Developmental Neurobiology

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.

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.

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.

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.

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.

Vestibular neurons are core elements of the pathways involved in vestibulo-motor functions, such as vestibulo-spinal and vestibulo-ocular reflexes. To meet behavioral needs, electrophysiological neuronal properties are adequately adapted to the sensory-motor computation sustaining these distinct vestibular reflexes. During frog metamorphosis, there is a complete reorganization of the posturo-locomotor system while the oculomotor system remains minimally changed, probably associated to so far unknown changes in vestibular neuronal properties. We used this unique model to investigate the central developmental mechanisms underlying such a reconfiguration of vestibular-associated behaviors. Central vestibular neurons exhibit two types of electrophysiological phenotypes: tonic neurons with a continuous discharge and phasic neurons with a transitory discharge mainly due to the activation of Kv1.1 channel. Electrophysiological recordings and Kv1.1 immunolabeling of vestibulospinal (VS) and vestibulo-ocular (VO) neurons at both larval and juvenile stages revealed that the majority of VS neurons exhibited a tonic discharge in larvae but a phasic discharge in juvenile, while VO neurons remained mainly tonic throughout development. Changes in phasic and tonic neurons proportions in VS population are partly explained by neurogenesis. But we provide evidences that an electrophysiological phenotype switch is a concomitant developmental mechanism participating in the maturation of these central vestibular neurons. All together our results showed that the maturation process in central vestibular neuronal groups is highly related to the metamorphosis-induced remodeling of vestibulo-motor functions they are involved in, with the ultimate purpose of ensuring an adequate adaptation of neuronal elements properties to the developmental changes of behavioral constrains.

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) and gland cells from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica planula larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the early gastrula presumptive endoderm generate a substantial pool of nematoblasts, a few of which migrate and differentiate in the planula ectoderm. Some neurogenic and neuronal markers, however, showed a markedly distinct expression profile, developing within a basal layer of the aboral/lateral ectoderm during gastrulation. Embryo bisection and lineage tracing experiments confirmed that sensory neurons and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic neurons derive from i-cells. Knockdown and inhibitor treatments revealed steps in neuron and nematocyte development regulated by Wnt-{beta}-catenin. We conclude that two distinct neurogenesis pathways operate during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. Summary statementDuring embryogenesis in the hydrozoan Clytia neural cell types derive both from Piwi/Nanos expressing "i-cells" and from ectodermal delamination during gastrulation.

Hippocampal area CA2 has recently emerged as a critical region for social recognition memory. Furthermore, this understudied region has been implicated in psychiatric diseases and neurodegenerative diseases. There has been accumulating evidence indicating that the pyramidal neurons (PNs) in area CA2 exhibit functional specializations that correlate with somatic position in stratum pyramidale (sp). In this study, we investigated the morphological differences in dendritic architecture of CA2 PNs with a focus on the radial gradient, i.e., along the deep-superficial axis of the sp. We conducted a comprehensive morphological analysis including Sholl intersection profiles, branching order distributions, root angle distributions, and dendritic cable lengths. We found that CA2 PNs have fewer oblique dendrites and a larger number of tuft-like dendrites as compared to CA1 PNs. Furthermore, within the CA2 population, we found that many of the dendritic structural features gradually changed along the radial axis from deep to superficial somatic location, indicating a continuum of dendritic morphology rather than two sharply defined subtypes of pyramidal neurons. This morphological characterization may serve as a starting point to better understand the corresponding functional organization of CA2. The gradual difference between deeper and superficial CA2 PNs suggests a continuum of their computational capabilities beyond two binary functional classes. In briefUsing several methods, we examine the dendritic morphology of over 130 CA2 and CA1 pyramidal neurons and find that many properties such as the cable length and terminal numbers of the dendritic arbors vary as a with the location of the soma in the pyramidal layer. HighlightsO_LIWe use scholl analysis, graph theory and machine learning techniques to quantify the different dendritic morphologies of CA2 pyramidal neurons. C_LIO_LIMany properties of CA2 pyramidal neuron apical dendrites vary as a function of somatic location in the pyramidal layer. C_LIO_LIMore superficial CA2 pyramidal neurons have longer oblique apical dendrites, and shorter tuft dendrites. C_LI

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.

Octopamine is involved in a variety of different physiological and behavioral mecha-nisms in Drosophila melanogaster. Throughout the life cycle of the fruit fly, from the larva to the adult, octopaminergic neurons in both the central and the peripheral nerv-ous system target a multitude of neurons and even non-neuronal tissues, making it challenging to analyze individual mechanisms of octopamine function. One approach to deconstructing this complex system is to examine the postsynaptic components of signal transmission. In Drosophila, octopamine interacts with six distinct G-protein-coupled receptors. For some of these receptors, expression maps and functional im-plications have been described. In contrast, other receptors have been neglected, partly due to the lack of suitable genetic tools. Here, for the first time, we compiled a complete set of mutant lines of all known octopamine receptors, all generated using the same genetic tool, the recently established Trojan Exon system. It integrates the Gal4/UAS binary expression strategy while simultaneously impairing receptor func-tion. This enabled us to generate a comprehensive anatomical map of receptor ex-pression in the larva and, at the same time, analyze the function of individual octopa-mine receptors during larval development, chemosensory perception and locomotion. All octopamine receptors (Oamb, Oct2R, Oct{beta}1R, Oct{beta}2R, Oct{beta}3R, and Oct-TyrR) showed extensive signal in the central nervous system. The same was found for the peripheral nervous system, with the exception of Oct{beta}2R, which showed pronounced expression in the somatic muscles. We also observed a previously undescribed role of Oct{beta}1R, Oct{beta}3R, and Oct-TyrR in larval hatching and in the survival of larvae and pupae. Molecular evaluation of the Trojan Exon octopamine lines supports our analy-sis. In addition, we combined the experimental results with gene expression data from the different development stages of Drosophila melanogaster and from different tis-sues and cell populations throughout the body. Overall, we compiled, analyzed and validated a complete set of octopamine lines which, together with gene expression analysis, provides a basis for further functional studies on the larval octopaminergic system.

The functional organization of the teleost telencephalic pallium remains poorly understood, particularly regarding the presence of modality-specific sensory domains and their topographic arrangement. Here, we used in vivo wide-field voltage-sensitive dye imaging to map sensory-evoked neural activity across the dorsal surface of the telencephalic pallium of adult goldfish. Somatosensory, auditory, gustatory, and visual stimulation revealed distinct, modality-specific domains primarily located within the dorsomedial (Dm) and dorsolateral (Dl) pallium. Within Dm, somatosensory and auditory stimuli activated partially overlapping territories in the caudal subregion (Dm4), exhibiting clear somatotopic and tonotopic organization along the mediolateral axis. Gustatory stimulation selectively engaged Dm3, where different tastants activated spatially distinct but partially overlapping domains. A more rostral subregion (Dm2) responded only to high-intensity somatosensory stimulation, suggesting involvement in processing negatively valenced inputs. Visual stimulation activated a circumscribed area within the dorsolateral pallium (Dld2),that closely matched cytoarchitectural boundaries. Pharmacological blockade of ionotropic glutamate receptors markedly reduced sensory-evoked responses, indicating that these maps depend on glutamatergic synaptic transmission. Together, these findings show that the goldfish pallium contains distinct, spatially organized sensory representations and a refined internal functional architecture. This organization suggests that pallial topographic sensory maps may not be exclusive to mammals and birds. Based on these results, we propose that dorsomedial and dorsolateral pallial regions may be functionally comparable to components of the mammalian mesocortical network, more than to the pallial amygdala or the neocortex. This framework provides a new perspective on pallial organization in teleosts and contributes to understanding the evolutionary origins of the vertebrate pallium. HIGHLIGHTSO_LIVoltage-sensitive dye imaging was used to map sensory responses in the goldfish pallium. C_LIO_LIDistinct sensory areas for somatosensory, auditory, gustatory, and visual modalities were identified. C_LIO_LISome sensory regions in Dm show topographically organized maps. C_LIO_LIFunctional segregation suggests a complex, non-diffuse pallial organization. C_LIO_LIFindings support a novel hypothesis linking Dm and Dld to mammalian mesocortical regions. C_LI

BackgroundChoroid plexus (ChP) produces cerebrospinal fluid (CSF), and regulates brain development and adult subventricular zone (SVZ) neurogenesis, but its role in hippocampal subgranular zone (SGZ) neurogenesis in adulthood and early postnatal stages is not well understood. Current tools to directly manipulate neonatal ChP/CSF volume are very limited, representing an urgent need in the field. MethodsWe first discovered the specific "leaky" expression of DTR gene in the ChP of adult ROSA26-iDTR mice which can be used to specifically ablate ChP in adult brain that generated robust and long-lasting ablation of ChP and reduction of CSF volume. In this study, we the effectiveness of ROSA26-iDTR allele in ablating neonatal ChP. We also developed a novel AAV2/5-CMV-DTR vector with validated ChP tropism in both neonatal and adult mice, which induces substantial CSF loss in both neonates and adult mice. With both the ROSA26-iDTR genetic and AAV2/5-DTR viral-mediated ChP ablation in young adults and at defined postnatal ages, we quantified ventricular CSF volume by MRI and characterized postnatal neurogenesis. Doublecortin-positive (DCX+) neuroblasts, Ki67+ proliferating cells, and TUNEL+ apoptotic cells were quantified in SVZ and SGZ using confocal microscopy and machine learning-assisted cell counting. ResultsWe show that ROSA26-iDTR-mediated ChP ablation is inefficient before postnatal day 10, suggesting that this line may be of limited utility for CSF reduction in the early neonatal period before P10. P3-5 Dtx treatment of a previously used dosage of 20ng/g dosage did not lead to a reduction in CSF volume. Higher dosage of 40ng/gX3 Dtx dosage at p3-5 generated only moderate partial reduction of CSF in third ventricle and total CSF volume, with indication of toxicity associated with high Dtx dosage in general. In contrast, p10-12 injection of 20ng/gX3 Dtx led to robust CSF reduction. To target early neonatal days, AAV2/5 CMV-DTR virus shows high tropism for ChP epithelial cells and leads to near-complete ablation of CSF in neonatal brains. ChP/CSF loss in neonates or young adult mice leads to a substantial reduction of DCX+ cells at the SVZ but a moderate but significant reduction of SGZ DCX+ neuroblasts, without changes in Ki67+ or TUNEL+ cells. ConclusionsThis study reports a novel role of the ChP/CSF in maintaining the neuroblast pool in the neurogenic niches in both early postnatal and adult stages. Moreover, we expand the available tools to target the ChP and CSF production in the neonate, with potential uses in treating conditions such as neonatal hydrocephalus.

Neural circuit assembly relies on different neuronal subtypes coming together to form a functional circuit. The question of how the appropriate number of each subtype is integrated into an emerging circuit remains relatively unknown. To answer this question, we used the mouse retina to uncover the molecular mechanisms responsible for neuron subtype integration in a developing circuit. In the mammalian retina, bipolar neurons are a class of interneurons that relay visual information from photoreceptors to ganglion cells. Extensive studies have shown there are 15 distinct bipolar subtypes: 6 types of OFF cone bipolars, 8 types of ON cone bipolars, and 1 type of rod bipolar. During retinal development, bipolar neurons are born in excess and through programmed cell death, a precise number of each subtype remains to give rise to the retinal circuit. Although this process has been well-described, little is known about the key molecules responsible for bipolar subtype integration in the developing retina. Our work uncovered a new role for the autism-associated risk gene, Protocadherin 9 (Pcdh9) in bipolar subtype integration. Deletion of Pcdh9 using a floxed allele leads to loss of OFF and ON cone bipolars; however, disruption in the extracellular binding of Pcdh9 leads to selective loss of ON cone bipolars but not rod bipolars. Moreover, we found this later function of Pcdh9 is mediated by homophilic interactions between ON cone bipolars and their known synaptic partners. Taken together, our work revealed a new role for Pcdh9 in bipolar subtype integration during retinal development. SUMMARY STATEMENTNeural circuits are comprised of multiple neuronal subtypes where a specific number need to come together to give rise to a functional circuit. Although this is a critical process during neurodevelopment, little is known about the molecular mechanisms that determines the precise number of each subtype during circuit development. In the present study, we identified the autism risk gene, Protocadherin 9 as a critical molecule in subtype integration of bipolar neurons within the developing mouse retina. Using newly generated mouse lines, we found distinct requirements of Pcdh9 to promote survival in different bipolar subtypes during retinal circuit assembly. The significance of this work is that it shed lights into how different neuronal subtypes are integrated in nascent neural circuits.

The mesencephalic trigeminal nucleus (MTN) contains the proprioceptive sensory neurons that innervate mechanoreceptors in the jaw closing muscles. In the chick embryo, MTN neurons are the first neurons generated in the mesencephalon. They arise bilaterally adjacent to the roof plate and then extend their axons ventrally before projecting caudally towards the rhombencephalon. MTN axons remain in a mid - dorsoventral position and pioneer the lateral longitudinal fasciculus. Notably, MTN axons never cross the roof plate, raising the question of which mechanisms underlie this restriction. Here, we investigated the effects of tissue transplants on the guidance of MTN axons. We found that both the diencephalon and the notochord exert repulsive effects on MTN axons, which could partially explain their early trajectory. We have also analysed the potential roles of the guidance cues BMP2/4, GDF7, SLIT and NETRIN in MTN axon navigation, both in vivo and in vitro. We found no evidence for a role of BMP2/4 or GDF7 in directing MTN axons. However, SLIT-ROBO signaling was found to play a significant role. SLIT proteins are repulsive guidance cues expressed by roof and floor plate. Loss or reduced expression of ROBO2 led to aberrant axon meandering within the dorsal midbrain. Most axons eventually reoriented posteriorly, and only a small fraction crossed the roof plate. Unexpectedly, in the absence of ROBO2, MTN somata migrated into the roof plate, resulting in the loss of a defined roof plate region. Taken together, these results suggest that SLIT2-ROBO2 signaling not only prevents MTN axons from crossing the roof plate but also maintains MTN cell bodies adjacent to the roof plate. With regards to MTN neuron guidance, we conclude that additional roof plate - derived factors are likely to co-operate with SLIT proteins to prevent crossing of the roof plate. Another possibility could be that SLIT might signal through additional receptors.

A delicate balance between the quiescent and proliferative states of neural stem cells (NSCs) is important for neurogenesis and homeostasis. Histone deacetylase 4 (HDAC4) variants are associated with neurodevelopmental disorders, however, its role in early brain development remains elusive. In this study, we demonstrate that Drosophila HDAC4 plays a crucial role in neural stem cells (NSCs) reactivation and brain development. Depletion of HDAC4 results in notable defects in NSC reactivation, while its overexpression leads to premature reactivation. HDAC4 is SUMOylated at Lys902, which enhances its protein stability by preventing HDAC4 from undergoing ubiquitin-proteasome-mediated degradation. Moreover, phosphorylation of HDAC4 by salt-inducible kinase 3 (SIK3), an AMPK-related kinase, allows cytoplasmic localization of HDAC4 and enhances the association between HDAC4 and Warts, a core kinase of the Hippo pathway. This HDAC4-Wts association inhibits Warts activity, and in turn, the inactivation of the Hippo pathway, triggering NSC reactivation. Finally, genetic epistasis experiments support the SIK3-HDAC4-Warts axis during NSC reactivation. In conclusion, our findings identify HDAC4 as a molecular switch that integrates SUMOylation, ubiquitination, and the Hippo pathway to govern NSC reactivation.

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

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.

Apuschkin et al. (2024) proposed a GPCR-based transcriptomic atlas for midbrain dopamine (DA) neuron subpopulations, including candidates such as Nmur1, Cckar, and Ffar4. To guide genetic targeting, these markers must reflect functional expression in adult DA neurons. Using in situ hybridization, Cre-dependent reporter lines, and both intracranial and systemic viral approaches, we find no evidence of adult Nmur1-mediated recombination in DA neurons, while Cckar-driven recombination is consistent with developmental expression only. Notably, Ffar4 expression overlaps extensively with Ntsr1 midbrain populations, indicating that it does not define a distinct DA neuron class. Furthermore, analysis of independent spatial transcriptomic datasets together with our MERFISH data shows that many proposed GPCR markers are not detectably expressed in adult DA neurons. These findings demonstrate that transcriptomic enrichment does not always yield reliable adult markers and highlight the need for functional validation prior to use in circuit targeting.

The axon initial segment (AIS), situated within the first 20-60 {micro}m of the axon, is essential for action potential generation and maintenance of axonal identity. Its structure relies on the beta ({beta})-IV-spectrin/AnkyrinG (AnkG) scaffold arranged periodically underneath the plasma membrane, harbouring diverse membrane proteins. Although a [~]190-nm cytoskeletal periodic organization is well established, the precise stoichiometry and spatial arrangement of AIS proteins within the [~]190-nm spatial period remain rudimentary, mostly for lack of sufficient spatial resolution and labelling efficiency. Here, using expansion microscopy and cryo-electron tomography, which overcome these technical limitations, we present data on the organization of the AnkG-associated complex within the [~]190-nm spatial period. We demonstrate that exactly two AnkG molecules with their C-termini separated by [~]80 nm are situated within each period. By contrast, the AnkG-associated cell-adhesion protein neurofascin-186 appears in clusters of varying sizes that are consistent with the periodic organisation of AnkG pairs, yet suggest a more complex molecular arrangement between the two molecules. Altogether, our novel approach provides new insights into AIS molecular organisation and protein stoichiometry.

To avoid image blurring, the vestibulo-ocular (VOR) and the optokinetic (OKR) reflexes stabilize gaze. In all vertebrates, the VOR is mediated via direct projections from the vestibular nuclei to the motor nuclei that control the extraocular muscles. Lampreys show three vestibular nuclei that are well characterized in terms of their projections and sensory inputs, but much less is known about their inputs from other brain regions and the connectivity between them. Using tracer injections and electrophysiological recordings, we show that the lamprey vestibular nuclei are largely interconnected, while their inputs from other brain regions are scarce. The main rostral areas projecting to the vestibular nuclei are the pretectum and the ventral tier of the thalamus, which send ipsilateral inputs to the three vestibular nuclei.