Frontiers in Neuroanatomy

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.

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CC) this has yet to be fully accomplished. The CC is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are highly debated. To address this, we conducted a multifaceted analysis of fiber- and cyto-architecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to the CC. We identified four distinct subregions within the CC, subdividing both the CL and the DEn into two. Additionally, we conducted brain-wide tracing of inputs to the entire CC using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of the CC and its subregions. Myelinated fibers were abundant in dorsal parts of the CL but absent in ventral parts, while parvalbumin labelled fibers occupied the entire CL. Calbindin staining revealed a central gap within the CL, which was also visible at levels anterior to the striatum. Furthermore, cells in the CL projecting to the retrosplenial-cortex were located within the myelin sparse area. By combining our own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework. Significance statementMice are a highly tractable model for studying the claustrum complex (CC). However, without a consensus on how to delineate the CC in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CC, to match the level of detail needed to study its functional properties. Using multiple strategies for identifying claustral borders, we created a comprehensive guide to delineate the CC and its subregions. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CC.

The zona incerta (ZI) supports diverse behaviors including binge feeding, sleep/wake cycles, nociception, and hunting. This diversity of functions can be attributed to the heterogenous neurochemicals, cytoarchitecture, and efferent connections that characterize the ZI. The ZI is predominantly GABAergic, but we recently identified a subset of medial ZI GABA cells that co-express dopamine (DA), as marked by the enzyme tyrosine hydroxylase (TH). While the role of GABA within the ZI is well studied, little is understood about the function of ZI DA cells. To identify potential roles of ZI DA cells we mapped the efferent fiber projections from Th-cre ZI cells. We first validated a Th-cre;L10-Egfp mouse line and found that medial Egfp ZI cells were more likely to co-express TH-immunoreactivity (TH-ir). We thus delivered a cre-dependent virus into the medial ZI of Th-cre or Th-cre;L10-Egfp mice and selected two injection cases for full brain mapping. We selected the cases with the lowest (17%) and highest (53%) percentage of colocalization between TH-ir and virus transfected cells labelled with DsRed. Overall, DsRed-labelled fibers were observed throughout the brain and were most prominent within motor-related regions of the midbrain (MBmot), notably the periaqueductal grey area and superior colliculus. We also observed considerable DsRed-labelled fibers within the polymodal cortex associated regions of the thalamus (DORpm), including the paraventricular thalamic nucleus and nucleus of reunions. Overall, ZI DA cells displayed a similar connectivity profile to ZI GABA cells, suggesting that ZI DA cells may perform synergistic or opposing functions at the same target sites. List of RRIDsAB_2201528, AB_10013483, AB_11177031, AB_2340593, AB_2315778, SCR_016477 Three key points- Tyrosine hydroxylase immunoreactivity was more prominent in the medial zona incerta - Intersection points from two Th-cre injection cases revealed common target regions - Dopaminergic zona incerta cells project predominantly to motor-related brain regions

The cortico-basal ganglia pathways that mediate vocal learning in zebra finches (Taeniopygia guttata) are localized in parallel circuits formed by CORE and SHELL subregions. These circuits traverse a specialized region of the basal ganglia essential for vocal learning (Area X), which includes intermixed striatal and pallidal neurons. The pallidal neurons within Area X exhibit analogs of mammalian direct and indirect pathways that may have opposing effects and thereby increase or inhibit thalamic activity respectively. Direct pallidal neurons of Area X send projections to the medial portion of the dorsolateral anterior thalamic nucleus (DLM), whereas indirect pallidal neurons form intrinsic connections onto DLM-projecting neurons. Expression of the transcription factor FoxP2 in the basal ganglia is necessary for normal vocal learning and production in both humans and songbirds. We used tract-tracing techniques to label direct pallidal Area X[->]DLM projection neurons and immunohistochemical techniques to label neurons expressing the transcription factor FoxP2 in adult and juvenile male zebra finches. Our results showed that DLM-projecting neurons did not express FoxP2 in either adults or juveniles. Measurements of nuclear sizes revealed a population of large neurons that expressed FoxP2 but were not retrogradely-labeled from DLM. A putative marker of striatal neurons (DARPP-32) did not co-localize with FoxP2 in many of these large neurons, suggesting that they form a class of indirect pallidal neurons. These findings offer FoxP2 as a possible marker for indirect pallidal neurons and support the existence of different subpopulations of neurons that correspond to direct and indirect pathways within Area X.

The posterior cingulate cortex (mainly area 23) in human and non-human primates is a critical component of the default mode network and is involved in many neurological and neuropsychiatric diseases such as Alzheimers disease, autism, depression, attention deficit hyperactivity disorder and schizophrenia. However, cingulate area 23 has not yet identified in rodents and other lower mammals and this makes modeling related circuits and diseases in rodents very difficult. Using a comparative approach and unique connectional patterns the present study has uncovered the location and extent of rodent equivalent of the primate cingulate area 23. Like in monkeys, area 23 but not adjoining retrosplenial and visual areas in the rats and mice displays strong reciprocal connections with the anteromedial thalamic nucleus. Rodent area 23 also reciprocally connects with the medial pulvinar and claustrum as well as with the anterior cingulate, granular retrosplenial, medial orbitofrontal, postrhinal, and visual and auditory association cortices. The rodent A23 also projects to the subcortical effectors such as the dorsal striatum, ventral lateral geniculate nucleus, zona incerta, pretectal nucleus, superior colliculus, periaqueductal gray, and brainstem reticular formation. All these connectional findings support the versatility of area 23 in the integration and modulation of multimodal information underlying spatial processing, episodic memory, self-reflection, attention, value assessment and many adaptive behaviors. Additionally, this study also suggests that the rodents can be used to model primate and human area 23 in future structural, functional, pathological and neuromodulation studies.

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.

Cryo-electron microscopy (cryo-EM) enables the study of protein complexes, cytoskeletal elements, and organelles in three dimensions without the use of chemical fixation. Most cryo-EM studies focus on vitreously frozen individual cells separated from their native tissue contexts. This reliance on imaging of single cells is primarily due to technical challenges associated with preparing fresh tissue sections at a thinness sufficient for visualization via cryo-EM. Highly heterogenous and specialized tissues, such as brain, are especially affected by this limitation as the cellular, subcellular, and synaptic milieus can significantly vary across neuroanatomical locations. To address this limitation, we established new instrumentation and a workflow that consists of: 1) high-pressure freezing of fresh brain tissue; 2) tissue trimming followed by cryo-focused ion beam milling via the H-bar approach to generate ultrathin lamellae; and 3) cryo-EM imaging. Here, we apply this workflow to visualize the fine ultrastructural details of organelles, as well as cytoskeletal and synaptic elements that comprise the cortical neuropil within fresh, unfixed mouse brain tissue. Moreover, we present initial studies that apply principles of the above workflow to the analysis of postmortem human brain tissue. Overall, our work integrates the strengths of cryo-electron microscopy and tissue-based approaches to produce a generalizable workflow capable of visualizing subcellular structures within complex tissue environments.

Studying neural mechanisms in complementary model organisms from different ecological niches in the same animal class can leverage the comparative brain analysis at the cellular level. To advance such a direction, we developed a unified brain atlas platform and specialized tools that allowed us to quantitatively compare neural structures in two teleost larvae, medaka (Oryzias latipes) and zebrafish (Danio rerio). Leveraging this quantitative approach we found that most brain regions are similar but some subpopulations are unique in each species. Specifically, we confirmed the existence of a clear dorsal pallial region in the telencephalon in medaka lacking in zebrafish. Further, our approach allows for extraction of differentially expressed genes in both species, and for quantitative comparison of neural activity at cellular resolution. The web-based and interactive nature of this atlas platform will facilitate the teleost communitys research and its easy extensibility will encourage contributions to its continuous expansion.

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.

The insula has been classically divided into vast granular, dysgranular and agranular sectors. Over the years, several distinct studies proposed subdivisions of these sectors, with however no consensus. We recently proposed a cyto- and myelo-architectonic partition in which each sector contained sharply delimited areas (Evrard et al. 2014 J Comp Neurol 522: 64-97). Some of these areas were further divided into distinct subareas with obvious functional implications. Here, we examined the spatial relationship between architectonic boundaries and tract-tracing labeling in the insula in the macaque monkey. Injections of neuronal tracers in distinct areas of the prefrontal or anterior cingulate cortices produced heterogeneous and discontinuous patterns of anterograde and retrograde labeling in the insula. These patterns were made of sharply delimited patches forming anteroposterior stripes across consecutive coronal sections. While the overall pattern of labeling varied with the injection site, the patches systematically coincided with specific architectonic subareas, particularly in the dysgranular insula. This unequivocally validates our prior architectonic partition and strongly supports the idea of a refined modular Bauplan of the primate insula. This modular organization may underlie a serial stream of integration of interoception with self-agency and social activities across distinct insulo-prefrontal processing units that need to be explored.

Birds undisputedly range amongst natures foremost navigators. To successfully navigate between breeding and wintering quarters, they, in addition to other natural orientation cues, rely on their ability to sense the Earths magnetic field. For this reason, migratory birds have become key model species for studying the sensory mechanisms underlying magnetic field-guided navigation, as evidenced by the identification of several brain regions believed to be involved in processing magnetic field information. However, there is as yet no readily accessible, high-resolution three-dimensional (3D) brain atlas to serve as a common reference within and across studies. Here we provide the neuroscience research community with the first freely available, digital, high-resolution (25 {micro}m), 3D bird brain atlas. It is based on light microscopy images from ten Eurasian blackcaps (Sylvia atricapilla), a night-migratory songbird widely used model species in magnetoreception and navigation research. We outline the individual steps for the creation of a brain atlas, from whole-brain imaging using serial-section, two-photon tomography, to the creation of an average template at an isotropic 25-{micro}m voxel size, and finally to brain area segmentation and annotation. In this first version of the atlas, we have mapped a total of 24 brain areas, including 6 principal compartments, 13 conspicuous anatomical subdivisions common to all bird species and 5 functionally defined areas of the visual and trigeminal sensory systems involved in processing magnetic field information. This atlas is accessible via the standardised BrainGlobe Atlas API, making it compatible with a growing suite of computational neuroanatomy tools provided by the BrainGlobe Initiative. This integration enables precise alignment of future experimental data to a common coordinate space, facilitating collaboration, data visualization and sharing. Furthermore, this resource enables the accurate localization and comparison of implanted devices, injection sites, and/or cell populations across individual brains, both within and across studies.

Nitric oxide (NO) is a diffusible neuromodulator with roles in synaptic plasticity and memory flexibility, exerting its primary effects via the enzyme soluble guanylate cyclase (sGC). Despite its well-documented functions in mammals and insects, little is known about the neuroanatomical distribution and functional relevance of NO in birds, particularly in relation to dopaminergic systems. This study used histochemical and immunohistochemical techniques to map the distribution of NO-synthesizing neurons--identified by NADPH-diaphorase (NADPH-d) and nNOS activity--and their relation to sGC and tyrosine hydroxylase (TH)-positive dopaminergic pathways in the pigeon brain. We found extensive NADPH-d labeling throughout forebrain, midbrain, and hindbrain regions. Among TH-positive midbrain structures, the locus coeruleus exhibited high colocalization with nNOS, while moderate colocalization was seen in the ventral tegmental area substantia grisea centralis and substantia nigra. Notably, a significant proportion of sGC-positive neurons was targeted by TH and NADPH-d positive fibres in the pigeon NCL. Our findings support the potential for NO-dopamine interactions in avian species, reminiscent of memory-related mechanisms in Drosophila melanogaster, and contribute to an understanding of conserved pathways that may underlie flexible learning and memory processing during navigation or related tasks across vertebrates. This work also offers insight into comparative NADPH-d distribution among avian species, with implications for aging, spatial learning, and memory formation.

The central nucleus (CeN) of the amygdala is an important afferent to the DA system that mediates motivated learning. We previously found that CeN terminals in nonhuman primates primarily overlap the elongated lateral VTA (parabrachial pigmented nucleus, PBP, A10), and retrorubral field(A8) subregion. Here, we examined CeN afferent contacts on cell somata and proximal dendrites of DA and GABA neurons, and distal dendrites of each, using confocal and electron microscopy (EM) methods, respectively. At the soma/proximal dendrites, the proportion of TH+ and GAD1+ cells receiving at least one CeN afferent contact was surprisingly similar (TH = 0.55: GAD1=0.55 in PBP; TH = 0.56; GAD1 =0.51 in A8), with the vast majority of contacted TH+ and GAD1+ soma/proximal dendrites received 1-2 contacts. Similar numbers of tracer-labeled terminals also contacted TH-positive and GAD1-positive small dendrites and/or spines (39% of all contacted dendrites were either TH- or GAD1-labeled). Overall, axon terminals had more symmetric (putative inhibitory) axonal contacts with no difference in the relative distribution in the PBP versus A8, or onto TH+ versus GAD1+ dendrites/spines in either region. The striking uniformity in the amygdalonigral projection across the PBP-A8 terminal field suggests that neither neurotransmitter phenotype nor midbrain location dictates likelihood of a terminal contact. We discuss how this afferent uniformity can play out in recently discovered differences in DA:GABA cell densities between the PBP and A8, and affect specific outputs. Significance statementThe amygdalas central nucleus (CeN) channels salient cues to influence both appetitive and aversive responses via DA outputs. In higher species, the broad CeN terminal field overlaps the parabrachial pigmented nucleus ( lateral A10) and the retrorubral field (A8). We quantified terminal contacts in each region on DA and GABAergic soma/proximal dendrites and small distal dendrites. There was striking uniformity in contacts on DA and GABAergic cells, regardless of soma and dendritic compartment, in both regions. Most contacts were symmetric (putative inhibitory) with little change in the ratio of inhibitory to excitatory contacts by region. We conclude that post-synaptic shifts in DA-GABA ratios are key to understanding how these relatively uniform inputs can produce diverse effects on outputs.

To better understand the cortical connections and organization of visual areas in galagos, we examined the interconnections among cortical visual fields and their relationships with posterior parietal cortex (PPC) and temporal regions associated with dorsal and ventral streams of visual processing. In five galagos, two to four distinguishable tracers were injected into different visual areas, allowing direct comparison of connection patterns within the same cases. To reveal distributions of labeled neurons for each injection, labeled cells were plotted from serial brain sections cut parallel to the flattened cortical surface and summed across sections to generate surface reconstructions. Alternate sections processed for cytoarchitectonic features were used to identify cortical borders, especially those of V1 and middle temporal visual area (MT). Overall, our results support the conclusion that regions of V2 and V3 represent the contralateral visual hemifield in parallel with V1 and with each other. However, dorsal V3, representing the lower visual hemifield, includes at least one discontinuity where representations of the upper visual field extend to the dorsal border of V2. This portion of V3 appears to belong to the dorsomedial visual area (DM), which extends rostrally from V2 into PPC. The dorsal part of the DL-V4 region receives projections from other parts of dorsolateral visual area (DL), central V1, V2 and V3, inferotemporal (IT) cortex, the MT complex, and PPC regions surrounding the intraparietal sulcus (IPS). More central portions of DL-V4 receive inputs from central representations of V1, V2, and V3, as well as from PPC regions lateral to the IPS, the MT complex, and upper IT cortex. The ventral part of DL receives projections from central V2, caudal PPC adjoining DM and ventral PPC, and from IT cortex. These patterns indicate that the DL-V4 region serves as a major node linking dorsal and ventral streams and likely includes more than one functionally distinct visual area. In addition, areas MT and DM show strong reciprocal connections with PPC, while the connections of IT cortex indicate that much of this region is visual in nature having strong connections with higher order visual areas and it is composed of multiple functionally specialized visual domains. Key pointsO_LIOrganization of the visual cortex in galagos is much like that in New World and Old Word monkeys. C_LIO_LIPatterns of cortical connections of early visual areas V1 and V2 support the view that dorsal V3 has a gap in the representation of the lower visual field that is occupied by the proposed dorsomedial visual area (DM). C_LIO_LIVisual areas DM and middle temporal visual area (MT) provide the major visual inputs to posterior parietal cortex (PPC) of the dorsal stream of visual processing for actions. C_LI

We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of series of tripartite synapses enveloped by astrocytic endfeet linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel with such specialized synapses suggests a mechanoreceptor role. In Golgi impregnated and 3D reconstructions of cerebral cortex and thalamus, series of tripartite synapses appear sequentially ordered in a tributary dendrite paralleled by synaptic outgrowths termed here golf club-like extensions apposed to a longitudinal crest from the capillary basal lamina. To facilitate identification of principal cell dendrites and arriving axons to these putative mechanosensory structures, we utilized the cerebellar cortex since it has a well known organization and observed that afferent fibers and interneurons display interactions with the capillary wall. Afferent mossy fiber rosettes and ascending granule cell axons and dendrites define pericapillary on passage interactions surrounded by endfeet. The ability of such structures to modulate synaptic transmission is supported by the presence of mRNA of the mechanosensitive channel Piezo 1 in the mossy fiber rosettes, pyramidal isocortical and thalamic neurons. This suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ.

The accessory olfactory bulb (AOB) plays a key role in processing chemical signals crucial for species-specific social and reproductive behaviors. While extensive research has focused on the vomeronasal system of laboratory rodents, less is known about wild species, particularly those that rely heavily on chemical communication. This study aims to characterize the morphological and neurochemical organization of the AOB in the fossorial water vole (Arvicola scherman), a subterranean rodent species from the family Cricetidae. We have employed histological techniques, including Nissl and hematoxylin staining, as well as immunohistochemical and lectin-histochemical markers, to assess the AOB structure. Our findings reveal that the AOB of the water vole exhibits a distinct laminar organization with prominent mitral cells in the mitral-plexiform layer, as well as dense labeling of periglomerular and short-axon cells in the glomerular layer. Lectin histochemistry further confirmed zonation patterns analogous to those seen in other rodent species. Immunohistochemical analysis demonstrated significant expression of PGP 9.5, suggesting its involvement in maintaining neuronal activity within the AOB. In contrast, the absence of SMI-32 labeling in the AOB, compared to its strong expression in the main olfactory bulb, highlights functional distinctions between these two olfactory subsystems. These structural and neurochemical characteristics suggest that the AOB of the fossorial water vole is adapted for enhanced processing of chemosensory signals, which may play a pivotal role in its subterranean lifestyle. Our results provide a foundation for future studies exploring the functional implications of these adaptations, including potential improvements in the integrated management of these vole populations.

Information on regional variation in cell numbers and densities in the CNS provides critical insight into structure, function, and the progression of CNS diseases. However, variability can be real or can be a consequence of methods that do not account for technical biases, including morphologic deformations, errors in the application of cell type labels and boundaries of regions, errors of counting rules and sampling sites. We address these issues of by introducing a workflow that consists of the following steps: 1. Magnetic resonance histology (MRH) to establish the size, shape, and regional morphology of the mouse brain in situ. 2. Light-sheet microscopy (LSM) to selectively label all neurons or other cells in the entire brain without sectioning artifacts. 3. Register LSM volumes to MRH volumes to correct for dissection errors and morphological deformations. 4. Implement novel protocol for automated sampling and counting of cells in 3D LSM volumes. This workflow can analyze the cells density of one brain region in less than 1 min and is highly replicable to cortical and subcortical gray matter regions and structures throughout the brain. We report deformation-corrected neuron (NeuN) counts and neuronal density in 13 representative regions in 5 C57B6/6J and 2 BXD strains. The data represent the variability among cases for the same brain region and across regions within case. Our data are consistent with previous studies. We demonstrate the application of our workflow to a mouse model of aging. This workflow improves the accuracy of neuron counting and the assessment of neuronal density on a region-by-region basis, with broad applications in how genetics, environment, and development across the lifespan impact brain structure.

The mammalian olfactory systems can be divided into several subsystems based on the anatomical location of their neuroreceptor cells and the family of receptors they express. The more in depth studied systems are the main olfactory system and the vomeronasal system, whose first integrative enters are the main and the accessory olfactory bulb, respectively. In addition, there is a range of olfactory subsystems which converge to the transition zone located between the main olfactory bulb and the accessory olfactory bulb., which has been termed as olfactory limbus (OL) and includes specialized glomeruli which receive uncanonical sensory afferences and interact with the MOB and AOB. Beyond the laboratory rodents, there is a lack of information regarding the olfactory subsystems of carnivores. We have focused on the specific study of the olfactory limbus of the fox, performing serial histological sections, general and specific histological stainings, including both double and simple immunohistochemical and lectin-histochemical labeling techniques. As a result, we have been able to determine that the OL of the fox shows an uncommon development with a high degree of development and complexity. This makes this species a novel mammalian model that could provide a wider understanding of non-canonical pathways involved in the processing of chemosensory cues.

Animal experiments reveal distinct GABAergic cell clusters within the dopaminergic midbrain regions in the rostromedial tegmental nucleus (RMTg) and retrorubral fields (RRF) that have yet to be clearly defined in humans. These neurons send prominent inhibitory projections to dopaminergic neurons in the substantia nigra and ventral tegmental area that impact motor, reward and threat processing. We have identified GABAergic RMTg and RRF cell clusters in 6{micro}m formalin-fixed paraffin-embedded transverse human midbrain sections from ten control cases obtained from the Sydney Brain Bank using immunohistochemistry for GABA and tyrosine hydroxylase. We determined the location and cell size of RMTg and RRF GABAergic neurons, further mapping these cell cluster in transverse 50{micro}m thick cresyl violet stained serial midbrain sections (every 750{micro}m) from previously published controls (Halliday et al. 1990a). GABAergic neurons were cytoarchitecturally distinct, with the largest GABAergic neurons in the RRF, followed by RMTg neurons which were larger than GABAergic neurons in the well-defined interpedunclular nucleus (Kruskal-Wallis test, p<0.0001). RMTg and RRF GABAergic neurons first appear in caudal transverse midbrain sections approximately 38mm above the obex. RMTg moves rostrally and medially from underneath the decussation of the superior cerebellar peduncle to just lateral to the interpeduncular nucleus. The RRF cluster also moves rostrally and medially to the parabrachial pigmented nucleus (PBP) just under the red nucleus. The GABAergic neurons in RMTg and RRF/PBP that modulate dopamine neuronal excitability have distinct morphologies in humans. Identifying these inhibitory neurons is key to evaluating their role in neurodegenerative diseases.