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.

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.

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.

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.

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.

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.

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.

Disruption of amyloid plaques integrity affects the soluble oligomers content from Alzheimer disease brains.

The implication of soluble Abeta in the Alzheimer's disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinico-pathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Tris-buffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III-IV and Braak V-VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/µg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD samples.

Early neuronal loss and axonal/presynaptic damage is associated with accelerated amyloid-ß accumulation in AßPP/PS1 Alzheimer's disease mice subiculum.

The progressive cognitive decline leading to dementia in Alzheimer's disease (AD) patients is the consequence of a severe loss of synapses and neurons affecting particular cell subpopulations in selected brain areas, with the subiculum being one of the earliest regions displaying severe atrophy and pathology. The lack of significant neuronal loss in most AD models is, in fact, the major shortcoming for the preclinical evaluation of drugs that could have greater potential in patients to alleviate or prevent this disease. In this study, using immunohistochemical and stereological approaches, we have analyzed the histopathological events in the subiculum of AßPP751SwedLondon/PS1M146L mice, a transgenic model that displays neuronal vulnerability at early ages in hippocampus and entorhinal cortex. Our results indicate that the subiculum is the earliest affected region in the hippocampus, showing a selective early loss of both principal neurons (28%) and SOM-positive interneurons (69%). In addition, our data demonstrate the existence of an early axonal and synaptic pathology, which may represent the beginning of the synaptic disruption and loss. These neurodegenerative processes occur in parallel, and closely related, with the onset and accelerated progression of the extracellular amyloid-ß deposition, thus suggesting plaques as major contributors of neuronal/axonal damage. Data reported here indicate that this AD model displays a selective AD-like neurodegenerative phenotype in highly vulnerable regions, including the subiculum, and therefore can be a very useful model for testing the therapeutic ability of potential compounds to protect neurons and ameliorate disease symptoms.

In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer's disease pathology.

BACKGROUND: Alzheimer's disease (AD) is characterized by the abnormal accumulation of extracellular beta-amyloid (Abeta) plaques, intracellular hyperphosphorylated tau, progressive synaptic alterations, axonal dystrophies, neuronal loss and the deterioration of cognitive capabilities of patients. However, no effective disease-modifying treatment has been yet developed. In this work we have evaluated whether chronic lithium treatment could ameliorate the neuropathology evolution of our well characterized PS1M146LxAPPSwe-London mice model. RESULTS: Though beneficial effects of lithium have been previously described in different AD models, here we report a novel in vivo action of this compound that efficiently ameliorated AD-like pathology progression and rescued memory impairments by reducing the toxicity of Abeta plaques. Transgenic PS1M146LxAPPSwe-London mice, treated before the pathology onset, developed smaller plaques characterized by higher Abeta compaction, reduced oligomeric-positive halo and therefore with attenuated capacity to induce neuronal damage. Importantly, neuronal loss in hippocampus and entorhinal cortex was fully prevented. Our data also demonstrated that the axonal dystrophic area associated with lithium-modified plaques was highly reduced. Moreover, a significant lower accumulation of phospho-tau, LC3-II and ubiquitinated proteins was detected in treated mice. Our study highlights that this switch of plaque quality by lithium could be mediated by astrocyte activation and the release of heat shock proteins, which concentrate in the core of the plaques. CONCLUSIONS: Our data demonstrate that the pharmacological in vivo modulation of the extracellular Abeta plaque compaction/toxicity is indeed possible and, in addition, might constitute a novel promising and innovative approach to develop a disease-modifying therapeutic intervention against AD.

Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus.

BACKGROUND: Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer's patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months) and aged (18 months) PS1(M146L)/APP(751sl) transgenic mice. RESULTS: Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. CONCLUSION: A progressive age-dependent cytoskeletal pathology along with a reduction of lysosomal and, in minor extent, proteasomal activity could be directly implicated in the progressive accumulation of APP derived fragments (and Abeta peptides) in parallel with the increase of BACE-1 and gamma-secretase activities. This retard in the APP metabolism seemed to be directly implicated in the synaptic Abeta accumulation and, in consequence, in the pathology progression between synaptically connected regions.

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.

Expression of somatostatin and neuropeptide Y in the embryonic, postnatal, and adult mouse amygdalar complex.

Recent developmental studies indicate that distinct neuronal subpopulations in the amygdala, including somatostatin (SOM)-containing neurons, originate from progenitor domains in the anterior entopeduncular area, thus suggesting a different origin from subpallial territories for amygdalar versus cortical SOM-expressing interneurons, the latter derived from the dorsal part of the medial ganglionic eminence. In this context, we carried out an immunohistochemical study analyzing spatiotemporal expression patterns for SOM- and neuropeptide Y (NPY)-containing neurons in the embryonic, postnatal, and adult mouse amygdala. Our results indicate that SOM- and NPY-immunoreactive cells are present in the amygdalar complex from embryonic day (E)12.5, and that these peptidergic cells seem to arise from the anterior entopeduncular area progenitor domain. From E12.5 on there was a notable increase in the number and immunoreactivity of cells containing these peptides in distinct territories of the amygdalar complex, reaching a peak around birth. The distribution pattern for NPY neurons was very similar to that of SOM neurons in most nuclei of the amygdala, although the number of NPY neurons was always lower than that of SOM. At postnatal ages a reduction in the number of immunoreactive cells is observed in most amygdalar nuclei, remaining then similar from P14 to the adult. We interpret this reduction of the number of immunoreactive neurons in relation to the increased immunoreactivity for axons that occurs postnatally. We also suggest that the anterior entopeduncular area-derived SOM- and NPY-containing neurons in pallial and subpallial amygdaloid nuclei become local interneurons and projection neurons, respectively.

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.