differences between ipsilateral and contralateral representations of the forelimb and hindlimb nerves (Fig. 1B) s'u-13. Some longitudinal zones are subdivided further, the divisions being based on the nerves represented and the origin of branching olivary axons 14'15. The concept of longitudinal organization suggests that stimulation of certain peripheral nerves will produce synchronous activity in longitudinal zones. Presumably, localization of tactile stimulation will be accomplished by activating a longitudinal zone or a portion of a zone that represents a dermatome supplied by a particular peripheral sensory nerve. However, some stimuli might produce a more complex pattern of activations than exciting simply a single zone, since a single climbing fiber can branch to contact many Purkinje cells that might be widely separated or in distinct zones or lobules ~6'17. The third organizational concept involves a patchlike arrangement where adjacent regions might contain representations of a noncontiguous body area and where patches of climbing fiber responses represent an assortment of receptive fields (Fig. 1C). The patch-like arrangement of the climbing fiber responses has been found consistently in studies employing natural tactile stimulation of the skin surface instead of electrical stimulation of nerves and using high resolution extracellular mapping procedures in cats anesthetized with sodium pentobarbital 18-21. Welker and associates 22 also describe a similar patch-like arrangement for the mossy fiber system.
© 1991,ElsevierSciencePublishersLtd,(UK) 0166-2236/91/$02.00
TINS, VOI. 14, NO. 6, 1991
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Fig. 1. Three organizational schemes of representation of the body are shown for Iobules III-VI of the anterior lobe. The boxed area on the diagram of the cerebellum of the cat (lower right) shows the region of Iobules IIl-Vl. (A) The body surface is represented as a continuous dermatomal sequence on the dorsal surface of the cerebellum. Each body area is represented in a specific cerebellar region. (Modified from Ref. 2 .) (B) Longitudinal zones (a, b, cl, c2, c3 and d) of the cat cerebellum are associated with short-latency afferent pathways of forelimb and hindlimb nerves. In this scheme, dermatomes of various sensory nerves would be represented in a certain longitudinal zone or zones. (Modified from Ref. 11 .) (C) Different areas of the skin surface are represented in a patch mosaic when tactile stimulation is used to elicit the responses. The parasagittal section [insert; the approximate location of which is indicated by the dashed line on the dorsal cerebellar surface in (C)] shows that the patches and overlapping patches extend into the deeper parts of the Iobules. Various body areas are represented in multiple patches, sometimes overlapping with other body areas. (Modified from Ref. 21.)
Within each patch there is a diversity of receptive fields that can include a mixture of large and small areas of skin, a blend of proximal and distal areas, or various combinations of glabrous and hairy surfaces. Each patch contains a unique combination of receptive fields, although some receptive fields are represented in multiple patches. The distribution and diversity of the climbing fiber receptive fields in various cortical regions suggest that most tactile stimuli would TINS, Vol. 14, No. 6, 1991
produce a complex pattern of cortical activity. For example, tactile stimulation of just the ventral (volar) surface of the forepaw would not be sufficient to drive all the climbing fiber responses of any longitudinal zone of the anterior lobe, but would activate a complex pattern of responses in several zones of the anterior lobe, as well as in various parts of the paramedian lobule, dorsal paraflocculus and posterior vel-mis21,23-25" 249
intermediate zone of lobules III-V of the anterior lobe and throughout • i/~ the caudal portion (sublobule C) of i the paramedian lobule. The all-ornothing properties of the climbing fiber response allowed us to define "., I' "-. I/ Ankle // the boundaries of the receptive \/x\ Dors // fields. Overlapping all the hindpaw receptive fields on a schematic / A " " ," drawing of the paw revealed that C "-,, / .'\ most receptive fields conform to the anatomical boundaries of the paw, and that most share common Tips/claws boundaries. Figure 2 shows four receptive fields (A-D) that are superimposed on the schematic drawing of the paw as projected onto a fiat surface (the dorsal, ventral, medial and lateral surfaces . . s ,' / on the same plane). The bound,i aries of several receptive fields L ',.,, / ~ntral Pad overlap. For example, the boundaries of receptive fields A, B and D I / B / Medial ~ Lateral share portions of the boundary of Fig. 2. Four examples of receptive fields of the hindpaw (A-D) are superimposed on a diagram of the central pad; receptive fields B the distal extremity as projected onto a plane, which allows all skin surfaces to be represented in the and D share a boundary with the same drawing, Most receptive fields share boundaries with other receptive fields and several medial-middle toe. Because most receptive fields are included within larger receptive fields. receptive fields share common boundaries with other receptive Overlapping receptive fields show fields, the paw can be described as being organized in asymmetrical regional distribution compartments of the skin surface. Compartments can To gain an understanding of regional variations in be defined as the smallest area of the skin that the the climbing fiber representations, we initially focused climbing fiber system can resolve 3, even though each on the receptive fields limited to the distal hindpaw a. compartment does not necessarily occur as a single High density recordings (with a 300-400 #m inter- receptive field. For the distal hindpaw, when 136 electrode penetration distance) were made in the receptive fields of the hindpaw are superimposed using the schematic drawing of the paw, then their common boundaries delineate 42 compartments, which are shown in the representation of the paw of Anterior Lobe Paramedian Lobule Figs 3-5. Most of the receptive fields are unions of two or more compartments. In Fig. 2, receptive field D is the union of the two middle toes, which also include the two toe pad compartments. Some receptive fields are also included completely in a larger receptive field. The toe pad of receptive field C is included in the receptive field of the two middle toes (D), which is included in the receptive field of the three lateral toes (B). Since stimulation of all parts of a receptive field can elicit a climbing fiber response, stimulation of the lateral-middle toe pad would also activate responses with receptive fields that include the lateral-middle toe pad. Inclusions provide an indication of the effectiveness Proportion ot C l i m b i n g Fil~er Responses of each area of the paw in activating a population of responses. A population of receptive fields can >0 >10 >20 >30 >40 >50 >60 preferentially include a particular compartment or set of neighboring compartments. Most hindpaw recepFig. :3. When 155 hindpaw receptive fields, obtained from climbing fiber tive fields of the paramedian lobule are concentrated responses in the rostral anterior lobe, and 198 hindpaw receptive fields from around the lateral-middle toe (Fig. 3). Thus, preferenthe paramedian Iobule were overlapped on the schematic drawing of the paw, tially stimulating the lateral-middle toe could synthe proportion of each compartment included in the total number of hindpaw receptive fields was revealed for each region. In the paramedian Iobule, the chronously activate a large subset of Purkinje cells in hindpaw receptive fields are centered around the three lateral toes, with the the paramedian lobule and a small set in the anterior highest proportion involving the lateral-middle toe. In the anterior lobe, the lobe, which contains a comparatively equal distrifields are concentrated around the three medial toes and involve compart- bution of compartments. In contrast, a stimulus ments of the lateral toe and the distal dorsal part of all toes. (See Fig. 2 for applied only to the medial toe could activate a rather details of the schematic representation of the paw.) small subset of responses in the paramedian lobule, A~
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Whereas the population of responses that could be activated in the anterior lobe would be similar in size to the population activated by stimulation of the lateral-middle toe. The two slightly separated stimuli could elicit strongly different balances of the activation of the paramedian lobule and anterior lobe.
Comparison of anterior lobe and paramedian lobule transformations of stimuli The transformation from a particular stimulus to a response ensemble in each region is characterized by its set of receptive fields. For this review, we assume that tactile stimulation of a skin area will synchronously activate all the climbing fiber responses with receptive fields that include the stimulated area. In actual fact, a more complex pattern is likely because of particular coupling mechanisms or the inhibition of inferior olivary neurons 26-~8, although the details of how these mechanisms influence afferent input are not yet understood. Because different sets of receptive fields exist in the anterior lobe and in the paramedian lobule, coincident tactile stimulation of various combinations of toes could result in different response ensembles activated in the two regions. The transformation of stimuli to the ventral surface of the hindpaw toes illustrates this point. For this illustration, we limited the receptive fields to the ventral surface of the four toes and did not consider the other compartments (such as pads, the area surrounding the pads, and toe tips). The first step in determining the transformation is to identify in each region the set of receptive fields that includes part or all of the ventral toe surfaces. In the anterior lobe, receptive fields exist for each of the four toes and for almost all neighboring combinations (left diagram in Fig. 4). The receptive fields are organized as an inverted pyramid - the four single Anterior Lobe
toes on top, then the three neighboring pairs (the lateral and lateral-middle, the two middle, and the medial-middle and medial toes), the two neighboring triples (lateral to medial-middle toes, medial to lateralmiddle toes) and, at the bottom, all four toes. This organization reflects an unbroken rule among receptive fields: whenever two or more toes on the same paw are involved in a receptive field, the toes are neighbors 29. In the paramedian lobule, receptive fields involving the medial toes are conspicuously absent, that is, no single medial or medial-middle toe, no medial pair, and no medial triple were identified (right diagram in Fig. 4). Stimulation of various combinations of the toes (a stimulus array) would produce different response ensembles in the two regions. We assume that the response to coincident stimulation of more than one toe is the union of responses to individual toe stimulation. For example, touching all four toes produces a response ensemble involving all the receptive fields that includes any of the toes. In the anterior lobe, touching all four toes would produce a response ensemble of all the possible receptive fields; in the paramedian lobule, the response ensemble would be only those receptive fields that include the lateral and the lateral-middle toes (Fig. 4). Because of its larger set of receptive fields, the anterior lobe can distinguish more stimulus combinations of the toes than can the paramedian lobule. Different stimulus arrays elicit different response patterns in the anterior lobe, but might elicit identical response patterns in the paramedian lobule. The possible combinations of stimuli to the ventral toes consist of 16 stimulus arrays. Each stimulus array elicits a specific response ensemble in the anterior lobe. In contrast, only seven different response ensembles are given for the 16 stimulus arrays in the paramedian lobule (boxes A-G in Fig. 5). Paramedian Lobule
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Fig. 4. A limited set of hindpa w receptive fields exempfifies differences between the distributions of receptive fields identified for the anterior lobe and the paramedian Iobule. The set is organized as an inverted pyramid: the top row is receptive fields of individual toe pads and the surroundinE hairy surface (the medial toe is on the left side whereas the lateral toe is on the right), the second row consists of pairs of toes, the third row shows three toes, and the bottom receptive field is of all four toes. Not encountered in the paramedian Iobule were any responses representing only the medial or medial-middle toes, the two medial toes, or the three medial toes (open circles). TINS, VoL 14, No. 6, 1991
251
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• _@@ (4iLl Fig. 5. With the inverted pyramid organization of the hindtoe receptive fields (shown in Fig. 4), various response ensembles could be elicited by stimulating (*) various combinations of toes. Each of the 16 different stimulus combinations of the toes could evoke a different response ensemble in the anterior lobe (AL), but the same stimulus combinations could evoke only seven different response ensembles (boxes A to G) in the paramedian Iobule (PML). For example, in box E, the four different stimulus combinations could evoke different response ensembles among the receptive fields of the anterior lobe but the same response ensemble in the paramedian Iobule, which are equivalent to each other. Thus, the hindtoe climbing fiber receptive fields of the anterior lobe can distinguish a greater diversity of stimulus combination than those of the paramedian Iobule.
Consider the stimulation of only the lateral toe (Fig. 5D). Four receptive field types (the lateral toe, the two lateral toes, the three lateral toes, and all four toes) include the lateral toe, so they would be activated by its stimulation in both the anterior lobe and paramedian lobule. The coincident stimulus combination of the medial toe with the lateral toe would activate the same four receptive fields that include the lateral toe, but the three other receptive fields that include the medial toe - the medial toe, the two medial toes, and the three medial toes - would be activated in the anterior lobe. Stimulation of either the lateral or the medial toe would activate the response with the receptive field of all four toes, but, in the paramedian 252
lobule, the response ensemble activated by the lateral toe would not be augmented by coincident stimulation of the medial toe. In the paramedian lobule, simultaneous stimulation of the medial and lateral toes is indistinguishable from stimulation of the lateral toe alone. These two stimulus arrays belong to a set that are equivalent with respect to the responses in the paramedian lobule, but each of the two stimuli elicits a distinct response ensemble in the anterior lobe. Within the seven response ensembles in the paramedian lobule, the different stimulus arrays are equivalent to each other. In addition to response ensembles for stimulation of the medial (Fig. 5C) and the lateral toes (Fig. 5D), the equivalent responses TINS, Vol. 14, No. 6, 1991
exist for stimulation of the medial-middle toe or the medial and medial-middle toes (Fig. 5A), for stimulation of lateral-middle toe alone or with combinations of the two medial toes (Fig. 5E), for stimulation of the two lateral toes with combinations of the two medial toes (Fig. 5F), or for stimulation of the lateral toe with the medial-middle toe or both medial toes (Fig. 5G).
Information processing in the cerebellum The balance of total climbing fiber activity between the anterior lobe and paramedian lobule can vary sharply depending on the stimulus location. There is also a difference in the representation of detail, with the paramedian lobule equating stimuli that are distinguished by the anterior lobe. Although these conclusions are based on assumptions that might be replaced in the future by more sophisticated information, knowing the features of the receptive fields is a valuable step towards understanding the transformation of neural activity carried out by the cerebellum. Ensemble activity, which we are currently studying, is expected to depend on the properties of individual olivary neurons, especially the long refractory period and the tendency to oscillate a°,al. These intrinsic electrophysiological properties of olivary neurons might influence how incoming stimuli are transformed into sets of climbing fiber responses. For example, the quick sequential stimulation of the lateral toe and then the lateral-middle toe would produce a different set of responses than the reverse stimulation sequence (stimulation of the lateral-middle toe followed by the lateral toe). Since the cells activated by the first stimulus are refractory when the second stimulus arrives, the set of responses for the second stimulus would not include responses with receptive fields that encompass the area of the first stimulus. The intrinsic electrical properties might also be modulated in ways not yet specified by the incoming stimuli. In such a case, repetition of a particular stimulus could produce a dynamic set of responses. The potential modulation of activity in cerebellar and vestibular nuclei by the cerebellar cortex also depends on the circuit properties, including the output from the local cerebella circuitry3z'a3, the interaction of various afferents within the nuclei 34, and the features of the corticonuclear connection 35. Since the olivo-corticonuclear projection displays, in general, a longitudinal organization 6'12'13'36, a different pattern of modulation will probably occur in the cerebellar nuclei if tactile stimuli elicit climbing fiber responses that are located within a limited region or longitudinal zone of the cerebellar cortex, as opposed to a pattern where responses are widely distributed, either rostrocaudaliy or mediolaterally. However, even within a longitudinal zone with common olivary input, the Purkinje cells do not necessarily project to the same part of the cerebellar nuclei 13,37. Further information is required before we can appreciate how the spatial characteristics of response ensembles at the level of the cerebellar cortex modulate the activity of cerebellar and vestibular nuclei. There is some evidence supporting the prediction that stimulation of different parts of the hindpaw will activate different ensembles of Purkinje cells, which will be reflected in the modulation of the nuclear activity. Eccles et al.3S observed that stimulation of TINS, Vol. 14, No. 6, 1991
various parts of the hindpaw produces variable patterns of activity in interpositus neurons. They reported, for example, that taps to the lateral hindtoe produced a response that was three times larger than the response to taps to the central pad or middle toe. The differential activation of response ensembles in the anterior lobe and paramedian lobule, and a concomitant differential modulation of the cerebellar nuclei, could contribute to postural adjustments in particular environmental conditions. For example, the postural reaction to perturbations during stance involves an increase in force of the lateral toes of each paw39. Such a change in force is likely to activate climbing fiber responses with receptive fields of the lateral toes, which would activate a higher proportion of responses in the paramedian lobule than in the anterior lobe. The response ensemble in the paramedian lobule would then modulate specific parts of the cerebellar nuclei. A change in direction of the perturbation would change the balance of activity between the various cerebellar nuclei, which could induce a prolonged postural change 4°. The postural change would complete the chain of transformations both in its detail and in its balance between cerebellar cortical regions.
Selected references 1 Erickson, R. P. (1984) Am. Sci. 72, 233-240 2 Snider, R. S. (1950)Arch. Neurol. Psychiatry 64, 196-219 3 McCollum, G. and Robertson, L. T. (1988) Neuroscience 27, 93-105 4 Brown, P. B. and Fuchs, J. L. (1975)J. Neurophysiol. 38, 1-9 5 Kaas, J. H., Nelson, R. J., Sur, M., Lin, C-S. and Merzenich, M. M. (1979) Science 204, 521-523 6 Voogd, J. and Bigar~, F. (1980) in The Inferior Olivary Nucleus: Anatomy and Physiology (Courville, J., de Montigny, C. and Lamarre, Y., eds), pp. 207-234, Raven Press 70scarsson, O. (1979) Trends Neurosci. 2, 143-145 80scarsson, O. (1980) in The Inferior Olivary Nucleus: Anatomy and Physiology (Courville, J., de Montigny, C. and Larnarre, Y., eds), pp. 278-289, Raven Press 9 Marani, E. and Voogd, J. (1977) J. Anat. 124, 335-345 10 Hawkes, R. and Leclerc, N. (1987)J. Comp. Neurol. 256, 29-41 11 Ekerot, C-F. and Larson, B. (1979) Exp. Brain Res. 36, 201-217 12 Trott, J. R. and Armstrong, D. M. (1987) Exp. Brain Res. 66, 318-338 13 Trott, J. R. and Armstrong, D. M. (1987) Exp. Brain Res. 68, 339-354 14 Andersson, G. and Oscarsson, O. (1978) Exp. Brain Res. 32, 565-579 15 Ekerot, C-F. and Larson, B. (1982) Exp. Brain Res. 48, 185-198 16 Armstrong, D. M., Harvey, R. J. and Schild, R. F. (1974) J. Comp. NeuroL 154, 287-302 17 Rosina, A. and Provini, L. (1987) J. Comp. Neurol. 256, 317-328 18 Eccles, J. C., Provini, L., Strata, P. and T~boF[kov~, H. (1968) Exp. Brain Res. 6, 195-215 19 Leicht, R., Rowe, M. J. and Schmidt, R. F. (1977) Exp. Brain Res. 27, 459-477 20 Miles, T. S. and Wiesendanger, M. (1975) J. Physiol. 245, 409-424 21 Robertson, L. T. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 281-320, Alan R. Liss 22 Welker, W. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 268-280, Alan R. Liss 23 Brons, J. F., Robertson, L. T. and Tong, G. (1990) Brain Res. 519, 243-248 24 Robertson, L. T. (1984) Soc. Neurosci. Abstr. 10, 538 25 Robertson, L. T. and Elias, S. A. (1988) Brain Res. 452, 97-104 253
Acknowledgements WethankDrCarol Pratt for the critical
review of the manuscript. This work was supported by NIH Grants NS18242 and N523209.
26 Llin&s, R., Baker, R. and Sotelo, C. (1974) J. Neurophysiol. 37, 560-571 27 Nelson, B. J. and Mugnaini, E. (1989) in The Ofivocerebellar System in Motor Control (Strata, P., ed.), pp. 86-107,
Springer-Verlag 28 de Zeeuw, C. I., Holstege, J. C., Ruigrok, T. J. H. and Voogd, J. (1989) in The Olivocerebellar System in Motor Control (Strata, P., ed.), pp. 111-116, Springer-Verlag 29 McCollum, G. and Robertson, L. T. (1987) Soc. Neurosci. Abstr. 13, 602 30 Bell, C. C. and Kawasaki, T. (1972) J. Neurophysiol. 35, 155--169 31 Llinb,s, R. and Yarom, Y. (1981) J. PhysioL 315, 569-584 32 Bishop, G. A., Blake, T. L. and O'Donoghue, D. L. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 29-56, Alan R. Liss
33 Ebner, T. J. and Bloedel, J. R. (1987) in New Concepts in Cerebellar Neurobiology (King, J. S., ed.), pp. 371-386, Alan R. Liss 34 Ito, M. (1984) The Cerebellum and Neural Control, Raven Press 35 Haines, D. E., Patrick, G. W. and Satrulee, P. (1982) in The Cerebellum - New Vistas (Palay, S. L. and Chan-Palay, V., eds), pp. 320-371, Springer-Verlag 36 Dietrichs, E. and Walberg, F. (1979) Anat. Embryol. 158, 13-39 37 Andersson, G. and Oscarsson, O. (1978) Exp. Brain Res. 32, 549-564 38 Eccles, J. C., Rosen, I., Scheid, P. and T~bo~ikov,~, H. (1974) J. NeurophysioL 37, 1438-1448 39 Macpherson, J. (1988)J. NeurophysioL 60, 204-217 40 Boylls, C. C. (1980)in Tutorials in Motor Behavior(Stelmach, G. E. and Requin, J., eds), pp, 83-94, Elsevier
Frombehaviorto molecules:an integratedapproachto the studyof neuropeptides N. T u b l i t z , D. B r i n k , K. S. B r o a d i e , P. K. Loi a n d A . W . S y l w e s t e r
Despite extensive information on many aspects of psptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory p@tides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes m target sensitivity and the distribution of the CAPcontaining neurons, including a set of peptidergic neurons that alter their transmitter pheno~pe postembryonically. Studies show that inositol 1,4,5USA. trisphosphate (IP3), linked to mtracellular Ca z+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.
N, Tublitz, D. Brink and P. K. toi are at the Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA, K. S. Broadieis at the Dept of Zoology, University of Cambridge, Cambridge CB23H, UK, and A. W. 5ylwester is at the Dept of Biology, University of Iowa, Iowa City, IA 52242,
It is now well documented that neuropeptides play a pivotal role in the control and proper expression of behavior in a wide range of organisms. Among the invertebrates, molting in insects is initiated by eclosion hormone 1, feeding in gastropod mollusks is regulated by bucculin 2, defensive postures in lobsters are triggered by proctolin 3, and egg hying in Aplysia is stimulated by egg laying hormone 4. In rats, drinking is activated by angiotensin II 5, maternal behavior is enhanced by oxytocin6, feeding is inhibited by bombesin and cholecystokinin 7'8, lordosis behavior is facilitated by LHRH 9"1°, and stereotyped grooming is induced by ACTH n. More complex, long-term behavioral changes, such as learning, memory, arousal, emotional states and schizophrenia, are also influenced by neuropeptides 12,13. Despite the extensive amount of literature on peptide-mediated behavior, there still remains a large gap in our knowledge of the connection between peptide effects on behavior and the cellular and molecular mechanisms underlying these responses. 254
For example, unequivocal identification of peptidergic neurons has been hampered in most cases by difficulties in satisfying many of the criteria for neurotransmitter identification that are routinely used to characterize non-peptidergic neurons. At present we have only a minimal understanding of the regulation of the activity of peptidergic neurons, and virtually nothing is known about the events surrounding the development and maturation of these unique neurons. Even in those areas where results have been forthcoming, e.g. the numerous reports on the sequences of peptide-coding genes 14, it has been difficult to apply this information to physiologically or behaviorally relevant issues. Because of these obstacles, it has become increasingly important to find experimental preparations in which this unique class of neural messenger can be investigated simultaneously at the behavioral, cellular and molecular levels. Such a preparation would accomplish three goals: (1) it would permit a complete examination of the functional relationship between peptides and behavior; (2) it would enable the identification of peptide-containing neurons and an analysis of their properties; and (3) it would allow the study of the molecular mechanisms that regulate peptide action at its targets. The tobacco hawkmoth, Manduca sexta, provides an excellent system for such an integrated analysis of neuropeptide function. This particular species has been the focus of intensive study over the past two decades, partly because of its large size, short life cycle (about six weeks), ease of growth in the laboratory, and an accessible CNS that contains relatively few neurons 15. As in other invertebrates, the CNS of M. sexta contains large, peptidergic neurons that are re-identifiable in different individuals and are amenable to a wide range of electrophysiological, biochemical, endocrinological, behavioral and molecular techniques. Their cell bodies are relatively large (30-50 ~tm in diameter) and frequently opalescent in situ, which greatly facilitates intracellular recordings and single cell dissections. Because many peptidergic neurons in M. sexta are neurosecretory in
© 1991,ElsevierSciencePublishersLtd,(UK) 0166- 2236/91/$0200
TINS, Vol. 14, No. 6, 1991
