Journal of Comparative Neurology

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.

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

Visual acuity spans more than a 100-fold range in mammals and yet the neural correlates of this perceptual gradient has not been fully evaluated. Furthermore, even though the known derived features of the human brain include specific changes to the cerebral cortex and the visual system in particular, no evolution of developing life histories approach has been quantitatively applied to metrics of cell composition. In this study, we present stereological estimates of neuron and glia density in V1 and granular layer 4 in a comparative sample of primates. We then integrate these data with the literature to construct a larger comparative dataset to test for phylogenetic relationships in mammalian visual system organization. Our examination revealed a primary relationship between acuity and neuron number along with secondary relationships among cell types tied to metabolic maintenance and support across the lifespan. Retinal metabolics along with phylogenetic position accounts for a large amount of V1 neuron density, which are further related to acuity while glia are related to longevity. These results identify a dissociation in the evolutionary developmental organization and senescence of V1 that map onto first principal parameters to explore the phylogenetic position and ecological pressures acting on the mammalian visual system. In accord with the literature, humans are revealed as outliers in glial support of neuronal metabolism across the lifespan. These findings provide evidence that mammalian visual cortex varies along at least two partially separable cellular dimensions in which visual resolution differs from lifelong maintenance. Summary and SignificanceIf the cellular organization of visual cortex is shaped by a single constraint or multiple independent pressures is debated. We used stereology to test whether V1 neurons and glia make separable contributions to visual performance across the mammal lifespan. V1 neuron density, together with brain size, predicts visual acuity. The glia-to-neuron ratio is alternatively associated with maximum lifespan after controlling for neuron density. Humans and chimpanzees have nearly identical V1 neuron densities, yet humans appear show substantially elevated glial investment across the literature. These findings suggest that mammalian visual cortex evolves under at least two partially separable pressures with neuron density and spatial resolution on one hand and glia investment to sustain function across life on the other.

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.

The peripheral auditory system of dolphins comprises specialised bony, fatty, vascular, and neural structures adapted for underwater hearing and diving physiology. These include the external ear canal, acoustic fat bodies, sinuses, and associated neurovascular networks, which together support sound conduction, protection, and possibly sensory functions. Despite advances in gross anatomical description, the detailed integration of these tissues, particularly the innervation, neurovascular organisation, and their functional implications, remains poorly understood. Previous studies have described the presence of sensory nerve formations and vascular plexuses, but their arrangement, connectivity, and relation to each other are unresolved. Here, we combine macroscopic dissection, DICE-{micro}CT, histology, and high-resolution confocal microscopy to characterise several neurovascular and sensory components of the dolphin peripheral auditory system in several delphinid species. Macroscopic dissection and DICE-{micro}CT revealed the traditional acoustic fat body distribution with detailed morphology of the posterolateral extension that is not well-known. The cranial nerve distribution, and specifically the mandibular nerve branching patterns, are described in detail. Confocal microscopy uncovered a stratified neurovascular plexus around the external ear canal with a complex sensory system comprising lamellar corpuscles, Merkel cell-neurite complexes, and intraepithelial nerve fibres. Notably, the lamellar corpuscles formed a continuous, three-dimensional neural network with frequent merging and splitting of axonal bundles, shared perineuria, and vascular integration, features not observed in previous studies. Our findings demonstrate that the dolphin external ear canal and surrounding structures form a sophisticated, multimodal somatosensory organ, integrating structural, vascular, and neural specialisations likely adapted for proprioceptive mechanosensation in the aquatic environment. This study provides insights into the integration of the various components of the peripheral hearing apparatus. Future studies integrating anatomical, electrophysiological, and biomechanical approaches are needed to fully elucidate these adaptations.

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.

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.

The evolutionary origin of nervous systems in animals remains elusive and is largely hidden from the fossil record. Ctenophores, one of the earliest-branching animals possessing neurons, are instrumental to our understanding of nervous system origin, and a few rare ctenophore fossils preserve traces of nervous tissue as carbonaceous remains. Cambrian ctenophores appear to exhibit a more diverse neuroanatomy than that of modern species, suggesting secondary loss in extant ctenophores. However, much remains unknown about the origin and ontogeny giving rise to the structural organization of modern ctenophore nervous systems. Here, by investigating the neural anatomy of the ctenophore Mnemiopsis leidyi during development, we identified a ladder-like nerve net (LNN) beneath the comb rows that converges into condensed neurites and connects to the aboral organ. Examination of carbon-rich areas of Ctenorhabdotus capulus, an extinct ctenophore from the Burgess Shale, reveals a pattern similar to that of M. leidyi, consistent with a shared neural organization. Furthermore, M. leidyi exhibits a condensed comb nerve, resembling the longitudinal nerve preserved in the Cambrian ctenophore Fasciculus vesanus and the giant axon of extant Euplokamis dunlapae. Our study reveals conserved evolutionary constraints shaping nervous system architectures linked to locomotory organs and indicates that the different modes of nervous system organization observed in Cambrian ctenophores are variably retained in modern species.

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.

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.

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.

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.

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.

Animals often aggregate information from multiple different sensory modalities to accurately assess and react to a stimulus. It is often assumed that cross-modality integration mostly occurs at high-level processing centers, such as the mammalian cortex or insect mushroom bodies. However, we hypothesized that integration could occur relatively early in the sensory pathways. The insect antennal lobe is one such location, receiving direct inputs from the antennae via the antennal nerve. These inputs are highly multimodal, including olfactory, mechanosesnory, and gustatory information, all of which are relevant to foraging honeybees (Apis mellifera). Here we assess integration by recording electrophysiological spike data within the honeybee antennal lobe while exposing the bee to various combinations of wind speed and odor concentration. This paper accompanies another publication by Joseph Reed and Mainak Patel approaching the same question from a modelling perspective, where their model corroborates our data and vice versa. Together, we show that integration occurs within this early layer of processing, while also demonstrating the complex relationship of these two closely-linked stimuli. SIGNIFICANCEAccurate perception depends on the brains ability to combine information from multiple senses, commonly thought belonging to high level of information processing. Using the European honeybee, Apis mellifera, we show strong evidence that olfactory and mechanosensory signals interact at an early stage of neural processing, within the antennal lobe, producing stimulus representations closely-linked to the animals navigation and decision-making. By identifying a tractable model for early multisensory processing, this work offers broader insight into how animals construct reliable representations of their environment.

Nociception is an essential response for organisms to avoid potential harm and promote survival. Its molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion channels exhibiting selective peripheral expression and functional coupling that underpins nociception and pain modulation in complex organisms. However, the execution of protective behaviours triggered by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their investigation in invertebrate model organisms to increase understanding of animal nociception. Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous system, whose complexity suggests it might support pain-like states that exist in vertebrates. This possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. Despite this, there is poor characterisation of cephalopod molecular nociceptors. For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent expression in sensory tissues. We investigated the functional competence of these TRPVs by heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods.

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.

Top down signaling from the cortex to the hypothalamus is critical to link cognitive and emotional processing to homeostasis and motivation. This study investigates signaling from the medial prefrontal cortex (mPFC) to the posterior hypothalamus (PH), a region that modulates endocrine and autonomic stress responses and motivated behaviors. The function and anatomy of this circuit was examined with patch clamp electrophysiology and mapping studies in male and female rats. Spontaneous firing properties of PH neurons were determined in a cell-type specific manner by combining a transgenic glutamic acid decarboxylase-Cre rat with Cre-dependent colorswitch virus to determine postsynaptic cell-type identity. Overall, PH neurons were more excitable in females compared to males and, in both sexes, data indicated tonic inhibition within the PH, with significantly greater inhibition in males. Using Channelrhodopsin-assisted circuit mapping to query the mPFC-PH circuit, we found that a majority of PH neurons received input from the mPFC and mPFC synapses targeted glutamatergic cells over GABAergic PH cells. Retrograde tracing revealed more PH-projecting neurons in females, specifically within the tenia tecta and infralimbic regions of the mPFC, with significantly more stress-activated PH-projecting cells in the female prelimbic cortex. Anterograde tracing revealed, surprisingly, no sex differences in mPFC presynaptic terminal density in the PH, despite more PH-projecting cell bodies in the female mPFC. These data help to elucidate the sexual divergence in cortical-hypothalamic signaling and how cognitive and emotional information from the prefrontal cortex may differentially regulate homeostasis and motivation between sexes. Significance StatementNeural signaling between the prefrontal cortex and the hypothalamus is important for maintaining homeostasis, particularly during contextual challenges such as stressors. Here we find multiple aspects of sex-specific organization and neurophysiology in this circuitry. Excitatory inputs from the medial prefrontal cortex target both excitatory and inhibitory neurons within the posterior hypothalamic nucleus in both sexes. However, there are sex differences in the number of stress-activated neurons in the prefrontal cortex that innervate the posterior hypothalamus, as well as differences in hypothalamic inhibitory signaling and estrous cycle-dependent effects on neuronal excitability. Altogether, these data suggest that organizational, synaptic, and hormonal factors may contribute to sex-specific behavioral and physiological integration.

Male odor induces various behavioral and physiological responses across the reproductive cycle in female mice. Although male odor preference in females is reduced during pregnancy, how it changes across later stages of the reproductive cycle, including nursing and weaning, remains unclear. Here, we found that male odor preference is lost during pregnancy and nursing. To identify the olfactory systems involved in these changes, we examined neural activity using c-Fos immunohistochemistry. Male odor exposure during nursing increased neural activity in the accessory olfactory bulb and the posteroventral medial amygdala (MeApv), a key node of the accessory olfactory system, as well as in subdivisions of the central amygdala, but not in the ventromedial hypothalamus or the bed nucleus of the stria terminalis. Finally, lesions of the MeApv prevented the loss of male preference during nursing, indicating that the MeApv is required for suppression of male preference during this stage.

Proprioception and reflexive control of muscle tone depend on the activity of muscle spindles, specialized sensory receptors embedded deep within skeletal muscle that detect changes in muscle length. Their location and complex three-dimensional architecture have historically limited morphological analysis to techniques such as silver-impregnation, muscle teasing, or serial sectioning followed by volumetric reconstruction. Here, we describe a workflow for three-dimensional, in situ visualization of muscle spindles in the rabbit tenuissimus muscle, a preparation uniquely enriched in spindles and well suited for whole-mount imaging. The protocol combines fluorescent labeling of spindle sensory and motor innervation, including intrafusal {gamma} neuromuscular junctions labeled with -bungarotoxin, with immunolabeling and solvent-based optical clearing. Optically cleared tenuissimus muscles were compatible with both whole-mount confocal and light-sheet microscopy, enabling volumetric imaging of complete spindle structures and detailed visualization of Ia annulospiral endings at the spindle equator. This approach provides access to spindle morphology and connectivity at multiple spatial scales while avoiding physical sectioning and reconstruction. By enabling reproducible three-dimensional imaging of intact muscle spindles, this workflow offers a practical platform for studying spindle structure and plasticity in health and disease.

Triatomines are the vectors of Chagas disease, one of the main endemic diseases from South to North America, now expanding to other continents. These hemimetabolous insects have been considered poorly visual animals. However, recent findings challenge this idea. Here, we used Rhodnius prolixus as a model species to comprehensively characterize triatomine compound eyes. We found that in the adult stage, eye size significantly exceeds the dimensions predicted by the nymphal eye growth rate. Moreover, while the compound eye grows symmetrically in its dorsal and ventral directions throughout the nymphal instars, in the adult, the eye undergoes greater ventral growth, resulting in a dorsoventrally asymmetrical eye. By studying a bright pseudopupil induced by fluorescence in natural mutant animals, we observed no major differences in sampling resolution between the last nymphal instar and the adult stage. However, the adult eye possesses significantly larger ommatidia, particularly in its ventral region, shifting the area of highest sensitivity from the equatorial region in the nymphal instars to the ventral region in the adult. A similar eye growth pattern was observed in Triatoma infestans and Panstrongylus megistus. The analysis of photographic records from 39 species across 10 genera indicates that an asymmetrical eye is the predominant eye pattern in adult triatomines. Notable exceptions in wingless adults of Mepraia spinolai, reveal a tight association between possessing a large asymmetrical eye and the presence of wings. This suggests that vision might support triatomine dispersal flights among other visual behaviors. Significance StatementKissing bugs are hematophagous insects known for being the vectors of Chagas disease, one of the main endemic diseases in the Americas. Vision was not considered a relevant sensory system in these insects. Here, we show that their eyes increase in size beyond expected by ontogeny and become asymmetrical when transitioning from the last nymphal instar to the adult stage. The eyes undergo a ventral expansion that shifts the region of greatest light sensitivity from the equatorial zone in nymphs to the ventral region in adults. We found this asymmetrical eye only in winged kissing bugs, suggesting that vision supports flight. This is relevant in ecological and epidemiological terms since kissing bugs disperse by flight for habitat colonization and host-seeking.