Pravastatin reduces microvascular basal lamina damage following focal cerebral ischemia and reperfusion

Transient ischemia has been shown to damage the basal lamina of the cerebral microvasculature. Other studies proved statins to be beneficial to non-cerebral microvessels. The aim of this study was to determine whether pravastatin pretreatment ameliorates microvascular basal lamina damage following transient ischemia. Using the suture model, we subjected 15 rats to focal ischemia (3 h) and reperfusion (24 h). Rats received pravastatin (20 mg/kg/day) or saline for 4 weeks prior to the experiment. The outcome was determined by a behavior test and the infarct size. Collagen type IV, a marker for an intact basal lamina, and hemoglobin extravasation were measured by Western blot analysis. A ratio (in percentage) between ischemic and contralateral hemispheres was calculated. Pravastatin pretreatment resulted in a significantly better neurological outcome and reduced infarct size (15 +/- 0.5 and 59 +/- 10 mm(3), respectively) compared with controls (12.25 +/- 0.4 and 167 +/- 13 mm(3), respectively, P < 0.01 for both). In controls, loss of collagen type IV was seen in the basal ganglia and in the cortex (43 +/- 4 and 64 +/- 5%, respectively). Pravastatin prevented significant collagen loss (basal ganglia: 106 +/- 17%; cortex: 112 +/- 14%, P < 0.01 for both) and significantly reduced the hemoglobin extravasation compared with controls in the basal ganglia (198 +/- 49 vs. 553 +/- 47%, P < 0.01). Pravastatin pretreatment resulted in a reduction of microvascular basal lamina damage and hemoglobin extravasation following transient ischemia. Pravastatin seems to protect the cerebral microvascular system.     

Disappearance of the post-lesional laminin immunopositivity of brain vessels is parallel with the formation of gliovascular junctions and common basal lamina. A double-labelling immunohistochemical study

Previous studies revealed that during development the laminin immunopositivity gradually disappeared from the brain vessels, but temporarily re-appeared in them around lesions. The question of the present study was the correlation between the post-lesional vascular immunopositivity to laminin and the glial reaction. Following stab wounds, double fluorescent immunohistochemical labelling was performed against laminin (using a polyclonal antiserum against laminin 1) and glial fibrillary acidic protein. A number of vessels exhibited intense immunopositivity to laminin within the lesioned tissue. Where these laminin immunopositive vessels entered the perilesional brain substance, the astroglia formed contacts on them, and the separate vascular and glial basal laminae fused. The disappearance of the post-lesional laminin immunopositivity seemed to coincide with these phenomena. When monoclonal antibodies were applied against the beta1 and gamma1 laminin chains, vessels proved to be immunopositive at the lesion, but none in the intact brain tissue. No immunoreactivity was detected in the cases of alpha2 and beta2 chains. The results suggest that the disappearance of laminin immunopositivity may be attributed to that the epitopes become inaccessible for antibodies owing to the formation of gliovascular junctions and common basal lamina between astroglia and vessel. The possible role of an alteration in the laminin composition and the effect of fixation are discussed.     

Abnormal expression of heparan sulfate proteoglycan on basal lamina of muscle fibers in two Japanese patients with adhalin deficiency

We recently reported the selective reduction of the BI subunit of laminin in two Japanese patients with adhalin deficiency. We here investigated immunohistochemically the expression of other components of the extracellular matrix (ECM), including collagen type IV, heparan sulfate proteoglycan (HSPG), chondroitin-4-sulfate proteoglycan, decorin, and fibronectin in adhalin deficiency, compared with other types of muscular dystrophy. We found a reduction of HSPG on the basal lamina surrounding each muscle fiber in adhalin deficiency compared with HSPG in other diseases. This finding may be characteristic evidence of the disturbance of the sarcolemma-ECM interaction and the sarcolemmal instability in adhalin deficiency. Recently, a direct role of HSPG in fibroblast growth factor (FGF) signal transduction was demonstrated. Further investigation is required to determine if the dysfunction of FGF is relevant to the pathogenesis of adhalin deficiency.     

Muscarinic and purinergic Ca mobilizations in the neural retina of early embryonic chick

Acetylcholine and adenosine triphosphate (ATP) raise intracellular Ca²⁺ concentration via muscarinic receptors and P_2U purinoceptors by releasing Ca²⁺ from intracellular Ca²⁺ stores in the neural retina of early embryonic chick. The signal transduction mechanisms for the muscarinic and purinergic Ca²⁺ responses with fura-2 fluorescence measurements were studied. Li⁺ (1 mM), which inhibits phosphatidylinositol metabolism, enhanced both the Ca²⁺ rises to carbamylcholine (CCh, 30 μM), a muscarinic agonist, and ATP (200 μM). Thapsigargin (250 nM), an inhibitor of Ca²⁺-ATPase of inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores, abolished both the Ca²⁺ rises to CCh (100 μM) and ATP (500 μM). U-73122 (2 μM), an inhibitor of phospholipase C_b, suppressed the Ca²⁺ rise to ATP (500 μM), but its analog U-73343 (2 μM) did not suppress it. In contrast, both U-73122 and U-73343 suppressed the Ca²⁺ rise to CCh (100 μM). Pertussis toxin (250 ng/ml) suppressed the ATP-induced Ca²⁺ rise at least partly, whereas no inhibition was observed on the CCh-induced Ca²⁺ rise. Cross-talk occurred between the muscarinic and purinergic Ca²⁺ mobilizations but they were not occlusive. This study suggests that the muscarinic and purinergic Ca²⁺ mobilizations utilize IP₃-sensitive Ca²⁺ stores, but different signal transduction pathways are involved in between the muscarinic and purinergic Ca²⁺ responses.     

Expression of dystroglycan and the laminin-alpha 2 chain in the rat peripheral nerve during development

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha 2/beta 1/gamma 1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of beta-dystroglycan and the laminin-alpha 2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of beta-dystroglycan and the laminin-alpha 2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2.     

Fezf1 is required for penetration of the basal lamina by olfactory axons to promote olfactory development

In the development of the olfactory system, olfactory receptor neurons (ORNs) project their axons from the olfactory epithelium (OE) to the olfactory bulb (OB). The surface of the OB is covered by the central nervous system (CNS) basal lamina. To establish this connection, pioneer axons of the ORNs penetrate the CNS basal lamina at embryonic day 12.5 in mice. The importance of this penetration is highlighted by the Kallmann syndrome. However, little has been known about the molecular mechanism underlying this penetration process. Fezf1 (also called as Fez, Zfp312‐like, and 3110069A13Rik) is a C2H2‐type zinc‐finger gene expressed in the OE and hypothalamic region in mice. In Fezf1‐deficient mice, ORN axons (olfactory axons) do not reach the OB. Here we demonstrate that Fezf1‐deficient olfactory axons do not penetrate the CNS basal lamina in vivo, and the penetration activity of the axons in Matrigel is impaired in vitro. Coculture experiments using the OE and OB reveal that axonal projection of ORNs is rescued in Fezf1‐deficient mice in which the meninges including the CNS basal lamina are removed from the mutant OB. These data indicate that Fezf1 is required for the penetration of olfactory axons through the CNS basal lamina before they innervate the OB. J. Comp. Neurol. 515:565–584, 2009. © 2009 Wiley‐Liss, Inc.     

Expression, distribution and ultrastructural localization of the synapse-organizing molecule agrin in the mature avian retina

At the vertebrate neuromuscular junction the extracellular matrix molecule agrin is responsible for the formation, maintenance and regeneration of most if not all postsynaptic specializations. Several agrin isoforms are generated by alternative splicing which differ in their function and which are all expressed in the CNS. To analyse the role of agrin in the CNS, we investigated the expression and ultrastructural localization of agrin in the posthatched chick retina. In situ hybridization revealed the presence of agrin mRNA in all cellular layers of the mature retina, indicating that most if not all major retinal cell types synthesize agrin. Pan-specific as well as isoform-specific antiagrin antisera stained the optic fibre layer and the outer plexiform layer. However, only the pan-specific antiserum additionally stained the inner limiting membrane. Immunoelectron microscopy showed that in the optic fibre layer agrin was associated with ganglion cell axons and that at least part of this agrin corresponds to a neuronal isoform of agrin. In the outer plexiform layer, agrin was localized in the cleft between the photoreceptor terminals and the invaginating horizontal and bipolar cell dendrites. In the synapse-containing inner plexiform layer both antisera revealed punctate immunoreactivity. This staining corresponded to agrin concentrated in the synaptic cleft of conventional synapses as determined by preembedding immunoelectron microscopy. Agrin is thus concentrated at mature interneuronal synapses as it is at the neuromuscular junction, consistent with a role of agrin during formation and/or maintenance of synapses in the CNS.     

Dehydration-associated changes in the ventral glial limitans subjacent to the supraoptic nucleus include a reduction in the extent of the basal lamina but not astrocytic process shrinkage

In these studies we have investigated factors that might account for two previous observations of the ventral glial limitans subjacent to the supraoptic nucleus (SON-VGL) of dehydrated rats: (1) a reversible reduction in the thickness of the SON-VGL, and (2) a reversible reorientation of VGL astrocytes. Since components of the basal lamina influence both cell viability and polarity, we used electron microscopic sterology to determine the volume fraction of basal lamina in the SON-VGL. We further made extensive measurements of astrocytic process thickness to determine if cellular shrinkage is a factor in the thinning of the SON-VGL. While we found no evidence for changes in the thickness of astrocytic processes, there was a significant and reversible reduction in the extent of the basal lamina. These data suggest that the thinning of the VGL is due to complex biochemical events and is not merely an epiphenomenon of dehydration.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Role of the extracellular matrix in myelination of peripheral nerve.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.     

Maturational changes in laminin, fibronectin, collagen IV, and perlecan in germinal matrix, cortex, and white matter and effect of betamethasone

Germinal matrix is selectively vulnerable to hemorrhage in premature infants, and use of prenatal betamethasone is associated with a lower occurrence of germinal matrix hemorrhage. Because the major components of extracellular matrix of the cerebral vasculature-laminin, fibronectin, collagen IV, and perlecan-provide structural stability to blood vessels, we examined whether the expression of these molecules was decreased in the germinal matrix and affected by betamethasone. In both human fetuses and premature infants, fibronectin was significantly lower in the germinal matrix than in the cortical mantle or white matter anlagen. Conversely, laminin alpha1 gene expression was greater in the human germinal matrix compared with the cortical mantle or white matter. Expression of alpha1- and alpha2(IV) collagen chains increased with advancing gestational age. Low-dose prenatal betamethasone treatment enhanced fibronectin level by 1.5-2-fold whereas a high dose reduced fibronectin expression by 2-fold in rabbit pups. Because fibronectin provides structural stability to the blood vessels, its reduced expression in the germinal matrix may contribute to the fragility of germinal matrix vasculature and the propensity to hemorrhage in premature neonates.     

Heterogeneity of muscarinic cholinergic receptors in the developing chick embryo retina

Heterogeneity of muscarinic cholinergic receptors was investigated in chick embryo retina throughout development and in chicks immediately after hatching. The presence of a homogeneous receptor population was evidenced by antagonist binding. The affinity of antagonists increased up to day 14 of incubation, when synaptogenesis occurs. After this stage, it remained substantially unchanged. The number of receptors increased in embryos until hatching. On the contrary, agonists, such as acetylcholine and carbachol, bound to two (high- and low-affinity) binding sites. Through development, the affinity of both significantly increased until day 14, further substantiating the hypothesis of a maturation of the receptor pattern which precedes synapse formation. Muscarinic cholinergic binding seems to identify 3 critical steps in retinal neuronal development. The first is between 7 and 9 days of incubation, the second when synaptogenesis occurs and the third after initiation of function.     

Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet,an increased number of ganglion cells,and aberrant axonal outgrowth

Bacterial collagenase was injected into the vitreous of the eye of chick and quail embryos. Immunocytochemical and ultrastructural studies revealed that the collagenase dissolved the retinal basal lamina of the injected eye. The basal lamina disruption was first detectable 1 hour after enzyme injection and was complete within 3 hours. With further development, the retinal basal lamina was not reestablished; newly developing neuroepithelium in the peripheral retina, however, generated an intact basal lamina. Western blot analysis showed that Clostridial collagenase degraded various collagens but spared noncollagenous proteins. Basal lamina disruption of embryonic day 3 to 6 retinae led to the retraction of the end feet of the neuroepithelial cells, caused an increase in the number of Islet-1⁺ cells (most likely ganglion cells), an increase in the thickness of the optic fiber layer, and aberrant growth of optic axons on their way toward the optic disc. None of these changes were observed when retinal basal laminae were disrupted at later stages of development. The present data demonstrate that the retinal basal lamina, by anchoring the neuroepithelial cells to the pial surface of the retina, has an important function in the development of the normal cytoarchitecture of this structure. It is proposed that the altered extracellular environment in the vitreal part of the retina, resulting in the retraction of the neuroepithelial end feet, is responsible for the increased number of Islet-1⁺ cells and the aberrant axonal navigation. J. Comp. Neurol. 397:89–104, 1998. © 1998 Wiley-Liss, Inc.     

Tangential migration of cells from the basal to the dorsal telencephalic regions in the chick

The evolutionary relationship between telencephalic regions of the avian and mammalian brains has been a long-standing issue in comparative neuroanatomy. Based on various criteria, a number of homologous regions have been proposed. Recent studies in mammals have shown that basal regions of the telencephalon give rise to neurons that migrate dorsally and populate the cerebral cortex. In the present study we demonstrate that, similar to mammals, neurons from a ventricular region of the palaeo-striatal complex - the dorsal subpallial sulcus - of the chick telencephalon migrate dorsally to populate the developing pallium. Further characterization of these cells revealed that they express the neurotransmitter gamma-aminobutyric acid, but not the calcium-binding protein calbindin. These findings provide evidence that the mouse and chick basal regions are not only homologous in terms of gene expression patterns and connectivity, but they both also contribute inhibitory interneurons to dorsal regions of the developing telencephalon.     

Onset of expression of the alpha subunit of Ca2+/calmodulin-dependent protein kinase II and a novel related protein in the developing retina

Calcium-calmodulin-dependent protein kinase II is an abundant protein in the nervous system and has been associated with many aspects of neuronal function, including events related to synaptic transmission. The purpose of this study is to correlate the onset of expression of this kinase with a specific developmental event in retinal morphogenesis using a monoclonal antibody to the 50-kDa alpha-subunit. Microscopy showed the antigen to be associated with the plexiform layers of the retina. Western blots demonstrated that the onset of expression of the alpha-subunit coincided in time with the initial formation of the plexiform layers. However, the onset of expression of the 50-kDa alpha-subunit was preceded by the earlier embryonic appearance of a related 82.5-kDa antigen that was recognized by the antibody. The amount of this latter protein declined as the amount of the alpha-subunit increased in retinal homogenates. Although this related 82.5 kDa protein disappeared from blots of retinal homogenates after embryonic d 14, it could be detected in concentrated supernatant fractions isolated from the retinae of hatched chicks. Microscopy showed that a subset of retinal cells and their processes contained this antigen in early embryonic chicks. Finally, the 50 kDa alpha-subunit of kinase II and the 82.5 kDa novel antigen were shown to be separable by differential centrifugation.     

Basal lamina deficiency of Schwann cells induced by β-aminopropionitrile (BAPN) in rat dorsal root ganglion culture

In cultures of rat dorsal root ganglion (DRG) treated with β-aminopropionitrile (BAPN), the following abnormalities were observed. Abnormalities in the size and the shape of collagen fibers; longer periodicity of collagen fibers in BAPN (70 ± 5nm) than in control (66 ± 6nm) (P < 0.025); lack of basal lamina of both unmyelinated and myelinated Schwann cells; and abundant amorphous materials in the interstitial area between fibroblasts or peripheral cells and Schwann cells associated with neurites. Detachment of the explant from the collagen substratum also occurred and showed dose dependency. In contrast to the alterations of connective tissue, the neurites were well preserved in this experimental regimen.     

Temporal progression of angiogenesis and basal lamina deposition after contusive spinal cord injury in the adult rat

After spinal cord injury (SCI), the absence of an adequate blood supply to injured tissues has been hypothesized to contribute to the lack of regeneration. In this study, blood vessel changes were examined in 28 adult female Fischer 344 rats at 1, 3, 7, 14, 28, and 60 days after a 12.5 g x cm NYU impactor injury at the T9 vertebral level. Laminin, collagen IV, endothelial barrier antigen (SMI71), and rat endothelial cell antigen (RECA-1) immunoreactivities were used to quantify blood vessel per area densities and diameters in ventral gray matter (VGM), ventral white matter (VWM), and dorsal columns (DC) at levels ranging 15 mm rostral and caudal to the epicenter. This study demonstrates an angiogenic response, defined as SMI71/RECA-1-immunopositive endothelial cells that colocalize with a robust deposition of basal lamina and basal lamina streamers, 7 days after injury within epicenter VGM. This angiogenesis diminishes concurrent with cystic cavity formation. GAP43- and neurofilament- (68 kDa and 210 kDa) immunopositive fiber outgrowth was associated with these new blood vessels by day 14. Between 28 and 60 days after injury, increases in SMI71-immunopositive blood vessel densities were observed in the remaining VWM and DC with a corresponding increase in vessel diameters up to 15 mm rostral and caudal to the epicenter. This second angiogenesis within VWM and DC, unlike the acute response observed in VGM, did not correspond to any previously described changes in locomotor behaviors in this model. We propose that therapies targeting angiogenic processes be directed at the interval between 3 and 7 days after SCI.     

Distribution and development of VIP immunoreactive neurons in the spinal cord of the embryonic and newly hatched chick

The distribution and development of vasoactive intestinal polypeptide (VIP) immunoreactive elements were studied in the spinal cord of embryonic and newly hatched chicks with the indirect immunofluorescence method. VIP neurons were first detectable in the presumed dorsal horn at stages 27-28 (incubation day 5). Subsequently they increased in number, and by stage 39 (day 12) many occurred in lamina I, in the nucleus of the dorsolateral funiculus, and in the lateral portion of the neck of the dorsal horn throughout the cord. However, at the thoracic level many were also situated lateral to the central canal, with their processes running to the ipsilateral lateral and contralateral ventral funiculi. The pattern described above remained visible in both embryonic and colchicine-pretreated newly hatched chicks. During development, VIP fibers appeared later than cell bodies. In the gray matter, they were mainly scattered in the intermediate zone, especially around the central canal at all levels examined. In the white matter, however, longitudinal fibers were observed in the lateral funiculus throughout the cord, but mostly at the cervical level, though some also occurred in the ventral funiculus. This finding supports the idea that spinal VIP neurons might project rostrally via the lateral funiculus. In addition, no VIP immunoreactivity was found in the spinal ganglia, but examination of the sympathetic paravertebral ganglia showed immunoreactivity as described by others.     

Ommatidial type-specific interphotoreceptor connections in the lamina of the swallowtail butterfly, Papilio xuthus

The eye of the butterfly Papilio xuthus contains a random array of three types of ommatidia (types I-III), each bearing nine photoreceptors, R1-R9. Of the six spectral classes of photoreceptors identified, types I, II, and III ommatidia contain four, three, and two classes, respectively: the ommatidia are thus spectrally heterogeneous. The photoreceptors send their axons to the lamina where, together with some large monopolar cells (LMCs), the nine from a single ommatidium contribute to a module called a lamina cartridge. We recently reported that among different photoreceptor axon terminals visualized by confocal microscopy, the number and length of axon collaterals differ for different spectral receptors, suggesting that lamina circuits are specific for each ommatidial type. Here we studied the distribution of synapse-like structures in the cartridges, first characterizing a photoreceptor by measuring its spectral sensitivity and then injecting Lucifer yellow (LY). We subsequently histologically identified the type of ommatidium to which the injected photoreceptor belonged, cut serial ultrathin sections of the entire lamina, labeled these with anti-LY immunocytochemistry, and then localized synapse-like structures. We found numerous interphotoreceptor contacts both within and between cartridges, the combination of which was again specific for the ommatidial type. R3 and R4, which are green-sensitive photoreceptors in all ommatidia, have thick axons lacking collaterals. We found that these cells exclusively make contacts with LMCs and not with photoreceptors. We therefore presume that R3 and R4 construct a system for motion vision, whereas other randomly distributed spectral types provide inputs for color vision.     

Disruption of the pial basal lamina during early avian embryonic development inhibits histogenesis and axonal pathfinding in the optic tectum

Bacterial collagenase was injected into the ventricular cavity of the optic tectum of chick and quail embryos. Histological examination up to 6 days after enzyme injection revealed that the collagenase disrupted the pial basal lamina, which was evident by the fragmented distribution of basal lamina proteins at the pial surface of the midbrain and the brainstem. Although the disrupted basal lamina was not reestablished at later stages of development, the pial basal lamina of the newly developing neuroepithelium in the caudal part of the tectum was continuous and intact. Western blot analysis showed that the collagenase digested collagens but spared noncollagenous proteins. The disruption of the pial basal lamina caused the neuroepithelial cells to retract their pial end feet and caused tectal axons to exit the brain tissue into the adjacent mesenchyme. The vertical migration of neuroblasts to the pial layers of the tectum was inhibited, leading to a disruption of the tectal histogenesis. In the developing optic pathways, retinal axons were misguided at the optic chiasma and terminated in the head mesenchyme instead of the tectum. None of the abnormalities in histogenesis and axonal pathways were observed when the basal lamina was disrupted at a later stage of embryonic development. The present experiments demonstrate that the pial basal lamina has an important function during brain morphogenesis in restricting axons to the brain, providing an anchoring of the neuroepithelial cells to the pial surface, and allowing the formation of a defined cytoarchitecture of the brain. J. Comp. Neurol. 397:105–117, 1998. © 1998 Wiley-Liss, Inc.