Branching patterns of corticospinal axon arbors in the rodent

Despite extensive study of corticospinal connections in a variety of species, little is known about the detailed morphology of corticospinal axon arbors. Results in previous studies of primates based on intra-axonal filling with horseradish peroxidase (HRP) staining of a limited sample of fibers suggest that corticospinal arbors branch widely to multiple motoneuronal pools. To determine whether this pattern of corticospinal connectivity is present in nonprimate species as well, we studied the branching patterns of corticospinal axon arbors in a rodent species, the golden hamster. The axons were labeled by iontophoretic injection of Phaseolus vulgaris-leucoagglutinin (PHA-L) into small regions of the forelimb and hindlimb sensorimotor cortex, and immunohistochemistry with the peroxidase-antiperoxidase (PAP) method was used to reveal fine details of terminal arbors within the cervical and lumbar enlargements of the spinal cord. As in higher mammals, corticospinal connections are topographically organized. Moreover, corticospinal axons arising from somatosensory cortex project primarily to the dorsal horn, whereas those from motor cortex terminate most heavily in the ventral horn. This differential projection pattern, not previously demonstrated in rodents, implies functional differences between somatosensory and motor components of the corticospinal pathway. Reconstruction of corticospinal arbors in the ventral horn showed that in both cervical and lumbar spinal cord segments, axons branch widely into interneuronal regions. A surprising number appear to extend into motoneuron cell groups, and some of these axons branch into multiple motoneuronal pools. Widely divergent corticospinal axons that branch to multiple motoneuron pools have been shown to mediate activity in functionally related muscle groups of the primate forearm. The present results suggest that in other species, such as the rodent, a similar divergence of corticospinal arbors may also function to facilitate activity in subsets of muscles.     

Development of specificity in corticospinal connections by axon collaterals branching selectively into appropriate spinal targets

Corticospinal projections in adult rodents arise exlusively from layer V neurons in the sensorimotor cortex. These neurons are topographically organized in their connections to spinal cord targets. Previous studies in rodents have shown that the mature distribution pattern of corticospinal neurons develops during the first 2 weeks postnatal from an initial widespread pattern that includes the visual cortex to a distribution restricted to the sensorimotor cortex. To determine whether specificity in corticospinal connections also emerges from an intially diffuse set of projections, we have studied the outgrowth of corticospinal axons and the formation of terminal arbors in developing hamsters. The sensitive fluorescent tracer 1, 1′, dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorat (DiI) was used to label corticospinal axons from the visual cortex or from small regions of the forelimb or hindlimb sensorimotor cortex in living animals at 4–17 days postnatal. Initially axon outgrowth was imprecise. Some visual cortical axons extended transiently beyond their permanent targets in the pontine nuclei, by growing through the pyramidal decussation and in some cases extending as far caudally as the lumbar enlargement. Forelimb sensorimotor axons also extended past their targets in the cervical enlargement, in many cases growing in the corticospinal tract to lumbar levels of the cord. By about 17 days postnatal these misdirected axons or axon segments were withdrawn from the tract. Despite these errors in axon trajectories within the corticospinal tract, terminal arbors branching into targets in the spinal gray matter were topographically appropriate from the earliest stages of innervation. Thus visula cortical axons never formed connections in the spinal cord, forelimb sensorimotor axons arborized only in the cervical enlargement, and hindlimb cortical axons terminated only in the lumbar cord at all stages of development examined. Corticospinal arbors formed from collaterals that extended at right angles from the shafts of primary axons, most likely by the process of interstitial branching after the primary growth cone had extended past the target. Once collaterals extended into the spinal gray matter, highly branched terminal arbors formed within 2–4 days, beginning at about 4 and 8 days postnatal for the cervical and lumbar enlargements, respectively. These results show that specificity in connectivity is achieved by selectivty growth of axon collaterals in to appropriate spinal targets from the beginning and not by the later remodeling of intially diffuse connections. In contrast, errors occur in the initial outgrowth of axons in the corticospinal tract, which are subsequently corrected. Copyright © 1994 Wiley-Liss, Inc.     

Pre‐synaptic and post‐synaptic neuronal activity supports the axon development of callosal projection neurons during different post‐natal periods in the mouse cerebral cortex

Callosal projection neurons, one of the major types of projection neurons in the mammalian cerebral cortex, require neuronal activity for their axonal projections [H. Mizuno et al. (2007) J. Neurosci., 27, 6760–6770; C. L. Wang et al. (2007) J. Neurosci., 27, 11334–11342]. Here we established a method to label a few callosal axons with enhanced green fluorescent protein in the mouse cerebral cortex and examined the effect of pre‐synaptic/post‐synaptic neuron silencing on the morphology of individual callosal axons. Pre‐synaptic/post‐synaptic neurons were electrically silenced by Kir2.1 potassium channel overexpression. Single axon tracing showed that, after reaching the cortical innervation area, green fluorescent protein‐labeled callosal axons underwent successive developmental stages: axon growth, branching, layer‐specific targeting and arbor formation between post‐natal day (P)5 and P9, and the subsequent elaboration of axon arbors between P9 and P15. Reducing pre‐synaptic neuronal activity disturbed axon growth and branching before P9, as well as arbor elaboration afterwards. In contrast, silencing post‐synaptic neurons disturbed axon arbor elaboration between P9 and P15. Thus, pre‐synaptic neuron silencing affected significantly earlier stages of callosal projection neuron axon development than post‐synaptic neuron silencing. Silencing both pre‐synaptic and post‐synaptic neurons impaired callosal axon projections, suggesting that certain levels of firing activity in pre‐synaptic and post‐synaptic neurons are required for callosal axon development. Our findings provide in‐vivo evidence that pre‐synaptic and post‐synaptic neuronal activities play critical, and presumably differential, roles in axon growth, branching, arbor formation and elaboration during cortical axon development.     

Specificity of corticospinal axon arbors sprouting into denervated contralateral spinal cord.

Previous studies have reported considerable plasticity in the rodent corticospinal pathway in response to injury. This includes sprouting of intact axons from the normal pathway into the contralateral spinal cord denervated by an early corticospinal lesion. We carried out the present study to obtain detailed information about the time course, origin, and degree of specificity of corticospinal axons sprouting in response to denervation. Hamsters (Mesocricetus auratus) ranging in age from 5 to 23 days received unilateral lesions of the left medullary pyramidal tract. Two weeks after the lesion, small regions of the right sensorimotor cortex opposite the lesion were injected with the plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After a further 2 week survival period, immunohistochemistry was carried out on frozen sections of the fixed brains and spinal cords. Detailed morphological analysis of PHA-L labeled corticospinal axons revealed that sprouting from the intact corticospinal pathway into the contralateral denervated spinal cord occurred only at local spinal levels and not at the pyramidal decussation. Arbors sprouting into the denervated cord frequently arose from corticospinal axons that branched into the normal side of the cord as well. Sprouting was maximal after early lesions (5 days) and declined with lesions at later ages up to 19 days. Sprouting corticospinal axons arborized with the same degree of functional and topographic specificity as previously reported for normal corticospinal arbors (Kuang and Kalil: J. Comp. Neurol. 292:585-598, '90), such that axons arising from somatosensory cortex projected only to the dorsal horn, those from motor cortex innervated only the ventral horn, and normal forelimb and hindlimb topography was preserved. Sprouting fibers also had normal branching patterns. Parallel studies of developing corticospinal arbors showed that sprouting could not be attributed to maintenance or expansion of early bilateral connections. These results suggest that local signals, most likely similar to those governing normal corticospinal development, elicit corticospinal sprouting and determine specificity of axon arbors.     

Somatosensory cortex of the neonatal pig: I. Topographic organization of the primary somatosensory cortex (SI)

The cerebral cortex of adult mammals contains several somatotopic representations of the body surface. Although the organization of the various somatosensory cortices of numerous species of adult animals has been elucidated, data on the somatosensory representations of fetal and neonatal animals are limited. As part of an investigation into the perinatal development of the somatosensory cortices, it was necessary to delineate the organization of the somatosensory cortices of the perinatal pig. This study presents the topographical organization of the primary somatosensory cortex (SI) of the perinatal pig. Multiunit microelectrode mapping methods were used to produce topographic maps of SI from barbiturate anesthetized pigs ranging in age from 7 days preterm to 2 months postpartum. It was demonstrated that the overall organization of this region of cortex was similar to that of other mammals: a somatotopic projection of predominantly the contralateral body surface was delineated in which the hindlimb is represented medially and the face laterally across the cortex. A disproportionately enlarged rostrum representation was mapped in detail, and multiple representations of the rostrum, face, and mouth were found. Several of these representations exhibited bilateral and ipsilateral input. The SI trunk and hindlimb representations were located on the medial wall of the hemisphere; these representations were small but their presence refutes speculation that ungulates do not have a complete body representation in SI.     

Variations in conduction velocity and excitability following single and multiple impulses of visual callosal axons in the rabbit

The present study examined the conduction properties of 75 visual callosal axons of the awake rabbit. These axons were studied by measuring latency to antidromic activation of cell bodies following midline callosal and/or contralateral cortical stimulation. Seventy-three of 75 neurons (axon conduction velocities = 0.3 to 12.9 m/sec) demonstrated decreases in antidromic latency and threshold to a test stimulus which followed an antecedent conditioning stimulus at appropriate intervals. Control experiments indicated that (i) the latency and threshold variations resulted from prior impulse conduction along the axon, and (ii) the latency decrease reflected an increase in conduction velocity along the main axon trunk. The maximum magnitude of the latency decrease for different axons ranged from 3 to 22% of control values, and the duration ranged from 18 to 169 msec. The duration of the latency decrease was greater for slowly conducting axons than for fast conducting axons. Latency increases to an antidromic test stimulus occurred for up to several minutes following a train of antidromic conditioning pulses. Antidromic latency and threshold shifts were also observed in somatosensory callosal axons and in some corticotectal axons.     

Morphology of single intracellularly stained axons terminating in area 3b of macaque monkeys

We have studied the morphology of single thalamocortical axons innervating area 3b of postcentral somatosensory cortex in macaque monkeys. We recorded from axons in the white matter below the representation of the hand in postcentral cortex in two monkeys (Macaca fascicularis) by using micropipettes filled with horseradish peroxidase (HRP). When an axon was recorded, we delineated its receptive field and determined its modality, and if cutaneous, whether it was slowly or rapidly adapting (SA or RA). We then impaled the axon and injected it with HRP. We recorded and successfully injected many more RA than SA axons, possibly because of differences in their true proportions. The RA axonal arbors varied in mediolateral extent from 350 to 800 microns with a mean of 600 microns. One of the RA axons gave rise to four separate arbors spanning 2.5-3.0 mm of cortex. The single SA axon we recovered was 370 microns in width. We suggest that the individual terminal zones underlie the columnar parcellation of the somatosensory cortex. The presence of arbors spanning several such columns suggests that all regions within the arbor may not be equally effective in driving cortical cells under normal conditions, and such arbors may provide the substrate for a cortical response to alterations in the pattern of input.     

Morphology and synaptic connections of ultrafine primary axons in lamina I of the spinal dorsal horn: candidates for the terminal axonal arbors of primary neurons with unmyelinated (C) axons

Neurons in Rexed's lamina I have the bulk of their dendritic arbors confined within this lamina. This study examines the morphology and synaptic connections of primary axons which generate axonal endings in lamina I of the spinal dorsal horn and are in position to deliver their inputs directly to lamina I neurons. Primary axons were made visible for light and electron microscopical study by applying horseradish peroxidase (HRP) to the severed central stumps of cervical and lumbar dorsal roots and allowing sufficient time for the orthograde movement of the HRP into the terminal axonal arbors. Golgi preparations provided supplementary light microscopical views of these axons. Lamina I receives the terminal arborization of two very different kinds of primary axons. One of these generates many ultrafine endings along unbranched, long rostrocaudally oriented, strand-like collaterals which arise from thin parent branches in Lissauer's tract. In view of these thin parent branches, most ultrafine primary axons are considered to be unmyelinated (C) primary axons. The second kind of primary axon generates large caliber endings on branched collaterals. These arise from relatively thick parent branches in Lissauer's tract which, on the basis of their size, are considered to be myelinated (A delta) primary axons. The scalloped endings of both primary axons lie in the interior of glomeruli where they form axodendritic synapses on small dendritic shafts and spines. It is at these synapses that these two kinds of primary axons are thought to transfer nociceptive and thermal inputs directly to the dendritic arbors of lamina I neurons. Transmitter release at these axodendritic synapses in response to primary inputs can be modified, probably diminished or inhibited, by synaptic events within the glomeruli from at least three sources. Synaptic vesicle-containing dendrites form dendroaxonic synapses on primary endings and two kinds of axons form axoaxonic synapses either on primary endings or on the intervaricose segments of the primary axons.     

Computational Structure of Visual Callosal Axons

We analysed the activation profiles obtained by simulating invasion of an orthodromic action potential in eleven anterogradely filled and serially reconstructed terminal arbors of callosal axons originating and terminating in areas 17 and 18 of the adult cat. This was done in order to understand how geometry relates to computational properties of axons. In the simulation, conduction from the callosal midline to the first bouton caused activation latencies of 0.9-3.2 ms, compatible with published electrophysiological values. Activation latencies of the total set of terminal boutons varied across arbors between 0.3 and 2.7 ms. Arbors distributed boutons in tangentially segregated terminal columns spanning one or, more often, several layers. Individual columns of one axon were frequently activated synchronously or else within a few hundred microseconds of each other. Synchronous activation of spatially separate columns is achieved by: (i) long primary or secondary branches of similar calibre running nearly parallel to each other for several millimetres; (ii) variations in the calibre of branches serially fed to separate columns by the same primary or secondary branch; (iii) exchange of high-order or preterminal branches across columns. The long, parallel branches blatantly violate principles of axonal economy. Simulated alterations of the axonal arbors indicate that similar spatiotemporal patterns of activity could, in principle, be obtained by less axon-costly architectures. The structure of axonal arbors, therefore, may not be determined solely by the type of spatiotemporal activation profiles it achieves in the cortex but also by other constraints, in particular those imposed by developmental mechanisms.     

Development of callosal connections in the sensorimotor cortex of the hamster.

To investigate the development of corpus callosal connectivity in the hamster sensorimotor cortex, we have used the sensitive axonal tracer 1,1 dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI), which was injected either in vivo or in fixed brains of animals 3-6 days postnatal. First, to study changes in the overall distribution of developing callosal afferents we made large injections of DiI into the corpus callosal tract. We found that the anterogradely labeled callosal axons formed a patchy distribution in the contralateral sensorimotor cortex, which was similar to the pattern of adult connectivity described in earlier studies of the rodent corpus callosum. This result stands in contrast to previous retrograde studies of developing callosal connectivity which showed that the distribution of callosal neurons early in development is homogeneous and that the mature, patchy distribution arises later, primarily as a result of the retraction of exuberant axons. The initial patchy distribution of callosal axon growth into the sensorimotor cortex described in the present study suggests that exuberant axons destined to be eliminated do not enter the cortex. In addition, small injections of DiI into developing cortex resulted in homotopic patterns of callosal topography in which reciprocal regions of sensorimotor cortex are connected, as has been shown in the adult. Second, to study the radial growth of callosal afferents we followed the extension of individual callosal axons into the developing cortex. We found that callosal axons began to invade the contralateral cortex on about postnatal day 3, with little or no waiting period in the callosal tract. Callosal afferents then advanced steadily through the cortex, never actually invading the cortical plate but extending into layers on the first day that they could be distinguished from the cortical plate. The majority of callosal axons grew radially through the cortex and did not exhibit substantial branching until postnatal day 8, the age when the cortical plate disappears and callosal afferents reach the outer layer of cortex. This mode of radial growth through cortex prior to axon branching could serve to align callosal afferents with their radial or columnar targets before arborizing laterally.     

Terminal arborizations of retinotectal axons in the bullfrog

In the optic tectum of Rana catesbeiana four laminae of myelinated fibers in the superficial zone of the optic tectum (laminae B, D, F, and G: Potter, ′69) are identified as retinal axons on evidence from patterns of degeneration following contralateral eye removal. After survival times of 5 to 22 days Cajal's block method II shows either fragmentation or abnormal beading of the axons in the four laminae and the paraffin-Nauta method shows coarse granules, representing axonal debris, in these laminae. Golgi impregnations of terminal arborizations arising from fibers in laminae B, D, F, and G show three major types of branching patterns. The arbors are horizontally flattened and are elongated in the same direction as the parent fibers. Densely branched (DB) arbors have stem fibers in laminae B and D and are distributed within the same and adjacent laminae. Widely branched (WB) and thin, straight (TB) arbors have stem fibers in laminae F and G and are distributed mainly in the two laminae of origin with some overlap into adjacent layers of cells and fibers. The horizontal dimensions of DB arbors are 30 μ–70 μ by 100 μ–200 μ, whereas the WB arbors are more variable in overall dimensions. The TB arbors consist of long thin stem axons that give off infrequent terminal specializations in the form of forked or spiny appendages. Numerous terminal forks are present on the WB arbors but are rare on the DB arbors. Swellings and varicosities, that probably represent synaptic specializations, are particularly numerous on the long tertiary branches of DB arbors.     

Double-labeling study of axonal branching within the lateral reticulocerebellar projection in the rat

Collateral axonal branching to the cerebellum from the lateral reticular nucleus (LRN) was studied in the rat by using the fluorescent double-labeling technique. Following injection of Fast Blue (FB) into the cerebellar cortex, followed 3 days later by injection of Nuclear Yellow (NY) into a different region of the cortex, single- and double-labeled cells were found within the LRN. Most LRN-cerebellar projections were bilateral with ipsilateral preponderance, except for the projection to the paramedian lobule, which was completely ipsilateral. The dorsolateral area of the magnocellular division of the LRN contained cells whose axons branch to terminate in the rostral anterior lobe and the caudal part of the ipsilateral paramedian lobule (hindlimb areas of the cerebellar cortex), while the medial area of the LRN contained cells that supply, via collateral axonal branching, the caudal area of the contralateral anterior lobe and the rostral part of the ipsilateral paramedian lobule (forelimb areas of the cerebellum). Branched LRN-cerebellar axons projected to both hemispheres and to both sides of the caudal anterior lobe. No axonal branching was evident in the LRN-cerebellar projection to the rostral anterior lobe. The projection to the anterior and posterior lobe vermis also contained collateral axonal branching.     

Staining of regenerated optic arbors in goldfish tectum: progressive changes in immature arbors and a comparison of mature regenerated arbors with normal arbors

Individual optic arbors, normal and regenerated, were stained via anterograde transport of HRP and viewed in tectal whole mounts. Camera lucida drawings were made of 119 normal optic arbors and of 242 regenerated arbors from fish 2 weeks to 14 months postcrush. These arbors were analyzed for axonal trajectory, spatial extent in the horizontal plane, degree of branching, number of branch endings, average depth, and degree of stratification. Normal optic arbors ranged in size from roughly 100 to 400 microns across in a continuous distribution, had an average of 20 branch endings with average of fifth-order branching, and were highly stratified into one of three planes within the major optic lamina (SO-SFGS). Small arbors arising from fine-caliber axons terminated in the most superficial plane of SO-SFGS; large arbors from coarse axons terminated in the superficial and middle planes; and medium arbors from medium-caliber axons terminated in the middle and deep planes of SO-SFGS, as well as deeper in the central gray and deep white layers. Arbors from central tectum tended to be much more tightly stratified than those in the periphery. No other differences between central and peripheral arbors were noted. Mature regenerated arbors (five months or more postcrush) were normal in their number of branch endings, order of branching, and depth of termination. Their branches covered a wider area of tectum, partially because of their early branching and abnormal trajectories of branches. Axonal trajectories were often abnormal with U-turns and tortuos paths. Fine-, medium-, and coarse-caliber axons were again present and gave rise to small, medium, and large arbors at roughly the same depths as in the normals. There was frequently a lack of stratification in the medium and large arbors, which spanned much greater depths than normal. Overall, however, regenerates reestablished nearly normal morphology except for axonal trajectory and stratification. Early in regeneration, the arbors went through a series of changes. At 2 weeks postcrush, regenerated axons had grown branches over a wider-than-normal extent of tectum, though they were sparsely branched and often tipped with growth cones. At 3 weeks, the branches were more numerous and covered a still wider extent (average of five times normal), many covering more than half the tectal length or width. At 4-5 weeks smaller arbors predominated, although a few enlarged arbors were present for up to 8 weeks. Additional small changes occurred beyond 8 weeks as the arbors became progressively more normal in appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization,Development and Enucleation-induced Alterations in the Visual Callosal Projection of the Hamster: Single Axon Tracing with Phaseolus vulgaris leucoagglutinin and Di-I

The distribution of callosal axons interconnecting lateral area 17 and medial area 18 of the rodent's occipital cortex is dramatically altered by neonatal enucleation, but it is not known how this manipulation affects the morphology of individual callosal axons or whether the enucleation-induced changes in this pathway reflect maintenance of a transient developmental state by these fibres. In the present study, these questions were addressed by tracing the individual callosal axons in normal adult and neonatally enucleated adult hamsters with Phaseolus vulgaris leucoagglutinin (PHAL) and by anterograde labelling of developing callosal axons with the carbocyanine dye, Di-I. In normal adults, injections of PHAL into the region of the 17 - 18a border produced dense labelling in all layers in the region of the contralateral 17 - 18a border. Larger injections resulted in callosal labelling that extended across the lateral one-half of area 17, primarily in layers I and V. Thirty-four callosal axons from normal adult hamsters were reconstructed through all the cortical laminae. Most of these had very simple terminal arbors. They gave off short collaterals in the infragranular layers and branched more extensively in the uppermost part of layer II - III and in lamina I. Small injections of PHAL into the occipital cortex of neonatally enucleated adult hamsters resulted in labelled axons throughout most of areas 17 and 18a in the contralateral hemisphere. The terminal arbors of most individual callosal axons in eyeless hamsters were not appreciably different from those in sighted animals. However, 26.8% of 28 fibres reconstructed through all cortical laminae in the neonatally enucleated hamsters had much more widespread branches than any of the axons recovered from normal hamsters. As a result, the average total length of the callosal axons from the blinded hamsters was significantly greater than that for such fibres from the sighted animals. Anterograde labelling with Di-I demonstrated axons in the anterior commissure and anterior part of the corpus callosum on P-0. Labelled fibres extended into the white matter underlying the occipital cortex on P-1 and entered the cortical plate on P-2. Some of these axons reached into the marginal layer. Many developing callosal axons had short branches in the white matter, but generally extended only a single collateral into the cortical grey matter. Callosal axons in perinatal animals branched very little within the cortex and, in this respect, resembled fibres labelled with PHAL in adult hamsters. These results support the conclusion that the expanded tangential distribution of the occipital callosal projection in neonatally enucleated adult hamsters results, at least in part, from individual axons with abnormally widespread terminal arbors which are not present in large numbers at any time during normal development.     

D-[3H]aspartate retrograde labelling of callosal and association neurones of somatosensory areas I and II of cats

Experiments were carried out on cats to ascertain whether corticocortical neurones of somatosensory areas I (SI) and II (SII) could be labelled by retrograde axonal transport of D-[3H]aspartate (D-[3H]Asp). This tritiated enantiomer of the amino acid aspartate is (1) taken up selectively by axon terminals of neurones releasing aspartate and/or glutamate as excitatory neurotransmitter, (2) retrogradely transported and accumulated in perikarya, (3) not metabolized, and (4) visualized by autoradiography. A solution of D-[3H]Asp was injected in eight cats in the trunk and forelimb zones of SI (two cats) or in the forelimb zone of SII (six cats). In order to compare the labelling patterns obtained with D-[3H]Asp with those resulting after injection of a nonselective neuronal tracer, horseradish peroxidase (HRP) was delivered mixed with the radioactive tracer in seven of the eight cats. Furthermore, six additional animals received HRP injections in SI (three cats; trunk and forelimb zones) or SII (three cats; forelimb zone). D-[3H]Asp retrograde labelling of perikarya was absent from the ipsilateral thalamus of all cats injected with the radioactive tracer but a dense terminal plexus of anterogradely labelled corticothalamic fibres from SI and SII was observed, overlapping the distribution area of thalamocortical neurones retrogradely labelled with HRP from the same areas. D-[3H]Asp-labelled neurones were present in ipsilateral SII (SII-SI association neurones) in cats injected in SI. In these animals a bundle of radioactive fibres was observed in the rostral portion of the corpus callosum entering the contralateral hemisphere. There, neurones retrogradely labelled with silver grains were present in SI (SI-SI callosal neurones). Association and callosal neurones labelled from SI showed a topographical distribution similar to that of neurones retrogradely labelled with HRP. The laminar patterns of corticocortical neurones labelled with D-[3H]Asp or with HRP were also similar, with one exception. In the inner half of layer II, SII-SI association neurones and SI-SI callosal neurones labelled with the radioactive marker were much less numerous than those labelled with HRP. In cats injected in SII, D-[3H]Asp retrogradely labelled cells were present in ipsilateral SI (SI-SII association neurones). Their topographical and laminar distribution overlapped that of neurones labelled with HRP but, as in cats injected in SI, association neurones labelled with silver grains were unusually rare in the inner layer III.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synapses made by axons of callosal projection neurons in mouse somatosensory cortex: emphasis on intrinsic connections

This is one of a series of papers aimed at identifying the synaptic output patterns of the local and distant projections of subgroups of pyramidal neurons. The subgroups are defined by the target site to which their main axon projects. Pyramidal neurons in areas 1 and 40 of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of these neurons ("intrinsic" terminals) were identified in somatosensory areas 1 and 40, and their distribution and synaptic connectivity were examined. Also examined were the synaptic connections of "extrinsic" callosal axon terminals labeled by lesion induced degeneration consequent to the severing of callosal fibers. A post-lesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by HRP. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where they formed only asymmetrical synapses. Layers II and III contained the highest concentrations of both types of callosal axon terminal. Analyses of serial thin sections through layers II and III in both areas 1 and 40 yielded similar results: 97% of the extrinsic (277 total sample) and of the intrinsic (1215 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal contrasts with the proportion of asymmetrical, axospinous synapses that occur in the surrounding neuropil where only about 80% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of both extrinsic and intrinsic axonal pathways in the cortex (White and Keller, 1989: Cortical Circuits; Boston: Birkhauser): in all instances, axonal pathways are highly selective for the types of elements with which they synapse.     

Interhemispheric connections of somatosensory cortex in the flying fox

The interhemispheric connections of somatosensory cortex in the gray-headed flying fox (Pteropus poliocephalus) were examined. Injections of anatomical tracers were placed into five electrophysiologically identified somatosensory areas: the primary somatosensory area (SI or area 3b), the anterior parietal areas 3a and 1/2, and the lateral somatosensory areas SII (the secondary somatosensory area) and PV (pairetal ventral area). In two animals, the hemisphere opposite to that containing the injection sites was explored electrophysiologically to allow the details of the topography of interconnections to be assessed. Examination of the areal distribution of labeled cell bodies and/or axon terminals in cortex sectioned tangential to the pial surface revealed several consistent findings. First, the density of connections varied as a function of the body part representation injected. For example, the area 3b representation of the trunk and structures of the face are more densely interconnected than the representation of distal body parts (e.g., digit 1, D1). Second, callosal connections appear to be both matched and mismatched to the body part representations injected in the opposite hemisphere. For example, an injection of retrograde tracer into the trunk representation of area 3b revealed connections from the trunk representation in the opposite hemisphere, as well as from shoulder and forelimb/wing representations. Third, the same body part is differentially connected in different fields via the corpus callosum. For example, the D1 representation in area 3b in one hemisphere had no connections with the area 3b D1 representation in the opposite hemisphere, whereas the D1 representation in area 1/2 had relatively dense reciprocal connections with area 1/2 in the opposite hemisphere. Finally, there are callosal projections to fields other than the homotopic, contralateral field. For example, the D1 representation in area 1/2 projects to contralateral area 1/2, and also to area 3b and SII. J. Comp. Neurol. 402:538–559, 1998. © 1998 Wiley-Liss, Inc.     

Synapses of extrinsic and intrinsic origin made by callosal projection neurons in mouse visual cortex

Neurons in areas 17/18a and 17/18b of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of labeled neurons (intrinsic terminals) were identified in the border regions of area 17 with areas 18a and 18b, and their distribution and synaptic connectivity were determined. Also examined were the synaptic connections of extrinsic callosal axon terminals labeled by lesion-induced degeneration consequent to the severing of callosal fibers. A postlesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by horseradish peroxidase. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where, with rare exception, they formed asymmetrical synapses. Layers II and III contained the highest concentrations of intrinsic and extrinsic callosal axon terminals. Analyses of serial thin sections through layers II and III in both areas 17/18a and 17/18b yielded similar results: 97% of the intrinsic (1,412 total sample) and of the extrinsic (414 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus, the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal (97%) contrasts with the proportion of asymmetrical axospinous synapses that occurs in the surrounding neuropil where about 64% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of callosal projection neurons in mouse somatosensory cortex, and lends additional weight to the hypothesis that axonal pathways are highly selective for the types of elements with which they synapse.     

Development of layer-specific axonal arborizations in mouse primary somatosensory cortex

In the developing neocortex, pyramidal neurons use molecular cues to form axonal arbors selectively in the correct layers. Despite the utility of mice for molecular and genetic studies, little work has been done on the development of layer-specific axonal arborizations of pyramidal neurons in mice. We intracellularly labeled and reconstructed the axons of layer 2/3 and layer 5 pyramidal neurons in slices of primary somatosensory cortex from C57Bl6 mice on postnatal days 7-21. For all neurons studied, the development of the axonal arborizations in mice follows a pattern similar to that seen in other species; laminar specificity of the earliest axonal branches is similar to that of mature animals. At P7, pyramidal neurons are very simple, having only a main descending axon and few primary branches. Between P7 and P10, there is a large increase in the total number of axonal branches, and axons continue to increase in complexity and total length from P10 to P21. Unlike observations in ferrets, cats, and monkeys, two types of layer 2/3 pyramidal neurons are present in both mature and developing mice; cells in superficial layer 2/3 lack axonal arbors in layer 4, and cells close to the layer 4 border have substantial axonal arbors within layer 4. We also describe axonal and dendritic arborization patterns of three pyramidal cell types in layer 5. The axons of tall-tufted layer 5 pyramidal neurons arborize almost exclusively within deep layers while tall-simple, and short layer 5 pyramidal neurons also project axons to superficial layers.     

Callosal connections of the somatic sensory areas II and IV in the cat

The homotopic and heterotopic callosal connections in the forelimb representations of the second (SII) and fourth (SIV) somatic sensory areas of cats were investigated by means of the axonal transport of horseradish peroxidase (HRP) in conjunction with microelectrode recording. The tracer was injected in the electrophysiologically identified hand and/or digit zone of SII (six cats) or SIV (four cats). The homotopic area in the contralateral hemisphere was explored with microelectrodes in five animals (three injected in SII and two in SIV) to map neuronal receptive fields. The aim was to correlate in the same experimental case the topography of labelled callosal neurons with the physiological map of the forelimb. Labelled cells and recording sites were plotted on planar maps reconstructed with the aid of a computer from serial coronal sections from the anterior ectosylvian gyrus. After SII injections, labelled callosal neurons were observed throughout the forelimb representation in the contralateral area, but in the tangential plane their distribution was uneven. Each somatotopic zone composing the forelimb map, that is, the arm, hand, and digit zones, contained several subzones in which callosal neurons were either dense or rare. Microelectrode explorations showed that receptive fields mapped from callosal and relatively acallosal subzones representing the same body part were similar in extent and location. After SIV injections, labelled callosal neurons were observed throughout the forelimb and proximal body representation of the contralateral area. Although slight regional variations in the density of labelled cells were apparent, no subzones bare of callosal labelling were observed in SIV. In both SII and SIV, callosal neurons were concentrated mainly in layer III, but a significant number was also evident in the infragranular layers. After HRP injections in the digit zone of SII or SIV, labelled cell bodies were also observed in heterotopic areas of the contralateral hemisphere. Most of these neurons were clustered in the medial bank of the coronal sulcus and in two other heterotopic cortical regions lying, respectively, in the anterior suprasylvian sulcus and in the lateral branch of the ansate sulcus. Some callosal cells interconnecting SII and SIV were also labelled. The results show that the distal forelimb zones in SII and SIV are callosally connected with the respective homotopic zones and with several somatosensory fields located heterotopically in the contralateral hemisphere.