Formation of Specific Efferent Connections in Organotypic Slice Cultures from Rat Visual Cortex Cocultured with Lateral Geniculate Nucleus and Superior Colliculus

Cells in the cerebral cortex project to many distant regions in the brain. Each cortical target receives input from a specific population of cells which have a characteristic morphology and which are located in a distinct cortical layer. In an attempt to learn about the mechanisms by which this stereotypic output pattern is generated during development, we have studied the formation of cortical projections in an in vitro system. Slices from developing rat visual cortex were cocultured with slices from the superior colliculus, the major target of cells in layer 5, and the lateral geniculate nucleus, the major target of cells in layer 6. Cortical neurons which established connections with tectal and thalamic explants were retrogradely labelled with fluorescent dyes. It was found that, in vitro , different populations of neurons project to these two targets, and that the laminar position and cellular morphology of the projecting cells were similar to their in vivo counterparts. These specific connections were established when the target explants were placed either next to the white matter or next to the pial side of cortical slice cultures. The axons of cells projecting to ectopic positioned explants reoriented their trajectories and grew through the cortical grey matter directly towards their targets. Thus subcortical targets exert an orienting effect specifically on their innervating cells and attract growing axons of the appropriate cells at a distance. These results suggest that different targets release different molecules that act selectively on specific populations of neurons. Therefore, chemotropic guidance is likely to play a significant role in the development of specific connections between cortical neurons and their target areas.     

Mutation of the BiP/GRP78 gene causes axon outgrowth and fasciculation defects in the thalamocortical connections of the mammalian forebrain

Proper development of axonal connections is essential for brain function. A forward genetic screen for mice with defects in thalamocortical development previously isolated a mutant called baffled. Here we describe the axonal defects of baffled in further detail and identify a point mutation in the Hspa5 gene, encoding the endoplasmic reticulum chaperone BiP/GRP78. This hypomorphic mutation of BiP disrupts proper development of the thalamocortical axon projection and other forebrain axon tracts, as well as cortical lamination. In baffled mutant brains, a reduced number of thalamic axons innervate the cortex by the time of birth. Thalamocortical and corticothalamic axons are delayed, overfasciculated, and disorganized along their pathway through the ventral telencephalon. Furthermore, dissociated mutant neurons show reduced axon extension in vitro. Together, these findings demonstrate a sensitive requirement for the endoplasmic reticulum chaperone BiP/GRP78 during axon outgrowth and pathfinding in the developing mammalian brain. J. Comp. Neurol. 521:677–696, 2013. © 2012 Wiley Priodcals, Inc.     

Addition of tetrodotoxin alters the morphology of thalamocortical axons in organotypic cocultures

Living organotypic cocultures of rat thalamic and cortical explants were used to examine the effects of blocking action potential activity on the morphological development of axons in the mammalian neocortex. Studies in vivo have suggested that blocking sodium channel-dependent activity influences the growth characteristics of thalamocortical axons during development. We have extended these observations by using an in vitro system that affords more direct observational analysis of the early events of axonal growth in an accessible cellular environment DiI-labeled thalamocortical axons grow exuberantly into the target cortex and establish axonal connections that reflect the events of early thalamocortical afferent development. Within these cocultures, the morphological features of DiI-labeled axons can be readily distinguished. Tracings of thalamocortical axons were quantitated with respect to number, length, and termination pattern of axonal branches, as well as number of varicosities. Addition of the voltage-dependent sodium channel blocker, tetrodotoxin, to cocultures did not change the general pattern of thalamocortical axonal ingrowth or the average length of collateral branches of these axons. However, in the presence of tetrodotoxin, axons were more highly branched, with an increased number of varicosities as compared to untreated cocultures. This pattern of axonal growth and branching may reflect the activity-dependent fine-tuning and trimming of collaterals that occur as thalamic afferents begin to refine their cortical territory. Our observations in thalamocortical cocultures are consistent with the view that neuronal activity modulates the pattern of axonal growth and development. © 1996 Wiley-Liss, Inc.     

Differential regulation of thalamic and cortical axonal growth by hepatocyte growth factor/scatter factor

The initial axonal projections between the cerebral cortex and thalamus are established during embryogenesis. Chemoattractants and repellents are thought to provide specific guidance cues for directional growth of these pathways. Hepatocyte growth factor/scatter factor (HGF/SF) serves as an attractant for developing motor neurons, and its distribution in embryonic pallidum, pallium and thalamus suggests a similar role in forebrain development. We examined the effectiveness of HGF/SF in regulating thalamic and cortical neuronal growth using in vitro assays. HGF/SF increased neurite outgrowth of thalamic, but not cortical neurons, grown in dissociated cultures or as explants. HGF/SF also exhibited a chemoattractant property for thalamic axons, promoting the extension of neurites towards an HGF/SF source. These experiments demonstrate HGF/SF has the capacity to selectively direct thalamocortical projections into an intermediate target, the pallidum, and eventually to their final cortical destination.     

Local connections of excitatory neurons to corticothalamic neurons in the rat barrel cortex

Corticothalamic projection neurons in the cerebral cortex constitute an important component of the thalamocortical reciprocal circuit, an essential input/output organization for cortical information processing. However, the spatial organization of local excitatory connections to corticothalamic neurons is only partially understood. In the present study, we first developed an adenovirus vector expressing somatodendritic membrane-targeted green fluorescent protein. After injection of the adenovirus vector into the ventrobasal thalamic complex, a band of layer (L) 6 corticothalamic neurons in the rat barrel cortex were retrogradely labeled. In addition to their cell bodies, fine dendritic spines of corticothalamic neurons were well visualized without the labeling of their axon collaterals or thalamocortical axons. In cortical slices containing retrogradely labeled L6 corticothalamic neurons, we intracellularly stained single pyramidal/spiny neurons of L2-6. We examined the spatial distribution of contact sites between the local axon collaterals of each pyramidal neuron and the dendrites of corticothalamic neurons. We found that corticothalamic neurons received strong and focused connections from L4 neurons just above them, and that the most numerous nearby and distant sources of local excitatory connections to corticothalamic neurons were corticothalamic neurons themselves and L6 putative corticocortical neurons, respectively. These results suggest that L4 neurons may serve as an important source of local excitatory inputs in shaping the cortical modulation of thalamic activity.     

Intrinsic properties of the developing motor cortex in the rat: in vitro axons from the medial somatomotor cortex grow faster than axons from the lateral somatomotor cortex

The axons that originate in the medial somatomotor cortex of the rat depart, during development, after those from the lateral somatomotor cortex, yet they arrive in the cervical spinal cord first. Either the medially originating axons elongate faster, or the laterally originating ones pause along the descent pathway. To investigate the presence of an intrinsic difference of the axonal elongation velocity between the lateral and medial somatomotor cortical areas, we cultured explants taken from these areas for 2 days, and measured the length of the outgrowth. After 2 days the explants were surrounded by a radiate corona of axons of which the longest measured 1.95 mm. A significant difference was detected between the medial and lateral somatomotor cortical areas in vitro. Axons originating from explants taken from the medial somatomotor cortical area are, after 2 days in culture, on average 0.16 mm longer than those from the lateral somatomotor cortical area. Though the observed difference is not large enough to allow for the overtaking observed in vivo, it does indicate that intrinsic differences exist within the developing rat somatomotor cortex. This in turn indicates that intrinsic cortical traits not only influence regionalization and targeting behavior of cortical projection neurons, but also their axonal elongation speed.     

The early development of thalamocortical and corticothalarnic projections

The early development ofthalamocortical and corticothalarnic projections in hamsters was studied to compare the specificity and maturation of these pathways, and to identify potential sources of information for specification of cortical areas. The cells that constitute these projections are both generated prenatally in hamsters and they make reciprocal connections. Fluorescent dyes (Dil and DiA) were injected into the visual cortex or lateral geniculate nucleus in fixed brains of fetal and postnatal pups. Several issues in axonal development were examined, including timing of axon outgrowth and target invasion, projection specificity, the spatial relationship between the two pathways, and the connections of subplate cells. Thalarnic projections arrive in the visual cortex 2 days before birth and begin to invade the developing cortical plate by the next day. Few processes invade inappropriate cortical regions. By postnatal day 7 their laminar position is similar to mature animals. By contrast, visual cortical axons from subplate and layer 6 cells reach posterior thalamus at l day after birth in small numbers. By 3 days after birth many layer 5 cell projections reach the posterior thalamus. On postnatal day 7, there is a sudden increase in the number of layer 6 projections to the thalamus. Surprisingly, these layer 6 cells are precisely topographically mapped with colabeied thalarnic afferents on their first appearance. Subplate cells constitute'a very small component of the corticothalarnic projection at all ages. Double injections of Dil and DiA show that the corticofugal and thalamocortical pathways are physically separate during development. Corticofugal axons travel deep in the intermediate zone to the thalarnic axons and are separate through much of the internal capsule. Their tangential distribution is also distinct. The early appearance of the thalamocortical pathway is consistent with an organizational role in the specification of some features of cortical cytoarchitecture. The specific initial projection of thalamocortical axons strongly suggests the recognition of particular cortical regions. The physical Separation of these two pathways limits the possibility for exchange of information between these Systems except at their respeetive targets. © 1993 Wiley-Liss, Inc.     

Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex

Autoradiographic, axonal degeneration, and horseradish peroxidase fiber tracing methods were employed to investigate the organization, development and potential plasticity of the thalamocortical projection to the somatic sensory cortex of the rat. In the adult animal, thalamocortical terminals are concentrated primarily in layers I and IV and in the upper part of layer VI. Fibers terminating in layers IV and VI arise from a different thalamic region than those terminating in layer I. Discrete clusters of fibers and terminals 250–450 μm wide are distributed only to the parts of the SI cortex containing dense aggregates of layer IV granule cells and not to the intervening, less granular and commissurally connected zones. At birth, thalamocortical fibers have invaded the deep part of the developing SI cortex and are concentrated in the upper part of layer VI. Between the age of two and three days, an additional concentration of fibers appears in the part of the cortical plate which will become layer IV. Layer IV is clearly recognizable by three days of age and the dense granule cell aggregates appear in it no more than one day later. The ingrowth of commissural fibers (Wise and Jones, '76) lags behind that of thalamic fibers. The mature commissural fiber pattern is not established until the age of seven days. After removal of the developing thalamocortical system by thalamotomy in newborn rats, subsequent investigation of the commissural system in the adult showed that no commissural fibers or terminals had invaded either laminae or zones of the cortex deprived of thalamic input. Similarly, commissurotomy at birth was not followed by sprouting of thalamic fibers into zones or laminae deprived of commissural connections. The connectional specificity observed in these neocortical fiber systems contrasts markedly with the plasticity of connections reported in allocortical systems. Removal of thalamocortical afferents before they attain their definitive distribution does not radically effect the overall development of the dense granule cell aggregates in layer IV. Within the aggregates, however, subsidiary features such as the “barrels” fail to appear. This finding suggests that certain elements of cortical architecture such as the dense granule cell aggregates are independent of thalamic afferents while others, such as the barrels, result from the interaction of the developing thalamocortical fibers and/or terminals with maturing neurons.     

Reduction of early thalamic input alters adult corticocortical connectivity

The functional specificity of mammalian isocortex requires that precise connections be established between cortical areas and their targets. While recent studies of cortical development have focused on intrinsic specification, the role of extrinsic factors has received considerably less attention. In the present study, we examined how early removal of thalamic input affects the development of visual corticocortical connections. Hamster pups received ablations of visual thalamic nuclei on the day of birth. At 30 days of age, an injection of horseradish peroxidase (HRP) was placed into the area of cortex deafferented by the early thalamic ablation to retrogradely label adult corticocortical connections. Ablated animals displayed a significant increase in the number of corticocortical connections compared to control animals. The increased connectivity in ablated animals was primarily due to a significant increase in the number of corticocortical projections arising from non-visual areas. These results demonstrate that an intact thalamocortical projection is necessary for the development of normal cortical connectivity.     

Ultrastructural Evidence for Synaptic Interactions between Thalamocortical Axons and Subplate Neurons

Thalamic axons are known to accumulate in the subplate for a protracted period prior to invading the cortical plate and contacting their ultimate targets, the neurons of layer 4. We have examined the synaptic contacts made by visual and somatosensory thalamic axons during the transition period in which axons begin to leave the subplate and invade the cortical plate in the ferret. We first determined when geniculocortical axons leave the subplate and begin to grow into layer 4 of the visual cortex by injecting 1,1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine (Dil) into the lateral geniculate nucleus (LGN). By birth most LGN axons are still confined to the subplate. Over the next 10 days LGN axons grow into layer 4, but many axons retain axonal branches within the subplate. To establish whether thalamic axons make synaptic contacts within the subplate, the anterograde tracer PHA-L was injected into thalamic nuclei of neonatal ferrets between postnatal day 3 and 12 to label thalamic axons at the electron microscope level. The analysis of the PHA-L injections confirmed the Dil data regarding the timing of ingrowth of thalamic axons into the cortical plate. At the electron microscope level, PHA-L-labelled axons were found to form synaptic contacts in the subplate. The thalamic axon terminals were presynaptic primarily to dendritic shafts and dendritic spines. Between postnatal days 12 and 20 labelled synapses were also observed within layer 4 of the cortex. The ultrastructural appearance of the synapses did not differ significantly in the subplate and cortical plate, with regard to type of postsynaptic profiles, length of postsynaptic density or presynaptic terminal size. These observations provide direct evidence that thalamocortical axons make synaptic contacts with subplate neurons, the only cell type within the subplate possessing mature dendrites and dendritic spines; they also suggest that functional interactions between thalamic axons and subplate neurons could play a role in the establishment of appropriate thalamocortical connections.     

Development of Signals Influencing the Growth and Termination of Thalamocortical Axons in Organotypic Culture

Explants of embryonic or postnatal rat cortex, organotypically cultured in serum-free medium, maintain their structural integrity and their upper layers continue to mature. Coculture of portions of embryonic thalamus with cortical slices taken at different ages reveals a temporal cascade of cortical signals. (1) Slices of occipital cortex taken at E19 or earlier stimulate axonal outgrowth from explants of embryonic lateral geniculate nucleus but do not allow the fibers to invade. (2) In cortical slices taken after E19 but before P2, thalamic axons enter the slice, from any direction, and extend radially across the entire depth of the cortical plate without branching or terminating. (3) In slices taken after P2, fibers slow down, arborize, and terminate in the maturing layer 4 of the cortex. If the thalamic explant is placed against the pial surface of the cortical slice, axons still enter and branch in the same layer. These findings imply that the developing cortex expresses a diffusible growth-promoting factor and then itself becomes growth permissive, and finally the maturing layer 4 expresses a "stop signal." In triple cocultures of one thalamic explant with a "choice" of two neighboring slices, thalamic axons will not invade slices of cerebellum but behave indistinguishably in response to slices from any region of the hemisphere. Thus the initial tangential distribution of the thalamic projection in vivo (which is achieved by about E16) is unlikely to be controlled by regional variation in signals produced by the cortex. When cortical slices were precultured alone for 7-14 days before the addition of an explant of embryonic thalamus for 4 further days of coculture, the pattern of innervation was more appropriate to the chronological age of the slice than the age at which it was first taken. Thus the timing of the cascade of cortical properties is at least partly intrinsically determined. This sequence of expression of these signals suggests that they play a part in vivo in controlling the outgrowth of thalamic fibers, their accumulation under the cortical plate, their invasion of the plate, and their arborization in layer 4.     

Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.     

Thalamocortical connections in newborn mice

Thalamocortical axons reach the developing neocortex and become distributed within the cortical subplate during the third week of gestation. The present study is an analysis of the organization of connections that link thalamus and cortical subplate (corresponding to future layers V and VI) at birth. This age antedates the ascent of thalamic axons to contact cells of the supragranular layers, their principal targets in the adult cortex. At birth thalamic nuclear subdivisions are explicit; field-characteristic cytoarchitectonic features, relating principally to the infragranular layers, delineate the majority of neocortical fields. The projection of principal relay nuclei upon the majority of fields of the cerebral convexity has been mapped by means of retrograde transport of HRP. Nucleus-to-field interrelationships as well as topologic order of the overall thalamic projection prove to be identical to that in the adult animal. The neonatal projection appears to be somewhat more divergent than that of the adult.     

Pathfinding and target selection by developing geniculocortical axons.

During development of the mammalian cerebral cortex, thalamic axons must grow into the telencephalon and select appropriate cortical targets. In order to begin to understand the cellular interactions that are important in cortical target selection by thalamic axons, we have examined the morphology of axons from the lateral geniculate nucleus (LGN) as they navigate their way to the primary visual cortex. The morphology of geniculocortical axons was revealed by placing the lipophilic tracer Dil into the LGN of paraformaldehyde-fixed brains from fetal and neonatal cats between embryonic day 26 (E26; gestation is 65 d) and postnatal day 7 (P7). This morphological approach has led to three major observations. (1) As LGN axons grow within the intermediate zone of the telencephalon toward future visual cortex (E30-40), many give off distinct interstitial axon collaterals that penetrate the subplate of nonvisual cortical areas. These collaterals are transient and are not seen postnatally. (2) There is a prolonged period during which LGN axons are restricted to the visual subplate prior to their ingrowth into the cortical plate; the first LGN axons arrive within visual subplate by E36 but are not detected in layer 6 of visual cortex until about E50. (3) Within the visual subplate, LGN axons extend widespread terminal branches. This represents a marked change in their morphology from the simple growth cones present earlier as LGN axons navigate en route to visual cortex. The presence of interstitial collaterals suggests that there may be ongoing interactions between LGN axons and subplate neurons along the entire intracortical route traversed by the axons. From the extensive branching of LGN axons within the visual subplate during the waiting period, it appears that they are not simply "waiting." Rather, LGN axons may participate in dynamic cellular interactions within the subplate long before they contact their ultimate target neurons in layer 4. These observations confirm the existence of a prolonged waiting period in the development of thalamocortical connections and provide important morphological evidence in support of the previous suggestion that interactions between thalamic axons and subplate neurons are necessary for cortical target selection.     

The organization of thalamic connections

Connections ascending to the thalamus. Contrary to classical opinion, all thalamic nuclei receive extrathalamic afferents. Segregation or convergence within a topographically defined nucleus represent two modalities of thalamic afferents. In addition, certain topographically organized thalamic afferents possess "privileged" or primary "targets" in the thalamic nucleus while others possess supplementary "targets" in other thalamic nuclei (see cerebellar, pallidal and spinothalamic projections). Ascending connections from several brain stem structures can converge on the same nucleus or diverge to several thalamic nuclei. Thalamic connections with the telencephalon. Methods for determining axonal transport have demonstrated that all thalamic nuclei, with the exception of the reticular nucleus and the ventral part of the lateral geniculate body, project towards the cerebral cortex. Four nuclear complexes can be recognized in the cat as a function of the different modalities of localization, concentration and lamination of the projections towards the cortex and the central grey nuclei. In general, the thalamocortical connections have reciprocal ipsilateral corticothalamic projections originating in the infragranular layers of the cerebral cortex. The reticular nucleus and the ventral part of the lateral geniculate body, which is not projected to the cerebral cortex, are exceptions. Each cortical area receives a "privileged" connection from a thalamic nucleus and a supplementary connection- from one or several other thalamic nuclei. The "privileged" connections usually pass to the fourth and third layers of the neocortex, and sometimes also to the first layer. In contrast, the supplementary connections pass to different superficial or deep cortical layers. Each nucleus is formed of subunits which possess different hodologic and topographic characteristics as a function of the nucleus considered. Convergence or divergence of thalamocortical and corticothalamic projections on the different thalamic nuclei, as well as the laminar distribution of efferents in the cerebral cortex, are related strictly to the hodologic organization of different cellular subunits constituting the nuclei. Concentration or diffusion of thalamic projections on cerebral cortex is related more to the single or multiple projection of cell populations belonging to a thalamic nucleus than to widespread collateralization of thalamocortical axons.