Morphological and functional changes in TRPM8‐expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8‐expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8^BAC‐EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low‐threshold, robust responses to cooling. The remaining TRPM8⁺ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low‐firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8⁺ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low‐background activity and abnormal responsiveness to cooling pulses. These morpho‐functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age‐induced abnormal tearing and to the high incidence of DED in elderly people.     

Developmental characterization of Zswim5 expression in the progenitor domains and tangential migration pathways of cortical interneurons in the mouse forebrain

GABAergic interneurons play an essential role in modulating cortical networks. The progenitor domains of cortical interneurons are localized in developing ventral forebrain, including the medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), preoptic area (POA), and preoptic hypothalamic border domain (POH). Here, we characterized the expression pattern of Zswim5, an MGE-enriched gene in the mouse forebrain. At E11.5–E13.5, prominent Zswim5 expression was detected in the subventricular zone (SVZ) of MGE, POA, and POH, but not CGE of ventral telencephalon where progenitors of cortical interneurons resided. At E15.5 and E17.5, Zswim5 expression remained in the MGE/pallidum primordium and ventral germinal zone. Zswim5 mRNA was markedly decreased after birth and was absent in the adult forebrain. Interestingly, the Zswim5 expression pattern resembled the tangential migration pathways of cortical interneurons. Zswim5-positive cells in the MGE appeared to migrate from the MGE through the SVZ of LGE to overlying neocortex. Indeed, Zswim5 was co-localized with Nkx2.1 and Lhx6, markers of progenitors and migratory cortical interneurons. Double labeling showed that Ascl1/Mash1-positive cells co-expressed Zswim5. Zswim5 expressing cells contained none or at most low levels of Ki67 but co-expressed Tuj1 in the SVZ of MGE. These results suggest that Zswim5 is immediately upregulated as progenitors exiting cell cycle become postmitotic. Given that recent studies have elucidated that the cell fate of cortical interneurons is determined shortly after becoming postmitotic, the timing of Zswim5 expression in early postmitotic interneurons suggests a potential role of Zswim5 in regulation of neurogenesis and tangential migration of cortical interneurons.     

Expression of adiponectin receptors in the brain of adult zebrafish and mouse: Links with neurogenic niches and brain repair

Adiponectin and its receptors (adipor) have been initially characterized for their role in lipid and glucose metabolism. More recently, adiponectin signaling was shown to display anti-inflammatory effects and to participate in brain homeostasis and neuroprotection. In this study, we investigated adipor gene expression and its regulation under inflammatory conditions in two complementary models: mouse and zebrafish. We demonstrate that adipor1a, adipor1b, and adipor2 are widely distributed throughout the brain of adult fish, in neurons and also in radial glia, behaving as neural stem cells. We also show that telencephalic injury results in a decrease in adipor gene expression, inhibited by an anti-inflammatory treatment (Dexamethasone). Interestingly, adiponectin injection after brain injury led to a consistent decrease (a) in the recruitment of microglial cells at the lesioned site and (b) in the proliferation of neural progenitors, arguing for a neuroprotective role of adiponectin. In a comparative approach, we investigate Adipor1 and Adipor2 gene distribution in the brain of mice and demonstrated their expression in regions shared with fish including neurogenic regions. We also document Adipor gene expression in mice after middle cerebral artery occlusion and lipopolysaccharide injection. In contrast to zebrafish, these inflammatory stimuli do no impact cerebral adiponectin receptor gene expression in mouse. This work provides new insights regarding adipor expression in the brain of fish, and demonstrates evolutionary conserved distribution of adipor with mouse. This is the first report of adipor expression in adult neural stem cells of fish, suggesting a potential role of adiponectin signaling during vertebrate neurogenesis. It also suggests a potential contribution of inflammation in the regulation of adipor in fish.     

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30 min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca²⁺ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3 Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.     

Expression of tachykinin3 and related reproductive markers in the brain of the African cichlid fish Astatotilapia burtoni

Neurokinin B, encoded by the tachykinin3 gene, plays a crucial role in regulating reproduction in mammals via KNDy neurons and interaction with GnRH. Previous work in teleost fishes has focused on hypothalamic tac3 expression for its role in reproduction, but detailed studies on extra-hypothalamic tac3 expression are limited. Here, we identified two tac3 genes in the social African cichlid fish Astatotilapia burtoni, only one of which produces a functional protein containing the signature tachykinin motif. In situ hybridization for tac3a mRNA identified cell populations throughout the brain. Numerous tac3a cells lie in several thalamic and hypothalamic nuclei, including periventricular nucleus of posterior tuberculum, lateral tuberal nucleus (NLT), and nucleus of the lateral recess (NRL). Scattered tac3-expressing cells are also present in telencephalic parts, such as ventral (Vv) and supracomissural (Vs) part of ventral telencephalon. In contrast to other teleosts, tac3 expression was absent from the pituitary. Using double-fluorescent staining, we localized tac3a-expressing cells in relation to GnRH and kisspeptin cells. Although no GnRH-tac3a colabeled cells were observed, dense GnRH fibers surround and potentially synapse with tac3a cells in the preoptic area. Only minimal (<5%) colabeling of tac3a was observed in kiss2 cells. Despite tac3a expression in many nodes of the mesolimbic reward system, it was absent from tyrosine hydroxylase (TH)-expressing cells, but tac3a cells were located in areas with dense TH fibers. The presence of tac3a-expressing cells throughout the brain, including in socially relevant brain regions, suggest more diverse functions beyond regulation of reproductive physiology that may be conserved across vertebrates.     

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting I_h and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.     

Multiparametric assessment of the impact of opsin expression and anesthesia on striatal cholinergic neurons and auditory brainstem activity

Recent developments in genetic engineering have established murine models that permit the selective control of cholinergic neurons via optical stimulation. Despite copious benefits granted by these experimental advances, the sensory physiognomy of these organisms has remained poorly understood. Therefore, the present study evaluates sensory and neuronal response properties of animal models developed for the study of optically induced acetylcholine release regulation. Auditory brainstem responses, fluorescence imaging, and patch clamp recording techniques were used to assess the impact of viral infection, sex, age, and anesthetic agents across the ascending auditory pathway of ChAT-Cre and ChAT-ChR2(Ai32) mice. Data analyses revealed that neither genetic configuration nor adeno-associated viral infection alters the early stages of auditory processing or the cellular response properties of cholinergic neurons. However, anesthetic agent and dosage amount profoundly modulate the response properties of brainstem neurons. Last, analyses of age-related hearing loss in virally infected ChAT-Cre mice did not differ from those reported in wild type animals. This investigation demonstrates that ChAT-Cre and ChAT-ChR2(Ai32) mice are viable models for the study of cholinergic modulation in auditory processing, and it emphasizes the need for prudence in the selection of anesthetic procedures.     

Organization of the catecholaminergic systems in the brain of lungfishes,the closest living relatives of terrestrial vertebrates

Lungfishes are a group of sarcopterygian fishes currently considered the closest living relatives of tetrapods, and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. Catecholaminergic systems in the brain are among the most carefully analyzed neurotransmitter systems in the brain of most vertebrate groups. Their organization shows major shared characteristics, although traits particular to each vertebrate class have also been found, primarily between anamniotes and amniotes. Given the relevance of lungfishes in evolutionary terms, the present study provides the first comprehensive and detailed map of the catecholaminergic structures in the brain of two representative species of lungfishes, an African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry. Distinct groups of catecholaminergic cells were observed in the olfactory bulb, pallium, and preoptic area of the telencephalon, and the subpallium is devoid of these cells. Hypothalamic and diencephalic groups were detected and, in particular, the dopaminergic nucleus of the periventricular organ was evidenced with dopamine antibodies but not with anti‐tyrosine hydroxylase. A well developed mesostriatal system was revealed formed by conspicuous groups of dopamine cells in the midbrain tegmentum and profuse innervation of the subpallium. Comparison of these results with those from other classes of vertebrates shows numerous common traits shared by most groups and also highlights particular features in lungfishes different from actinopterygian fishes that resemble those of amphibians and amniotes.     

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS‐expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS‐immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα‐immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP^Vglut2, EYFP^Vgat, and GFP^Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.     

A comparative study of the neural stem cell niche in the adult hypothalamus of human,mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non‐human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co‐expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.     

Differential expression of neurexin genes in the mouse brain

Synapses, highly specialized membrane junctions between neurons, connect presynaptic neurotransmitter release sites and postsynaptic ligand-gated channels. Neurexins (Nrxns), a family of presynaptic adhesion molecules, have been characterized as major regulators of synapse development and function. Via their extracellular domains, Nrxns bind to different postsynaptic proteins, generating highly diverse functional readouts through their postsynaptic binding partners. Not surprisingly given these versatile protein interactions, mutations and deletions of Nrxn genes have been identified in patients with autism spectrum disorders, intellectual disabilities, and schizophrenia. Therefore, elucidating the expression profiles of Nrxns in the brain is of high significance. Here, using chromogenic and fluorescent in situ hybridization, we characterize the expression patterns of Nrxn isoforms throughout the brain. We found that each Nrxn isoform displays a unique expression profile in a region-, cell type-, and sensory system-specific manner. Interestingly, we also found that αNrxn1 and αNrxn2 mRNAs are expressed in non-neuronal cells, including astrocytes and oligodendrocytes. Lastly, we found diverse expression patterns of genes that encode Nrxn binding proteins, such as Neuroligins (Nlgns), Leucine-rich repeat transmembrane neuronal protein (Lrrtms) and Latrophilins (Adgrls), suggesting that Nrxn proteins can mediate numerous combinations of trans-synaptic interactions. Together, our anatomical profiling of Nrxn gene expression reflects the diverse roles of Nrxn molecules.     

Exorhodopsin and melanopsin systems in the pineal complex and brain at early developmental stages of Atlantic halibut (Hippoglossus hippoglossus)

The complexity of the nonvisual photoreception systems in teleosts has just started to be appreciated, with colocalization of multiple photoreceptor types with unresolved functions. Here we describe an intricate expression pattern of melanopsins in early life stages of the marine flat fish Atlantic halibut (Hippoglossus hippoglossus), a period when the unpigmented brain is directly exposed to environmental photons. We show a refined and extensive expression of melanopsins in the halibut brain already at the time of hatching, long before the eyes are functional. We detect melanopsin in the habenula, suprachiasmatic nucleus, dorsal thalamus, and lateral tubular nucleus of first feeding larvae, suggesting conserved functions of the melanopsins in marine teleosts. The complex expression of melanopsins already at larval stages indicates the importance of nonvisual photoreception early in development. Most strikingly, we detect expression of both exorhodopsin and melanopsin in the pineal complex of halibut larvae. Double‐fluorescence labeling showed that two clusters of melanopsin‐positive cells are located lateral to the central rosette of exorhodopsin‐positive cells. The localization of different photopigments in the pineal complex suggests that two parallel photoreceptor systems may be active. Furthermore, the dispersed melanopsin‐positive cells in the spinal cord of halibut larvae at the time of hatching may be primary sensory cells or interneurons representing the first example of dispersed high‐order photoreceptor cells. The appearance of nonvisual opsins early in the development of halibut provides an alternative model for studying the evolution and functional significance of nonvisual opsins. J. Comp. Neurol. 522:4003–4022, 2014. © 2014 Wiley Periodicals, Inc.     

Anatomy of the lobula complex in the brain of the praying mantis compared to the lobula complexes of the locust and cockroach

The praying mantis is an insect which relies on vision for capturing prey, avoiding being eaten and for spatial orientation. It is well known for its ability to use stereopsis for estimating the distance of objects. The neuronal substrate mediating visually driven behaviors, however, is not very well investigated. To provide a basis for future functional studies, we analyzed the anatomical organization of visual neuropils in the brain of the praying mantis Hierodula membranacea and provide supporting evidence from a second species, Rhombodera basalis, with particular focus on the lobula complex (LOX). Neuropils were three‐dimensionally reconstructed from synapsin‐immunostained whole mount brains. The neuropil organization and the pattern of γ‐aminobutyric acid immunostaining of the medulla and LOX were compared between the praying mantis and two related polyneopteran species, the Madeira cockroach and the desert locust. The investigated visual neuropils of the praying mantis are highly structured. Unlike in most insects the LOX of the praying mantis consists of five nested neuropils with at least one neuropil not present in the cockroach or locust. Overall, the mantis LOX is more similar to the LOX of the locust than the more closely related cockroach suggesting that the sensory ecology plays a stronger role than the phylogenetic distance of the three species in structuring this center of visual information processing.     

Apparent absence of hypothalamic cholinergic neurons in the common ostrich and emu: Implications for global brain states during sleep

We examined the presence/absence and parcellation of cholinergic neurons in the hypothalami of five birds: a Congo grey parrot (Psittacus erithacus), a Timneh grey parrot (P. timneh), a pied crow (Corvus albus), a common ostrich (Struthio camelus), and an emu (Dromaius novaehollandiae). Using immunohistochemistry to an antibody raised against the enzyme choline acetyltransferase, hypothalamic cholinergic neurons were observed in six distinct clusters in the medial, lateral, and ventral hypothalamus in the parrots and crow, similar to prior observations made in the pigeon. The expression of cholinergic nuclei was most prominent in the Congo grey parrot, both in the medial and lateral hypothalamus. In contrast, no evidence of cholinergic neurons in the hypothalami of either the ostrich or emu was found. It is known that the expression of sleep states in the ostrich is unusual and resembles that observed in the monotremes that also lack hypothalamic cholinergic neurons. It has been proposed that the cholinergic system acts globally to produce and maintain brain states, such as those of arousal and rapid-eye-movement sleep. The hiatus in the cholinergic system of the ostrich, due to the lack of hypothalamic cholinergic neurons, may explain, in part, the unusual expression of sleep states in this species. These comparative anatomical and sleep studies provide supportive evidence for global cholinergic actions and may provide an important framework for our understanding of one broad function of the cholinergic system and possible dysfunctions associated with global cholinergic neural activity.     

Monosynaptic retrograde tracing of neurons expressing the G‐protein coupled receptor Gpr151 in the mouse brain

GPR151 is a G‐protein coupled receptor for which the endogenous ligand remains unknown. In the nervous system of vertebrates, its expression is enriched in specific diencephalic structures, where the highest levels are observed in the habenular area. The habenula has been implicated in a range of different functions including behavioral flexibility, decision making, inhibitory control, and pain processing, which makes it a promising target for treating psychiatric and neurological disease. This study aimed to further characterize neurons expressing the Gpr151 gene, by tracing the afferent connectivity of this diencephalic cell population. Using pseudotyped rabies virus in a transgenic Gpr151‐Cre mouse line, monosynaptic afferents of habenular and thalamic Gpr151‐expressing neuronal populations could be visualized. The habenular and thalamic Gpr151 systems displayed both shared and distinct connectivity patterns. The habenular neurons primarily received input from basal forebrain structures, the bed nucleus of stria terminalis, the lateral preoptic area, the entopeduncular nucleus, and the lateral hypothalamic area. The Gpr151‐expressing neurons in the paraventricular nucleus of the thalamus was primarily contacted by medial hypothalamic areas as well as the zona incerta and projected to specific forebrain areas such as the prelimbic cortex and the accumbens nucleus. Gpr151 mRNA was also detected at low levels in the lateral posterior thalamic nucleus which received input from areas associated with visual processing, including the superior colliculus, zona incerta, and the visual and retrosplenial cortices. Knowledge about the connectivity of Gpr151‐expressing neurons will facilitate the interpretation of future functional studies of this receptor.     

Spatial and temporal expression profiles of urocortin 3 mRNA in the brain of the chicken (Gallus gallus)

Urocortin 3 (UCN3) is a neuropeptide believed to regulate stress‐coping responses by binding to type 2 corticotropin‐releasing hormone receptors. Here, we report the cloning and brain distribution of UCN3 mRNA in a sauropsid—the chicken, Gallus gallus. Mature chicken UCN3 is predicted to be a 40‐amino acid peptide showing high sequence similarity to human (93%), mouse (93%), and Xenopus (88%) UCN3. During the last third of embryonic development, UCN3 mRNA levels changed differentially in the various brain parts. In all brain parts, UCN3 mRNA levels tended to increase toward hatching, except for caudal brainstem, where a gradual decrease was observed during the last week of embryonic development. In cerebellum, a rapid increase in gene expression occurred between embryonic days 17 and 19. Using in situ hybridization, UCN3 mRNA was found to be expressed predominantly in the hypothalamus, pons, and medulla of posthatch chick brains, but not in some areas that are among the main expression sites in rodents, such as the brain areas where in mammals the median preoptic nucleus and the medial amygdala are located. This suggests that the roles of UCN3 in chicken, and perhaps sauropsids in general, are not all identical to those in rodents.