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

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.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

Recent studies have shown that symbiotic bacteria can have drastic effects on host neurobiology, but few simple, accessible models currently exist in which to study these interactions. Hawaiian bobtail squid (Euprymna scolopes) participate in a binary symbiosis with the bacterium Vibrio fischeri, a population of which resides in a specialized hindgut-derived organ called the light organ. Upon colonization by V. fischeri, the light organ undergoes transcriptional changes that suggest neurons are impacted by the initiation of symbiosis, but the nascent light organs innervation has remained uncharacterized. Here, we show that the light organ-associated nervous system (LONS) in hatchling E. scolopes is a remarkably complex segment of the peripheral nervous system. The LONS is largely plexiform and originates from two primary nerves connected by a local commissure. The abundance of synapsin-like immunoreactivity (-lir) indicates that the lobe plexus is highly interconnected. We also highlight a small number of serotonin-lir neurites that innervate the anterior appendages whose developmental fate may be directly affected by symbiont-driven light organ morphogenesis. Finally, we present evidence that a limited but diverse population of neurons reside within the light organ and are often located near internal symbiont-interacting structures. This description of the E. scolopes LONS serves to provide a foundation from which to investigate how beneficial bacterial symbionts affect host peripheral neurobiology in a tractable model system.

The SCN1A gene encodes NaV1.1, a voltage-gated sodium channel protein that is necessary for neuronal excitability and whose loss-of-function mutations cause Dravet syndrome, a treatment-resistant childhood onset epilepsy. Gene replacement strategies for this syndrome are challenged by the large size of SCN1A and difficulty achieving stable cellular expression. Lentiviral vectors (LVVs) offer sufficient packaging capacity and genomic integration for defective SCN1A gene replacement. Here, we evaluated LVV-mediated delivery of different engineered SCN1A transgene sequences in human cells. LVV-transduced cells expressed full-length NaV1.1 protein that trafficked to the membrane and produced functional sodium currents. However, SCN1A transgene expression declined over time despite stable vector copy number, indicating post-integration regulatory limitations. Expression efficiency varied by SCN1A transgene sequence, with a codon-optimized variant showing higher expression despite lower LVV copy number. Treatment with sodium butyrate, a histone deacetylase inhibitor, significantly enhanced SCN1A transgene expression and partially rescued expression decay in a sequence-dependent manner. Incorporation of a ubiquitous chromatin opening element (UCOE) upstream of the promoter to maintain expression resulted in a trend of increased expression and increased responsiveness to butyrate. These findings demonstrate that sequence-specific and epigenetic factors may influence expression of large transgenes following lentiviral delivery, highlighting key challenges and design considerations for therapeutic SCN1A transgene expression.

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

Voltage-gated sodium channels (VGSCs) are conventionally described as heterotrimers composed of one alpha and two beta subunits. However, the patterns of co-expression of alpha- and beta-subunits in neurons remain unclear. In the present study, we report that alpha- (Nav1.1, Nav1.2, and Nav1.6) and beta- (beta-1 and beta-2) subunits are densely expressed in axon initial segments (AISs) of neurons in the neocortex, hippocampus and cerebellum at postnatal days 14-15 (P14-15) and 8-9 weeks (8-9W). These distributions are largely unique and partially overlapping among brain regions. Notably, in the neocortex and hippocampus, AISs of presumptive parvalbumin-positive inhibitory neurons are positive for Nav1.1 and beta-1, whereas those of excitatory ones are positive for Nav1.2 and beta-2. Similarly, AISs of cerebellar basket cells, which are inhibitory neurons, are positive for Nav1.1 and beta-1, whereas those of granule cells, which are excitatory neurons, are positive for Nav1.2 and beta-2. Nav1.6 is expressed in many of these neurons. Some subunits exhibited distinct distribution patterns at the two postnatal stages analyzed, possibly because of their developmental changes of subcellular localizations. Taken together, these results indicate that combinations of VGSC subunits are largely unique among different neuronal subpopulations. These findings provide a useful reference for understanding the distribution and interactions of VGSC subunits in the brain.

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

In adult superficial dorsal horn, 90% of Reelin (Reln+) and 70% of Disabled-1 (Dab1+) neurons co-express the transcription factor LIM-homeobox 1-beta (Lmx1b+) and therefore are glutamatergic neurons. Here we asked if embryonic Reln+Lmx1b+ and Dab1+Lmx1b+ dorsal horn neurons are derived from Lmx1b-expressing early-born dI5 or late-born dILB dorsal neurons. On Embryonic day (E)11.5, Reln+ and Dab1+ neurons appear to be part of the migration of early-born dI5 Lmx1b-expressing neurons. Between E12.5-E15.5, the lateral Reln+Lmx1b+ and Dab1+Lmx1b+ neurons migrate circumferentially along the rim of what will become the superficial dorsal horn, whereas medial Reln+Lmx1b+ and Dab1+Lmx1b+ neurons move into the dorsal midline and then migrate into lamina V. The small, late-born dILB Reln+Lmx1b+ and Dab1-Lmx1b+ neurons fill the superficial dorsal horn. In Reln mutants, large Dab1+Lmx1b+ neurons were mispositioned in lamina I and at the border between the superficial and deep dorsal horn. To confirm the identity of the circumferential and midline Reln+Lmx1b+ and Dab1+Lmx1b+ neurons, we asked if they expressed the transcription factor Zfhx3, a marker of dI5 projection neurons. We detected examples of Reln+Lmx1b+Zfhx3+ and Dab1+Lmx1b+Zfhx3+ projection neurons that migrated along the outer rim of the superficial dorsal horn and others that migrated from the midline into lamina V. Taken together, our study demonstrates that the larger Reln+Lmx1b+Zfhx3+ and Dab+Lmx1b+Zfhx3+ neurons represent two subsets of dI5 projections neurons, whereas smaller Reln+Lmx1b+ and Dab1+Lmx1b+ neurons concentrated in lamina II are likely dILB interneurons.

Serotonin is one of the main neuromodulators in the brain, involved in regulating mood, complex behaviors and sensory input. Serotonin reaches primary somatosensory cortex (S1) via axons of neurons located in the dorsal raphe nucleus (DRN). DRN neurons can be modulated, amongst others, by reward, sensory stimulation, or movement but the activity pattern of serotonergic neurons targeting S1 is not known. Therefore, it is unclear under which circumstances serotonin is released in S1. Here, we expressed GCaMP8 in serotonergic neurons of the DRN to analyze the activity of their axons in S1 using two-photon Ca2+-imaging. Cluster analysis of axonal activities suggests that one to four functional groups of serotonergic axon segments project to a 0.3 mm2 horizontal plane of S1. We show that activity in serotonergic axons is strongly driven by reward and weakly by sensory stimulation of the whiskers. Movement, however, is preceded by a modulation, up and down, of the serotonergic signal seconds before the running onset. In summary, rewards and sensory stimulation lead to activity in serotonergic axons which is likely to adjust signal processing in S1 upon these events. The serotonergic signal changes seconds before movement onset probably preparing the neural network in S1 for the state change that accompanies running.

The cerebellar nuclei form the main output structures of the cerebellum and are composed of a deeply conserved set of cell types. Two excitatory cell classes, Class-A and -B, are present in each cerebellar nucleus and mediate all excitatory output of the cerebellum. To provide genetic access to these cell types, here we identified Acan as a marker gene for Class-B cells and generated a knock-in Acan-P2A-Cre mouse line. We demonstrate that this Acan-Cre line selectively labels Class-B neurons in the cerebellar nuclei and validate its use in viral projection tracing. This new mouse line provides a valuable genetic tool to study cerebellar nuclei organization and function.

Among mammals, spiny mice (Acomys spp.) exhibit the unique capacity to regenerate parts of their nervous system. Studying this phenomenon has the potential to reveal new targets that can slow or halt human neurodegenerative disorders. Unfortunately, research tools (e.g., transgenic lines, gene delivery vehicles) are lacking compared to those available for other rodent models. Here, we tested systemic adeno-associated viral vectors (AAVs) in Acomys dimidiatus and identified three promising candidates: X1.1, CAP-Mac, and MaCPNS1. Characterizing their tropism following intravenous delivery, we found that in the brain, MaCPNS1 and X1.1 primarily transduced astrocytes. In the peripheral nervous system, MaCPNS1 efficiently transduced dorsal root ganglia, axon bundles of the ear pinnae, and enteric neurons throughout the gastrointestinal tract. As a proof-of-concept, we used MaCPNS1 to chemogenetically modulate the activity of enteric neurons, successfully decreasing gastric motility in vivo and increasing colonic motility ex vivo. We expect these findings to enable functional studies of the uniquely regenerative nervous system of Acomys, which may in turn help advance neuroregenerative therapeutics for humans. Summary StatementIdentification of an AAV tool to efficiently deliver transgenes to the central and peripheral nervous systems of spiny mice enables functional studies of the nervous system in a mammalian model of regeneration.

1.Functional neuronal circuits in the vertebrate retina emerge through coordinated developmental events, yet the timeline by which bipolar cells acquire visual feature selectivity remains unclear. A major barrier is the limited genetic access to bipolar subtypes during early postnatal stages. Recent comprehensive transcriptomic study points to Igfn1 as a molecular marker for type-7 bipolar cells (BC 7), a subtype that exhibits direction-selective glutamate releases in adult. Here, we generated an Igfn1iCre knock-in mouse line and characterized Igfn1-positive cell morphology from postnatal day 4(P4) to adult using Cre-dependent tdTomato reporter mice. We found Igfn1-positive cells in the inner retina by P12-P15, predominantly labelling bipolar cells and some amacrine populations. At P15, about 71% of labelled bipolar cells stratified their axons in the S4 sublamina of the inner plexiform layer, consistent with BC 7 morphology. In adult retina, the widespread Igfn1-labelling appears slightly dominated in amacrine cells. To validate these observations, we analysed Igfn1 expression in the Mouse Retina Cell Atlas and confirmed strong Igfn1 enrichment in BC 7 and expression in additional retinal cell types, mirroring experimental results. Overall, these results reveal Igfn1iCre as a potential developmental tool for BC 7 access in the retina.

The retinal topography of mammals reflects significant influences of the visual environment. In diurnal species, local specializations, such as the visual streak (VS) for panoramic vision and the area centralis or fovea for binocular vision, play a key role in optimizing visual perception and species viability. While the location of these sites is typically considered the retinal center, the definition of a "central retina" is less clear in nocturnal species. In mice, the most frequently used model in ophthalmologic research, the location of a central retina is hardly discernible in retinal images, neither in retinal structure (OCT sections) nor in vascular organization (SLO and angiography). In this study, we compare the murine retina with that of a diurnal rodent, the Mongolian gerbil (MG). We found that the S-opsin transitional zone (OTZ), a region characterized by the change from S-to M-opsin dominance along the dorsoventral opsin gradient in mice, has a similar relative position in the retina to the VS in the Mongolian gerbil, suggesting an evolutionary positional homology between these regions. Further, since the S-opsin-dominant region is optimized for visualizing the sky and the M-opsin-dominant region for visualizing the ground, the OTZ in between -much like the VS- naturally points toward the horizon. We therefore propose considering the OTZ as the position of a "central retinal area" in mice. Determining the anatomical-physiological center is particularly important to obtain meaningful relative measures such as averages across different retinal areas, as the common referencing to the optic nerve head (ONH) in mice does not take into account retinal organization and the eccentric position of the functional center.

Human brain tissue preserved in biorepositories is foundational for the structural, cellular, and biomolecular research necessary for a mechanistic understanding of neurological diseases. Realizing the research potential of these valuable resources requires well-characterized research-relevant tissue that can be efficiently identified by investigators and incorporated into the conceptual and computational frameworks of interdisciplinary research. Several large-scale efforts to improve research reliability and reproducibility have sought to characterize and annotate the processes by which these samples are collected, yet limited progress has been made on standardizing spatial information for these samples. Biorepositories systematically collect brain tissue according to a brain sampling protocol (BSP) that differs between institutions, yet explicit spatial information regarding the samples may not be documented in standard operating procedures (SOPs). The amount of anatomical location details available to investigators are inconsistent across biorepositories and typically lack sufficient anatomical precision to ensure correspondence with samples from other biorepositories or research relevant brain regions specified by neuroimaging, functional, or disease-susceptibility criteria. Here, we introduce a pipeline for developing a Spatial Atlas for Mapping Protocol Locations of Ex vivo Samples (SAMPLES), which uses a neuroimaging framework to create a 3D representation of a BSP through a metrically precise digital instantiation of the procedures for brain extraction, segmentation, slicing, and sampling on a modern digital brain template. SAMPLES incorporates modern neuroinformatics conventions to create explicit 3D labels of BSP-defined samples that can be interactively visualized with freely available neuroimaging software. We illustrate the pipeline by developing an atlas for the protocol from the University of Washington BioRepository and Integrated Neuropathology laboratory (UW BRaIN SAMPLES). By providing an explicit, computable reference, SAMPLES atlases can support the efficient identification, referencing, and utilization of postmortem samples for interdisciplinary research. These capabilities enable biorepository workflows, data harmonization across biorepositories, and integration with antemortem neuroimaging.

The design of the classical fluorescence microscope has undergone few changes since the 1970s-1980s, when Ploemopak modules with filter cubes became widespread. Most of these changes have been in the replacement of mercury and xenon lamps with LED illuminators in the 2010s. However, this does not mean that this stable design cannot be improved upon. New method: The implementation of a vibrating optical fiber, positioned using a micromanipulator and connected to any suitable type of laser, enables a full spectrum of fluorescence research. This work presents an advanced version of the Ellis concept, in which light is delivered directly onto the sample, rather than into the filter cube (technical novelty).To confirm the functionality of the microscope, vibrational slices of mouse brain stained with three fluorescent markers (B3-PPC, DiI and DiD) covering most of the visible spectrum were examined. The fiber-optic illumination system eliminates the need for bulky and obsolete high-voltage plasma arc lamp units without compromising image quality (confirmed by the USAF 1951 test and SDNR assessment on fluorescent beads). Furthermore, the optical fiber mounted on manipulators is convenient and easy to integrate, for example, into stereomicroscopes for scanning large brain tissue samples.

Dopamine signaling through dopamine 1 receptors (D1R) and dopamine 2 receptors (D2R) regulates hippocampal synaptic plasticity underlying learning and memory, yet their subcellular localization within the hippocampus is unknown. Here we performed electron microscopic immunocytochemistry to elucidate the distribution of D1R and D2R in subregions of the mouse hippocampus. In CA1 and CA3 stratum radiatum (SR), D1R- and D2R-immunoreactivity was found primarily on pyramidal cell dendritic spines and unmyelinated axons, and to a lesser extent in axon terminals and glia. In both regions, D1R-labeled terminals formed predominantly asymmetric (excitatory-type) synapses on dendritic spines, whereas D2R-labeled terminals formed mainly symmetric (inhibitory-type) synapses on pyramidal cell dendritic shafts. In the dentate gyrus (DG) hilus, D1R-labeling was almost exclusively found in unmyelinated axons and glia. D2R immunoreactivity in the hilus similarly was present in unmyelinated axons and glia but was also detected in dendritic spines originating from mossy cells and in terminals forming symmetric synapses. These findings indicate that dopamine receptors are positioned to influence excitatory and inhibitory signaling in the murine hippocampus. As D1R and D2R exert opposing effects on neuronal signaling, their localization on pyramidal neuron compartments provides a structural substrate for bidirectional modulation of synaptic plasticity and pyramidal cell activity. In addition, the presence of D2Rs on inhibitory terminals contacting pyramidal neurons and hilar interneurons suggests a role in regulating inhibitory circuitry within the hippocampus.

The basal ganglia (BG) form anatomically and functionally segregated yet integrative parallel circuits, but the molecular mechanisms specifying them remain unclear. We immunohistochemically mapped the expression of three {delta}2-protocadherin ({delta}2-PCDH) cell adhesion molecules--PCDH10, PCDH17, and PCDH19--in the BG of macaques. Within the striatum, each PCDH exhibited regional gradients of expression along the rostro-caudal and ventromedial-dorsolateral axes. The three PCDHs showed complementary distributions that continuously delineated molecular boundaries corresponding to functional subdivisions in a graded fashion. Such complementary distributions were also observed in the BG output nuclei. Given that neurons expressing the same {delta}2-PCDH in distinct BG structures preferentially connect with each other, the three {delta}2-PCDH expression patterns could define functional territories within parallel BG circuits. Together, the complementary expression of PCDH10, PCDH17, and PCDH19 broadly align with the distinct BG circuits, respectively, suggesting molecular codes underlying the segregated yet integrative parallel organization of the primate BG.