Evidence of cell‐nonautonomous changes in dendrite and dendritic spine morphology in the met‐signaling–deficient mouse forebrain

Human genetic findings and murine neuroanatomical expression mapping have intersected to implicate Met receptor tyrosine kinase signaling in the development of forebrain circuits controlling social and emotional behaviors that are atypical in autism‐spectrum disorders (ASD). To clarify roles for Met signaling during forebrain circuit development in vivo, we generated mutant mice (Emx1^Cre/Met^fx/fx) with an Emx1‐Cre‐driven deletion of signaling‐competent Met in dorsal pallially derived forebrain neurons. Morphometric analyses of Lucifer yellow‐injected pyramidal neurons in postnatal day 40 anterior cingulate cortex (ACC) revealed no statistically significant changes in total dendritic length but a selective reduction in apical arbor length distal to the soma in Emx1^Cre/Met^fx/fx neurons relative to wild type, consistent with a decrease in the total tissue volume sampled by individual arbors in the cortex. The effects on dendritic structure appear to be circuit‐selective, insofar as basal arbor length was increased in Emx1^Cre/Met^fx/fx layer 2/3 neurons. Spine number was not altered on the Emx1^Cre/Met^fx/fx pyramidal cell populations studied, but spine head volume was significantly increased (∼20%). Cell‐nonautonomous, circuit‐level influences of Met signaling on dendritic development were confirmed by studies of medium spiny neurons (MSN), which do not express Met but receive Met‐expressing corticostriatal afferents during development. Emx1^Cre/Met^fx/fx MSN exhibited robust increases in total arbor length (∼20%). As in the neocortex, average spine head volume was also increased (∼12%). These data demonstrate that a developmental loss of presynaptic Met receptor signaling can affect postsynaptic morphogenesis and suggest a mechanism whereby attenuated Met signaling could disrupt both local and long‐range connectivity within circuits relevant to ASD. J. Comp. Neurol. 518:4463–4478, 2010. © 2010 Wiley‐Liss, Inc.     

Developmental expression of Bis protein in the cerebral cortex and hippocampus of rats

Bis (Bcl-2 interacting death suppressor), identified as a Bcl-2-binding protein, has been suggested to have diverse functions in addition to binding to Bcl-2, thereby regulating cell death. To investigate the potential role of Bis in the developing brain, the spatiotemporal expression of Bis protein was studied in the rat forebrain during prenatal and early postnatal development using immunohistochemistry. Initial expression of Bis was detected in the medial telencephalic wall of the lateral ventricle, the area most likely corresponded to the cortical hem from the earliest age examined (E13). There was an abrupt increase of immunoreactive neurons in the cortex and hippocampus during the first postnatal week, which declined thereafter. Two populations of Bis-immunoreactive neurons can be clearly distinguished in the developing forebrain: a population of differentiating and postmitotic neurons coexpressing Bis and microtubule-associated protein-2 (MAP-2), and a population of neurons with the characteristic morphology of Cajal-Retzius cells located exclusively in the marginal zone/layer I of the cortex and in the hippocampal equivalents of the marginal zone. The latter neurons were colabeled with reelin, a marker for Cajal-Retzius cells. While Bis expression in the cerebral cortex and hippocampus exists only transiently by P14, considerable expression was found to be maintained in the rostral migratory stream and the subventricular zone of the lateral ventricle, where Bis-immunoreactive cells were glutamine synthetase-positive glial cells. Our results suggest that Bis may contribute to the developmental processes, including the differentiation and maturation of specific neuronal populations in relation to Bcl-2 in the developing rat forebrain.     

Development of glucocorticoid receptor regulation in the rat forebrain: implications for adverse effects of glucocorticoids in preterm infants

Glucocorticoids are the consensus treatment to avoid respiratory distress in preterm infants but there is accumulating evidence that these agents evoke long-term neurobehavioral deficits. Earlier, we showed that the developing rat forebrain is far more sensitive to glucocorticoid-induced disruption in the fetus than in the neonate. Feedback regulation of glucocorticoid receptors (GRs) is an essential homeostatic mechanism and we therefore examined the development of GR downregulation in the perinatal period. Pregnant rats or newborn pups were given dexamethasone daily (gestational days 17–19, postnatal days 1–3, or postnatal days 7–9), ranging from doses below that recommended for use in preterm infants (0.05 mg/kg) to therapeutic doses (0.2 or 0.8 mg/kg). Twenty-four hours after the last injection, we determined forebrain GR protein by Western blotting. Although postnatal dexamethasone treatment downregulated GRs at all doses, the fetal forebrain failed to show any decrement and instead exhibited slight GR upregulation. In controls, forebrain GR levels also showed a large increment over the course from late gestation through the second postnatal week, despite the fact that circulating glucocorticoid levels increase substantially during this period. Our results suggest that GR homeostasis develops primarily postnatally and that fetal inability to downregulate GRs in the face of exogenous glucocorticoid administration plays a role in the vulnerability of key neural circuits to developmental disruption. Since this developmental phase in the rat corresponds to the critical period in which glucocorticoids are used in preterm infants, adverse effects on brain development may be inescapable.     

Development of muscarinic receptor subtypes in the forebrain of the mouse

Cholinergic mechanisms are involved in the regulation of developmental events in the nervous system. Muscarinic cholinergic receptors are thought to be the predominant mediator of cholinergic neurotransmission in the forebrain; however, their developmental role is less well understood. The present study takes advantage of subtype-specific antibodies to muscarinic receptor proteins to investigate the cellular localization of the subtypes in developing mouse forebrain. Receptor protein expression wag assessed between postnatal day (PND) 5 and adulthood by immunocytochemical methods with antibodies to ml, m2, and m4 receptors, the most abundant subtypes in rodent brain. We have found dramatic developmental changes in the distribution of all three receptors. In the adult mouse, ml and m2 receptor immunoreactivity displayed complementary staining patterns in most forebrain areas with m4 sharing simiarities in pattern with both ml and m2. Furthermore, each receptor was expressed transiently in gray matter areas or fiber bundles at various developmental stages. The m4 receptor was also expressed in developing blood vessels. Such transient immunoreactivity was usually associated with times and areas of dynamic morphogenesis, thus suggesting distinct roles for the receptor subtypes in ontogenetic events. © 1995 Wiley-Liss, Inc.     

Allelic specificity of Ube3a Expression In The Mouse Brain During Postnatal Development

Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type–specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele‐specific Ube3a protein expression throughout postnatal brain development in the mouse, a species that exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS‐like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a‐expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele‐specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS. J. Comp. Neurol. 522:1874–1896, 2014. © 2013 Wiley Periodicals, Inc.     

Expression of vascular endothelial growth factor receptor‐3 mRNA in the rat developing forebrain and retina

Vascular endothelial growth factor receptor (VEGFR)‐3, a receptor for VEGF‐C and VEGF‐D, is expressed in neural progenitor cells, but there has been no comprehensive study of its distribution in the developing brain. Here, the temporal and cell‐specific expression of VEGFR‐3 mRNA was studied in the developing rat forebrain and eye. Expression appeared along the ventricular and subventricular zones of the lateral and third ventricles showing ongoing neurogenesis as early as embryonic day 13 but was progressively down‐regulated during development and remained in the subventricular zone and rostral migratory stream of the adult forebrain. VEGFR‐3 expression was also detectable in some differentiating and postmitotic neurons in the developing cerebral cortex, including Cajal‐Retzius cells, cortical plate neurons, and subplate neurons. Expression in the subplate increased significantly during the early postnatal period but was absent by postnatal day 14. It was also highly expressed in nonneural tissues of the eye during development, including the retinal pigment epithelium, the retinal ciliary margin, and the lens, but persisted in a subset of cells in the pigmented ciliary epithelium of the adult eye. In contrast, there was weak or undetectable expression in the early neural retina, but a subset of retinal neurons in the postnatal and mature retina showed intense signals. These unique spatiotemporal mRNA expression patterns suggest that VEGFR‐3 might mediate the regulation of both neurogenesis and adult neuronal function in the rat forebrain and eye. J. Comp. Neurol. 518:1064–1081, 2010. © 2009 Wiley‐Liss, Inc.     

Dynamic expression of the Slit‐Robo GTPase activating protein genes during development of the murine nervous system

We investigated the expression of the three known Slit‐Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit‐Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues. J. Comp. Neurol. 513:224–236, 2009. © 2009 Wiley‐Liss, Inc.     

Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia.

Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2'-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.     

The effects of neonatal basal forebrain lesions oncognition : towards understanding the developmental roleof the cholinergic basal forebrain

Abnormal development of the cholinergic basal forebrain has been implicated innumerous developmental disabilities such as Rett Syndrome and Down Syndrome. This reviewsummarizes recent data using two rodent animal models that involve interrupting cholinergic basalforebrain projections on postnatal day 1 and postnatal day 7 when basal forebrain fibers arebeginning to innervate their neocortical and hippocampal targets, respectively. In one model,electrolytic lesions in mice aimed at the basal forebrain on postnatal day 1 transiently reducecholinergic markers in neocortex which induce permanent alterations in neocortical anatomy thatcorrelate with impairments on cognitive tasks. Furthermore, the lesion effects are sex dependent.In another model, 192 IgG saporin lesions in rats on postnatal day 7 permanently reducecholinergic markers in neocortex and hippocampus, and result in mild impairments in spatialprocessing, acquisition and exploratory activities. These data suggest that during the firstpostnatal week of development the cholinergic basal forebrain system is critical for normalneocortical differentiation and, possibly synaptogenesis in neural circuits that will be important forspatial memory and acquisition of spatial data. During the second postnatal week of development,the cholinergic basal forebrain system appears to take on a role largely similar to its adult role inselective attention and processing of new information. These studies also suggest strongly thatinterrupting cholinergic basal forebrain innervation of neocortex and hippocampus leads toanatomical and neurochemical abnormalities that may serve as neural substrates for some of thecognitive deficits seen in disorders such as Rett Syndrome and Down Syndrome.     

Signaling by hepatocyte growth factor in neurons is induced by pharmacological stimulation of synaptic activity

Activity-dependent signaling by growth factors is hypothesized to link synaptic activity to structural and functional modifications of neurons. The receptor tyrosine kinase Met and its ligand, hepatocyte growth factor (HGF), are clustered at excitatory synapses and may regulate aspects of excitatory synaptic function, as HGF increases expression of excitatory synaptic proteins, enhances their clustering at sites along dendrites, and increases current through the NMDA receptor. In this article, we test for secretion or activation of HGF and for activation of Met in response to pharmacological stimulation of synaptic activity. Stimulation of dissociated hippocampal neuron cultures with glutamate caused increased immunocytochemical staining against HGF on nonpermeabilized cells. Glutamate treatment also decreased the amount of pro HGF and increased the amount of the proteolytically-activated HGF in immunoblots of neuron culture lysates, and increased the levels of activated HGF in culture media. Stimulation of neuron cultures with glutamate or bicuculline induced autophosphorylation of Met on dendrites and the soma of neurons. Pretreatment of neurons with glutamate receptor inhibitors prior to glutamate treatment blocked autophosphorylation of Met. These results suggest that HGF can participate in activity-dependent signaling in neurons.     

A new synaptic player leading to autism risk: Met receptor tyrosine kinase

The validity for assigning disorder risk to an autism spectrum disorder (ASD) candidate gene comes from convergent genetic, clinical, and developmental neurobiology data. Here, we review these lines of evidence from multiple human genetic studies, and non-human primate and mouse experiments that support the conclusion that the MET receptor tyrosine kinase (RTK) functions to influence synapse development in circuits relevant to certain core behavioral domains of ASD. There is association of both common functional alleles and rare copy number variants that impact levels of MET expression in the human cortex. The timing of Met expression is linked to axon terminal outgrowth and synaptogenesis in the developing rodent and primate forebrain, and both in vitro and in vivo studies implicate this RTK in dendritic branching, spine maturation, and excitatory connectivity in the neocortex. This impact can occur in a cell-nonautonomous fashion, emphasizing the unique role that Met plays in specific circuits relevant to ASD.     

Developmental changes in the amount of glial fibrillary acidic protein in three regions of the rat brain

Glial fibrillary acidic (GFA) protein, extractable in 50 mM phosphate buffer, pH 8, was measured in the olfactory bulbs, forebrain and cerebellum of the rat during development using a double antibody radioimmunoassay. Each brain region showed a different pattern of development for GFA protein. At birth GFA protein per mg protein was highest in olfactory bulbs followed by forebrain and cerebellum, and these amounted to 15, 10 and 8% of the adult values, respectively. The relative increase in GFA protein was more marked during the first 2 postnatal weeks than in the following 7 weeks after birth. When values were expressed per brain region, the developmental increase in the amount of GFA protein from birth to adulthood was about 100-fold in olfactory bulbs, 85-fold in forebrain and 485-fold in cerebellum. The patterns of developmental increases in GFA protein and in glutamine synthetase activity, another protein enriched in astrocytes, were similar in the forebrain and olfactory bulbs, but differed markedly in the cerebellum. The major increase in content of the GFA protein during development was found to correspond with the maturation of astrocytes rather than with their proliferation; however, a small but significant amount of GFA protein acquired at an early age may be related to increase in astroglial cell numbers in the cerebellum.     

Insights into the complex influence of 5-HT signaling on thalamocortical axonal system development

The topographic organization of the thalamocortical axons (TCAs) in the barrel field (BF) in the rodent primary somatosensory cortex results from a succession of temporally and spatially precise developmental events. Prenatally, growth and guidance mechanisms enable TCAs to navigate through the forebrain and reach the cortex. Postnatally, TCAs grow into the cortex, and the refinement of their terminal arborization pattern in layer IV creates barrel-like structures. The combined results of studies performed over the past 20 years clearly show that serotonin (5-hydroxytryptamine; 5-HT) signaling modulates these pre- and early postnatal developmental processes. In this context, 5-HT signaling can purposely be described as 'modulating' rather than 'controlling' because developmental alterations of 5-HT synthesis, uptake or degradation either have a dramatic, moderate or no effect at all on TCA pathway and BF formation. In this review we summarize and compare the outcomes of diverse pharmacological and genetic manipulations of 5-HT signaling on TCA pathway and BF formation, in an attempt to understand these discrepancies.     

Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons

Mice lacking serotonin receptor 1A (Htr1a) display increased anxiety behavior that depends on the expression of the receptor in the forebrain during the third to fifth postnatal weeks. Within the forebrain, Htr1a is prominently expressed in the soma and dendrites of CA1 pyramidal neurons of the hippocampus and these cells undergo rapid dendritic growth and synapse formation during this period. Consistent with a possible role of Htr1a in synaptic maturation, CA1 pyramidal neurons in the knockout mice show increased ramification of oblique dendrites. These findings suggest that Htr1a may shape hippocampal circuits by directly modulating dendritic growth. Here we show that pharmacological blockade of the receptor during the third to fifth postnatal weeks is sufficient to reproduce the increased branching of oblique dendrites seen in knockout mice. Using dissociated hippocampal cultures we demonstrate that serotonin functions through Htr1a to attenuate the motility of dendritic growth cones, reduce their content of filamentous actin and alter their morphology. These findings suggest that serotonin modulates actin cytoskeletal dynamics in hippocampal neurons during a limited developmental period to restrict dendritic growth and achieve a long‐term adjustment of neural connectivity.     

Expression of the BMP antagonist Dan during murine forebrain development

Dan (Differential screening-selected gene aberrative in neuroblastoma, also known as N03) is a member of a class of glycoproteins shown to be secreted inhibitors of the transforming growth factor-beta (TGF-beta) and bone morphogenic protein pathways. We examined Dan expression during murine forebrain development from embryonic day 10.5 to postnatal day 14 and found that Dan was expressed in highly specific spatiotemporal patterns. In early telencephalic development, Dan is highly expressed in the fibroblasts covering the cortex. From E12.5-E14.5, Dan is also weakly expressed in a region of neuroepithelium at the medial margin of the telencephalon called the cortical hem. From E17.5 on, Dan is expressed strongly in CA3 pyramidal neurons of the hippocampus as well as in the developing thalamus and amygdala. To determine if Dan expression is correlated with the expression of any of its known ligand targets, we examined the expression of GDF-5, -6 and -7 in the forebrain and found that GDF-5 is expressed in Cajal-Retzius cells in Layer I of cortex, immediately adjacent to the expression of Dan in the meninges.