Showing papers in "The Journal of Comparative Neurology in 2021"

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

Abstract: The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain

Convergence of peptidergic and non‐peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn

Abstract: Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long-standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1, and Nav1.7 proteins stained the entirety of lamina 1-2, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

The entorhinal cortex of the monkey: VI. Organization of projections from the hippocampus, subiculum, presubiculum, and parasubiculum

Abstract: The organization of projections from the macaque monkey hippocampus, subiculum, presubiculum, and parasubiculum to the entorhinal cortex was analyzed using anterograde and retrograde tracing techniques. Projections exclusively originate in the CA1 field of the hippocampus and in the subiculum, presubiculum, and parasubiculum. The CA1 and subicular projections terminate most densely in Layers V and VI of the entorhinal cortex, with sparser innervation of the deep portion of Layers III and II. Entorhinal projections from CA1 and the subiculum are topographically organized such that a rostrocaudal axis of origin is related to a medial-to-lateral axis of termination. A proximodistal axis of origin in CA1 and distoproximal axis in subiculum are related to a rostrocaudal axis of termination in the entorhinal cortex. The presubiculum sends a dense, bilateral projection to caudal parts of the entorhinal cortex. This projection terminates most densely in Layer III with sparser termination in Layers I, II, and V. The same parts of entorhinal cortex receive a dense projection from the parasubiculum. This projection terminates in Layers III and II. Both presubicular and parasubicular projections demonstrate the same longitudinal topographic organization as the projections from CA1 and the subiculum. These studies demonstrate that: (a) hippocampal and subicular inputs to the entorhinal cortex in the monkey are organized similar to those described in nonprimate species; (b) the topographic organization of the projections from the hippocampus and subicular areas matches that of the reciprocal projections from the entorhinal cortex to the hippocampus and the subicular areas.

Functional organization and connectivity of the dorsal column nuclei complex reveals a sensorimotor integration and distribution hub

Abstract: The dorsal column nuclei complex (DCN-complex) includes the dorsal column nuclei (DCN, referring to the gracile and cuneate nuclei collectively), external cuneate, X, and Z nuclei, and the median accessory nucleus. The DCN are organized by both somatotopy and modality, and have a diverse range of afferent inputs and projection targets. The functional organization and connectivity of the DCN implicate them in a variety of sensorimotor functions, beyond their commonly accepted role in processing and transmitting somatosensory information to the thalamus, yet this is largely underappreciated in the literature. To consolidate insights into their sensorimotor functions, this review examines the morphology, organization, and connectivity of the DCN and their associated nuclei. First, we briefly discuss the receptors, afferent fibers, and pathways involved in conveying tactile and proprioceptive information to the DCN. Next, we review the modality and somatotopic arrangements of the remaining constituents of the DCN-complex. Finally, we examine and discuss the functional implications of the myriad of DCN-complex projection targets throughout the diencephalon, midbrain, and hindbrain, in addition to their modulatory inputs from the cortex. The organization and connectivity of the DCN-complex suggest that these nuclei should be considered a complex integration and distribution hub for sensorimotor information.

Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice

Abstract: The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Efferent projections of CGRP/Calca-expressing parabrachial neurons in mice.

Abstract: The parabrachial nucleus (PB) is composed of glutamatergic neurons at the midbrain-hindbrain junction. These neurons form many subpopulations, one of which expresses Calca, which encodes the neuropeptide calcitonin gene-related peptide (CGRP). This Calca-expressing subpopulation has been implicated in a variety of homeostatic functions, but the overall distribution of Calca-expressing neurons in this region remains unclear. Also, while previous studies in rats and mice have identified output projections from CGRP-immunoreactive or Calca-expressing neurons, we lack a comprehensive understanding of their efferent projections. We began by identifying neurons with Calca mRNA and CGRP immunoreactivity in and around the PB, including populations in the locus coeruleus and motor trigeminal nucleus. Calca-expressing neurons in the PB prominently express the mu opioid receptor (Oprm1) and are distinct from neighboring neurons that express Foxp2 and Pdyn. Next, we used Cre-dependent anterograde tracing with synaptophysin-mCherry to map the efferent projections of these neurons. Calca-expressing PB neurons heavily target subregions of the amygdala, bed nucleus of the stria terminalis, basal forebrain, thalamic intralaminar and ventral posterior parvicellular nuclei, and hindbrain, in different patterns depending on the injection site location within the PB region. Retrograde axonal tracing revealed that the previously unreported hindbrain projections arise from a rostral-ventral subset of CGRP/Calca neurons. Finally, we show that these efferent projections of Calca-expressing neurons are distinct from those of neighboring PB neurons that express Pdyn. This information provides a detailed neuroanatomical framework for interpreting experimental work involving CGRP/Calca-expressing neurons and opioid action in the PB region.

Projections of ipRGCs and conventional RGCs to retinorecipient brain nuclei.

Abstract: Retinal ganglion cells (RGCs), the output neurons of the retina, allow us to perceive our visual environment. RGCs respond to rod/cone input through the retinal circuitry, however, a small population of RGCs are in addition intrinsically photosensitive (ipRGCs) and project to unique targets in the brain to modulate a broad range of subconscious visual behaviors such as pupil constriction and circadian photoentrainment. Despite the discovery of ipRGCs nearly two decades ago, there is still little information about how or if conventional RGCs (non-ipRGCs) target ipRGC-recipient nuclei to influence subconscious visual behavior. Using a dual recombinase fluorescent reporter strategy, we showed that conventional RGCs innervate many subconscious ipRGC-recipient nuclei, apart from the suprachiasmatic nucleus. We revealed previously unrecognized stratification patterns of retinal innervation from ipRGCs and conventional RGCs in the ventral portion of the lateral geniculate nucleus. Further, we found that the percent innervation of ipRGCs and conventional RGCs across ipsi- and contralateral nuclei differ. Our data provide a blueprint to understand how conventional RGCs and ipRGCs innervate different brain regions to influence subconscious visual behaviors.

Anatomy and function of retinorecipient arborization fields in zebrafish.

Abstract: In 1994, Burrill and Easter described the retinal projections in embryonic and larval zebrafish, introducing the term "arborization fields" (AFs) for the retinorecipient areas. AFs were numbered from 1 to 10 according to their positions along the optic tract. With the exception of AF10 (neuropil of the optic tectum), annotations of AFs remained tentative. Here we offer an update on the likely identities and functions of zebrafish AFs after successfully matching classical neuroanatomy to the digital Max Planck Zebrafish Brain Atlas. In our system, individual AFs are neuropil areas associated with the following nuclei: AF1 with the suprachiasmatic nucleus; AF2 with the posterior parvocellular preoptic nucleus; AF3 and AF4 with the ventrolateral thalamic nucleus; AF4 with the anterior and intermediate thalamic nuclei; AF5 with the dorsal accessory optic nucleus; AF7 with the parvocellular superficial pretectal nucleus; AF8 with the central pretectal nucleus; and AF9d and AF9v with the dorsal and ventral periventricular pretectal nuclei. AF6 is probably part of the accessory optic system. Imaging, ablation, and activation experiments showed contributions of AF5 and potentially AF6 to optokinetic and optomotor reflexes, AF4 to phototaxis, and AF7 to prey detection. AF6, AF8 and AF9v respond to dimming, and AF4 and AF9d to brightening. While few annotations remain tentative, it is apparent that the larval zebrafish visual system is anatomically and functionally continuous with its adult successor and fits the general cyprinid pattern. This study illustrates the synergy created by merging classical neuroanatomy with a cellular-resolution digital brain atlas resource and functional imaging in larval zebrafish.

Novel circular RNA 2960 contributes to secondary damage of spinal cord injury by sponging miRNA-124.

Abstract: Recent studies have shown that circular RNAs (circRNAs) are involved in many human diseases, but their roles in secondary damage after spinal cord injury (SCI) remain unclear. In the current study circRNA sequencing was performed in the damaged tissues of SCI rats on the seventh day after injury, and related molecular mechanisms were investigated. Quantitative PCR validations of molecules that exhibited significantly altered expression in SCI mice were performed. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to assess differentially expressed circRNAs. A novel circRNA-2960 was the most significantly upregulated in the SCI group. It could downregulate its target molecule miRNA-124, then exacerbate the inflammatory response and induce apoptosis at the lesion site. Disrupting circRNA-2960 expression promoted recovery of tissues affected by secondary SCI damage. The results of the present study may provide new insight into the mechanisms of secondary injury in SCI, and a new molecular marker for the diagnosis and treatment of SCI.

LacZ-reporter mapping of Dlx5/6 expression and genoarchitectural analysis of the postnatal mouse prethalamus.

Abstract: We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.

Somatosensory innervation of healthy human oral tissues.

Abstract: The oral somatosensory system relays essential information about mechanical stimuli to enable oral functions such as feeding and speech The neurochemical and anatomical diversity of sensory neurons across oral cavity sites have not been systematically compared To address this gap, we analyzed healthy human tongue and hard-palate innervation Biopsies were collected from 12 volunteers and underwent fluorescent immunohistochemistry (≥2 specimens per marker/structure) Afferents were analyzed for markers of neurons (βIII tubulin), myelinated afferents (neurofilament heavy, NFH), and Merkel cells and taste cells (keratin 20, K20) Hard-palate innervation included Meissner corpuscles, glomerular endings, Merkel cell-neurite complexes, and free nerve endings The organization of these somatosensory endings is reminiscent of fingertips, suggesting that the hard palate is equipped with a rich repertoire of sensory neurons for pressure sensing and spatial localization of mechanical inputs, which are essential for speech production and feeding Likewise, the tongue is innervated by afferents that impart it with exquisite acuity and detection of moving stimuli that support flavor construction and speech Filiform papillae contained end bulbs of Krause, as well as endings that have not been previously reported, including subepithelial neuronal densities, and NFH+ neurons innervating basal epithelia Fungiform papillae had Meissner corpuscles and densities of NFH+ intraepithelial neurons surrounding taste buds The differing compositions of sensory endings within filiform and fungiform papillae suggest that these structures have distinct roles in mechanosensation Collectively, this study has identified previously undescribed neuronal endings in human oral tissues and provides an anatomical framework for understanding oral mechanosensory functions

Location and arrangement of campaniform sensilla in Drosophila melanogaster

Abstract: Sensory systems provide input to motor networks on the state of the body and environment. One such sensory system in insects is the campaniform sensilla (CS), which detect deformations of the exoskeleton arising from resisted movements or external perturbations. When physical strain is applied to the cuticle, CS external structures are compressed, leading to transduction in an internal sensory neuron. In Drosophila melanogaster, the distribution of CS on the exoskeleton has not been comprehensively described. To investigate CS number, location, spatial arrangement, and potential differences between individuals, we compared the front, middle, and hind legs of multiple flies using scanning electron microscopy. Additionally, we imaged the entire body surface to confirm known CS locations. On the legs, the number and relative arrangement of CS varied between individuals, and single CS of corresponding segments showed characteristic differences between legs. This knowledge is fundamental for studying the relevance of cuticular strain information within the complex neuromuscular networks controlling posture and movement. This comprehensive account of all D. melanogaster CS helps set the stage for experimental investigations into their responsivity, sensitivity, and roles in sensory acquisition and motor control in a light-weight model organism.

An open access mouse brain flatmap and upgraded rat and human brain flatmaps based on current reference atlases.

Abstract: Here we present a flatmap of the mouse central nervous system (CNS) (brain) and substantially enhanced flatmaps of the rat and human brain. Also included are enhanced representations of nervous system white matter tracts, ganglia, and nerves, and an enhanced series of 10 flatmaps showing different stages of rat brain development. The adult mouse and rat brain flatmaps provide layered diagrammatic representation of CNS divisions, according to their arrangement in corresponding reference atlases: Brain Maps 4.0 (BM4, rat) (Swanson, The Journal of Comparative Neurology, 2018, 526, 935-943), and the first version of the Allen Reference Atlas (mouse) (Dong, The Allen reference atlas, (book + CD-ROM): A digital color brain atlas of the C57BL/6J male mouse, 2007). To facilitate comparative analysis, both flatmaps are scaled equally, and the divisional hierarchy of gray matter follows a topographic arrangement used in BM4. Also included with the mouse and rat brain flatmaps are cerebral cortex atlas level contours based on the reference atlases, and direct graphical and tabular comparison of regional parcellation. To encourage use of the brain flatmaps, they were designed and organized, with supporting reference tables, for ease-of-use and to be amenable to computational applications. We demonstrate how they can be adapted to represent novel parcellations resulting from experimental data, and we provide a proof-of-concept for how they could form the basis of a web-based graphical data viewer and analysis platform. The mouse, rat, and human brain flatmap vector graphics files (Adobe Reader/Acrobat viewable and Adobe Illustrator editable) and supporting tables are provided open access; they constitute a broadly applicable neuroscience toolbox resource for researchers seeking to map and perform comparative analysis of brain data.

Peculiar protrusions along tanycyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma

Abstract: Tanycytes are highly specialized ependymal cells that line the bottom and the lateral walls of the third ventricle. In contact with the cerebrospinal fluid through their cell bodies, they send processes into the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. In the present work, we combined transgenic and immunohistochemical approaches to investigate the neuroanatomical associations between tanycytes and neural cells present in the hypothalamic parenchyma, in particular in the arcuate nucleus. The specific expression of tdTomato in tanycytes first allowed the observation of peculiar subcellular protrusions along tanycyte processes and at their endfeet such as spines, swelling, en passant boutons, boutons, or claws. Interestingly, these protrusions contact different neural cells in the brain parenchyma including blood vessels and neurons, and in particular NPY and POMC neurons in the arcuate nucleus. Using both fluorescent and electron microscopy, we finally observed that these tanycyte protrusions contain ribosomes, mitochondria, diverse vesicles, and transporters, suggesting dense tanycyte/neuron and tanycyte/blood vessel communications. Altogether, our results lay the neuroanatomical basis for tanycyte/neural cell interactions, which will be useful to further understand cell-to-cell communications involved in the regulation of neuroendocrine functions.

Neuroarchitecture of the central complex in the brain of the honeybee: Neuronal cell types.

Abstract: The central complex (CX) in the insect brain is a higher order integration center that controls a number of behaviors, most prominently goal directed locomotion. The CX comprises the protocerebral bridge (PB), the upper division of the central body (CBU), the lower division of the central body (CBL), and the paired noduli (NO). Although spatial orientation has been extensively studied in honeybees at the behavioral level, most electrophysiological and anatomical analyses have been carried out in other insect species, leaving the morphology and physiology of neurons that constitute the CX in the honeybee mostly enigmatic. The goal of this study was to morphologically identify neuronal cell types of the CX in the honeybee Apis mellifera. By performing iontophoretic dye injections into the CX, we traced 16 subtypes of neuron that connect a subdivision of the CX with other regions in the bee's central brain, and eight subtypes that mainly interconnect different subdivisions of the CX. They establish extensive connections between the CX and the lateral complex, the superior protocerebrum and the posterior protocerebrum. Characterized neuron classes and subtypes are morphologically similar to those described in other insects, suggesting considerable conservation in the neural network relevant for orientation.

Topographic gradients define the projection patterns of the claustrum core and shell in mice

Abstract: The claustrum is densely connected to the cortex and participates in brain functions such as attention and sleep. Although some studies have reported the widely divergent organization of claustrum projections, others describe parallel claustrocortical connections to different cortical regions. Therefore, the details underlying how claustrum neurons broadcast information to cortical networks remain incompletely understood. Using multicolor retrograde tracing we determined the density, topography, and co-projection pattern of 14 claustrocortical pathways, in mice. We spatially registered these pathways to a common coordinate space and found that the claustrocortical system is topographically organized as a series of overlapping spatial modules, continuously distributed across the dorsoventral claustrum axis. The claustrum core projects predominantly to frontal-midline cortical regions, whereas the dorsal and ventral shell project to the cortical motor system and temporal lobe, respectively. Anatomically connected cortical regions receive common input from a subset of claustrum neurons shared by neighboring modules, whereas spatially separated regions of cortex are innervated by different claustrum modules. Therefore, each output module exhibits a unique position within the claustrum and overlaps substantially with other modules projecting to functionally related cortical regions. Claustrum inhibitory cells containing parvalbumin, somatostatin, and neuropeptide Y also show unique topographical distributions, suggesting different output modules are controlled by distinct inhibitory circuit motifs. The topographic organization of excitatory and inhibitory cell types may enable parallel claustrum outputs to independently coordinate distinct cortical networks.

Axo-axonic Synapses: Diversity in Neural Circuit Function.

Abstract: The chemical synapse is the principal form of contact between neurons of the central nervous system. These synapses are typically configured as presynaptic axon terminations onto postsynaptic dendrites or somata, giving rise to axo-dendritic and axo-somatic synapses, respectively. Beyond these common synapse configurations are less-studied, non-canonical synapse types that are prevalent throughout the brain and significantly contribute to neural circuit function. Among these are the axo-axonic synapses, which consist of an axon terminating on another axon or axon terminal. Here, we review evidence for axo-axonic synapse contributions to neural signaling in the mammalian nervous system and survey functional neural circuit motifs enabled by these synapses. We also detail how recent advances in microscopy, transgenics, and biological sensors may be used to identify and functionally assay axo-axonic synapses.

Basic quantitative morphological methods applied to the central nervous system.

Abstract: Generating numbers has become an almost inevitable task associated with studies of the morphology of the nervous system. Numbers serve a desire for clarity and objectivity in the presentation of results and are a prerequisite for the statistical evaluation of experimental outcomes. Clarity, objectivity, and statistics make demands on the quality of the numbers that are not met by many methods. This review provides a refresher of problems associated with generating numbers that describe the nervous system in terms of the volumes, surfaces, lengths, and numbers of its components. An important aim is to provide comprehensible descriptions of the methods that address these problems. Collectively known as design-based stereology, these methods share two features critical to their application. First, they are firmly based in mathematics and its proofs. Second and critically underemphasized, an understanding of their mathematical background is not necessary for their informed and productive application. Understanding and applying estimators of volume, surface, length or number does not require more of an organizational mastermind than an immunohistochemical protocol. And when it comes to calculations, square roots are the gravest challenges to overcome. Sampling strategies that are combined with stereological probes are efficient and allow a rational assessment if the numbers that have been generated are "good enough." Much may be unfamiliar, but very little is difficult. These methods can no longer be scapegoats for discrepant results but faithfully produce numbers on the material that is assessed. They also faithfully reflect problems that associated with the histological material and the anatomically informed decisions needed to generate numbers that are not only valid in theory. It is within reach to generate practically useful numbers that must integrate with qualitative knowledge to understand the function of neural systems.

Evolution of the speech‐ready brain: The voice/jaw connection in the human motor cortex

Abstract: A prominent model of the origins of speech, known as the "frame/content" theory, posits that oscillatory lowering and raising of the jaw provided an evolutionary scaffold for the development of syllable structure in speech. Because such oscillations are non-vocal in most non-human primates, the evolution of speech required the addition of vocalization onto this scaffold in order to turn such jaw oscillations into vocalized syllables. In the present functional MRI study, we demonstrate overlapping somatotopic representations between the larynx and the jaw muscles in the human primary motor cortex. This proximity between the larynx and jaw in the brain might support the coupling between vocalization and jaw oscillations to generate syllable structure. This model suggests that humans inherited voluntary control of jaw oscillations from ancestral species, but added voluntary control of vocalization onto this via the evolution of a new brain area that came to be situated near the jaw region in the human motor cortex. This article is protected by copyright. All rights reserved.

Distribution and overlap of entorhinal, premotor, and amygdalar connections in the monkey anterior cingulate cortex

Abstract: The anterior cingulate cortex (ACC) is important for decision-making as it integrates motor plans with affective and contextual limbic information. Disruptions in these networks have been observed in depression, bipolar disorder, and post-traumatic stress disorder. Yet, overlap of limbic and motor connections within subdivisions of the ACC is not well understood. Hence, we administered a combination of retrograde and anterograde tracers into structures important for contextual memories (entorhinal cortex), affective processing (amygdala), and motor planning (dorsal premotor cortex) to assess overlap of labeled projection neurons from (outputs) and axon terminals to (inputs) the ACC of adult rhesus monkeys (Macaca mulatta). Our data show that entorhinal and dorsal premotor cortical (dPMC) connections are segregated across ventral (A25, A24a) and dorsal (A24b,c) subregions of the ACC, while amygdalar connections are more evenly distributed across subregions. Among all areas, the rostral ACC (A32) had the lowest relative density of connections with all three regions. In the ventral ACC, entorhinal and amygdalar connections strongly overlap across all layers, especially in A25. In the dorsal ACC, outputs to dPMC and the amygdala strongly overlap in deep layers. However, dPMC input to the dorsal ACC was densest in deep layers, while amygdalar inputs predominantly localized in upper layers. These connection patterns are consistent with diverse roles of the dorsal ACC in motor evaluation and the ventral ACC in affective and contextual memory. Further, distinct laminar circuits suggest unique interactions within specific ACC compartments that are likely important for the temporal integration of motor and limbic information during flexible goal-directed behavior.

Light sheet microscopy of the gerbil cochlea

Abstract: Light sheet fluorescence microscopy (LSFM) provides a rapid and complete three-dimensional image of the cochlea. The method retains anatomical relationships-on a micrometer scale-between internal structures such as hair cells, basilar membrane (BM), and modiolus with external surface structures such as the round and oval windows. Immunolabeled hair cells were used to visualize the spiraling BM in the intact cochlea without time intensive dissections or additional histological processing; yet material prepared for LSFM could be rehydrated, the BM dissected out and reimaged at higher resolution with the confocal microscope. In immersion-fixed material, details of the cochlear vasculature were seen throughout the cochlea. Hair cell counts (both inner and outer) as well as frequency maps of the BM were comparable to those obtained by other methods, but with the added dimension of depth. The material provided measures of angular, linear, and vector distance between characteristic frequency regions along the BM. Thus, LSFM provides a unique ability to rapidly image the entire cochlea in a manner applicable to model and interpret physiological results. Furthermore, the three-dimensional organization of the cochlea can be studied at the organ and cellular level with LSFM, and this same material can be taken to the confocal microscope for detailed analysis at the subcellular level.

Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata.

Abstract: Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kolliker-Fuse nucleus (KFn), the medullary Botzinger complex (BotC), pre-BotC, or caudal midline raphe nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.

Neural anatomy of echinoid early juveniles and comparison of nervous system organization in echinoderms

Abstract: The echinoderms are a phylum of marine deuterostomes characterized by the pentaradial (five fold) symmetry of their adult bodies. Due to this unusual body plan, adult echinoderms have long been excluded from comparative analyses aimed at understanding the origin and evolution of deuterostome nervous systems. Here, we investigated the neural anatomy of early juveniles of representatives of three of the five echinoderm classes: the echinoid Paracentrotus lividus, the asteroid Patiria miniata, and the holothuroid Parastichopus parvimensis. Using whole mount immunohistochemistry and confocal microscopy, we found that the nervous system of echinoid early juveniles is composed of three main structures: a basiepidermal nerve plexus, five radial nerve cords connected by a circumoral nerve ring, and peripheral nerves innervating the appendages. Our whole mount preparations further allowed us to obtain thorough descriptions of these structures and of several innervation patterns, in particular at the level of the appendages. Detailed comparisons of the echinoid juvenile nervous system with those of asteroid and holothuroid juveniles moreover supported a general conservation of the main neural structures in all three species, including at the level of the appendages. Our results support the previously proposed hypotheses for the existence of two neural units in echinoderms: one consisting of the basiepidermal nerve plexus to process sensory stimuli locally and one composed of the radial nerve cords and the peripheral nerves constituting a centralized control system. This study provides the basis for more in-depth comparisons of the echinoderm adult nervous system with those of other animals, in particular hemichordates and chordates, to address the long-standing controversies about deuterostome nervous system evolution.

Defining vitamin D receptor expression in the brain using a novel VDRCre mouse.

Abstract: Vitamin D action has been linked to several diseases regulated by the brain including obesity, diabetes, autism, and Parkinson's. However, the location of the vitamin D receptor (VDR) in the brain is not clear due to conflicting reports. We found that two antibodies previously published as specific in peripheral tissues are not specific in the brain. We thus created a new knockin mouse with cre recombinase expression under the control of the endogenous VDR promoter (VDRCre ). We demonstrated that the cre activity in the VDRCre mouse brain (as reported by a cre-dependent tdTomato expression) is highly overlapping with endogenous VDR mRNAs. These VDR-expressing cells were enriched in multiple brain regions including the cortex, amygdala, caudate putamen, and hypothalamus among others. In the hypothalamus, VDR partially colocalized with vasopressin, oxytocin, estrogen receptor-α, and β-endorphin to various degrees. We further functionally validated our model by demonstrating that the endogenous VDR agonist 1,25-dihydroxyvitamin D activated all tested tdTomato+ neurons in the paraventricular hypothalamus but had no effect on neurons without tdTomato fluorescence. Thus, we have generated a new mouse tool that allows us to visualize VDR-expressing cells and to characterize their functions.

Progression of basal ganglia pathology in heterozygous Q175 knock-in Huntington's disease mice.

Abstract: We used behavioral testing and morphological methods to detail the progression of basal ganglia neuron type-specific pathology and the deficits stemming from them in male heterozygous Q175 mice, compared to age-matched WT males. A rotarod deficit was not present in Q175 mice until 18 months, but increased open field turn rate (reflecting hyperkinesia) and open field anxiety were evident at 6 months. No loss of striatal neurons was seen out to 18 months, but ENK+ and DARPP32+ striatal perikarya were fewer by 6 months, due to diminished expression, with further decline by 18 months. No reduction in SP+ striatal perikarya or striatal interneurons was seen in Q175 mice at 18-months, but cholinergic interneurons showed dendrite attenuation by 6 months. Despite reduced ENK expression in indirect pathway striatal perikarya, ENK-immunostained terminals in globus pallidus externus (GPe) were more abundant at 6 months, and remained so out to 18 months. Similarly, SP-immunostained terminals from striatal direct pathway neurons were more abundant in globus pallidus internus and substantia nigra at 6 months, and remained so at 18 months. FoxP2+ arkypallidal GPe neurons and subthalamic nucleus neurons were lost by 18 months but not prototypical PARV+ GPe neurons or dopaminergic nigral neurons. Our results show that striatal projection neuron abnormalities and behavioral abnormalities reflecting them develop between 2 and 6 months of age in Q175 male heterozygotes, indicating early effects of the HD mutation. The striatal pathologies resemble those in human HD, but are less severe at 18 months than even in premanifest HD.

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow.

Abstract: Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRβ or NG2 promoter (Pdgfrβ-CreRha, Pdgfrβ-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRβ line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.

Sympathetic innervation of inguinal white adipose tissue in the mouse.

Abstract: Adipose tissue plays an important role in metabolic homeostasis and its prominent role as endocrine organ is now well recognized. Adipose tissue is controlled via the sympathetic nervous system (SNS). New viral, molecular-genetic tools will soon allow a more detailed study of adipose tissue innervation in metabolic function, yet, the precise anatomical extent of preganglionic and postganglionic inputs to the inguinal white adipose tissue (iWAT) is limited. Furthermore, several viral, molecular-genetic tools will require the use of cre/loxP mouse models, while the available studies on sympathetic iWAT innervation were established in larger species. In this study, we generated a detailed map for the sympathetic innervation of iWAT in male and female mice. We adapted iDISCO tissue clearing to process large, whole-body specimens for an unprecedented view of the natural abdominal SNS. Combined with pseudorabies virus retrograde tracing from the iWAT, we defined the preganglionic and postganglionic sympathetic input to iWAT. We used fluorescence-guided anatomical dissections of sympathetic nerves in reporter mice to further clarify that postganglionic axons connect to iWAT via lateral cutaneous rami (dorsolumbar iWAT portion) and the lumbar plexus (inguinal iWAT portion). Importantly, these rami carry axons that branch to iWAT, as well as axons that travel further to innervate the skin and vasculature, and their functional impact will require consideration in denervation studies. Our study may serve as a comprehensive map for future experiments that employ virally driven neuromodulation techniques to predict anatomy-based viral labeling.

Variations on a theme: Morphological variation in the secondary eye visual pathway across the order of Araneae.

Abstract: Spiders possess a wide array of sensory-driven behaviors and therefore provide rich models for studying evolutionary hypotheses about the relationship between brain morphology, sensory systems, and behavior. Despite this, only a handful of studies have examined brain variation across the order of Araneae. In this study, I present descriptions of the gross brain morphology for 19 families of spiders that vary in eye morphology. Spiders showed the most variation in the secondary eye visual pathway. Based on this variation, spiders could be categorized into four groups. Group 1 spiders had small, underdeveloped laminae, no medullae, and no mushroom bodies. Group 2 spiders had large laminae, no medullae and large mushroom bodies. Group 3 spiders had laminae and some evidence of reduced medullae and mushroom bodies. Group 4 spiders had the most complex systems, with large laminae, medullae formed from optical glomeruli, and robust mushroom bodies. Within groups, there was large variation in the shape and size of individual regions, indicating possible variation in neuronal organization. The possible evolutionary implications of the loss of a dedicated olfactory organ in spiders and its effects on the mushroom body are also discussed.

A novel telencephalon-opto-hypothalamic morphogenetic domain coexpressing Foxg1 and Otp produces most of the glutamatergic neurons of the medial extended amygdala.

Abstract: Deficits in social cognition and behavior are a hallmark of many psychiatric disorders. The medial extended amygdala, including the medial amygdala and the medial bed nucleus of the stria terminalis, is a key component of functional networks involved in sociality. However, this nuclear complex is highly heterogeneous and contains numerous GABAergic and glutamatergic neuron subpopulations. Deciphering the connections of different neurons is essential in order to understand how this structure regulates different aspects of sociality, and it is necessary to evaluate their differential implication in distinct mental disorders. Developmental studies in different vertebrates are offering new venues to understand neuronal diversity of the medial extended amygdala and are helping to establish a relation between the embryonic origin and molecular signature of distinct neurons with the functional subcircuits in which they are engaged. These studies have provided many details on the distinct GABAergic neurons of the medial extended amygdala, but information on the glutamatergic neurons is still scarce. Using an Otp-eGFP transgenic mouse and multiple fluorescent labeling, we show that most glutamatergic neurons of the medial extended amygdala originate in a distinct telencephalon-opto-hypothalamic embryonic domain (TOH), located at the transition between telencephalon and hypothalamus, which produces Otp-lineage neurons expressing the telencephalic marker Foxg1 but not Nkx2.1 during development. These glutamatergic cells include a subpopulation of projection neurons of the medial amygdala, which activation has been previously shown to promote autistic-like behavior. Our data open new venues for studying the implication of this neuron subtype in neurodevelopmental disorders producing social deficits.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

Abstract: As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.