Bilateral cavitations of ganglionic eminence: a fetal MR imaging sign of halted brain development.

SUMMARY: Ganglionic eminence is the main transitory proliferative structure of the ventral telencephalon in human fetal brain and it contributes for at least 35% to the population of cortical interneurons; however data on the human GE anomalies are scarce. We report 5 fetal MR imaging observations with bilateral symmetric cavitations in their GE regions resembling an inverted open C shape and separating the GE itself form the deeper parenchyma. Imaging, neuropathology, and follow-up features suggested a malformative origin. All cases had in common characteristics of lissencephaly with agenesis or severe hypoplasia of corpus callosum of probable different genetic basis. From our preliminary observation, it seems that GE cavitations are part of conditions which are also accompanied by severe cerebral structure derangement.

In vivo detection of cortical abnormalities in BCNU-treated rats, model of cortical dysplasia, using manganese-enhanced magnetic resonance imaging.

The 1-3-bis-chloroethyl-nitrosurea (BCNU)-treated rats represent a good model of cortical dysplasia (CD), as proved by the presence of some histological alterations similar to those observed in human CD, including cortical thinning, laminar disorganization, and heterotopia. The cortical cytoarchitectonics of BCNU-treated rats has been widely investigated by means of histological procedures, immunocytochemistry, and in situ hybridization techniques, implying the sacrifice of the animals. With the aim of identifying brain alterations in vivo to have the possibility of performing longitudinal studies, we used both conventional T(2)-weighted magnetic resonance imaging (MRI) and manganese-enhanced MRI (MEMRI). Though the T(2)-weighted MRI showed the gross anatomical landmarks of BCNU-treated rats, only following Mn(2+) administration T(1)-weighted MRI did reveal the brain cytoarchitectonics both of control and BCNU-treated rats. In particular, changes in MEMRI signal depicted the laminar architecture of control rats while BCNU-treated cortex showed no appreciable changes in MEMRI contrast, consistent with their abnormal cortical lamination. Furthermore, in the treated animals MEMRI revealed hyperintense signals corresponding to heterotopia, as shown by the comparison between MEMRI images and Thionin staining and calbindin immunocytochemistry from the same animals. The qualitative findings obtained with MEMRI were semi-quantitatively confirmed by image segmentation of grey matter. Overall, these data show that MEMRI can be used as a non-invasive technique to investigate cortical alterations in animal models of CD in vivo, giving the possibility to perform longitudinal studies, such as electrophysiological recordings or behavioural investigations.

Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex.

BACKGROUND: Gap junctions are specialized channels composed of several connexins, membrane proteins that mediate electrical and metabolic coupling between cells. Previous data have suggested that changes in the expression of Cx43, the main astrocytic Cx isoform, may be involved in seizure activity in human epileptic tissue. However, Cx43 has never been examined in focal cortical dysplasia (FCD) and in other human refractory epilepsies. METHODS: We analyzed Cx43 protein localization and Cx43 mRNA levels in surgical specimens of cortex from a cohort of patients with intractable epilepsy vs control nonepileptic tissue. Samples had neuropathologically defined diagnosis of cryptogenic epilepsy or epilepsy secondary to FCD. RESULTS: Cx43 immunoreactivity, which labeled punctate elements, did not reveal distinctive features in cryptogenic epilepsy and FCD type IA and IIA. A peculiar pattern of immunolabeling was instead observed in FCD type IIB, in which large aggregates of Cx43-immunopositive puncta were clustered around subsets of balloon cells and astrocytes. Further characterization revealed that these balloon cells do not express markers of precursor cells, such as CD34. Quantitative real-time reverse transcriptase PCR showed elevated levels of Cx43 transcript in a subgroup (25%) of cryptogenic epilepsy specimens compared to control and FCD ones. CONCLUSIONS: Our study points out that a rearrangement of Cx43-positive elements is part of abnormal tissue organization in FCD type IIB, and that cryptogenic epilepsies include forms with increased Cx43 mRNA expression. The data implicate functional consequences of altered Cx43 expression, and therefore of altered gap junctional coupling, in abnormal network properties of subtypes of human refractory epilepsies.

Development of cortical malformations in BCNU-treated rat, model of cortical dysplasia.

Cortical dysplasia (CD) comprises a wide range of cerebral cortex alterations ranging from severe brain malformations to local disruption of the cortical structure. Most hypotheses focused on the role of embryonic/perinatal development insults as the main cause for the majority of CD. Rats with prenatal exposure to BCNU (1-3-bis-chloroethyl-nitrosurea) represent an injury-based model and reproduce many anatomical features seen in human patients with CD, such as altered cortical layering and the presence of heterotopia and dysmorphic/heterotopic neurons. With the aim to investigate the formation and evolution of CD during development, we analysed the expression of a panel of layer-specific genes (Nurr1, Er81, Ror-ß and Cux2, markers of layers VI, V, IV and superficial layers, respectively) in BCNU-treated cortices from E17 to postnatal day 14. By means of appropriate immunohistochemical markers, we also analysed the structural organization of embryonic ventricular zone and of glial and axonal fibres, substrates supporting radial and tangential migration, respectively. The main results of the present study are: (i) the ventricular zone appeared disorganized and the neuroependyma was partially disrupted; (ii) radial glia scaffold and tangential fibres were deeply disarranged, thus explaining the neuronal migration defects; (iii) cortical heterotopia were detectable by E19, whereas periventricular heterotopia were detectable after birth; (iv) both cortical and periventricular heterotopia showed a pseudo-laminar structure, with cells of the upper cortical layers in the core of the nodules and cells of layer IV and V at their border; (v) the distribution of GABAergic cells was altered since the embryonic stages, as a consequence of the derangement of tangential fibres. Our analysis sheds light on how a malformed cortex develops after a temporally discrete environmental insult and adds additional knowledge on specific aspects of the etiopathogenesis of CD.

Joubert syndrome with bilateral polymicrogyria: clinical and neuropathological findings in two brothers.

Joubert syndrome (JS) is characterized by hypotonia, ataxia, developmental delay, and a typical neuroimaging finding, the so-called "molar tooth sign" (MTS). The association of MTS and polymicrogyria (PMG) has been reported as a distinct JS-related disorder (JSRD). So far, five patients have been reported with this phenotype, only two of them being siblings. We report on one additional family, describing a living child with JS and PMG, and the corresponding neuropathological picture in the aborted brother. No mutations were detected in the AHI1 gene, the only so far associated with the JS + PMG phenotype. Moreover, linkage analysis allowed excluding all known gene loci, suggesting further genetic heterogeneity.

Layer-specific genes reveal a rudimentary laminar pattern in human nodular heterotopia.

OBJECTIVE: To define distinctive features of nodular heterotopia in specimens derived from drug-resistant patients with epilepsy by evaluating mRNA expression of three different layer-specific markers: Rorbeta, Er81, and Nurr1. METHODS: We analyzed the expression profile of these genes, recognized as markers mainly expressed in layer IV for Rorbeta, in layer V for Er81, and in layer VI for Nurr1, in surgical samples from 14 epileptic patients, using in situ hybridization. Six patients had subcortical nodular heterotopia and 8 patients were controls. The intrinsic organization of nodular formations and of the overlaying neocortex was assessed. RESULTS: In all patients, the 3 selected genes showed high cortical laminar specificity. In subcortical nodular heterotopia, the different gene expression profiles revealed a rudimentary laminar organization of the nodules. In the overlaying cortex, fewer cells expressed the 3 genes in the appropriate specific layer as compared to controls. CONCLUSIONS: These data provide new insights into possible ontogenetic mechanisms of nodular heterotopia formation and show the potential role of layer-specific markers to elucidate the neuropathology of malformations of cortical development.

Expression of layer-specific markers in the adult neocortex of BCNU-Treated rat, a model of cortical dysplasia.

The experimental model of cortical dysplasia (CD) obtained by administering carmustine (1-3-bis-chloroethyl-nitrosurea [BCNU]) in pregnant rat uterus mimics the histopathological abnormalities observed in human CD patients: altered cortical layering, and presence of heterotopia and dysmorphic/heterotopic neurons. To investigate further the cortical layering disruption and the neuronal composition of heterotopia in BCNU-exposed cortex, we analyzed the expression pattern of the transcription factors Nurr1, Er81, Ror-beta, and Cux2 (respectively specific markers of layers VI, V, IV and superficial layers) in the cortical areas of BCNU-treated rats by means of in situ hybridization, and compared the findings with those observed in adult control rats. Combining in situ hybridization and immunohistochemistry we also investigated the origin of dysmorphic or heterotopic neurons. The main results of the present study are (i) the analysis of cortical layer thickness revealed that the cortical thinning in the BCNU model was prevalently restricted to the superficial layers; (ii) in cortical and periventricular heterotopia, the prevalent presence of superficial layer neurons in the internal areas, and deeper layer neurons in a more peripheral region, demonstrated a rudimentary pattern of laminar organization in nodule formation; and (iii) the Er81 signal in the dysmorphic and heterotopic pyramidal neurons located in layers I/II showed that they belong to layer V. These results shed light on the disorganization of the laminar architecture of the BCNU model by providing correlations with normal cortical layering and revealing the ontogenesis of heterotopia and heterotopic/dysmorphic neurons. They also provide strong evidence of the usefulness of layer-specific markers in investigating the neuropathology of CD, thus opening up the possibility of expanding their application to human neuropathology.

GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons.

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.

Sequential antibodies to potassium channels and glutamic acid decarboxylase in neuromyotonia.

A patient with thymoma-associated neuromyotonia and voltage-gated potassium channel (Kv1.2 and Kv1.6) antibodies by immunoprecipitation and rat brain immunolabeling was treated successfully with immunoadsorption and cyclophosphamide. Curiously, glutamic acid decarboxylase antibodies, absent at onset, appeared later. Stiff-person syndrome was absent, but fast blink reflex recovery suggested enhanced brainstem excitability. The range of antibodies produced in thymoma-associated neuromyotonia is richer, and the timing of antibody appearance more complex, than previously suspected.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

Morphological organization of somatosensory cortex in Otx1(-/-) mice.

Knock-out Otx1 mice show brain hypoplasia, spontaneous epileptic seizures and abnormalities of the dorsal region of the neocortex. We investigated structural alterations in excitatory and inhibitory circuits in somatosensory cortex of Otx1(-/-) mice by immunocytochemistry using light, confocal and electron microscopy. Immunostaining for non-phosphorylated neurofilament SMI311 and subunit 1 of the NMDA receptor - used as markers of pyramidal neurons - showed reduced layer V pyramidal cells and ectopic pyramidal cells in layers II and III of the mutant cortex. Immunostaining for calcium-binding proteins calbindin, calretinin and parvalbumin - markers of non-overlapping types of GABAergic interneurons - showed no differences between wild-type and knock-out cortex for calbindin and calretinin neurons, while parvalbumin neurons were only patchily distributed in Otx1(-/-) cortex. The pattern of positivity of the GABAergic marker glutamic acid decarboxylase in Otx1(-/-) cortex was also altered and similar to that of parvalbumin. GABA transporter 1 immunoreactivity was greater in Otx1(-/-) than wild-type; quantitation of structures immunoreactive for this transporter in layer V showed that they were increased overall in Otx1(-/-) but the density of inhibitory terminals on pyramidal neurons in the same layer labeled with this transporter was similar to that in wild-type mice. No differences in the distribution or intensity of the glial markers GABA transporter 3 or glial fibrillary acidic protein were found. The defects found in the cortical GABAergic system of the Otx1(-/-) mouse can plausibly explain the cortical hyperexcitability that produces seizures in these animals.

Potentially epileptogenic dysfunction of cortical NMDA- and GABA-mediated neurotransmission in Otx1-/- mice.

Knockout Otx1 mice present a microcephalic phenotype mainly due to reduced deep neocortical layers and spontaneous recurrent seizures. We investigated the excitable properties of layer V pyramidal neurons in neocortical slices prepared from Otx1-/- mice and age-matched controls. The qualitative firing properties of the neurons of Otx1-/- mice were identical to those found in wild-type controls, but the proportion of intrinsically bursting (IB) neurons was significantly smaller. This is in line with the lack of the Otx1 gene contribution to the generation and differentiation of neurons destined for the deep neocortical layers, in which IB neurons are located selectively in wild-type rodents. The pyramidal neurons recorded in Otx1-/- mice responded to near-threshold electrical stimulation of the underlying white matter, with aberrant polysynaptic excitatory potentials often leading to late action potential generation. When the strength of the stimulus was increased, the great majority of the Otx1-/- neurons (78%) responded with a prominent biphasic inhibitory postsynaptic potential that was significantly larger than that observed in the wild-type mice, and was often followed by complex postinhibitory depolarizing events. Both late excitatory postsynaptic potentials and postinhibitory excitation were selectively suppressed by NMDA receptor antagonists, but not by AMPA antagonists. We conclude that the cortical abnormalities of Otx1-/- neocortex due to a selective loss of large projecting neurons lead to a complex rearrangement of local circuitry, which is characterized by an excess of N-methyl-d-aspartate-mediated polysynaptic excitation that is counteracted by GABA-mediated inhibition in only a limited range of stimulus intensity. Prominent postsynaptic inhibitory potentials may also act as a further pro-epileptogenic event by synchronizing abnormal excitatory potentials.

Cajal-Retzius cell density as marker of type of focal cortical dysplasia.

Cajal-Retzius cells, identified using calretinin antiserum, were studied in layer I (LI) of adult human temporal cortex from epileptic patients with Taylor's focal cortical dysplasia and architectural dysplasia, in comparison with normal cortex. Both types of dysplasia showed LI hypercellularity, but only in architectural dysplasia was the density of Cajal-Retzius cells significantly increased. A subset of Cajal-Retzius cells were reelin immunoreactive, but none were GABA positive. These findings suggest that differences in the persistence of Cajal-Retzius cells, which probably reflect different types of alteration during brain development, can assist in characterizing different forms of cortical dysplasia.

Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.

During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.

Labeling of rat neurons by anti-GluR3 IgG from patients with Rasmussen encephalitis.

The authors report the immunocytochemical localization in rat brain of affinity-purified anti-GluR3 (glutamate receptor) antibodies from two patients with Rasmussen encephalitis (RE) and from immunized rabbits. The distribution of immunolabeling was similar using antibodies from rabbits and patients with RE. No electrophysiologic responses were elicited from acutely dissociated kainate-responsive neurons isolated from rat brain when these antibodies were applied. These findings show that anti-GluR3 antibodies purified from patients with RE bind to specific regions of the CNS but do not act through an excitotoxic mechanism.

Expression of KIF3C kinesin during neural development and in vitro neuronal differentiation.

KIF3A, KIF3B and KIF3C are kinesin-related motor subunits of the KIF3 family that associate to form the kinesin-II motor complex in which KIF3C and KIF3B are alternative partners of KIF3A. We have analysed the expression of Kif3 mRNAs during prenatal murine development. Kif3c transcripts are detectable from embryonic day 12.5 and persist throughout development both in the CNS and in some peripheral ganglia. Comparison of the expression patterns of the Kif3 genes revealed that Kif3c and Kif3a mRNAs colocalize in the CNS, while only Kif3a is also present outside the CNS. In contrast, Kif3b is detectable in several non-neural tissues. We have also performed immunocytochemical analyses of the developing rat brain and have found the presence of the KIF3C protein in selected brain regions and in several fibre systems. Using neuroblastoma cells as an in vitro model for neuronal differentiation, we found that retinoic acid stimulated the expression of the three Kif3 and the kinesin-associated protein genes, although with different time courses. The selective expression of Kif3c in the nervous system during embryonic development and its up-regulation during neuroblastoma differentiation suggest a role for this motor during maturation of neuronal cells.

Parvalbumin and GABA in the developing somatosensory thalamus of the rat: an immunocytochemical ultrastructural correlation.

The calcium binding protein parvalbumin (PV) is widely distributed in the mammalian nervous system and its relationship with GABAergic neurons differs within thalamic nuclei and animal species. In the rat somatosensory thalamus PV immunoreactive (ir) neurons were found only in the GABAergic reticular thalamic nucleus (RT), while a dense PVir neuropil is present in the ventrobasal complex (VB). In this study the distribution and relationship of PV and GABA were investigated in RT and VB during postnatal development at electron microscopic level. The pre-embedding immunoperoxidase detection of PV was combined with the post-embedding immunogold localization of GABA. In RT, at all developmental ages, neuronal cell bodies, dendrites and rare axonal terminals were both PVir and GABAir. In VB during the first postnatal week several small vesicle-containing profiles were double-labelled and some of them were identifiable as synaptic terminals. From postnatal day 7 (P7) to P9 the medial part of VB was more intensely PVir than the lateral one and some differences in the sequence of maturation of PVir terminals were noted between these two VB subdivisions. Single-labelled PVir profiles were first observed at P8, whereas single-labelled PVir terminals appeared at P12 and at P15 they became more frequent and larger, showing the typical morphology of ascending afferents described in adult VB. These results demonstrate the late expression of PV and acquisition of adult morphology in ascending terminals of rat VB during postnatal development in comparison with the innervation arising from the GABAergic RT.

Organization of radial and non-radial glia in the developing rat thalamus.

The organization of glia and its relationship with migrating neurons were studied in the rat developing thalamus with immunocytochemistry by using light, confocal, and electron microscopy. Carbocyanine labeling in cultured slice of the embryonic diencephalon was also used. At embryonic day (E) 14, vimentin immunoreactivity was observed in radial fascicles spanning the neuroepithelium and extending from the ventricular zone to the lateral surface of the diencephalic vesicle. Vimentin-immunopositive fibers orthogonal to the radial ones were also detected at subsequent developmental stages. At E16, radial and non-radial processes were clearly associated with migrating neurons identified by the neuronal markers calretinin and gamma-aminobutyric acid. Non-radial glial fibers were no longer evident by E19. Radial fibers were gradually replaced by immature astrocytes at the end of embryonic development. In the perinatal period, vimentin immunoreactivity labeled immature astrocytes and then gradually decreased; vimentin-immunopositive cells were only found in the internal capsule by the second postnatal week. Glial fibrillary acidic protein immunoreactivity appeared at birth in astrocytes of the internal capsule, but was not evident in most of the adult thalamic nuclei. Confocal and immunoelectron microscopy allowed direct examination of the relationships between neurons and glial processes in the embryonic thalamus, showing the coupling of neuronal membranes with both radial and non-radial glia during migration. Peculiar ultrastructural features of radial glia processes were observed. The occurrence of non-radial migration was confirmed by carbocyanine-labeled neuroblasts in E15 cultured slices. The data provide evidence that migrating thalamic cells follow both radial and non-radial glial pathways toward their destination.

Synaptic properties of neocortical neurons in epileptic mice lacking the Otx1 gene.

PURPOSE: The murine homeobox-containing Otx gene is required for correct nervous system and sense organ development. Otx1-/1 mice obtained by replacing Otx with the lac Z gene show developmental abnormalities of the cerebellum, mesencephalon, and cerebral cortex associated with spontaneous epileptic seizures (1). The epileptogenic mechanisms accounting for these seizures were investigated by means of electrophysiological recordings made from neocortical slices. METHODS: The 400-microm slices were prepared from the somatosensory cortex of Otx1-/- and Otx1+/+ mice, and the current clamp intracellular recordings were obtained from layer V pyramidal neurons by means of pipettes containing K+ acetate 1.5 mol/L and biocytin 2% (pH 7.3). RESULTS: Synaptic responses could be evoked in the neocortical pyramidal neurons by electrically stimulating the underlying white matter. gamma-Aminobutyric acid A/B-mediated inhibitory postsynaptic potentials were more pronounced in the Otx1-/- than in the control pyramidal neurons from the earliest postnatal period; multisynaptic excitatory postsynaptic potentials were significantly more expressed in the Otx1-/- mice also at the end of the first postnatal month, when they were only rarely encountered in controls. CONCLUSION: Excessive excitatory amino acid-mediated synaptic driving may lead to a hyperexcitable condition that is responsible for the epileptic manifestations occurring in Otx1-/- mice. This excess of excitation is not counteracted by well-developed gamma-aminobutyric acid activity, which seems to be involved in the synchronization of cell discharges. Our ongoing and more extensive comparative analysis of the mutants and controls should help to clarify the way in which the putative rearrangement taking place in Otx1-/- neocortex may lead to the excitatory hyperinnervation of layer V pyramidal neurons.