Frontiers in Neuroanatomy

Brain banks provide small tissue samples fixed in neutral-buffered-formalin (NBF), but human anatomy teaching laboratories could provide full brains fixed with solutions that are more appropriate for gross anatomy such as a saturated salt solution (SSS) or an alcohol-formaldehyde solution (AFS). Advanced aging and prolonged exposure to aldehydes are known to enhance brain tissue autofluorescence (AF), limiting the efficacy of immunofluorescence (IF) procedures. We have previously shown by IF staining the antigenicity preservation in mouse brains fixed with the three solutions. We now aimed to compare the quality of IF staining in human brains fixed with SSS, AFS and NBF. In addition, we compared the efficiency of AF quenching methods, namely the application of SudanBlackB (SBB) and the treatment of sections with sodium borohydride (NaBH4). Blocks of neocortex were extracted from 18 brains (NBF=6, SSS=6, AFS=6) and cut into 40{micro}m sections. Neurons (anti-NeuN, AlexaFluor-488) and astrocytes (anti-GFAP, AlexaFluor-555) were revealed with IF after an antigen retrieval protocol, while two treatments (SBB or NaBH4) were used to quench AF. We then assessed the degree of AF (criteria: background or cell AF) and the immunostaining quality with excitation wavelengths of 488nm, 555nm and 647nm. Brains fixed with all three solutions showed well-labeled astrocytes, whereas neurons werent always stained, but this was not associated to the fixative solution. The overall AF intensity was similar in sections from brains fixed with all three solutions. Finally, the SBB treatment was the most effective at reducing AF in all specimens. Given the similarity in AF and antigenicity assessment across the three solutions, we conclude that brains fixed with SSS and AFS could be good alternatives for NBF-fixed specimens in the context of IF experiments processed with a SBB protocol. Highlights- Immunofluorescence staining is feasible in brains fixed with anatomy labs solutions - GFAP is less affected by fixation than NeuN - Autofluorescence can be reduced by Sudan Black treatment

The ultrastructural analysis of postmortem brain tissue can provide important insights into cellular architecture and disease-related changes. For example, connectomics studies offer a powerful emerging approach for understanding neural circuit organization. However, electron microscopy (EM) data is difficult to interpret when the preservation quality is imperfect, which is common in brain banking and may render it unsuitable for certain research applications. One common issue is that EM images of postmortem brain tissue can have an expansion of regions that appear to be made up of extracellular space and/or degraded cellular material, which we call ambiguous interstitial zones. In this study, we report a method to assess whether EM images have ambiguous interstitial zone artifacts in a cohort of 10 postmortem brains with samples from each of the cortex and thalamus. Next, in matched samples from the contralateral hemisphere of the same brains, we evaluate the structural preservation quality of light microscopy images, including immunostaining for cytoskeletal proteins. Through this analysis, we show that on light microscopy, cell membrane morphology can be largely maintained, and neurite trajectory visualized over micrometer distances, even in specimens for which there are ambiguous interstitial zone artifacts on EM. Taken together, our analysis may assist in maximizing the usefulness of donated brain tissue by informing tissue selection and preparation protocols for various research goals.

Creating a high-resolution brain atlas in diverse species offers crucial insights into general principles underlying brain function and development. A volume electron microscopy approach to generate such neural maps has been gaining importance due to advances in imaging, data storage capabilities, and data analysis protocols. Sample preparation remains challenging and is a crucial step to accelerate the imaging and data processing pipeline. Here, we introduce several replicable methods for processing the brains of the gastropod mollusc, Berghia stephanieae for volume electron microscopy. Although high-pressure freezing is the most reliable method, the depth of cryopreservation is a severe limitation for large tissue samples. We introduce a BROPA-based method using pyrogallol and methods to rapidly process samples that can save hours at the bench. This is the first report on sample preparation and imaging pipeline for volume electron microscopy in a gastropod mollusc, opening up the potential for connectomic analysis and comparisons with other phyla.

Volume electron microscopy (vEM) has become a powerful tool for 3D ultrastructural analysis of neural circuits, yet its application to human brain organoids remains limited, particularly for connectomic studies. Here, we established a comprehensive and scalable workflow for applying vEM to human cortical organoids, integrating correlative light and electron microscopy, large-area SEM mosaic imaging, focused ion beam-scanning electron microscopy (FIB-SEM), and transmission electron microscopy (TEM) validation. By systematically comparing two embedding protocols in use, we demonstrated that the DeFelipe and Fairen (1993)/Cano-Astorga et al. (2024) method provides optimal compatibility with toluidine blue-stained semithin sectioning and enables reliable synapse segmentation and neurite tracing. In contrast, the Deerinck et al. (2010) protocol offers enhanced membrane contrast but limits postsynaptic density visualization. Using FIB-SEM imaging of peripheral, neuropil-like regions of cortical organoids, we achieved accurate 3D reconstruction of synapses, neurites and intracellular organelles, enabling quantitative assessment of synaptic apposition surfaces, neurite trajectories, and organelle distribution across defined cellular compartments. Together, our results demonstrate for the first time the feasibility of micro-connectomic reconstruction in human cortical organoids at nanometer resolution. This methodological framework expands the applicability of vEM to organoid systems and provides a robust foundation for future studies of human brain development, disease modeling, and therapeutic evaluation at the synaptic and subcellular level.

Postmortem human brains stored in brain banks are important research resources to study the mechanisms underlying normal brain functions as well as various neurodegenerative disorders. Immunohistochemical (IHC) and histochemical (HC) staining have been used to examine human brains post-fixed in neutral-buffered formalin (NBF) for months, years, and even decades. As such, it is essential to establish the effects of prolonged post-fixation in NBF on both IHC and HC stains. Previously, we found that prolonged NBF post-fixation resulted in differential effects on IHC and HC staining on postmortem brains. In this study, we further examined the effects of prolonged post-fixation on IHC stains targeting 6 antigens and 2 HC stains of known biomarkers of cerebrovascular diseases in prefrontal cortex of human brains post-fixed for 1, 5, 10, 15, and 20 years. The IHC targets included microvasculature markers of the blood brain barrier (Collagen-IV and Claudin-5), a type III intermediate filament marker (Vimentin), an activated microglia marker (CD68), a biomarker for oligodendrocytic myelin proteolipid protein (PLP) and a marker for iron accumulation (Ferritin). The HC included Massons Trichrome Stain (MTS) and Bielschowsky silver stain (BSS). We found that staining intensities of Ferritin, Vimentin, Collagen-IV and BSS decreased with prolonged post-fixation, while no significant differences were observed in the staining intensity of other markers. Hence, these differential alterations should be taken into consideration when interpreting the results from processed tissues with prolonged post-fixation. We recommend performing IHC and HC staining for human brains with the same post-fixation times to offset any impact on downstream neuropathological analyses, as well as adding the post-fixation duration as a covariate in the analysis.

Studies of whole brain cryopreservation are rare but are potentially important for a variety of applications. It has been demonstrated that ultrastructure in whole rabbit and pig brains can be cryopreserved by vitrification (ice-free cryopreservation) after prior aldehyde fixation, but fixation limits the range of studies that can be done by neurobiologists, including studies that depend upon general molecular integrity, signal transduction, macromolecular synthesis, and other physiological processes. We now show that whole brain ultrastructure can be preserved by vitrification without prior aldehyde fixation. Rabbit brain perfusion with the M22 vitrification solution followed by vitrification, warming, and fixation showed an absence of visible ice damage and overall structural preservation, but osmotic brain shrinkage sufficient to distort and obscure neuroanatomical detail. Neuroanatomical preservation in the presence of M22 was also investigated in human cerebral cortical biopsies taken after whole brain perfusion with M22. These biopsies did not form ice upon cooling or warming, and high power electron microscopy showed dehydrated and electron-dense but predominantly intact cells, neuropil, and synapses with no signs of ice crystal damage, and partial dilution of these samples restored normal cortical pyramidal cell shapes. To further evaluate ultrastructural preservation within the severely dehydrated brain, rabbit brains were perfused with M22 and then partially washed free of M22 before fixation. Perfusion dilution of the brain to 3-5M M22 resulted in brain re-expansion and the re-appearance of well-defined neuroanatomical features, but rehydration of the brain to 1M M22 resulted in ultrastructural damage suggestive of preventable osmotic injury caused by incomplete removal of M22. We conclude that both animal and human brains can be cryopreserved by vitrification with predominant retention of ultrastructural integrity without the need for prior aldehyde fixation. This observation has direct relevance to the feasibility of human cryopreservation, for which direct evidence has been lacking until this report. It also provides a starting point for perfecting brain cryopreservation, which may be necessary for lengthy space travel and could allow future medical time travel.

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for the preservation of fixed whole brains using graded immersion cryoprotection and subzero temperature storage, which is one type of a more general approach that we refer to as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose. We used CT imaging to track cryoprotectant penetration, finding that with the use of our protocol, approximately 10 months is required to reach equilibration throughout whole human brains. In our initial histological validation, we found that insufficient equilibration time prior to freezing led to apparent ice crystal artifacts seen on ultrastructural imaging of the white matter. After refining the protocol to allow adequate diffusion time, histologic data at both the light and electron microscopic levels showed preserved cellular architecture and ultrastructure after the process of cryoprotectant loading, freezer storage, and unloading. This protocol can be implemented using laboratory freezers or freezer rooms and provides a degree of resilience against freezer failures because the morphology of the fixed tissue is expected to remain preserved long-term in the fluid state even if rewarmed. Our approach may be valuable for laboratories seeking to enhance the long-term preservation of antigenicity in large brain tissue specimens for future research applications.

Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of whole-cell-dissociated-suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and fluorescence-activated cell sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis.

Neuromodulatory systems regulate neural circuits across broad regions of the brain, and disruption of the dopaminergic system contributes to psychiatric and neurodegenerative disorders. Engineered viral vectors have been used to target the neuromodulatory systems of the nonhuman primate brain (Y. Chen et al., 2023; El-Shamayleh et al., 2016; Gray et al., 2010; Lerchner et al., 2014; Perez et al., 2022). However, a conspicuous obstacle to the isolation and modulation of specific pathways is the inability of many retrogradely infecting viruses to transduce dopaminergic (DA) cells efficiently (Tervo et al., 2016; Cushnie et al., 2020; Weiss et al., 2020). We compare the DA neuron retrograde transduction efficacy of four viral vectors after injection into the striatum of nonhuman primates (NHP). Selectivity was assessed by comparing the neuronal co-expression of fluorescent reporter protein and tyrosine-hydroxylase (TH) antibody in substantia nigra pars compacta (SNc). The rabies pseudotyped lentiviral vector, FuG-B2, produced superior retrograde transduction of DA cells to FuG-C or FuG-E. AAV2.retro was the least effective.

An automated ultra-microtome capable of sectioning thousands of ultrathin sections onto standard TEM slot grids was developed and used to section: a complete Drosophila melanogaster first-instar larva, three sections per grid, into 4,866 34-nm-thick sections with a cutting and pickup success rate of 99.74%; 30 microns of mouse cortex measuring roughly 400 um x 2000 um at 40 nm per slice; and a full adult Drosophila brain and ventral nerve column into 9,300 sections with a pickup success rate of 99.95%. The apparatus uses optical interferometers to monitor a reference distance between the cutting knife and multiple sample blocks. Cut sections are picked up from the knife-boat water surface while they are still anchored to the cutting knife. Blocks without embedded tissue are used to displace tissue-containing sections away from the knife edge so that the tissue regions end up in the grid slot instead of on the grid rim.

BACKGROUNDOligodendroglia encompass oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs). In the grey matter of the cortex, nearly all OPCs divide slowly, yet they dont differentiate solely into mature OLs, leaving the exact role of these OPCs in the grey matter enigmatic. METHODSOligodendroglia, including OPCs, were traced using the Sox10 Cre-ERT2 reporter mice. We compared the effect of tamoxifen dissolved in different solvents on the fate of Sox10 cells. We also compared the effect of tamoxifen dosage on the fate of Sox10 cells. The differentiation of labeled red fluorescent protein (RFP) cells was analyzed using immunofluorescence staining. RESULTSTwo groups of RFP cells, type A Sox10 (Sox10-A) cells, and type B Sox10 (Sox10-B) cells, were identified in the cortex, striatum, hippocampus, thalamus, and hypothalamus. Sox10-A cells differentiate into platelet-derived growth factor receptor-{beta} (PDGFR{beta})+, CD13+ pericytes, and smooth muscle myosin heavy chain 11 (MYH11) + smooth muscle cells when the mice received ethanol or high-dose tamoxifen. Sox10-B cells transform into glutamatergic neurons when the mice received high-dose tamoxifen. Sox10-B cells include perineurona OPCs and OLs. CONCLUSIONSThis investigation provides evidence that a substantial proportion of oligodendroglia in the grey matter serve as mural cell precursors and neuronal precursors. These two phenomena may contribute to our understanding of neurodegenerative diseases. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/549127v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1b7bef9org.highwire.dtl.DTLVardef@5af54borg.highwire.dtl.DTLVardef@19e3d45org.highwire.dtl.DTLVardef@1c881f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

NADPH diaphorase (N-d) is used to a histochemical identification of subgroup of neuronal cells. Beside regular intracellular N-d positivity, membrane-related positivity revealed as a specialized staining pattern in the pigeon brain stem. In the investigation of the nervous system of homing pigeons (Columba livia) with N-d staining, we found a specialized structure, which temporally was termed as N-d tubular glomerular body/structure or as T-J body related to the last name of authors. This N-d positive specialization constituted by tubular components bilaterally located in the medial to the lemniscus spinalis in the medulla oblongata. The tubular components were moderate staining. T-J body was a longitudinal oriented structure of 2400 m with N-d staining. N-d positive tubular components were twisted and intermingled together. Beside the young adult pigeons, T-J body s were also consistently detected in the aged pigeons. Membrane-related staining were also detected in the other rostral nuclei in the brain stem. With discussion and review of related scientific literatures, T-J body was considered as a new anatomical structure or a new feature of the existent nucleus. In summary, beside N-d intracellular distribution, there were other three N-d membrane-related localizations: mini-aggregation, patch-aggregation, and arrangement along tubular unit.

A vital question in neuroscience is if and how efficiently cellular models may be differentiated into functional neuronal cells in culture. Despite the frequent use of the human neuroblastoma cell line SH-SY5Y, differentiation protocols vary extensively, with the most common being differentiation via the addition of retinoic acid and brain-derived neurotrophic factor. However, due to the lack of a reliable evaluation method, their adequacy as synaptic models remains unclear. Here, we investigate whether SH-SY5Y cells constitute a functional model for synaptic studies by phenotypically and ultra structurally analyzing synaptogenesis in SH-SY5Y cells subjected to different differentiation protocols. Electron microscopy (EM) techniques, including conventional EM, cryo-EM, and cryo-electron tomography, were systematically applied to characterize synaptogenesis in SH-SY5Y cells. Further characterization was performed through immunostaining and functional assays, such as live exocytosis assays and whole-cell patch-clamp electrophysiology. Despite exhibiting some presynaptic-like features, differentiated SH-SY5Y cells do not form morphologically or functionally complete synapses under the conditions tested. Immunostaining results were consistent with previous findings, showing synaptic markers. However, functional investigations did not detect synaptic activity. High-throughput EM analyses revealed an absence of synaptic structures in these cells. Additionally, an alternative differentiation approach incorporating additional neurotrophic factors promoted the formation of pre-synaptic-like compartments containing synapse-like synaptic vesicles (SVLVs). Though these SVLVs exhibited pleomorphic size distributions, differing from typical synaptic vesicles, and lacked connectors. These findings emphasize the need for cautious interpretation of results derived from SH-SY5Y cells when studying molecular synaptic architecture or neurodegenerative diseases.

Understanding the synaptic characteristics of each cortical layer is essential for elucidating the functional architecture of each brain region. In the current study, we made a detailed quantitative comparison of the synaptic structure in the predominantly input layers of primate primary visual cortex (layer 4C) and in the predominant output layer (layer 3B) using focused ion beam scanning electron microscopy (FIB/SEM). We quantified the synaptic density in each layer, classified synaptic boutons according to their number of synapses and mitochondrial content, and quantified key morphometric parameters, including bouton volume, postsynaptic density (PSD) area and morphology, volume occupied by mitochondria, and postsynaptic targets. Our results revealed that for all the layers there is a higher proportion of single-synapse boutons without mitochondria. Multisynaptic boutons containing mitochondria (MSBm+)-- which likely correspond to TC terminals --were significantly more abundant in the thalamocortical recipient layers 4C and 4C{beta}. These MSBm+ boutons were also larger, more likely to contact dendritic spines, and contained more mitochondria than other bouton categories. In contrast, layer 3B, displayed a lower prevalence of MSBm+ boutons, these boutons were smaller than those in layer 4C and made fewer synapses. These findings highlight laminar differences in bouton architecture and support the idea that TC synapses are structurally adapted to support high synaptic efficacy. Together, our data provide a detailed quantitative framework for understanding the synaptic organization of primate V1, with implications for sensory processing and cortical circuit function.

Until relatively recently, the cerebellum was considered primarily as modulator of fine motor functions. This has changed during the last 20 years, and now the cerebellum has been shown to be involved in learning and cognition. With this renewed interest comes the need to better understand and potentially modify its input-output connections. In this pilot study, we tested the efficacy of a canine adenovirus type 2 (CAV-2) vector in the cerebellum of a nonhuman primate (NHP). Consistent with other reports, we found a preferentially transduction of cells with neuron-like morphology at the site of injection and efficient retrograde transport into several structures in the midbrain. These data will help identify cerebellar circuits and may lay the foundation for studies of human pathologies, such as ataxias, autism, and schizophrenia.

Retrosplenial area 29e, which was a cortical region described mostly in earlier rodent literature, is often included in the dorsal presubiculum (PrSd) or postsubiculum (PoS) in modern literature and commonly used brain atlases. Recent anatomical and molecular studies have revealed that retrosplenial area 29e belongs to the superficial layers of area prostriata, which in primates is found to be important in fast analysis of quickly moving objects in far peripheral visual field. As in primates, the prostriata in rodents adjoins area 29 (granular retrosplenial area), area 30 (agranular retrosplenial area), medial visual cortex, PrSd-PoS, parasubiculum (PaS) and postrhinal cortex (PoR). The present study aims to reveal the chemo-architecture of the prostriata versus PrSd-PoS or PaS by means of a systematic survey of gene expression patterns in adult and developing mouse brains. First, we find many genes that display differential expression across the prostriata, PrSd-PoS and PaS and that show obvious laminar expression patterns. Second, we reveal subsets of genes that selectively express in the dorsal or ventral parts of the prostriata, suggesting the existence of at least two subdivisions. Third, we detect some genes that shows differential expression in the prostriata of postnatal mouse brains from adjoining regions, thus enabling identification of the developing area prostriata. Fourth, gene expression difference of the prostriata from the medial visual cortex and PoR is also observed. Finally, molecular and connectional features of the prostriata in rodents and non-human primates are discussed and compared.

Myelin formation by oligodendrocytes is essential for the regulation of the conduction velocity and proper brain function. To ensure accurate information processing in response to experiences such as sensory stimuli and learning, oligodendrocytes adjust their number and morphology. In addition, oligodendrocyte morphology changes with senescence and in the presence of neurodegenerative diseases. Thus, visualizing oligodendrocytes and analyzing their morphology is crucial for understanding how our brains change under such conditions. Herein, we describe the methods for labeling and analyzing the morphologies of individual oligodendrocytes in mouse white matter at the light microscopic level. PDGFRa-CreERT2:Tau-mGFP and PLP-CreERT2:Tau-mGFP mice enable us to visualize and analyze later-born or early-born oligodendrocyte morphology. In addition, sparse oligodendrocyte labeling with attenuated rabies virus expressing GFP enables the visualization and morphological analysis of individual oligodendrocytes in various brain white matter regions without the need for transgenic animals. Furthermore, the combination with immunostaining in thick tissues enables the identification of labeled oligodendrocytes and myelin sheaths, as well as their interactions with neuronal axons. These methods are suitable for revealing how oligodendrocytes adapt their morphologies depending on environmental stimuli or pathological conditions.

AbstractO_ST_ABSBackgroundC_ST_ABSGene therapies are promising for diseases previously considered incurable. Adeno-associated virus serotype 9 (AAV9) demonstrates remarkable tropism for motor neurons (MNs) and represents an exciting candidate to target genetic causes of motor neuron diseases like amyotrophic lateral sclerosis (ALS). However, systemic delivery risks immunogenicity and off-target effects, therefore localised delivery to the CNS is advantageous. New methodWe assessed MN transduction in wild-type mice using AAV9-controlled, cytomegalovirus-promoter driven, enhanced GFP expression. Intra-cisterna magna (ICM) and intra-cerebroventricular (ICV) methods were compared. Four weeks post-delivery, GFP positivity in MN and astrocytes were quantified via immunohistochemical approaches and viral genome copy number determined by qPCR. ResultsAll delivery methods achieved high MN transduction in lumbar spinal cord (>68%). Unilateral ICV delivery provided the highest and most consistent levels (89 {+/-} 3%), and minimal peripheral viral copies. ICV delivery resulted in higher astrocytic transduction, most notably in the cortex. Brainstem MN transduction was high with all methods (>55%). We failed to find evidence of neuronal transduction in motor cortex. Viral genome copies trended higher in spinal cord and brainstem with ICV approaches, however further work is required to understand how bilateral delivery leads to such profound increases. Comparison to existing methodsWhilst several routes of administration into cerebrospinal fluid exist, direct comparisons for targeting MNs in vivo remain limited. ConclusionsOverall, consideration of gene therapy delivery methods is critical to ensure that the most appropriate administration route is chosen to reach MNs effectively, selectively, and at high levels to exact biological effects. HighlightsO_LICSF delivery of AAV9 targets >68% of lumbar spinal cord (SC) motor neurons C_LIO_LIUnilateral ICV yields high and consistent lumbar SC motor neuron transduction (>88%) C_LIO_LIUnilateral ICV results in the lowest number of peripheral viral copies C_LIO_LIICV targets significantly more astrocytes, particularly in cortex C_LIO_LIBilateral ICV leads to significantly higher viral copies in the liver C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/662642v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bd83a5org.highwire.dtl.DTLVardef@df63aorg.highwire.dtl.DTLVardef@a09142org.highwire.dtl.DTLVardef@1a0887a_HPS_FORMAT_FIGEXP M_FIG C_FIG

To achieve cell type-specific gene expression, using target cell-tropic AAV capsids is advantageous. However, their tropism across brain cell types remains unexplored in non-human primates. We assessed the tropism of nine AAV serotype capsids (AAV1, 2, 5, 6, 7, 8, 9, rh.10 (rh10), and DJ) on marmoset cerebral cortical cell types. Marmoset cerebral cortex was injected with different serotype AAVs expressing enhanced GFP (EGFP) by the ubiquitous chicken {beta}-actin hybrid (CBh) promoter. After 4 weeks, all nine AAV capsid vectors, especially AAV9 and AAVrh10, caused highly neuron-selective EGFP expression. Some AAV capsids, including AAV5, caused EGFP expression in oligodendrocytes to a lesser extent, with minimal or no expression in astrocytes and microglia. Different ubiquitous CMV and CAG promoters showed similar neuron-predominant transduction. Conversely, all nine AAV capsid vectors with the astrocyte-specific hGFA(ABC1D) promoter selectively transduced astrocytes, except AAV5, which transduced oligodendrocytes modestly. Oligodendrocyte-specific mouse myeline basic protein (mMBP) promoter in AAV5 vectors transduced oligodendrocytes specifically and efficiently. Our results suggest optimal combinations of capsids and promoters for cell type-specific expression: AAV9 or AAVrh10 and ubiquitous CBh, CMV, or CAG promoter for neuron-specific transduction; AAV2 or 7 and hGFA(ABC1D) promoter for astrocyte-specific transduction; and AAV5 and mMBP promoter for oligodendrocyte-specific transduction.

Although commonly known as rapid and easy to use methodology, Golgi staining requires a range of staining solutions, impregnation periods, concentrations and slicing variables. The use of this methodology can help researchers identify and label individual neuronal components within the extended circuitry. The original Golgi stain technique, developed by Camillo Golgi in 1873, is a silver staining method that enabled scientists to visualize individual neurons in their entirety within nervous tissue for the first time. publications featuring the Golgi staining technique utilise cryostat or microtome slicing, with the combination of a readily purchased kit which comes with a cost and limited morphological detail. Here, we describe an optimised Golgi staining methodology that specifically targets the major drawbacks of traditional protocols; prolonged and inconsistent impregnation, slice fragility during sectioning, and variable visualization of fine dendritic structures. Through modest adjustments to impregnation duration and temperature, fixation, and vibratome sectioning conditions, this low-cost and simple protocol improves staining reliability, facilitates robust slicing without specialized embedding, and supports detailed analysis of neuronal morphology throughout the central nervous system. We validate our optimised protocol using tissue from on-going animal studies of pain and treatment. Representative images illustrate typical staining patterns, characterised by sparse background and high signal-to-noise ratio, facilitating unbiased neuronal tracing and analysis.