Phosphorylation-site mutagenesis of the growth-associated protein GAP- 43 modulates its effects on cell spreading and morphology

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane- association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP- 43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin- containing cortical cytoskeleton in a regulatable manner.     

Effects of alterations of the vibrissae-related organization of thalamocortical axons upon the organization and outgrowth of intracortical connections in the barrelfield of the rat

Previous studies have shown that intracortical projections in layer IV of the vibrissae representation of primary somatosensory cortex (S-I) are arrayed in a pattern complementary to that of thalamocortical axons (TCAs). Elevation of cortical serotonin (5-HT) in rats during the first postnatal week results in a transient disruption of the vibrissae-related pattern of TCAs and layer IV neurons in S-I. The present study examines the influence of elevated cortical 5-HT levels and the attendant loss of vibrissae-related TCA clusters on the organization of S-I intracortical connections. Cortical 5-HT was elevated in neonatal rats via chronic injections of clorgyline from birth until P-6. Animals were euthanized on P-6 or allowed to survive an additional 4 days without further clorgyline treatment. Distributions of TCAs and intracortical axons were assessed via application of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Di-A) to the thalamic radiations and directly into the cortical barrelfield, respectively. Chronic administration of clorgyline resulted in a loss of the vibrissae-related organization of TCAs in layer IV of S-I. There was also a loss of the complementary pattern of intracortical projections in layer IV of this region. Discontinuation of clorgyline treatment resulted in a return of the vibrissae-related pattern of TCAs as well as the complementary pattern of intracortical projections. These results are consistent with the conclusion that the normal organization of intracortical projections in this region of S-I depends on the presence of the orderly array of TCAs.     

Effects of alterations of the vibrissae-related organization of thalamocortical axons upon the organization and outgrowth of intracortical connections in the barrelfield of the rat

Previous studies have shown that intracortical projections in layer IV of the vibrissae representation of primary somatosensory cortex (S-I) are arrayed in a pattern complementary to that of thalamocortical axons (TCAs). Elevation of cortical serotonin (5-HT) in rats during the first postnatal week results in a transient disruption of the vibrissae-related pattern of TCAs and layer IV neurons in S-I. The present study examines the influence of elevated cortical 5-HT levels and the attendant loss of vibrissae-related TCA clusters on the organization of S-I intracortical connections. Cortical 5-HT was elevated in neonatal rats via chronic injections of clorgyline from birth until P-6. Animals were euthanized on P-6 or allowed to survive an additional 4 days without further clorgyline treatment. Distributions of TCAs and intracortical axons were assessed via application of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Di-A) to the thalamic radiations and directly into the cortical barrelfield, respectively. Chronic administration of clorgyline resulted in a loss of the vibrissae-related organization of TCAs in layer IV of S-I. There was also a loss of the complementary pattern of intracortical projections in layer IV of this region. Discontinuation of clorgyline treatment resulted in a return of the vibrissae-related pattern of TCAs as well as the complementary pattern of intracortical projections. These results are consistent with the conclusion that the normal organization of intracortical projections in this region of S-I depends on the presence of the orderly array of TCAs.     

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.     

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn²⁺ into the prefrontal cortex indicated that DAT KO mice have a truncated Mn²⁺ distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn²⁺ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here.     

Characterization of factors regulating lamina-specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.     

Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.     

Polarized targeting of peripheral membrane proteins in neurons

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.     

Mice Lacking GD3 Synthase Display Morphological Abnormalities in the Sciatic Nerve and Neuronal Disturbances during Peripheral Nerve Regeneration

The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration.     

Co-expression of GAP-43 and nNOS in avulsed motoneurons and their potential role for motoneuron regeneration

Neuronal nitric oxide synthase (nNOS) is induced after axonal injury. The role of induced nNOS in injured neurons is not well established. In the present study, we investigated the co-expression of nNOS with GAP-43 in spinal motoneurons following axonal injury. The role of induced nNOS was discussed and evaluated. In normal rats, spinal motoneurons do not express nNOS or GAP-43. Following spinal root avulsion, expression of nNOS and GAP-43 were induced and colocalized in avulsed motoneurons. Reimplantation of avulsed roots resulted in a remarkable decrease of GAP-43- and nNOS-IR in the soma of the injured motoneurons. A number of GAP-43-IR regenerating motor axons were found in the reimplanted nerve. In contrast, the nNOS-IR was absent in reimplanted nerve. These results suggest that expression of GAP-43 in avulsed motoneurons is related to axonal regeneration whereas nNOS is not.     

Distribution of GAP-43-immunoreactive structures in the human fetal amygdala.

The growth-associated protein GAP-43 is a developmentally regulated protein which is involved in the formation of neuronal contacts. In immunohistochemical studies, GAP-43 is detected within axons during their elongation; thus a fibrous immunoreactivity is visible. After axonal growth is completed there is a shift from a fibrous to a punctate immunoreactivity. The latter has been shown to correlate with synaptogenesis. In the amygdala of the 5th gestational month, a fibrous GAP-43-immunoreactivity is seen in the basolateral nuclei, whereas the corticomedial nuclei exclusively show a punctate immunoreactivity. In the 7th month, all amygdaloid nuclei display immunoreactive puncta, but no fibers. In the 9th month GAP-43-immunoreactivity is no longer visible within the amygdala. The results demonstrate the differential distribution of GAP-43-immunoreactive structures in the amygdaloid nuclei. The nuclear specific immunostaining and its changes may indicate the sequential appearance of the monoaminergic innervation of the amygdala, as GAP-43 is known to occur in monoaminergic systems. Nuclei involved in high levels of the cortical processing hierarchy such as the lateral or basal nucleus display a late occurrence of GAP-43-immunoreactivity. In general, anti-GAP-43 has been shown to be an appropriate tool to investigate axonal growth and synaptogenesis in the developing human brain.     

Signaling mechanisms of daidzein-induced axonal outgrowth in hippocampal neurons

We aim to study the mechanisms underlying the neurotrophic effect of daidzein (Dz) in hippocampal neurons. Dz-enhanced axonal outgrowths manifested growth cone formation and increased immunostaining intensity of growth-associated protein 43 (GAP-43) in growth cones. Consistent with this, Dz increased GAP-43 phosphorylation and its membrane translocation without affecting total GAP-43 levels. In the presence of Dz, significant increase in the immunoreactivity for estrogen receptor (ER) β, but not ERα, was observed on the membrane of cell bodies and growing axons. Dz also induced the activation of protein kinase C α (PKCα), which was inhibited by the ICI182,780 pretreatment. Similarly, Dz-promoted axonal elongation was blocked by ICI182,780 and Gö6976. Moreover, Dz-stimulated activation of GAP-43 was specifically abolished by Gö6976, suggesting PKCα being the upstream effector of GAP-43. Taken together, our data suggest that Dz triggers an ERβ/PKCα/GAP-43 signaling cascade to promote axonal outgrowths in cultured hippocampal neurons.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.Key Words: motor cortex, neuronal transplantation, embryonic cells, GFP, GFAP, PLP     

Evidence that descending cortical axons are essential for thalamocortical axons to cross the pallial-subpallial boundary in the embryonic forebrain

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors.     

Characterization of factors regulating lamina‐specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer‐specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina‐specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol‐linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 56–68, 2000     

Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path

Thalamocortical axons (TCAs), which originate in dorsal thalamus, project ventrally in diencephalon and then dorsolaterally in ventral telencephalon to their target, the neocortex. To elucidate potentially key decision points in TCA pathfinding and hence the possible localization of guidance cues, we used DiI-tracing to describe the initial trajectory of TCAs in mice. DiI-labeled TCAs extend ventrally on the lateral surface of ventral thalamus. Rather than continuing this trajectory onto the lateral surface of the hypothalamus, TCAs make a sharp lateral turn into ventral telencephalon. This behavior suggests that the hypothalamus is repulsive and the ventral telencephalon attractive for TCAs. In support of this hypothesis, we find that axon outgrowth from explants of dorsal thalamus is biased away from hypothalamus and toward ventral telencephalon when cocultured at a distance in collagen gels. The in vivo DiI analysis also reveals a broad cluster of retrogradely labeled neurons in the medial part of ventral telencephalon positioned within or adjacent to the thalamocortical pathway prior to or at the time TCAs are extending through it. The axons of these neurons extend into or through dorsal thalamus and appear to be coincident with the oppositely extending TCAs. These findings suggest that multiple cues guide TCAs along their pathway from dorsal thalamus to neocortex: TCAs may fasciculate on the axons of ventral telencephalic neurons as they extend through ventral thalamus and the medial part of ventral telencephalon, and chemorepellent and chemoattractant activities expressed by hypothalamus and ventral telencephalon, respectively, may cooperate to promote the turning of TCAs away from hypothalamus and into ventral telencephalon.