Corticofugal axons from adjacent 'barrel' columns of rat somatosensory cortex: cortical and thalamic terminal patterns

The cortical representations of the vibrissae of the rat form a matrix in which each whisker has its own area of cortex, called a 'barrel'. The afferent pathways from the periphery travel first to the trigeminal nuclei and thence via the ventroposteromedial thalamus (VPM) to the cortical barrels have been described in detail. We have studied the output from barrels by filling adjacent areas of the primary somatosensory cortex (SI) with either Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) and demonstrating the course and terminations of the axons that arise within the barrel fields. The method not only dramatically illustrates the previously described corticothalamic pathway to VPM but also demonstrates a strict topography in the cortical afferents to the thalamic reticular nucleus (RT). Cells supplying the RT projection are found below the barrels in layer IV. Connections to the posterior thalamus, on the other hand, have no discernible topography and are derived from cortical areas surrounding the barrels. Thus the outputs of these 'septal' areas return to the region from which they receive thalamic input. The corticocortical connections are also visible in the same material. Contralateral cortical connections arise from the cells of the septa between barrels. The projections to secondary somatosensory area (SII) are mirror images of the barrel pattern in SI with rather more overlap but nonetheless a recognisable topography.     

Branching of individual somatosensory cerebropontine axons in rat: evidence of divergence

The cerebral cortex conveys major input to the granule cell layer of the cerebellar hemispheres by way of the pontine nuclei. Cerebrocortical projections terminate in multiple, widely distributed clusters in the pontine nuclei. This clustered organization is thought to provide the transition between the different organizational principles of the cerebrum and cerebellum, and indicates that parallel processing occurs at multiple sites in the pontine nuclei. At a cellular level, however, it is unknown whether individual cerebropontine neurons target pontocerebellar cells located in different clusters or not. We have employed anterograde axonal tracing and 3D computerized reconstruction techniques to characterize the branching pattern and morphology of individual cerebropontine axons from the primary somatosensory cortex (SI). Our findings show that 43% of the cerebrobulbar fibers arising from SI whisker representations provide two or three fibers entering the pontine nuclei, whereas 39% have only one fiber, and the remaining 18% do not project to the pontine nuclei. Thus, it appears that a majority of cerebropontine axons originating in SI whisker representations diverge to contact multiple, separated pontocerebellar cells. Further, 84% of the somatosensory cerebropontine fibers are collateral branches from cerebrobulbar and/or cerebrospinal parent fibers, while 16% are direct cerebropontine projections without a further descending projection. A range of thicknesses of the fibers entering the pontine nuclei were observed, with collaterals of corticobulbar fibers having the smallest diameter. Taken together, these findings may be related to previously described separate cerebropontine transmission lines with different properties.     

Receptors with opposing functions are in postsynaptic microdomains under one presynaptic terminal

Fast excitatory synaptic transmission through vertebrate autonomic ganglia is mediated by postsynaptic nicotinic acetylcholine receptors (nAChRs). We demonstrate a unique postsynaptic receptor microheterogeneity on chick parasympathetic ciliary ganglion neurons-under one presynaptic terminal, nAChRs and glycine receptors formed separate but proximal clusters. Terminals were loaded with [3H]glycine via the glycine transporter-1 (GlyT-1), which localized to the cholinergic presynaptic terminal membrane; depolarization evoked [3H]glycine release that was calcium independent and blocked by the GlyT-1 inhibitor sarcosine. Ganglionic synaptic transmission mediated by nAChRs was attenuated by glycine. Coexistence of separate clusters of receptors with opposing functions under one terminal contradicts Dale's principle and provides a new mechanism for modulating synaptic activity in vivo.     

Local erythropoietin signaling enhances regeneration in peripheral axons

Erythropoietin (EPO) and its receptor (EPO-R), mediate neuroprotection from axonopathy and apoptosis in the peripheral nervous system (PNS). We examined the impact and potential mechanisms of local EPO signaling on regenerating PNS axons in vivo and in vitro. As a consequence of injury, peripheral nerve axons and DRG neurons have a marked increase in the expression of EPO and EPO-R. Local delivery of EPO via conduit over 2 weeks to rat sciatic nerve following crush injury increased the density and maturity of regenerating myelinated axons growing distally from the crush site. In addition, EPO also rescued retrograde degeneration and atrophy of axons. EPO substantially increased the density and intensity of calcitonin gene-related peptide (CGRP) expression within outgrowing axons. Behavioral improvements in sensorimotor function also occurred in rats exposed to near nerve EPO delivery. EPO delivery led to decreased nuclear factor kappaB (NFkB) activation but increased phosphorylation of Akt and STAT3 within nerve and dorsal root ganglia neurons indicating rescue from an injury phenotype. Spinal cord explant studies also demonstrated a similar dose-dependent effect of EPO upon motor axonal outgrowth. Local EPO signaling enhances regenerating peripheral nervous system axons in addition to its known neuroprotection. Exogenous EPO may have a therapeutic role in a large number of peripheral nerve diseases through its impact on regeneration.     

The distribution of intrinsic cortical axons in area 3b of cat primary somatosensory cortex

Summary The morphology of single neurons in area 3b of cat primary somatosensory (SI) cortex was examined after horseradish peroxidase (HRP) injections. Neurons were labeled either by intracellular injection of HRP following intracellular recording or by small extracellular iontophoretic HRP injections. Both pyramidal and nonpyramidal neurons were labeled and reconstructed from serial sections. Their axons had local, interlaminar and interareal patterns of termination. Most neurons formed local axonal fields around their cell bodies and dendrites. Pyramidal neurons in cortical layer IV sent axons up into layers II and III, neurons in layers II and III sent axons down to layer V, and layer V neurons sent axons to layer VI as well as back to the upper layers. Layer VI neurons sent axons back to the upper cortical layers in a unique bowl-shaped pattern. The horizontal distribution of axons of pyramidal cells in layer III was extremely widespread. Axons of layer III neurons in area 3b terminated within 3b and area 1, but not in other areas of SI. Layer III neurons in area 1 distributed axon collaterals to all fields of SI as well as projecting a main axon to motor cortex. In general, the axon collaterals of area 3b pyramidal cells outside layer III remained confined to area 3b. Most of the nonpyramidal neurons labeled were basket cells in layers III and VI. These neurons formed dense axonal fields around their cell bodies, and none of their axons could be followed into the underlying white matter. The results of the present study demonstrate that area 3b somatosensory cortical neurons and their axons are vertically organized in a manner similar to that reported for other sensory cortical areas. They also show that widespread horizontal connections are formed by pyramidal neurons of layer III, and that these horizontal axons can travel for great distances in the cortical grey matter.     

Localization of ionotropic glutamate receptors in peripheral axons of human skin

Recent observations suggest that glutamate is important in sensory transduction in the periphery, contributing to peripheral sensitization of nociceptors and the hyperalgesia that accompanies inflammation. This study examined the presence of ionotropic glutamate receptors N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid (AMPA) and kainate (KA) in normal human hairy skin (n=6) using immunohistochemistry at the electron microscopic level. Analysis of labeled axons at the dermal-epidermal junction demonstrated that 26. 9+/-2, 19.5+/-3 and 18.5+/-1% of the axons analyzed were labeled for subunits of the NMDA, AMPA or KA receptors, respectively. An occasional Schwann cell process was labeled for either NMDA or KA receptors. The findings support the hypothesis that glutamate and its ionotropic receptors may play a role in the periphery in sensory processing in humans.     

Regeneration of olfactory sensory axons into transplanted segments of peripheral nerve

Isolated segments of cervical sympathetic trunk were transplanted onto the olfactory mucosa and bulb in adult rats, and olfactory nerve fascicles were sectioned in the region of the transplant. Olfactory axons, presumably arising from newly-formed sensory neurons, were observed to grow into the transplanted segments of peripheral nerve and were ensheathed by Schwann cells of the transplant. The axons were ensheathed as large bundles containing many axons in direct contact, an arrangement characteristic of the normal olfactory nerves, and not of the normal sympathetic trunk. However, single Schwann cell ‘units’ were each surrounded by basal lamina, an arrangement typical of the sympathetic trunk, not of olfactory nerves.The results indicate that olfactory axons, like other peripheral axons, are capable of directing some aspects of the manner in which they are ensheathed by Schwann cells.     

EphA4 is necessary for spatially selective peripheral somatosensory topography

Somatosensation is the primary sensory modality employed by rodents in navigating their environments, and mystacial vibrissae on the snout are the primary conveyors of this information to the murine brain. The layout of vibrissae is spatially stereotyped and topographic connections faithfully maintain this layout throughout the neuraxis. Several factors have been shown to influence general vibrissal innervation by trigeminal neurons. Here, the role of a cell surface receptor, EphA4, in directing position‐dependent vibrissal innervation is examined. EphA4 is expressed in the ventral region of the presumptive whisker pad and EphA4^?/? mice lack the ventroposterior‐most vibrissae. Analyses reveal that ventral trigeminal axons are abnormal, failing to innervate emerging vibrissae, and resulting in the absence of a select group of vibrissae in EphA4^?/? mice. EphA4's selective effect on a subset of whiskers implicates cell‐based signaling in the establishment of position‐dependent connectivity and topography in the peripheral somatosensory system. Developmental Dynamics 239:630–638, 2010. © 2009 Wiley‐Liss, Inc.     

Adaptive responses of monkey somatosensory cortex to peripheral and central deafferentation

This study deals with two kinds of activity-dependent phenomena in the somatosensory cortex of adult monkeys, both of which may be related: (1) mutability of representational maps, as defined electrophysiologically; (2) alterations in expression of genes important in the inhibitory and excitatory neurotransmitter systems. Area 3b of the cerebral cortex was mapped physiologically and mRNA levels or numbers of immunocytochemically stained neurons quantified after disrupting afferent input peripherally by section of peripheral nerves, or centrally by making lesions of increasing size in the somatosensory thalamus. Survival times ranged from a few weeks to many months.Mapping studies after peripheral nerve lesions replicated results of previous studies in showing the contraction of representations deprived of sensory input and expansion of adjacent representations. However, these changes in representational maps were in most cases unaccompanied by significant alterations in gene expression for calcium calmodulin-dependent protein kinase isoforms, for glutamic acid decarboxylase, GABA(A) receptor subunits, GABA(B) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-D-aspartate (NMDA) receptor subunits.Mapping studies after lesions in the ventral posterior lateral nucleus (VPL) of the thalamus revealed no changes in cortical representations of the hand or fingers until >15% of the thalamic representation was destroyed, and only slight changes until approximately 45% of the representation was destroyed, at which point the cortical representation of the finger at the center of a lesion began to shrink. Lesions destroying >60% of VPL resulted in silencing of the hand representation. Although all lesions were associated with a loss of parvalbumin-immunoreactive thalamocortical fiber terminations, and of cytochrome oxidase staining in a focal zone of area 3b, no changes in gene expression could be detected in the affected zone until >40-50% of VPL was destroyed, and even after that changes in mRNA levels or in numbers of GABA-immunoreactive neurons in the affected zone were remarkably small.The results of these studies differ markedly from the robust changes in gene expression detectable in the visual cortex of monkeys deprived of vision in one eye. The results confirm the view that divergence of the afferent somatosensory pathways from periphery to cerebral cortex is sufficiently great that many fibers can be lost before neuronal activity is totally silenced in area 3b. This divergence is capable of maintaining a high degree of cortical function in the face of diminishing inputs from the periphery and is probably an important element in promoting representational plasticity in response to altered patterns of afferent input.     

Rescue and regeneration of injured peripheral nerve axons by intrathecal insulin

Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both alpha and beta insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor alpha subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.     

Different multiple regeneration capacities of motor and sensory axons in peripheral nerve

Abstract After peripheral nerve injury, axons often project sprouts from the node of Ranvier proximal to the damage site. It is well known that one parent axon can sprout and maintain several regenerating axons. If enough endoneurial tubes in the distal stump are present for the regenerating axons to grow along, then the number of mature myelinated nerve fibers in the distal stump will be greater than the number in the proximal stump. "Multiple regeneration" is used to describe this phenomenon in the peripheral nerve. According to previous studies, a prominent nerve containing many axons can be repaired by the multiple regenerating axons sprouting from another nerve that contains fewer axons. Most peripheral nerves contain a mixture of myelinated motor and sensory axons as well as unmyelinated sensory and autonomic axons. In this study, a multiple regeneration animal model was developed by bridging the proximal common peroneal nerve with the distal common peroneal nerve and the tibial nerve. Differences in the multiple regeneration ratio of motor and sensory nerves were evaluated using histomorphometry one month after ablating the dorsal root ganglion (DRGs) and ventral roots, respectively. The results suggest that the motor nerves have a significantly larger multiple regeneration ratio than the sensory nerves at two different time points.     

Morphological and electrophysiological characteristics of somatosensory thalamocortical axons studied with intra-axonal staining and recording in the cat

Summary The intracortical arborizations of thalamocortical fibers arising from the ventroposterolateral (VPL) nucleus in the cat were studied following intra-axonal injections of horseradish peroxidase (HRP). The axons were impaled 1.5 to 3 mm below the surface of the cortex, identified electrophysiologically by stimulating the VPL nucleus and functionally by stimulating the somatic receptive field with natural stimuli. Many of the results obtained in a previous study using similar techniques (Landry and Deschênes 1981) were confirmed by the present experiments. Fibers activated by cutaneous stimulation arborized either in area 3b or 1 but some did send branches to both areas. Also, the intracortical arborization of a rapidly adapting cutaneous afferent fiber in area 2 is described. The size and tangential extent of the fiber in area 2 are similar to those arborizing in other areas of the primary somatosensory cortex and consist of multiple patches separated by uninvaded gaps. One fiber activated by stimulation of deep tissue receptors gave rise to two bushes that arborized along a rostrocaudal axis exclusively in area 3b. Terminal boutons and varicosities were found mostly in layers VI, IV, the bottom third of III and the upper portion of V, but some fibers did send a few collateral branches to layer II and the bottom part of layer I. The results suggest that in the forelimb representation, the same modality and submodality can be recorded in more than one cytoarchitectonic area but that areas 3b, 1 and 2 should not be considered as a single functionally homogeneous area. Counts of terminals suggest that a single fiber arborizing in area 1 makes as many as 3 times the number of synapses made in area 2 or 3b. Since fibers appear to be modality and submodality specific, if convergence of modality, submodality and/or body areas occur in the cortex, then this must be preferentially, but not exclusively, done by thalamic fibers of different functions which arborize in the same cytoarchitectonic area and synapse upon a shared postsynaptic target. In the same experiments intra-axonal recordings revealed the presence of two hyperpolarizing afterpotentials elicited by a preceding action potential. The first afterpotential was associated with a decrease in excitability of the fiber and an increase in membrane resistance. Passage of depolarizing current through the microelectrode was necessary to demonstrate the second afterpotential. These afterpotentials may affect the integrative properties of the axons by modifying impulses originating in the thalamus.Supported by grants from MRC and FRSQ to P. Landry and R. W. Dykes, and an FCAC studentship to P. Diadori     

Expansion of stimulus-evoked metabolic activity in monkey somatosensory cortex after peripheral denervation

Summary The 2-deoxy-glucose (2DG) technique was used to study changes in stimulus-evoked metabolic activity in the somatosensory cortex of the squirrel monkeySaimiri sciureus after unilateral digit amputation. Two to 52 weeks after digit 2 on the left hand was removed, a somatic stimulus was applied to digit 3 bilaterally. In area 3b corresponding to the deafferented side of the brain, the area of stimulus-evoked metabolic activity was greater than that on the opposite, control side of the brain within the same animal. The extent of the topographic projection map of 2DG label in area 3b on the deafferented side of the brain was 1.92 to 4.75 times greater than that on the control side. There was no difference, however, in the topographical area of stimulus-evoked metabolic activity between the left and right somatosensory cortices in a normal, unoperated animal. These data suggest that the changes in functional organization observed using electrophysiological recordings in somatosensory cortex after peripheral denervation may have a metabolic substrate.     

The long-term effects of a single injection of taxol upon peripheral nerve axons

Summary To study the long-term effects of a single injection of the microtubule stabilizing drug taxolin vivo, the compound was injected into rat sciatic nerve and the ensuing morphological changes followed for 8–25 weeks after injection. In accord with previously published works, taxol-induced giant axonal bulbs were common and were most marked at 8–10 weeks. From 12 weeks onwards these giant axons decreased in diameter with concomitant remyelination. By 20 weeks axonal bulbs could not be seen. The recovery of axons from taxol intoxication began 8–12 weeks after injection with the growth of axonal sprouts, longitudinally and laterally, from the distal aspect of the proximal stump. During recovery, from 12 weeks onwards, axons showed apparent reorganization of the axoplasmic cytoskeleton where microtubules diminished and neurofilaments became more numerous. By 16 weeks only small groups of microtubules remained, often encircling a mitochondrion. By 25 weeks taxol-treated nerves showed no apparent taxol-induced changes. A common ultrastructural finding up to 16 weeks was the appearance within axons of tubular profiles covered by a double membrane. These structures were sometimes arranged as crystalloid aggregates. The diameter of these profiles was 85 nm, they were most common at 12 weeks and it is proposed that they may be derived from mitochondria. The present results show taxol to have a long-lasting and local effect upon axoplasmic organizationin vivo. The cytoskeletal reorganization described supports the concept of the differential movement of axoplasmic neurofilaments and that neurofilaments stabilize axonal structures.     

Development of terminal arbors of retino-geniculate axons in the kitten—I. Light microscopical observations

The maturation of terminal arbors of retino-geniculate axons was studied in normal kittens from 1 to 8 postnatal weeks. Horseradish peroxidase injected into the optic tract rostral to the lateral geniculate nucleus gave a dense fill of cut axons and their terminals, resembling results obtained by the Golgi methods. At 1 and 2 weeks postnatal, the overall size and extent of axon arbors is not significantly different than in the adult. However, terminal branches of axon arbors at this age give rise to high variable endings. They terminate in finely divided sprays of finger-like extensions and filopodia bearing small spikes rather than the characteristic clusters and strands of crenulated terminals of adult axons. The bases of these sprays are broad and irregular in contour, with foliate growth-cone-like structures occurring at the ends of some branches. At 3 weeks postnatal, terminal swellings become thicker and more crenulated in contour. By 5 to 6 weeks, axon arbors have adult-like terminals with respect to their size, shape and arrangement, although entire branches may still be immature with irregular terminal swellings. Small slightly indented terminals are also seen for the first time. By 8 weeks, axon arbors are generally mature, but occasionally have a bizarre and immature arrangement of fine extensions or growth-cone-like tips.Although these observations do not establish whether reduction of branches or of terminals takes place during postnatal maturation, they demonstrate that kitten retino-geniculate axon terminal arbors are highly immature and undergo considerable changes during the period optimal for induction of sprouting by eye enucleation. The morphogenetic maturation of terminal branches that begins at 3 weeks also marks a decline in their sprouting capacity, even though remodeling of terminals is not complete until after 8 weeks of age.     

Comparison of cytokine expression profile during Wallerian degeneration of myelinated and unmyelinated peripheral axons

Changes in cytokine and chemokine expression during Wallerian degeneration have been studied using nerve transection models, which result in denervation of both myelinating and non-myelinating Schwann cells. Cytokine and chemokine response of non-myelinating Remak Schwann cells to loss of their axons is unknown. In this study, we compared the expression profile of various cytokines and chemokines in distal nerves after capsaicin-induced degeneration of unmyelinated axons to Wallerian degeneration induced by nerve transection. Upregulation of MCP-1, IL-2, IL-6 and IL-10 were seen in both groups but IL-1ß and LIF were primarily upregulated in Wallerian degeneration of the whole nerve and not in capsaicin-induced degeneration of unmyelinated axons. The activated macrophage response, as measured by an increase in ED-1 immunostaining, was more prominent in the transected sciatic nerves compared to capsaicin-treated nerves. These findings indicate that there are differences in the cytokine and chemokine response of myelinating and non-myelinating Schwann cells to loss of their axons, and add to a growing body of literature that points to greater heterogeneity among Schwann cells.