Morphological characteristics of astrocytes of the fastigial nucleus.

Astrocytes are a diverse and morphologically complex class of glial cells restricted to the central nervous system which have been implicated in the modulation of neuronal activity. The cerebellum is involved in planning movements and motor learning. Within the cerebellum three deep cerebellar nuclei (dentate, interposed and fastigial) provide the sole neuronal output. The fastigial nucleus participates in saccadic and vestibular function, and recent evidence disclosed neuronal projections to cognitive, affective, and motor areas. However, thus far there are no reliable descriptions of the distribution and morphological classifications of astrocytes in this nucleus. This work aims to describe the characteristics of astrocytes of the fastigial nucleus based on the expression of GFP in a transgenic mouse model.

Reply to Sheval, E.V. Comment on "Piña et al. Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence. Int. J. Mol. Sci. 2022, 23, 1426".

We have carefully read the interesting explanatory comment by Eugene V. Sheval [...].

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay.

Loss of ferritin-positive microglia relates to increased iron, RNA oxidation, and dystrophic microglia in the brains of aged male marmosets.

Microglia are cells that protect brain tissue from invading agents and toxic substances, first by releasing pro-inflammatory cytokines, and thereafter by clearing tissue by phagocytosis. Microglia express ferritin, a protein with ferroxidase activity capable of storing iron, a metal that accumulates in brain during aging. Increasing evidence suggests that ferritin plays an important role in inflammation. However, it is not known if ferritin/iron content can be related to the activation state of microglia. To this end, we aimed to delineate the role of ferritin in microglia activation in a non-human primate model. We analyzed brains of male marmosets and observed an increased density of ferritin+ microglia with an activated phenotype in hippocampus and cortex of old marmosets (mean age 11.25 ± 0.70 years) compared to younger subjects. This was accompanied by an increased number of dystrophic microglia in old marmosets. However, in aged subjects (mean age 16.83 ± 2.59 years) the number of ferritin+ microglia was decreased compared to old ones. Meanwhile, the content of iron in brain tissue and cells with oxidized RNA increased during aging in all hippocampal and cortical regions analyzed. Abundant amoeboid microglia were commonly observed surrounding neurons with oxidized RNA. Notably, amoeboid microglia were arginase1+ and IL-10+, indicative of a M2 phenotype. Some of those M2 cells also presented RNA oxidation and a dystrophic phenotype. Therefore, our data suggest that ferritin confers protection to microglia in adult and old marmosets, while in aged subjects the decline in ferritin and the increased amount of iron in brain tissue may be related to the increased number of cells with oxidized RNA, perhaps precluding the onset of neurodegeneration.

The Tiny Drosophila Melanogaster for the Biggest Answers in Huntington's Disease.

The average life expectancy for humans has increased over the last years. However, the quality of the later stages of life is low and is considered a public health issue of global importance. Late adulthood and the transition into the later stage of life occasionally leads to neurodegenerative diseases that selectively affect different types of neurons and brain regions, producing motor dysfunctions, cognitive impairment, and psychiatric disorders that are progressive, irreversible, without remission periods, and incurable. Huntington's disease (HD) is a common neurodegenerative disorder. In the 25 years since the mutation of the huntingtin (HTT) gene was identified as the molecule responsible for this neural disorder, a variety of animal models, including the fruit fly, have been used to study the disease. Here, we review recent research that used Drosophila as an experimental tool for improving knowledge about the molecular and cellular mechanisms underpinning HD.

A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.

Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

Ferritin Assembly in Enterocytes of Drosophila melanogaster.

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated.

GABAρ expression in the medial nucleus of the trapezoid body.

The Calyx of Held (CoH) synapse is the largest synapse in mammals. It is located in the medial nucleus of the trapezoid body (MNTB) and forms part of the auditory pathway. Modest GABAergic signaling is present in the CoH before hearing onset, when glutamatergic transmission predominates. In mice, after postnatal day 12, the absolute strength of glycinergic transmission increases markedly, while GABAergic signaling remains constant. The persistent GABAergic transmission in the MNTB is mediated by a slowly desensitizing component. In this study we recorded GABA-mediated responses from postsynaptic principal neurons (PPNs) of the MNTB and found that they are sensitive to TPMPA, suggesting the involvement of GABAρ subunits. RT-PCR and immunohistofluorescence in the MNTB confirmed GABAρ expression in PPNs. Interestingly, GABAρ3 was present only before hearing onset, and there was a switch to GABAρ1 and GABAρ2 expression in adult animals.

Expression of GABAρ receptors in the neostriatum: localization in aspiny, medium spiny neurons and GFAP-positive cells.

GABAergic transmission in the neostriatum plays a central role in motor coordination, in which a plethora of GABA-A receptor subunits combine to modulate neural inhibition. GABAρ receptors were originally described in the mammalian retina. These receptors possess special electrophysiological and pharmacological properties, forming a characteristic class of ionotropic receptors. In previous studies, we suggested that GABAρ receptors are expressed in the neostriatum, and in this report we show that they are indeed present in all the calretinin-positive interneurons of the neostriatum. In addition, they are located in calbindin-positive interneurons and projection neurons that express the dopamine D(2) receptor. GABAρ receptors were also located in 30% of the glial fibrillary acidic protein-positive cells, and may therefore also contribute to gliotransmission. Quantitative reverse transcription-PCR suggested that the mRNAs of this receptor do not express as much as in the retina, and that GABAρ2 is more abundant than GABAρ1. Electrophysiological recordings in brain slices provided evidence of neurons expressing a cis-4-aminocrotonic acid-activated, 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid-sensitive ionotropic GABA receptor, indicating the presence of functional GABAρ receptors in the neostriatum. Finally, electron-microscopy and immunogold located the receptors mainly in perisynaptic as well as in extrasynaptic sites. All these observations reinforce the importance of GABAρ receptors in the neostriatum and contribute to the diversity of inhibitory regulation in this area.

The GABA(A)ρ receptors in hippocampal spontaneous activity and their distribution in hippocampus, amygdala and visual cortex.

A bicuculline-resistant and TPMPA-sensitive GABAergic component was identified in hippocampal neurons in culture and in acute isolated brain slices. In both preparations, total GABAergic activity showed two inactivation kinetics: fast and slow. RT-PCR, in situ hybridization (ISH) and immunohistochemistry detected expression of GABAρ subunits. Immunogold and electron microscopy indicated that the receptors are mostly extrasynaptic. In addition, by RT-PCR and immunofluorescence we found GABAρ present in amygdala and visual cortex.

Expression of GABArho subunits during rat cerebellum development.

In the present study, we provide evidence for the expression of all three GABA(C) receptor rho subunits through development of the rat cerebellum. Injection of cerebellum mRNA into frog oocytes gave rise to the expression of both GABA(A) and GABA(C) receptors. qRT-PCR of RNA isolated from postnatal developing cerebella showed that the expression of each rho subunit is relatively low, with a relative comparative expression of rho3>rho1>rho2. In situ hybridization and immunohistochemistry revealed a limited distribution of GABA(C) receptors in the Purkinje and Golgi neurons whereas electron microscopy detected the rho1 and rho2 subunits in the soma and dendritic tree of the Purkinje cells. The expression of GABA(C) receptors in the cerebellum adds a new dimension to the regulation of GABAergic neurotransmission and suggests further experiments to determine their functional consequences.