Barrels IV: Proceedings of a Satellite Symposium of the 1991 Society for Neuroscience Meeting

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John K. Chapin,* Theodore A. Henderson,? Mark F. Jacquin,t.' and Bradley G . Klein$ *Department of Physiology and Biophysics, Hahnemann University, Philadelphia, Pennsylvania 19102; tDepartment of Anatomy and Neurobiology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104; $Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Key words barrels, tngeminal, somatosensory, pattern formation

On November 9 and 10, 1991, a group of somatosensory and developmental neurobiologists met in New Orleans, Louisiana, to deliberate over recent findings pertaining to the somatosensory system-in particular, those portions devoted to processing of inputs from digitized receptor organs, such as the whiskers and fingers. The focus of the fourth annual Barrels Symposium (Barrels IV) was on signal transmission within the barrel cortex, peripheral injury-induced reorganization in the adult somatosensory cortex, the temporal sequence of developmental events contributing to whisker-related pattern formation, and potential roles for cytotactin in developing neural systems. The meeting consisted of four invited presentations, two poster sessions, and a keynote address. This article summarizes the major points offered in each of these events.

The major aim of the experiments reported was to characterize the transmission of sensory signals

1. To whom all correspondence should be addressed.

through such cortical circuits. Single neurons were recorded in different layers of the rat primary somatosensory (SI) cortex, and their latencies of response to displacement of single whiskers were measured. As would be expected, the shortest-latency responses (7 msec) were found in the barrels in layer IV, but also in layer Vb. Longer-latency responses were found in other layers. In layer Va, for example, the average latency was 10 msec. The overall order in which neurons in different layers were found to respond to such stimuli was as follows: ventroposteromedial (VPM) thalamus + IV, Vb + I11+ 11- Va- VI and adjacent columns. This divergent flow of information in the barrel cortex toward the deeper layers may be related to the fact that receptive field sizes are larger in the deeper layers. To test this notion, a neuron was recorded in the D1 barrel; its latency and magnitude of response to stimulation of the D2 whisker were measured; and, subsequently, a small lesion was made in the D2 barrel. This produced a decreased magnitude, and increased latency, of response to stimulation of the D2 whisker. Over several such experiments it was found that with an increasing percentage of destruction of the D2 barrel, less response was seen after stimulation of the D2 whisker. This general finding was typical of neurons in the SI cortex with multiwhisker receptive fields: Latencies of response to stimulation of their nonprincipal whiskers were 5-24 msec longer than the earliest responses measurable in the layer IV barrels. This marked variability in response latencies of SI cortex cells contrasted with the narrow range of latencies observed in the VPM thalamus (3-6 msec). The much broader distribution of latencies in the SI cortex is consistent with a model

Somatosensory and Motor Research, Vol. 9, No. 4, 1992, pp. 291 -295

Accepted June 10, 1992

Signal Transmission through the Adult Barrel Cortex (MICHAEL ARMSTRONG-JAMES , LONDON HOSPITAL MEDICAL COLLEGE)

How are sensory signals propagated through the circuitry intrinsic to the barrel cortex? Classical thinking on this subject has focused on vertical transmission within cortical columns. Inasmuch as barrels are examples of cortical columns, they are a good model for further study of this issue. It is known that only about 20% of excitatory synapses in a whisker barrel in the mouse derive from thalamocortical endings, and these are mainly localized in layer IV. In contrast, about 80% of the synapses in a barrel derive from other cortical neurons, emphasizing the importance of intracortical circuitry for sensory processing in the neocortex.

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CHAPIN ET AL.

in which sensory information is synaptically propagated from barrel to barrel. What, then, might be the route for this intracortical communication? To test this, comparisons were made of neuronal response latencies to stimulation of whiskers that are “near neighbors” versus “far neighbors” of the principal whisker for a given neuron. The response latencies to stimulation of adjacent whiskers lagged behind those to stimulation of the principal whisker by 5-10 msec. By contrast, the time lag observed when the stimulated whisker was two whiskers away was 11-20 msec, and when it was four whiskers away it was longer than 20 msec. From such time delays, one can calculate that the corticocortical path traversed by these sensory volleys would have conduction velocities on the order of 0.1 d s e c . Such slow conduction velocities suggest that this transmission may be carried via small unmyelinated fibers, multiple synaptic relays in the cortex, and/or a relatively indirect route. As evidence in support of the latter possibility, Diamond and Armstrong-James have recently shown that longerlatency sensory responses in the posterior thalamic nucleus, pars medialis (POm), are derived mainly from the SI cortex. Because the POm is known to project topographically back to the SI cortex, it may constitute a corticothalamocortical loop through which sensory information originating in one barrel may be propagated to adjacent barrels. Injury-Znduced Reorganization in Adult SZ Cortex (JOHN T. WALL, MEDICAL COLLEGE OF OHIO)

Three major questions were raised: (1) How is cortical somatotopic organization changed after injury to a nerve in the hand? (2) How does subcortical organization contribute to normal cortical somatotopy? (3) How does injury of a hand nerve affect the normal configuration of the somatosensory system to produce its reorganization? The first question has been addressed in several studies by different investigators using different mammalian species. The results have suggested that the somatosensory cortical response to a peripheral nerve injury follows a characteristic recovery cycle. First, the cortical area that has lost its normal cutaneous activation as a result of the lesion begins to respond to stimulation of skin areas surrounding the deaerented skin area. Thus the SI cortex exhibits substitution of an intact sensory periphery for that which was injured. Over a longer time course, the nerve that was injured regenerates. This results in a reversal of the changes wrought by the original injury. However, in cases where only a limited recovery of the peripheral innervation occurs, the body map in the SI cortex remains abnormal. 292

These phenomena are illustrated in experiments in which multiunits are recorded in area 3b of monkeys. The hand representation of this area is mapped by using a probe to activate low-threshold cutaneous receptors in either the glabrous skin of the palm and fingers, or the hairy skin of the dorsum. It has been found that the representation of the glabrous skin in area 3b is continuous, whereas the hairy skin is represented in small discontinuous patches. Most of the glabrous skin is innervated by the median nerve. Thus, when the median nerve is damaged, the normal cortical representation of the glabrous skin is replaced within a few days by a hairy skin representation. Inasmuch as these changes occur rapidly, an important question is whether such changes involve preexisting cortical or subcortical circuitry. To address this, the anatomical organization of hand aerents were studied with anterograde tracers. It was found that afferent terminations from the thumb and digits in the cuneate nucleus are organized in five separate rodshaped aggregates. A similarly patterned digit representation can be seen in the spinal dorsal horn, as visualized in horizontal sections. Furthermore, these terminal aggregates are colocalized with densities of cytochrome oxidase staining. Overall, these findings emphasize the existence of two parallel systems of inputs to similar cytochrome-oxidase-dense aggregates in the VPM thalamus. How, then, does injury of a particular hand nerve produce its effect on the SI cortex? To study the cutaneous innervation territories covered by peripheral nerves, multiaxon recordings were carried out in the median and radial nerves. The nerves were found to have a nonsomatotopic organization,resulting in a partial overlap between the innervation territories of the two nerves. Similarly, when horseradish peroxidase (HRP) was injected in these nerves, anterograde terminal lab eh g was observed across relatively large and partially overlapping regions, involving several of the somatotopically arranged aggregates in the dorsal horn and dorsal column nuclei. Finally, when the SI cortex was electrophysiologically mapped followed nerve stimulation, the median and ulnar nerves were found to drive cells in the areas responsive to the glabrous skin, while radial nerve stimulation activated neurons responsive to hairy skin. However, some overlap was apparent, supporting the notion that central nervous system (CNS) inputs from peripheral nerves have a core-fringe pattern of termination. Overall, these results suggest that the representation of the body in the SI cortex is consistent with the somatotopic organization of the body in the dorsal column nuclei, but that it also reflects to some extent the representation of the periphery in peripheral nerves.

BARRELS IV

These findings suggested the following proposal: When a nerve is injured, the somatosensory system produces an internal “image” of the injury. As a result, inputs from ‘’fringe”areas surrounding the injury become dominant. Expansion of the territories of these intact fringe areas is thus defined by the normal degree of overlap between these areas. Developmental Events Contributing to WhiskerRelated Pattern Formation (REHA ERZURUMLU,

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MASSACHUSETTS INSTITUTE OF TECHNOLOGY)

The temporal sequence of developmental events involved in whisker-related pattern formation was examined and correlated in a series of carefully agematched rats. In the cortex, the emergence of patterns was compared for several markers. DiI labeling showed that thalamocortical axons appeared to be the first to enter the cortex, following a lateral-to-medial gradient, and were penetrating the lower cortical plate by embryonic day 18 (E18). At about the time of birth (postnatal day 0, or PO), a pattern emerged by progressive restriction of initially widespread arbors. A face representation was evident first, followed by rows, and finally patches by the end of P1. In the same animals, tegmentocortical serotonergic (5-HT)aEerents, labeled immunohistochemically, had not penetrated the cortical white matter by E18. Postnatally, 5-HTaxons continued to lag roughly 1- 1.5 days behind thalamocortical axons in the emergence of whisker-related patterns. Intrinsic cortical structures appeared to develop patterns only after thalamocortical patterns were evident. On P1, the extracellular matrix protein, cytotactin, was distributed uniformly with only a hint of rows. Cytotactin patterns developed on P3 or P4 by a progressive loss of staining from barrel hollows. It was hypothesized that as cells concentrate in the emerging barrel walls and growing axons and dendrites concentrate in the hollows, patterns may be transferred to matrix proteins by the proteolytic degradation of such proteins in the hollows. There is some evidence from tissue culture that growth cones may have proteolytic activity. Trigeminal ganglion cells are born about E9.5 to El4 in mouse and about 1 day later in rat. Axons first contact vibrissae about Ell-E12, and primary aerents invade the brainstem about E13. The upper two rows of vibrissae develop from the lateral nasal process, and the lower three rows arise from the maxillary process. When different carbocyanine dyes were placed in the two processes at E12-E13, the labels remained segregated throughout the infraorbital nerve and trigeminal ganglion. That is, gross topographic order exists in the ganglion prior to naturally occurring cell death, which occurs approximately between El4 and birth. Moreover,

the dyes remained segregated in the brainstem, with lateral nasal axons entering the brainstem medially and maxillary process axons entering laterally. This segregation of axons precedes the differentiation of vibrissae and the birth of most brainstem trigeminal neurons. By E14, labeling of developing vibrissae in rows A and E with one dye, and in row C with another dye, resulted in alternating bands of labeled cells in the ganglion and alternating bands of fibers in the trigeminal spinal tract and nuclei, reflecting adult-like topography. Thus, the dorsoventral, or axial, topography of the system is set up very early, with the orientation of the map in each neural station possibly being under intrinsic control. The whisker-related pattern may be laid down by axon-axon interactions upon an intrinsic axial framework. Supporting observations include the following: (1) Postnatal lesions do not alter the orientation of central patterns, and (2) geniculocortical axons in anophthalmic mice are topographically organized, although the geniculate has no afferent input from the retina. An interesting sidelight was the observation that ingrowing trigeminal primary afferents formed large Tshaped growth-cone-like structures where they enter the brainstem. These structures averaged 125- 150 pm wide with growth cones at the end of each arm, suggesting an origin point for the primary afferent bifurcation. The signal for their formation may be chemical. Cytotactin in Developing Neural Systems (KATHRYN CROSSIN, ROCKEFELLER UNIVERSITY)

Cytotactin (also known as tenascin) is one of a diverse group of morphoregulatory proteins that includes cell surface molecules, integrins, and extracellular matrix proteins. By SDS PAGE, it separates into three bands at 190, 200, and 220 kilodaltons. Without reduction, it does not migrate in SDS at all. A chondroitin sulfate proteoglycan with a large core protein copurifies with it. Cytotactin has an interesting shape with six arms radiating from a central core. It has sequence homologies to epidermal growth factor repeats, fibronectin type 3 repeats, and fibrinogen. It also has fibrinogen-like calcium-binding sites. Cytotactin binds to its associated proteoglycan, and to fibrinogen, fibronectin, and calcium; it does not bind to itself or to laminin. Cytotactin is expressed in a rostrocaudal gradient during embryogenesis. During gastrulation, it is found in rostral epithelial extracellular matrix. During neuralation, it is found in rostral neural plate before it appears in caudal regions. Similarly, it first appears in rostral somites. Shortly after somites develop, cytotactin is limited to rostral portions of somites, colocalizing with the region of neural crest cell migration, whereas its associated proteoglycan is concentrated in caudal portions. 293

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CHAPIN ET AL.

However, extirpation or wounding of neural crest primordium can perturb the pattern of cytotactin without affecting the migration of surviving neural crest cells. Thus, the pattern of cytotactin distribution is correlated with crest cell migration, but is not required for it. In culture, neurons do not plate well on cytotactin, and crest cells do not migrate on cytotactin. When a mixed substrate of cytotactin and fibronectin is used, the inhibition of migration can be titrated with increasing relative amounts of cytotactin. In somites, cytotactin may slow crest cell migration, allowing other adhesive molecules to interact and initiate differentiation. Possible genetic determinants for cytotactin distribution are now being sought, and homologies to Drosophila patternforming proteins, antennapedia and engrailed, have been found upstream of the cytotactin gene. In the developing CNS, cytotactin is made predominantly by glia, whereas the proteoglycan is made predominantly by neurons. In the cerebellum, cytotactin heavily labels the radial glial fibers. Studies of slice cultures of cerebellum have shown that antibodies to certain adhesive proteins can interfere with migration of neurons from the external granule cell layer to the internal granule cell layer. Antibodies to neural-glia cell adhesion molecule (NgCAM) prevents cells from leaving the external layer; antibodies to cytotactin cause the cells to accumulate in the molecular layer, although they leave this external layer at appropriate times. However, antibodies to NgCAM have no effect on migration. In the developing barrel cortex, cytotactin is differentially distributed. It is initially uniform, but becomes selectively decreased in the barrel hollows. However, this pattern is transient, and staining is uniform by P14. Cauterizing a row of whiskers results in a uniform staining in the corresponding cortical area at all ages. Indications for the possible role of the transient expression of cytotactin in the barrel cortex can be derived from cell culture studies. When chick dorsal root ganglion cells are plated on promoting substrates, such as fibronectin or laminin, they develop abundant neurites. However, if a triangle of cytotactin is painted on these substrates, the neurites completely avoid it. Cytotactin by itself can cause growth cone collapse, and on mixed cytotactin-fibronectin substrates, neurite outgrowth is decreased in proportion to the relative amount of cytotactin. In contrast, cytotactin can enhance neurite outgrowth when it is combined with a permissive substrate, such as polylysine. Uncertainty remains whether cytotactin is a permissive or inhibitory molecule, but it seems certain that its activity depends on what other molecules are present. This property, which is referred to as “ambitrophism,” gives cytotactin a

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wide possibility of actions, depending on its environment. Posters

The majority of posters dealt with the following themes: (1) developmental anatomy of barrel cortex; (2) developmental reorganization of barrel cortex and subcortical trigeminal relays following prenatal or neonatal peripheral lesions or modifications of activity; (3) activity-dependent regulation of normal functional organization in the mature barrel cortex; and (4) computer models of functional organization in the trigeminal system. Although it is difficult to select from the many excellent presentations, some of the highlights included a poster by Schlaggar and O’Leary examining the emergence of barrel patterning of ventrobasal thalamic afferents in rat SI cortex by means of acetylcholinesterase staining. They showed that a periphery-related pattern could be discerned in SI as early as 1 hr postnatally, 2 days earlier than that shown with other markers. Senft and Christensen used labeling in fixed tissue along with serial confocal reconstruction to examine spatial interrelationships of ingrowing thalamic afTerents with cortical dendrites. They concluded that neurites are selectively remodeled to match (and form) emerging barrel boundaries. Leslie et al. used multiple fluorescent labeling in cortex and immunocytochemistry in brainstem to demonstrate that both nucleus raphe dorsalis and the median raphe nucleus contribute to the transient, highly patterned serotonin immunoreactivity seen in SI of perinatal rats. However, the raphe-cortical neurons responsible for the pattern are not topographically organized. Interestingly, Laywell and Steindler demonstrated that transient, glial-derived, tenascin barrel boundaries of perinatal mice can be resurrected by lesions of SI in the adult. Rovainen et al. examined the development of endothelid shear stress of arterioles in mouse somatosensory cortex. They concluded that a set point for shear stress is established before birth, and that this could be used to govern the growth and postnatal differentiation of cortical microcirculation. With regard to activity-dependent regulation of trigeminal system development (Chiaia et al.), tetrodotoxin (TTX)-induced suppression of SI activity up to P11 demonstrated that neither peripherally evoked nor spontaneous activity in rat SI is required for normal pattern formation. However, Akhtar and Land showed that sensory deprivation induced by whisker trimming from birth can alter zinc levels in barrel cortex. Zinc is thought to modulate neurotransmission at glutamate receptors. Jacquin et al. used whisker trimming from birth to demonstrate that reduced peripheral stimulation

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BARRELS IV

can mimic the functional alterations in subnucleus interpolaris cells produced by infraorbital nerve section. This group also provided evidence that blocking impulses in the neonatal infraorbital nerve with TTX or bupivacaine did not alter the organization of cytochrome oxidase patches in the barrel neuraxis. Using partial lesions of the whisker pad from El5 to birth and cytochrome oxidase staining, and partial lesions of the infraorbital nerve in newborn rats and HRP tracing, Bennett-Clarke et al. and Stansel et al., respectively, provided evidence for competitive interactions among developing primary afferents in the brainstem. Two presentations examined long-term behavioral consequences of neonatal or perinatal peripheral alterations. Hand et al. demonstrated that whisker sparing prior to P3, combined with associatively paired training, can influence whisker preference during maze navigatioc, as well as cortical representations revealed by the 2deoxyglucose method. Klein et al. used the formalin test to show that neonatal infraorbital nerve cut renders the adult rat incapable of detecting a noxious chemical stimulus delivered to the whisker pad. With regard to the regulation of normal activity in barrel cortex by exogenous influences, Land et al. used immunocytochemistry and high-performance liquid chromatography to provide evidence that day-to-day variations in whisker stimulation can regulate y-aminobutyric acid (GABA) and glutamic acid decarboxylase levels in barrel cortex. GABA-ergic neurons in barrel cortex have been shown to modulate receptive field properties of barrel neurons. Guic-Robles and Merzenich showed that selective whisker stimulation can expand the cortical representations of the corresponding whiskers. Videomicroscopy was used by Cox et al. to show that variations in whisker activity also regulate blood flow within vessels supplying the corresponding cortical barrel. Furthermore, using a nontrigeminal system, Stem et al. presented evidence that in lactating rats, the intense stimulation of the nipples on the ventrum corresponds to an overall increase in ventrum representation in SI, with a decrease in the size of individual ventrum receptive fields.

Two groups presented computer models simulating response properties within the trigeminal system. Gupta et al. used data from multiple recordings of single neurons at multiple regions of the somatosensory cortex and thalamus to develop a computer simulation of networks responsible for oscillatory firing patterns observed in vivo, under conditions of drowsiness, slow-wave sleep, and anesthesia. The model suggests that afferent inputs compete for control of activity within these networks, and that strong afferent inputs can override endogenous oscillations, whereas weak afferent inputs are suppressed by endogenous oscillations. A model presented by Doherty et al. demonstrated that the parallel trigeminal pathways through the principal sensory nucleus and interpolaris, which converge onto the VPM thalamus, are sufficient to account for the principal whisker (center) and nonprincipal whisker (surround) response organization recorded from thalamocortical neurons in vivo. Keynote Address

(MICHAEL MERZENICH, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO)

Dr.Merzenich presented a historical overview of barrels research, emphasizing the system’s unique features as a model system for studying how experience affects cortical circuitry. Recent data were presented pertaining to the dynamic modulation of receptive fields in the somatosensory cortex. In particular, the effects of varying types of peripheral deafferentation were described, as well as experiments assessing how patterned inputs from neighboring whiskers dictate response properties in barrel cortex. Statedependent mechanisms were also implicated from cortical recording experiments in lactating rats. These results suggest that continued study of cortical “plasticity” will yield new and important directions for future research. ACKNOWLEDGMENTS

The participants would like to acknowledge IBRO’s and FIDIA’s generous contributions toward travel fellowships for graduate student and Third World attendance at Barrels IV.

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Barrels IV: proceedings of a satellite symposium of the 1991 Society for Neuroscience meeting.

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