Insulin regulates neurovascular coupling through astrocytes.

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.

Human Pluripotent Stem Cell-Derived Neural Cells as a Relevant Platform for Drug Screening in Alzheimer's Disease.

Extracellular amyloid-beta deposition and intraneuronal Tau-laden neurofibrillary tangles are prime features of Alzheimer's disease (AD). The pathology of AD is very complex and still not fully understood, since different neural cell types are involved in the disease. Although neuronal function is clearly deteriorated in AD patients, recently, an increasing number of evidences have pointed towards glial cell dysfunction as one of the main causative phenomena implicated in AD pathogenesis. The complex disease pathology together with the lack of reliable disease models have precluded the development of effective therapies able to counteract disease progression. The discovery and implementation of human pluripotent stem cell technology represents an important opportunity in this field, as this system allows the generation of patient-derived cells to be used for disease modeling and therapeutic target identification and as a platform to be employed in drug discovery programs. In this review, we discuss the current studies using human pluripotent stem cells focused on AD, providing convincing evidences that this system is an excellent opportunity to advance in the comprehension of AD pathology, which will be translated to the development of the still missing effective therapies.

Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aß) peptide, fibrillary tangles of intraneuronal tau and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer's disease) mice and found specifically expressed in microglia associated with Aß plaques. Single-nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aß. Gal3 deletion decreased the Aß burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aß monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aß aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2-DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.

Soluble phospho-tau from Alzheimer's disease hippocampus drives microglial degeneration.

The role of microglial cells in the development and progression of Alzheimer's disease (AD) has not been elucidated. Here, we demonstrated the existence of a weak microglial response in human AD hippocampus which is in contrast to the massive microglial activation observed in APP-based models. Most importantly, microglial cells displayed a prominent degenerative profile (dentate gyrus > CA3 > CA1 > parahippocampal gyrus), including fragmented and dystrophic processes with spheroids, a reduced numerical density, and a significant decrease in the area of surveillance ("microglial domain"). Consequently, there was a substantial decline in the area covered by microglia which may compromise immune protection and, therefore, neuronal survival. In vitro experiments demonstrated that soluble fractions (extracellular/cytosolic) from AD hippocampi were toxic for microglial cells. This toxicity was abolished by AT8 and/or AT100 immunodepletion, validating that soluble phospho-tau was the toxic agent. These results were reproduced using soluble fractions from phospho-tau-positive Thy-tau22 hippocampi. Cultured microglial cells were not viable following phagocytosis of SH-SY5Y cells expressing soluble intracellular phospho-tau. Because the phagocytic capacity of microglial cells is highly induced by apoptotic signals in the affected neurons, we postulate that accumulation of intraneuronal soluble phospho-tau might trigger microglial degeneration in the AD hippocampus. This microglial vulnerability in AD pathology provides new insights into the immunological mechanisms underlying the disease progression and highlights the need to improve or develop new animal models, as the current models do not mimic the microglial pathology observed in the hippocampus of AD patients.

Bidirectional modulation of synaptic transmission by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) plays a key role in the modulation of synaptic plasticity and is an essential factor in learning and memory processes. However, during aging, IGF-I levels are decreased, and the effect of this decrease in the induction of synaptic plasticity remains unknown. Here we show that the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) at layer 2/3 pyramidal neurons (PNs) of the mouse barrel cortex is favored or prevented by IGF-I (10 nM) or IGF-I (7 nM), respectively, when IGF-I is applied 1 h before the induction of Hebbian LTP. Analyzing the cellular basis of this bidirectional control of synaptic plasticity, we observed that while 10 nM IGF-I generates LTP (LTPIGF-I) of the post-synaptic potentials (PSPs) by inducing long-term depression (LTD) of the inhibitory post-synaptic currents (IPSCs), 7 nM IGF-I generates LTD of the PSPs (LTDIGF-I) by inducing LTD of the excitatory post-synaptic currents (EPSCs). This bidirectional effect of IGF-I is supported by the observation of IGF-IR immunoreactivity at both excitatory and inhibitory synapses. Therefore, IGF-I controls the induction of Hebbian NMDAR-dependent plasticity depending on its concentration, revealing novel cellular mechanisms of IGF-I on synaptic plasticity and in the learning and memory machinery of the brain.

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer's mice hippocampus.

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer's disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1(M146L)/APP(751SL) mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin-cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aß oligomers were identified, the presence of A11-immunopositive Aß plaques also suggested a direct role of plaque-associated Aß oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.

Animal and Cellular Models of Alzheimer's Disease: Progress, Promise, and Future Approaches.

Alzheimer's disease (AD) is an incurable neurodegenerative disease affecting over 45 million people worldwide. Transgenic mouse models have made remarkable contributions toward clarifying the pathophysiological mechanisms behind the clinical manifestations of AD. However, the limited ability of these in vivo models to accurately replicate the biology of the human disease have precluded the translation of promising preclinical therapies to the clinic. In this review, we highlight several major pathogenic mechanisms of AD that were discovered using transgenic mouse models. Moreover, we discuss the shortcomings of current animal models and the need to develop reliable models for the sporadic form of the disease, which accounts for the majority of AD cases, as well as human cellular models to improve success in translating results into human treatments.

Plaque-Associated Oligomeric Amyloid-Beta Drives Early Synaptotoxicity in APP/PS1 Mice Hippocampus: Ultrastructural Pathology Analysis.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD.

Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells.

Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in >60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4+ OPCs can be purified and either cryopreserved or used for functional studies. As an example of the type of functional study for which the derived cells could be used, O4+ cells can be co-cultured with maturing hPSC-derived neurons in 96/384-well-format plates, allowing the screening of pro-myelinating compounds.

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer's disease model.

In Alzheimer's disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aß) deposits. However, the effect of microtubule stabilizing agents on Aß pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aß accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aß levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aß pathology.

Distinct disease-sensitive GABAergic neurons in the perirhinal cortex of Alzheimer's mice and patients.

Neuronal loss is the best neuropathological substrate that correlates with cortical atrophy and dementia in Alzheimer's disease (AD). Defective GABAergic neuronal functions may lead to cortical network hyperactivity and aberrant neuronal oscillations and in consequence, generate a detrimental alteration in memory processes. In this study, using immunohistochemical and stereological approaches, we report that the two major and non-overlapping groups of inhibitory interneurons (SOM-cells and PV-cells) displayed distinct vulnerability in the perirhinal cortex of APP/PS1 mice and AD patients. SOM-positive neurons were notably sensitive and exhibited a dramatic decrease in the perirhinal cortex of 6-month-old transgenic mice (57% and 61% in areas 36 and 35, respectively) and, most importantly, in AD patients (91% in Braak V-VI cases). In addition, this interneuron degenerative process seems to occur in parallel, and closely related, with the progression of the amyloid pathology. However, the population expressing PV was unaffected in APP/PS1 mice while in AD brains suffered a pronounced and significant loss (69%). As a key component of cortico-hippocampal networks, the perirhinal cortex plays an important role in memory processes, especially in familiarity-based memory recognition. Therefore, disrupted functional connectivity of this cortical region, as a result of the early SOM and PV neurodegeneration, might contribute to the altered brain rhythms and cognitive failures observed in the initial clinical phase of AD patients. Finally, these findings highlight the failure of amyloidogenic AD models to fully recapitulate the selective neuronal degeneration occurring in humans.

Dynamic patterns of colocalization of calbindin, parvalbumin and GABA in subpopulations of mouse basolateral amygdalar cells during development.

Calbindin cells represent a major interneuron subtype of the cortical/pallial regions, such as the basolateral amygdala, which are often analyzed in studies of tangential migration of interneurons from the subpallial ganglionic eminences to the pallium/cortex. However, previous evidence suggests that during development the calbindin cells may include more than one of the interneuron subtypes found in the adult pallium/cortex. Furthermore, in the adult basolateral amygdala, calbindin cells include a subpopulation of non-GABAergic (non-interneuron) cells. To better characterize these cells throughout development, in the present study we investigated the colocalization of calbindin, parvalbumin and GABA in cells of the mouse basolateral amygdala during late embryonic (E16.5) and several postnatal ages from birth until 4 weeks after birth (P0, P10 and P28). Our results indicate that CB, PV and GABA show a dynamic pattern of colocalization in cells of the mouse basolateral amygdalar nucleus throughout development. From E16.5 through P28, the majority of CB+ neurons and virtually all PV+ neurons are GABAergic. However, after P10, the percentage of GABAergic CB+ cells decline from 96% to 70%. Furthermore, while only 9% of CB+ neurons are PV+ at P10, this percentage raises to 42% at P28. At all postnatal ages studied, the majority of the PV+ cells are CB+, suggesting that PV+ interneurons develop postnatally mainly as a subpopulation within the CB+ cells of the basolateral amygdalar nucleus. These results are important for interpreting data from interneuron migration.

Efferent retinal projections visualized by immunohistochemical detection of the estrogen-related receptor beta in the postnatal and adult mouse brain.

Recently, a new nuclear receptor subfamily has been identified and referred to as estrogen-related receptors. This new group shares sequence similarity, target genes, co-regulatory proteins, and action sites with the estrogen receptors; however, natural estrogens are not estrogen-related receptors ligands. One of the receptors belonging to this group, estrogen-related receptor beta (ERRbeta), is essential for embryo development and is believed to be involved in estrogen-regulated pathways. In this study, we analyzed the presence of the ERRbeta protein in the mouse brain by means of immunohistochemistry, using a commercial polyclonal antibody against ERRbeta (Sigma, E0156). This study represents the first description dealing with the immunolocalization of ERRbeta in a mammalian brain. Our results revealed numerous ERRbeta immunoreactive fibers in the retinal efferent projections in the brain, which was in agreement with the presence of intense ERRbeta immunoreactivity in the cell bodies and axonal processes of the retinal ganglion cells. In both postnatal and adult brains, ERRbeta immunoreactive fibers were distributed in a pattern which perfectly matched the retinal efferent projections: optic tract, supraoptic commissure, hypothalamic suprachiasmatic nucleus, ventral and dorsal geniculate nuclei, pretectal nuclei, and superior colliculus. Due to reliable, fine, and complete staining of the retinal axons obtained with the anti-ERRbeta antibody (E0156), we suggest that this antibody could be used as a valuable tool for labeling the full retinofugal projections in postnatal or adult brains.

Distinct immunohistochemically defined areas in the medial amygdala in the developing and adult mouse.

The medial amygdala has been considered a subpallial structure, but various studies have shown that is a somewhat complex structure expressing both pallial and subpallial gene markers. In this regard, we analyzed the immunohistochemical expression of the neurotransmitter GABA, the vesicular glutamate transporter type 2 (VGLUT2), the neuronal nitric oxide synthase (nNOS), and the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the developing and adult mouse medial amygdala. From intermediate embryonic stages on, neurochemical data show a distinctive superficial region forming a band all along the medial amygdalar surface. This superficial band displays a strong VGLUT2-immunoreactive neuropil and numerous CR-immunoreactive fibers, as well as some nNOS-, CR- or CB-positive cells. In contrast, the superficial region of the posterior medial amygdala appears to be non-GABA immunoreactive. This band in the posterior medial amygdala matches a Tbr1-expressing territory. Our results also show differences between dorsal and ventral parts of the postnatal and adult posterior medial amygdala. Especially, a compact cell aggregate of nNOS immunoreactive cells was found in the ventral portion of the medial amygdala, whereas the dorsal part is occupied by scattered weakly stained cells. Comparison of our results with gene expression patterns and fiber-tracing studies, let us to propose that the superficial band is a pallial derivative, whereas the dorsal and ventral nuclei of the posterior medial amygdala receive each neurons from different subpallial compartments, and the latter one a subset of excitatory, pallial projection neurons.

Development and adult organization of the lateral part of the bed nucleus of the stria terminalis in the chicken.

The lateral part of the bed nucleus of the stria terminalis (BSTL) is a component of the subpallial amygdala located near the ventral sulcus of the lateral ventricle, but its limits have not been well defined in birds. In this study, we analyzed the expression patterns of a number of neurochemical markers: GABA, calbindin (CB), calretinin (CR), or neuronal nitric oxide synthase (nNOS), in the embryonic and adult chicken brain, to further characterize the organization of the avian BSTL. From embryonic day 16, it was possible to distinguish three different regions within BSTL on the basis of cytoarchitectonic and immunohistochemical features. A central region, referred to as lateral bed nucleus of the stria terminalis pars densocellularis (BSTLdc), is characterized by numerous tightly packed cell bodies, most of which are GABA-immunoreactive (ir), and two peripheral regions with lower cellular density displaying a moderate GABA expression, referred to as lateral bed nucleus of the stria terminalis, plexiform part 1 (BSTLp1) and plexiform part 2 (BSTLp2), respectively. In contrast to BSTLdc, both plexiform parts are characterized by the presence of many fibers and terminals immunoreactive for nNOS and CR, as well as some CR-ir scattered cells. A distinctive feature of BSTLp2 is a population of CB-ir cells embedded in a slightly CB-ir neuropil. Comparison of our immunohistochemical data with gene expression data suggests that BSTLdc and BSTLp1 are pallidal in nature, whereas BSTLp2 receives important contributions from the entopeduncular/preoptic area.

SOX10 Single Transcription Factor-Based Fast and Efficient Generation of Oligodendrocytes from Human Pluripotent Stem Cells.

Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.

Dual roles of Aß in proliferative processes in an amyloidogenic model of Alzheimer's disease.

Alzheimer's disease is a major neurodegenerative disorder that leads to severe cognitive deficits in the elderly population. Over the past two decades, multiple studies have focused on elucidating the causative factors underlying memory defects in Alzheimer's patients. In this regard, new evidence linking Alzheimer's disease-related pathology and neuronal stem cells suggests that hippocampal neurogenesis impairment is an important factor underlying these cognitive deficits. However, because of conflicting results, the impact of Aß pathology on neurogenesis/gliogenesis remains unclear. Here, we investigated the effect of Aß on neuronal and glial proliferation by using an APP/PS1 transgenic model and in vitro assays. Specifically, we showed that neurogenesis is affected early in the APP/PS1 hippocampus, as evidenced by a significant decrease in the proliferative activity due to a reduced number of both radial glia-like neural stem cells (type-1 cells) and intermediate progenitor cells (type-2 cells). Moreover, we demonstrated that soluble Aß from APP/PS1 mice impairs neuronal cell proliferation using neurosphere cultures. On the other hand, we showed that oligomeric Aß stimulates microglial proliferation, whereas no effect was observed on astrocytes. These findings indicate that Aß has a differential effect on hippocampal proliferative cells by inhibiting neuronal proliferation and triggering the formation of microglial cells.

Calcium-binding proteins, neuronal nitric oxide synthase, and GABA help to distinguish different pallial areas in the developing and adult chicken. I. Hippocampal formation and hyperpallium.

To better understand the formation and adult organization of the avian pallium, we studied the expression patterns of gamma-aminobutyric acid (GABA), calbindin (CB), calretinin (CR), and neuronal nitric oxide synthase (nNOS) in the hippocampal formation and hyperpallium of developing and adult chicks. Each marker showed a specific spatiotemporal expression pattern and was expressed in a region (area)-specific but dynamic manner during development. The combinatorial expression of these markers was very useful for identifying and following the development of subdivisions of the chicken hippocampal formation and hyperpallium. In the hyperpallium, three separate radially arranged subdivisions were present since early development showing distinct expression patterns: the apical hyperpallium (CB-rich); the intercalated hyperpallium (nNOS-rich, CB-poor); the dorsal hyperpallium (nNOS-poor, CB-moderate). Furthermore, a novel division was identified (CB-rich, CR-rich), interposed between hyper- and mesopallium and related to the lamina separating both, termed laminar pallial nucleus. This gave rise at its surface to part of the lateral hyperpallium. Later in development, the interstitial nucleus of the apical hyperpallium became visible as a partition of the apical hyperpallium. In the hippocampal formation, at least five radial divisions were observed, and these were compared with the divisions proposed recently in adult pigeons. Of note, the corticoid dorsolateral area (sometimes referred as caudolateral part of the parahippocampal area) contained CB immunoreactivity patches coinciding with Nissl-stained cell aggregates, partially resembling the patches described in the mammalian entorhinal cortex. Each neurochemical marker was present in specific neuronal subpopulations and axonal networks, providing insights into the functional maturation of the chicken pallium.

Immunohistochemical localization of the vesicular glutamate transporter VGLUT2 in the developing and adult mouse claustrum.

We studied the immunoreactive expression pattern for the vesicular glutamate transporter VGLUT2 in the embryonic, postnatal and adult mouse dorsal claustrum, at the light and electron microscopic levels. VGLUT2 immunoreactivity in the dorsal claustrum starts to be observed at E16.5, with a dramatic increase towards P0. At this age, abundant VGLUT2-immunoreactive axons and puncta are observed in all pallial regions, including the claustral complex. From the first postnatal week, VGLUT2 immunoreactivity declines in several telencephalic areas, including the pallium, but abundant VGLUT2-immunoreactive fine axons and puncta remain in the claustrum. Beginning at E18.5, VGLUT2 immunoreactivity within the claustrum shows a characteristic arrangement: a central part of the region is practically devoid of VGLUT2 immunoreactivity, and it is surrounded by plenty of immunoreactive axon terminals forming a shell around it. This core/shell arrangement of the VGLUT2 immunoreactivity resembles the complementary expression of parvalbumin and calretinin described in the mouse claustrum [Real, M.A., Dávila, J.C., Guirado, S., 2003. Expression of calcium-binding proteins in the mouse claustrum. J. Chem. Neuroanat. 25, 151-160]. We observed immunoreactive neuronal cell bodies as well in the dorsal claustrum, but only at P0. Electron microscopic analysis reveals that VGLUT2 immunoreactivity in the developing and adult dorsal claustrum consists predominantly of presynaptic boutons making asymmetric synaptic contacts. These VGLUT2-immunoreactive boutons are observed as early as E16.5 and may be related to thalamo-claustral incoming fibers.