Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling-2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper-responsive SOCS2 null (-/-) mice and GH non-responsive GH receptor null (GHR-/-) mice. While SOCS2-/- mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR-/- mice showed a 25% increase even though brain size was decreased. Interneuron sub-populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2-/- mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR-/- mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron : glia ratio was increased in GHR-/- mice and decreased in SOCS2-/- mice. The astrocytes in GHR-/- mice appeared smaller, while they were larger in SOCS2-/- mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR-/- mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2-/- neurons were larger than GHR-/- neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR-/- and SOCS2-/- mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.     

Two-dimensional assessment of cytoarchitecture in the anterior cingulate cortex in major depressive disorder, bipolar disorder, and schizophrenia: evidence for decreased neuronal somal size and increased neuronal density.

BACKGROUND: Abnormalities of cortical neuronal organization and reductions in neuronal somal size have been reported in schizophrenia. The purpose of this investigation was to assess patterns of neuronal and glial distribution in the anterior cingulate cortex (ACC) in major depressive disorder (MDD), schizophrenia, bipolar disorder (BPD), and normal control subjects (15 subjects per group). METHODS: Estimates for neuronal somal and glial nuclear size and density were obtained. We employed two-dimensional morphometric analysis to examine the location of neurons and glia in a 1000-microm-wide strip of cortex. RESULTS: A decreased clustering of neurons was seen in BPD (p =.001). No other group differences were observed in the clustering of neurons, glia, or of neurons about glia. Neuronal somal size was reduced in layer 5 in schizophrenia (18%, p =.001), BPD (16%, p <.001), and MDD (9%, p =.01). Neuronal density was increased in layer 6 in BPD (63%, p =.004) and schizophrenia (61%, p =.006) and in layer 5 in MDD (24%, p =.018) and schizophrenia (33%, p =.003). CONCLUSIONS: The results of this study indicate that reduced neuronal somal size and increased neuronal density in cortical layers 5 and 6 of the ACC may be key features of schizophrenia, MDD, and BPD.     

The NMDA receptor co-agonists, D-serine and glycine, regulate neuronal dendritic architecture in the somatosensory cortex

There is substantial evidence, both pharmacological and genetic, that hypofunction of the N-methyl-d-aspartate receptor (NMDAR) is a core pathophysiological feature of schizophrenia. There are morphological brain changes associated with schizophrenia, including perturbations in the dendritic morphology of cortical pyramidal neurons and reduction in cortical volume. Our experiments investigated whether these changes in dendritic morphology could be recapitulated in a genetic model of NMDAR hypofunction, the serine racemase knockout (SR-/-) mouse. Pyramidal neurons in primary somatosensory cortex (S1) of SR-/- mice had reductions in the complexity, total length, and spine density of apical and basal dendrites. In accordance with reduced cortical neuropil, SR-/- mice also had reduced cortical volume as compared to wild type mice. Analysis of S1 mRNA by DNA microarray and gene expression analysis revealed gene changes in SR-/- that are associated with psychiatric and neurologic disorders, as well as neurodevelopment. The microarray analysis also identified reduced expression of brain derived neurotrophic factor (BDNF) in SR-/- mice. Follow-up analysis by ELISA confirmed a reduction of BDNF protein levels in the S1 of SR-/- mice. Finally, S1 pyramidal neurons in glycine transporter heterozygote (GlyT1+/-) mutants, which display enhanced NMDAR function, had increased dendritic spine density. These results suggest that proper NMDAR function is important for the arborization and spine density of pyramidal neurons in cortex. Moreover, they suggest that NMDAR hypofunction might, in part, be contributing to the dendritic and synaptic changes observed in schizophrenia and highlight this signaling pathway as a potential target for therapeutic intervention.     

Increased expression of insulin-like growth factor-I (IGF-I) during embryonic development produces neocortical overgrowth with differentially greater effects on specific cytoarchitectonic areas and cortical layers

The in vivo actions of insulin-like growth factor-I (IGF-I) on the growth and development of the cerebral cortex were investigated in transgenic (Tg) mice that overexpress IGF-I in the brain, beginning as early as embryonic day (E) 13. Compared to non-Tg littermate controls, Tg mice at postnatal day (P) 12 exhibited significant increases in total cortical volume (31%) and in total neuron number (27%). The numerical density of neurons did not differ significantly between Tg and control mice, except in layer I. Comparing cytoarchitectonic areas in Tg mice, significantly greater increases in cortical volume were found for the motor cortex (42%), compared to somatosensory cortex (35%). Similarly, greater increases in total neuron number were found for motor cortex (44%) compared to somatosensory cortex (28%). Comparing individual cortical layers in Tg mice, the greatest increase in neuron number was found in layer I for both motor (93%) and somatosensory (76%) regions, followed by layer V (36-53%)>II/III (26-47%)>VI (26-37%)>IV (22-34%). Our results demonstrate that increased expression of IGF-I in vivo during embryonic and early postnatal development produces substantial overgrowth of the neocortex. IGF-I-mediated growth and development exhibits differential effects in some cytoarchitectonic areas and in lamina-specific neuron populations, most notably the neurons of layer I.     

Reduced glial cell density and neuronal size in the anterior cingulate cortex in major depressive disorder

BACKGROUND: Glial cells are more numerous than neurons in the cortex and are crucial to neuronal function. There is evidence for reduced neuronal size in schizophrenia, with suggestive evidence for reduced glial cell density in mood disorders. In this investigation, we have simultaneously assessed glial cell density and neuronal density and size in the anterior cingulate cortex in schizophrenia, major depressive disorder, and bipolar disorder. METHODS: We examined tissue from area 24b of the supracallosal anterior cingulate cortex in 60 postmortem brain specimens from 4 groups of 15 subjects, as follows: major depressive disorder, schizophrenia, bipolar disorder, and normal controls. Glial cell density and neuronal size and density were examined in all subjects using the nucleator and the optical disector. RESULTS: Glial cell density (22%) (P =.004) and neuronal size (23%) (P =.01) were reduced in layer 6 in major depressive disorder compared with controls. There was some evidence for reduced glial density in layer 6 (20%) (P =.02) in schizophrenia compared with controls, before adjusting for multiple layerwise comparisons, but there were no significant changes in neuronal size. There was no evidence for differences in glial density or neuronal size in bipolar disorder compared with controls. Neuronal density was similar in all groups to that found in controls. CONCLUSION: These findings suggest that there is reduced frontal cortical glial cell density and neuronal size in major depressive disorder.     

Abnormal neocortical development in mice lacking cGMP-dependent protein kinase I

Cyclic GMP-dependent protein kinase type I (cGKI) is a key signaling intermediate important for synaptic potentiation in the hippocampus and cerebellum, but its expression and function in cortical development have not been elucidated. The expression of cGKI in the developing mouse neocortex was evaluated by immunofluorescence labeling, and effect of cGKI deletion on cortical development was studied in adult cGKI knockout mice. cGKI was expressed at highest levels at embryonic stages in young neurons and radial glial fibers, corresponding to the major period of radial migration and laminar development of pyramidal neurons (embryonic day E13.5-E14.5), declining upon maturation (E17.5-postnatal day P28). The cerebral cortex of homozygous null mutant mice lacking cGKI exhibited heterotopic collections of neurons in the upper cortical layers and abnormal invaginations of layer I, in accord with a neuronal migration or positioning defect. Some cGKI mutant mice displayed defects in midline development resulting in partial fusion of cerebral hemispheres with adjacent neuronal heterotopias. Apical dendrites of cortical pyramidal neurons were misoriented in the cerebral cortex of cGKI null mutants, as shown in reporter mice expressing yellow fluorescent protein in layer V pyramidal neurons and by Golgi impregnation. These results demonstrate a role for cGKI signaling in cortical development related to neuronal migration/positioning that is important for dendritic orientation and connectivity.     

Nerve growth factor stimulates growth of cortical pyramidal neurons in young adult rats

The cytoarchitectonics of pyramidal neurons in the cerebral cortex of non-lesioned rats can be re-modeled by i.c.v. infusions of nerve growth factor (NGF). 4 months after the application of NGF, the pyramidal neurons in layers III and V of the motor cortex and layer V of the anterior cingulate cortex were analyzed and compared with pyramidal neurons from vehicle-treated rats. NGF-treated brains showed: (1) significant increase in dendritic branching in the basilar fields of the layer V, but not layer III, neurons; and (2) a significant increase in spine density in the terminal, but not proximal, dendritic branches. These findings indicated that, besides its known effects on forebrain cholinergic neurons, NGF produces a very generalized synaptic re-modeling involving the cells responsible for the major output of the cerebral cortex in the intact adult brain. © 1979 Elsevier Science B.V. All rights reserved.     

The laminar distribution and ultrastructure of fibers projecting from three thalamic nuclei to the somatic sensory-motor cortex of the opossum

The projections of the ventrobasal complex (VB), the ventrolateral complex (VL), and the central intralaminar nucleus (CIN) to the somatic sensory-motor (SSM) cortex of the Virginia opossum were studied with light and electron microscopic autoradiographic methods. VB, VL, and CIN have overlapping projections to SSM cortex and each one also projects to an additional cortical area. Unit responses to somatic sensory stimulation and the areal and laminar distribution of axons in cortex is different for VB, VL, and CIN, but the axons from each form similar round asymmetrical synapses, predominantly with dendritic spines. As in other mammals, VB units in the opossum have discrete, contralateral cutaneous receptive fields. VB projects somatotopically to SSM cortex and also projects to the second somatic sensory representation. Within the cortex, VB axons terminate densely in layer IV and the adjacent part of layer III. A few axons also terminate in the outermost part of layer I and the upper part of layer VI. Most VB axon terminate upon dendritic spines (86.6%), but they also contact dendritic shafts (10%) and neuronal cell bodies (3%). Neurons in VL have no reliable response to somatic stimulation under our recording conditions. VL projects to the SSM cortex and to the posterior parietal area. Throughout this entire projection field VL fibers terminate in layers I, III, and IV most densely, and sparsely in the other cortical layers. The density of termination in the mid-cortical laminae is quite sparse compared to VB, but the projection to layer I is considerably greater. Nearly all (93%) of VL axons contact dendritic spines, the remainder (7%) end dendritic shafts. CIN is a thalamic target of ascending medial lemniscal, cerebellar, spinal, and reticular formation axons. Neurons in CIN respond to stimulation restricted to a particular body part, but typically responses may be evoked from larger areas and at longer latencies than neurons in VB that are related to the same body part. CIN neurons require a firm tap or electrical stimulation within their receptive field to elicit a response in the anesthetized preparation. CIN axons terminate throughout the entire parietal cortex, but unlike VB and VL, CIN fibers end almost exclusively in the outer part of layer I. Approximately 21% of CIN fibers contact dendritic shafts in layer I, which is twice the percentage of shafts contacted by VL or VB axons. All of the other CIN synapses are formed with dendritic spines. These experiments demonstrate three different pathways to SSM cortex. The results suggest that each projection has a unique role in controlling the patterns of activity of neurons within the SSM cortex.     

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.     

Arg kinase regulates prefrontal dendritic spine refinement and cocaine-induced plasticity

Adolescence is characterized by vulnerability to the development of neuropsychiatric disorders including drug addiction, as well as prefrontal cortical refinement that culminates in structural stability in adulthood. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, although intracellular mechanisms are largely unknown. We characterized layer V prefrontal dendritic spine development and refinement in adolescent wild-type mice and mice lacking the cytoskeletal regulatory protein Abl-related gene (Arg) kinase. Relative to hippocampal CA1 pyramidal neurons, which exhibited a nearly linear increase in spine density up to postnatal day 60 (P60), wild-type prefrontal spine density peaked at P31, and then declined by 18% by P56-P60. In contrast, dendritic spines in mice lacking Arg destabilized by P31, leading to a net loss in both structures. Destabilization corresponded temporally to the emergence of exaggerated psychomotor sensitivity to cocaine. Moreover, cocaine reduced dendritic spine density in wild-type orbitofrontal cortex and enlarged remaining spine heads, but arg(-/-) spines were unresponsive. Local application of Arg or actin polymerization inhibitors exaggerated cocaine sensitization, as did reduced gene dosage of the Arg substrate, p190RhoGAP. Genetic and pharmacological Arg inhibition also retarded instrumental reversal learning and potentiated responding for reward-related cues, providing evidence that Arg regulates both psychomotor sensitization and decision-making processes implicated in addiction. These findings also indicate that structural refinement in the adolescent orbitofrontal cortex mitigates psychostimulant sensitivity and support the emerging perspective that the structural response to cocaine may, at any age, have behaviorally protective consequences.     

The effects of intrauterine growth retardation on synaptogenesis and mitochondrial formation in the cerebral and cerebellar cortices of fetal sheep

In an experimental model of growth retardation which involves the reduction of placental mass in sheep, we have investigated the effects of intrauterine deprivation on synaptogenesis, synaptic ultrastructure and mitochondrial formation in the cerebral and cerebellar cortices of fetal sheep (140 days gestation). In the growth-retarded fetus, the numerical density of synapses in layer I of the visual cortex was reduced by 17% (P<0.05) compared with controls but there was no detectable difference between the two groups in the density of parallel fibre-Purkinje cell synapses in the molecular layer of the cerebellum. The length and curvature of the postsynaptic density at synapses in both regions were not affected in growth retardation but the synaptic cleft was 13% wider in the cerebellum (P<0.05) in growth retardation compared with controls. The number of mitochondrial profiles per unit area of neuropil in the visual cortex was increased by 20% (P<0.01) in growth retardation and the electron density of the inner matrix increased but the average profile area was not affected. These findings show that intrauterine growth retardation affects some aspects of synaptic development in the cerebellum and the visual cortex. The increase in the number of mitochondrial profiles in the visual cortex of growthretarded fetuses might be an attempt by the cortical neurons to compensate for the reduced efficiency of aerobic metabolism in individual mitochondria.     

Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia

BACKGROUND: The pathophysiological characteristics of schizophrenia appear to involve altered synaptic connectivity in the dorsolateral prefrontal cortex. Given the central role that layer 3 pyramidal neurons play in corticocortical and thalamocortical connectivity, we hypothesized that the excitatory inputs to these neurons are altered in subjects with schizophrenia. METHODS: To test this hypothesis, we determined the density of dendritic spines, markers of excitatory inputs, on the basilar dendrites of Golgi-impregnated pyramidal neurons in the superficial and deep portions of layer 3 in the dorsolateral prefrontal cortex (area 46) and in layer 3 of the primary visual cortex (area 17) of 15 schizophrenic subjects, 15 normal control subjects, and 15 nonschizophrenic subjects with a psychiatric illness (referred to as psychiatric subjects). RESULTS: There was a significant effect of diagnosis on spine density only for deep layer 3 pyramidal neurons in area 46 (P = .006). In the schizophrenic subjects, spine density on these neurons was decreased by 23% and 16% compared with the normal control (P = .004) and psychiatric (P = .08) subjects, respectively. In contrast, spine density on neurons in superficial layer 3 in area 46 (P = .09) or in area 17 (P = .08) did not significantly differ across the 3 subject groups. Furthermore, spine density on deep layer 3 neurons in area 46 did not significantly (P = .81) differ between psychiatric subjects treated with antipsychotic agents and normal controls. CONCLUSION: This region- and disease-specific decrease in dendritic spine density on dorsolateral prefrontal cortex layer 3 pyramidal cells is consistent with the hypothesis that the number of cortical and/or thalamic excitatory inputs to these neurons is altered in subjects with schizophrenia.     

Cortical serotonin transporter density and verbal memory in individuals who stopped using 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"): preliminary findings

BACKGROUND: Although the popular drug 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") has been shown to damage brain serotonin (5-HT) neurons in animals, the fate and functional consequences of 5-HT neurons after MDMA injury are not known in humans. We investigated the long-term effects of MDMA use on cortical 5-HT neurons in humans and memory function, because brain 5-HT has been implicated in memory function. METHODS: Twenty-two recent MDMA users, 16 ex-MDMA users who had stopped using MDMA for more than 1 year, and 13 control subjects. The effects of MDMA use on cortical 5-HT neurons was studied by means of single-photon emission computed tomography with iodine 123-labeled 2beta-carbomethoxy-3beta-(4-iodophenyl) tropane ([(123)I]beta-CIT) by quantification of brain 5-HT transporter densities. Verbal memory performance was assessed with the Rey Auditory Verbal Learning Test. RESULTS: Mean cortical [(123)I]beta-CIT-labeled 5-HT transporter density was significantly lower in recent MDMA users than in controls (1.17 vs. 1.28 [-9%]) but not in ex-MDMA users (1.24 vs. 1.28 [-3%]). Recent and ex-MDMA users recalled significantly fewer words than did controls on the immediate recall (47.0 and 48.0 vs 60.0, respectively; P =.001) as well as the delayed recall (9.8 and 10.1 vs. 13.1, respectively; P =.003). Greater use of MDMA was associated with greater impairment in immediate verbal memory. However, memory performance was not associated with [(123)I]beta-CIT binding to cortical 5-HT transporters or duration of abstinence from MDMA. CONCLUSION: The present study suggests that, while the neurotoxic effects of MDMA on 5-HT neurons in the human cortex may be reversible, the effects of MDMA on memory function may be long-lasting.     

Insulin-like growth factor-I (IGF-I) inhibits neuronal apoptosis in the developing cerebral cortex in vivo

Increased expression of insulin-like growth factor-I (IGF-I) in embryonic neural progenitors in vivo has been shown to accelerate neuron proliferation in the neocortex. In the present study, the in vivo actions of (IGF-I) on naturally occurring neuron death in the cerebral cortex were investigated during embryonic and early postnatal development in a line of transgenic (Tg) mice that overexpress IGF-I in the brain, directed by nestin genomic regulatory elements, beginning at least as early as embryonic day (E) 13. The areal density of apoptotic cells (N(A), cells/mm2) at E16 in the telencephalic wall of Tg and littermate control embryos was determined by immunostaining with an antibody specific for activated caspase-3. Stereological analyses were conducted to measure the numerical density (N(V), cells/mm3) and total number of immunoreactive apoptotic cells in the cerebral cortex of nestin/IGF-I Tg and control mice at postnatal days (P) 0 and 5. The volume of cerebral cortex and both the N(V) and total number of all cortical neurons also were determined in both cerebral hemispheres at P0, P5 and P270. Apoptotic cells were rare in the embryonic telencephalic wall at E16. However, the overall N(A) of apoptotic cells was found to be significantly less by 46% in Tg embryos. The volume of the cerebral cortex was significantly greater in Tg mice at P0 (30%), P5 (13%) and P270 (26%). The total number of cortical neurons in Tg mice was significantly increased at P0 (29%), P5 (29%) and P270 (31%), although the N(V) of cortical neurons did not differ significantly between Tg and control mice at any age. Transgenic mice at P0 and P5 exhibited significant decreases in the N(V) of apoptotic cells in the cerebral cortex (31% and 39%, respectively). The vast majority of these apoptotic cells (> 90%) were judged to be neurons by their morphological appearance. Increased expression of IGF-I inhibits naturally occurring (i.e. apoptotic) neuron death during early postnatal development of the cerebral cortex to a degree that sustains a persistent increase in total neuron number even in the adult animal.     

Depletion of microglia in developing cortical circuits reveals its critical role in glutamatergic synapse development,functional connectivity,and critical period plasticity

Microglia populate the early developing brain and mediate pruning of the central synapses. Yet, little is known on their functional significance in shaping the developing cortical circuits. We hypothesize that the developing cortical circuits require microglia for proper circuit maturation and connectivity, and as such, ablation of microglia during the cortical critical period may result in a long-lasting circuit abnormality. We administered PLX3397, a colony-stimulating factor 1 receptor inhibitor, to mice starting at postnatal day 14 and through P28, which depletes >75% of microglia in the visual cortex (VC). This treatment largely covers the critical period (P19-32) of VC maturation and plasticity. Patch clamp recording in VC layer 2/3 (L2/3) and L5 neurons revealed increased mEPSC frequency and reduced amplitude, and decreased AMPA/NMDA current ratio, indicative of altered synapse maturation. Increased spine density was observed in these neurons, potentially reflecting impaired synapse pruning. In addition, VC intracortical circuit functional connectivity, assessed by laser scanning photostimulation combined with glutamate uncaging, was dramatically altered. Using two photon longitudinal dendritic spine imaging, we confirmed that spine elimination/pruning was diminished during VC critical period when microglia were depleted. Reduced spine pruning thus may account for increased spine density and disrupted connectivity of VC circuits. Lastly, using single-unit recording combined with monocular deprivation, we found that ocular dominance plasticity in the VC was obliterated during the critical period as a result of microglia depletion. These data establish a critical role of microglia in developmental cortical synapse pruning, maturation, functional connectivity, and critical period plasticity.     

Ablation of ErbB4 from excitatory neurons leads to reduced dendritic spine density in mouse prefrontal cortex

Dendritic spine loss is observed in many psychiatric disorders, including schizophrenia, and likely contributes to the altered sense of reality, disruption of working memory, and attention deficits that characterize these disorders. ErbB4, a member of the EGF family of receptor tyrosine kinases, is genetically associated with schizophrenia, suggesting that alterations in ErbB4 function contribute to the disease pathology. Additionally, ErbB4 functions in synaptic plasticity, leading us to hypothesize that disruption of ErbB4 signaling may affect dendritic spine development. We show that dendritic spine density is reduced in the dorsomedial prefrontal cortex of ErbB4 conditional whole‐brain knockout mice. We find that ErbB4 localizes to dendritic spines of excitatory neurons in cortical neuronal cultures and is present in synaptic plasma membrane preparations. Finally, we demonstrate that selective ablation of ErbB4 from excitatory neurons leads to a decrease in the proportion of mature spines and an overall reduction in dendritic spine density in the prefrontal cortex of weanling (P21) mice that persists at 2 months of age. These results suggest that ErbB4 signaling in excitatory pyramidal cells is critical for the proper formation and maintenance of dendritic spines in excitatory pyramidal cells. J. Comp. Neurol. 522:3351–3362, 2014. © 2014 Wiley Periodicals, Inc.     

Type 1 insulin‐like growth factor receptor signaling is essential for the development of the hippocampal formation and dentate gyrus

Type 1 insulin‐like growth factor receptor (IGF1R) signaling in neuronal development was studied in mutant mice with blunted igf1r gene expression in nestin‐expressing neuronal precursors. At birth [postnatal (P) day 0] brain weights were reduced to 37% and 56% of controls in mice homozygous (nes‐igf1r^?/?) and heterozygous (nes‐igf1r^?/Wt) for the null mutation, respectively, and this brain growth retardation persisted postnatally. Stereological analysis demonstrated that the volumes of the hippocampal formation, CA fields 1–3, dentate gyrus (DG), and DG granule cell layer (GCL) were decreased by 44–54% at P0 and further by 65–69% at P90 in nes‐igf1r^?/Wt mice. In nes‐igf1r^?/? mice, volumes were 29–31% of controls at P0 and, in the two mice that survived to P90, 6–19% of controls, although the hilus could not be identified. Neuron density did not differ among the mice at any age studied; therefore, decreased volumes were due to reduced cell number. In postnatal nes‐igf1r^?/Wt mice, the percentage of apoptotic cells, as judged by activated caspase‐3 immunostaining, was increased by 3.5–5.3‐fold. The total number of proliferating DG progenitors (labeled by BrdU incorporation and Ki67 staining) was reduced by ～50%, but the percentage of these cells was similar to the percentages in littermate controls. These findings suggest that 1) the postnatal reduction in DG size is due predominantly to cell death, pointing to the importance of the IGF1R in regulating postnatal apoptosis, 2) surviving DG progenitors remain capable of proliferation despite reduced IGF1R expression, and 3) IGF1R signaling is necessary for normal embryonic brain development. © 2009 Wiley‐Liss, Inc.     

In vivo effects of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal development of the central nervous system

The in vivo actions of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal brain development were investigated in transgenic (Tg) mice that overexpress IGF-I prenatally under the control of regulatory sequences from the nestin gene. Tg mice demonstrated increases in brain weight of 6% by embryonic day (E) 18 and 27% by postnatal day (P) 12. In Tg embryos at E16, the volume of the cortical plate was significantly increased by 52% and total cell number was increased by 54%. S-phase labeling with 5-bromo-2'-deoxyuridine revealed a 13-15% increase in the proportion of labeled neuroepithelial cells in Tg embryos at E14. In Tg mice at P12, significant increases in regional tissue volumes were detected in the cerebral cortex (29%), subcortical white matter (52%), caudate-putamen (37%), hippocampus (49%), dentate gyrus (71%) and habenular complex (48%). Tg mice exhibited significant increases in the total number of neurons in the cerebral cortex (27%), caudate-putamen (27%), dentate gyrus (69%), medial habenular nucleus (61%) and lateral habenular nucleus (36%). In the cerebral cortex and subcortical white matter of Tg mice, the total numbers of glial cells were significantly increased by 37% and 42%, respectively. The numerical density of apoptotic cells in the cerebral cortex, labeled by antibodies against active caspase-3, was reduced by 26% in Tg mice at P7. Our results demonstrate that IGF-I can both promote proliferation of neural cells in the embryonic central nervous system in vivo and inhibit their apoptosis during postnatal life.     

Leukemia inhibitory factor impairs structural and neurochemical development of rat visual cortex in vivo

Minipump infusions into visual cortex in vivo at the onset of the critical period have revealed that the proinflammatory cytokine leukemia inhibitory factor (LIF) delays the maturation of thalamocortical projection neurons of the lateral geniculate nucleus, and tecto-thalamic projection neurons of the superior colliculus, and cortical layer IV spiny stellates and layer VI pyramidal neurons. Here, we report that P12–20 LIF infusion inhibits somatic maturation of pyramidal neurons and of all interneuron types in vivo. Likewise, DIV 12–20 LIF treatment in organotypic cultures prevents somatic growth GABA-ergic neurons. Further, while NPY expression is increased in the LIF-infused hemispheres, the expression of parvalbumin mRNA and protein, Kv3.1 mRNA, calbindin D-28k protein, and GAD-65 mRNA, but not of GAD-67 mRNA or calretinin protein is substantially reduced. Also, LIF treatment decreases parvalbumin, Kv3.1, Kv3.2 and GAD-65, but not GAD-67 mRNA expression in OTC. Developing cortical neurons are known to depend on neurotrophins. Indeed, LIF alters neurotrophin mRNA expression, and prevents the growth promoting action of neurotophin-4 in GABA-ergic neurons. The results imply that LIF, by altering neurotrophin expression and/or signaling, could counteract neurotrophin-dependent growth and neurochemical differentiation of cortical neurons.     

Glutamate-positive neurons and axon terminals in cat sensory cortex: a correlative light and electron microscopic study

Immunocytochemical methods were used to perform a correlative light and electron microscopic study of neurons and axon terminals immunoreactive to the antiglutamate (Glu) serum of Hepler et al. ('88) in the visual and somatic sensory areas of cats. At the light microscopic level, numerous Glu-positive neurons were found in all layers except layer I of both cortical areas. On the basis of the dendritic staining of Glu-positive cells, two major morphological categories were found: pyramidal cells, which were the most frequent type of immunostained neuron, and multipolar neurons, which were more numerous in layer IV of area 17 than in any other layer. A large number of Glu-positive neurons, however, did not display dendritic labelling and were considered unidentified neurons. Counts of labelled neurons were performed in the striate cortex; approximately 40% were Glu-positive. Numerous lightly stained punctate structures were observed in all cortical layers: the majority of these Glu-positive puncta were in the neuropil. After resectioning the plastic sections for electron microscopy it was observed that: 1) the majority of neurons unidentifiable at light microscopic level were indeed pyramidal neurons except in layer IV of area 17, where many stained cells were probably spiny stellate neurons. Some Glu-positive neurons, however, exhibited clear ultrastructural features of nonspiny nonpyramidal cells; 2) all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all asymmetric synaptic contacts were labelled. The vast majority of postsynaptic targets of Glu-positive axons were unlabelled dendritic spines and shafts. The present results provide further evidence that Glu (or a closely related compound) is probably the neurotransmitter of numerous excitatory neurons in the neocortex.