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.     

Differential expression of protocadherin‐19, protocadherin‐17, and cadherin‐6 in adult zebrafish brain

Cell adhesion molecule cadherins play important roles in both development and maintenance of adult structures. Most studies on cadherin expression have been carried out in developing organisms, but information on cadherin distribution in adult vertebrate brains is limited. In this study we used in situ hybridization to examine mRNA expression of three cadherins, protocadherin‐19, protocadherin‐17, and cadherin‐6 in adult zebrafish brain. Each cadherin exhibits a distinct expression pattern in the fish brain, with protocadherin‐19 and protocadherin‐17 showing much wider and stronger expression than that of cadherin‐6. Both protocadherin‐19 and protocadherin‐17‐expressing cells occur throughout the brain, with strong expression in the ventromedial telencephalon, periventricular regions of the thalamus and anterior hypothalamus, stratum periventriculare of the optic tectum, dorsal tegmental nucleus, granular regions of the cerebellar body and valvula, and superficial layers of the facial and vagal lobes. Numerous sensory structures (e.g., auditory, gustatory, lateral line, olfactory, and visual nuclei) and motor nuclei (e.g., oculomotor, trochlear, trigeminal motor, abducens, and vagal motor nuclei) contain protocadherin‐19 and/or protocadherin‐17‐expressing cell. Expression of these two protocadherins is similar in the ventromedial telencephalon, thalamus, hypothalamus, facial, and vagal lobes, but substantially different in the dorsolateral telencephalon, intermediate layers of the optic tectum, and cerebellar valvula. In contrast to the two protocadherins, cadherin‐6 expression is much weaker and limited in the adult fish brain. J. Comp. Neurol. 523:1419–1442, 2015. © 2015 Wiley Periodicals, Inc.     

Comparative analysis of monoaminergic cerebrospinal fluid‐contacting cells in Osteichthyes (bony vertebrates)

Cerebrospinal fluid‐contacting (CSF‐c) cells containing monoamines such as dopamine (DA) and serotonin (5‐HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF‐c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5‐HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF‐c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF‐c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF‐c cells coexpress TH2 and TPH1 and contain both DA and 5‐HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF‐c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF‐c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF‐c cells, while teleosts have increased their relative number.     

Gustatory and general visceral centers and their connections in the brain of adult zebrafish: a carbocyanine dye tract‐tracing study

The central connections of the gustatory/general visceral system of the adult zebrafish (Danio rerio) were examined by means of carbocyanine dye tracing. Main primary gustatory centers (facial and vagal lobes) received sensory projections from the facial and vagal nerves, respectively. The vagal nerve also projects to the commissural nucleus of Cajal, a general visceral sensory center. These primary centers mainly project on a prominent secondary gustatory and general visceral nucleus (SGN/V) located in the isthmic region. Secondary projections on the SGN/V were topographically organized, those of the facial lobe mainly ending medially to those of the vagal lobe, and those from the commissural nucleus ventrolaterally. Descending facial lobe projections to the medial funicular nucleus were also noted. Ascending fibers originating from the SGN/V mainly projected to the posterior thalamic nucleus and the lateral hypothalamus (lateral torus, lateral recess nucleus, hypothalamic inferior lobe diffuse nucleus) and an intermediate cell‐ and fiber‐rich region termed here the tertiary gustatory nucleus proper, but not to a nucleus formerly considered as the zebrafish tertiary gustatory nucleus. The posterior thalamic nucleus, tertiary gustatory nucleus proper, and nucleus of the lateral recess gave rise to descending projections to the SGN/V and the vagal lobe. The connectivity between diencephalic gustatory centers and the telencephalon was also investigated. The present results showed that the gustatory connections of the adult zebrafish are rather similar to those reported in other cyprinids, excepting the tertiary gustatory nucleus. Similarities between the gustatory systems of zebrafish and other fishes are also discussed. J. Comp. Neurol. 525:333–362, 2017. © 2016 Wiley Periodicals, Inc.     

GABA‐like immunoreactivity in Biomphalaria: Colocalization with tyrosine hydroxylase‐like immunoreactivity in the feeding motor systems of panpulmonate snails

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ‐aminobutyric acid‐like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz‐Ríos, Oyola, & Miller, 2002), double‐labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli‐THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli‐THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design.     

The distribution of GAD67‐mRNA in the adult zebrafish (teleost) forebrain reveals a prosomeric pattern and suggests previously unidentified homologies to tetrapods

We used in situ hybridization on sections to examine the distribution of GAD67‐expressing cell populations in the entire forebrain of the adult zebrafish. GAD67 is predominantly expressed in the olfactory bulb (OB), all regions of the subpallium (including the dorsal, ventral, central, and lateral nucleus of the area ventralis [Vd, Vv, Vc, and Vl, respectively]), as well as preoptic (PPa, PPp, and PM), pretectal (PPd, PPv, PCN, PSp, and PSm), ventral (= pre‐) thalamic (I, VM, and VL), hypothalamic (Hr, Hi, and Hc), preglomerular (P, PGa, PGl, PGm, and RT), and posterior tubercular (TPp and TPm) nuclei. Only scattered GAD67‐expressing cells are seen in all pallial zones (Dm, Dd, Dc, Dl, and Dp) and in the previously unidentified bed nucleus of the stria medullaris (BNSM). The BNSM appears to be the adult teleostean derivative of the larval eminentia thalami (EmT). We identify the GAD67‐positive entopeduncular nucleus proper (EN) as being homologous to the entopeduncular nucleus of nonprimate mammals. GAD67 is strongly expressed in the anterior thalamic nucleus (A). The anterior thalamic nucleus is laterally bordered by a distinct GAD67‐expressing cell population, which we interpret as the previously unidentified reticular thalamic nucleus (RTN) of teleosts. Furthermore, we identified a GAD67‐positive thalamic nucleus, the intercalated nucleus (IC), which is sandwiched between the GAD67‐negative dorsal (DP) and central posterior (CP) thalamic nuclei. Overall, the distribution of GAD67‐expressing cells highly resembles the distribution of γ‐aminobutyric acid (GABA)/GAD67‐expressing cells found in the early zebrafish (teleost) forebrain and thus allows us to propose a prosomeric fate map of GABAergic cell populations. J. Comp. Neurol. 516:553–568, 2009. © 2009 Wiley‐Liss, Inc.     

Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish

A growing body of evidence supports hyperglycemia as a putative contributor to several brain dysfunctions observed in diabetes patients, such as impaired memory capacity, neural plasticity, and neurogenic processes. Thanks to the persistence of radial glial cells acting as neural stem cells, the brain of the adult zebrafish constitutes a relevant model to investigate constitutive and injury‐induced neurogenesis in adult vertebrates. However, there is limited understanding of the impact of hyperglycemia on brain dysfunction in the zebrafish model. This work aimed at exploring the impact of acute and chronic hyperglycemia on brain homeostasis and neurogenesis. Acute hyperglycemia was shown to promote gene expression of proinflammatory cytokines (il1β, il6, il8, and tnfα) in the brain and chronic hyperglycemia to impair expression of genes involved in the establishment of the blood–brain barrier (claudin 5a, zona occludens 1a and b). Chronic hyperglycemia also decreased brain cell proliferation in most neurogenic niches throughout the forebrain and the midbrain. By using a stab wound telencephalic injury model, the impact of hyperglycemia on brain repair mechanisms was investigated. Whereas the initial step of parenchymal cell proliferation was not affected by acute hyperglycemia, later proliferation of neural progenitors was significantly decreased by chronic hyperglycemia in the injured brain of fish. Taken together, these data offer new evidence highlighting the evolutionary conserved adverse effects of hyperglycemia on neurogenesis and brain healing in zebrafish. In addition, our study reinforces the utility of zebrafish as a robust model for studying the effects of metabolic disorders on the central nervous system. J. Comp. Neurol. 525:442–458, 2017. © 2016 Wiley Periodicals, Inc.     

Organization of alpha‐transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems

We employed an anti‐transducin antibody (Gαt‐S), in combination with other markers, to characterize the Gαt‐S‐immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt‐S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt‐S‐ and opsin‐ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García‐Fernández et al. (Cell and Tissue Research, 288, 267–278, 1997). Singly Gαt‐S‐ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt‐S‐ir fibers. The Gαt‐S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson‐Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164–E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt‐S‐ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5‐HT) or GABA do not co‐localize with Gαt‐S‐ir neurons although codistribution of fibers was observed. Codistribution of Gαt‐S‐ir fibers and isolectin‐labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal‐projecting Gαt‐S‐ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt‐S‐ir neurons, suggesting they might mediate non‐visual modulation by light in many systems.     

Cell‐specific and developmental expression of lectican‐cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015. © 2014 Wiley Periodicals, Inc.     

Expression profile of N‐cadherin and protocadherin‐19 in postnatal mouse limbic structures

Cadherins are a superfamily of calcium‐dependent cell adhesion molecules that are involved in brain development and organization. Previous genetic studies revealed that mutations in protocadherin‐19 (Pcdh19) lead to an epilepsy syndrome with a variable degree of cognitive disability. Seizure origins are located in the frontotemporal and limbic structures. Expression studies of Pcdh19 in mouse confirmed a widespread presence during brain development while the function and the pathogenesis of Pcdh19 are still unknown in mammals. The neuronal cadherin (N‐cadherin; Ncdh) is known for its important role in neurulation, brain development and regulation of synaptic function. Studies in zebrafish revealed that both cadherins can interact with each other in cell adhesion. We investigated the expression pattern of Pcdh19 and Ncdh in limbic structures at four postnatal stages of C57BL/6J mice by using double‐label in situ hybridization. Results confirm a strong expression of both, Ncdh and Pcdh19, in structures of the limbic system with overlapping expression patterns particularly within regions of the amygdala, the hippocampus and the ventral hypothalamus. A detailed analysis of the limbic system highlight clear expression boundaries between several nuclei and reveal the fine regulation of Pcdh19 and Ncdh expression during the first postnatal week. Most expression patterns of both cadherins remain constant with a few exceptions particularly between P2 and P5.     

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.     

The nucleus pretectalis principalis: A pretectal structure hidden in the mammalian thalamus

A defining feature of the amniote tecto-fugal visual pathway is a massive bilateral projection to the thalamus originating from a distinct neuronal population, tectal ganglion cells (TGCs), of the optic tectum/superior colliculus (TeO/SC). In sauropsids, the thalamic target of the tecto-fugal pathway is the nucleus rotundus thalami (Rt). TGCs axons collateralize en route to Rt to target the nucleus pretectalis principalis (PT), which in turn gives rise to bilateral projection to the TeO. In rodents, the thalamic target of these TGCs afferents is the caudal division of the pulvinar complex (PulC). No pretectal structures in receipt of TGC collaterals have been described in this group. However, Baldwin et al. (Journal of Comparative Neurology, 2011;519(6):1071–1094) reported in the squirrel a feedback projection from the PulC to the SC. Pulvino-tectal (Pul-T) cells lie at the caudal pole of the PulC, intermingled with the axonal terminals of TGCs. Here, by performing a combination of neuronal tracing, immunohistochemistry, immunofluorescence, and in situ hybridization, we characterized the pattern of projections, neurochemical profile, and genoarchitecture of Pul-T cells in the diurnal Chilean rodent Octodon degus. We found that Pul-T neurons exhibit pretectal, but not thalamic, genoarchitectonical markers, as well as hodological and neurochemical properties that match specifically those of the avian nucleus PT. Thus, we propose that Pul-T cells constitute a pretectal cell population hidden within the dorsal thalamus of mammals. Our results solve the oddity entailed by the apparent existence of a noncanonic descending sensory thalamic projection and further stress the conservative character of the tectofugal pathway.     

Cholinergic profiles in the Goettingen miniature pig (Sus scrofa domesticus) brain

Central cholinergic structures within the brain of the even‐toed hoofed Goettingen miniature domestic pig (Sus scrofa domesticus) were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) and the low‐affinity neurotrophin receptor, p75^NTR. ChAT‐immunoreactive (‐ir) perikarya were seen in the olfactory tubercle, striatum, medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis of Meynert, medial habenular nucleus, zona incerta, neurosecretory arcuate nucleus, cranial motor nuclei III and IV, Edinger‐Westphal nucleus, parabigeminal nucleus, pedunculopontine nucleus, and laterodorsal tegmental nucleus. Cholinergic ChAT‐ir neurons were also found within transitional cortical areas (insular, cingulate, and piriform cortices) and hippocampus proper. ChAT‐ir fibers were seen throughout the dentate gyrus and hippocampus, in the mediodorsal, laterodorsal, anteroventral, and parateanial thalamic nuclei, the fasciculus retroflexus of Meynert, basolateral and basomedial amygdaloid nuclei, anterior pretectal and interpeduncular nuclei, as well as select laminae of the superior colliculus. Double immunofluorescence demonstrated that virtually all ChAT‐ir basal forebrain neurons were also p75^NTR‐positive. The present findings indicate that the central cholinergic system in the miniature pig is similar to other mammalian species. Therefore, the miniature pig may be an appropriate animal model for preclinical studies of neurodegenerative diseases where the cholinergic system is compromised. J. Comp. Neurol. 525:553–573, 2017. © 2016 Wiley Periodicals, Inc.     

Isolation and characterization of melanopsin photoreceptors of atlantic salmon (Salmo salar)

Melanopsins constitute a recently described group of vertebrate opsin photoreceptors that are involved in nonvisual photoreception. Here we describe the identification of six melanopsin genes of Atlantic salmon (Salmo salar), a valuable teleost model for studying nonvisual photoreception and the basis of photoperiodism. The results show that genes belonging to two different groups, the mammalian‐like (Opn4m) and the Xenopus‐like (Opn4x) melanopsins have been duplicated in teleosts. In addition, two pairs of salmon duplicates were identified, presumably originating from the salmon lineage whole genome duplication event. The expression pattern of melanopsins was studied by in situ hybridization. The results show that Opn4m and Opn4x melanopsins are differentially expressed in the brain and retina, indicating a functional divergence. In the retina, Opn4m and Opn4x melanopsin are differentially expressed in ganglion, amacrine, and horizontal cells. In the brain, Opn4m is expressed in the dorsal thalamus and in the nucleus lateralis tuberis of the hypothalamus, which is closely connected to and involved in the regulation of pituitary function. Opn4x melanopsins are expressed in the dopaminergic, hypophysiotrophic cell population of the suporaoptic/chiasmatic nucleus and in the serotonergic cell population of the left habenula. The results suggest that melanopsin photoreceptors can be involved in signaling of photoperiodic information through multiple pathways, involving both the retina and possibly as deep‐brain photoreceptors directly transmitting photoperiodic information to the hypothalamus–pituitary axis. J. Comp. Neurol. 520:3727–3744, 2012. © 2012 Wiley Periodicals, Inc.     

Sushi repeat‐containing protein X‐linked 2: A novel phylogenetically conserved hypothalamo‐pituitary protein

Sushi repeat‐containing protein X‐linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type‐specific expression was verified by double‐labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2‐ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2‐ir cells were also found in the adenohypophysis. Light SRPX2‐ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo‐pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.     

Quercetin improves the activity of the ubiquitin‐proteasomal system in 150Q mutated huntingtin‐expressing cells but exerts detrimental effects on neuronal survivability

Quercetin, a strong free radical scavenger, is investigated for neuroprotective effects in a Neuro 2a cell line conditionally transfected with 16Q huntingtin (Htt) and 150Q Htt, which express the protein upon stimulation. Cells were protected from death by a 20‐µM dose of quercetin on the second day of Htt induction, but 30–100‐µM doses of the drug caused further toxicity in both 16Q and 150Q cells, as indicated by MTT assay and by significant reductions in the number of cells bearing neurites on the second day. A significant decrease in the number of cells containing aggregate was seen in induced 150Q cells treated with 20 µM but not for those treated with 40 or 50 µM quercetin up to 4 days of induction. Mutated Htt (mHtt)‐induced reduction in proteasomal activity of the ubiquitin‐proteasomal system (UPS) was significantly attenuated by 20 µM quercetin. However, neither mitochondrial membrane potential loss nor colocalization of 20S proteasome with mHtt aggregate was corrected by quercetin treatment. Our results imply that the neuroprotective effect of quercetin arises out of the upregulation of UPS activity, which causes a decrease in the number of mHtt aggregate‐harboring cells. The increased neurotoxicity could result from the continued association of mHtt with 20S proteasome and the failure of quercetin to correct mitochondrial membrane potential loss. These results suggest that, although quercetin at a low dose protects against mHtt‐mediated cell death, higher doses are toxic to the cells, clearly demarcating a narrow therapeutic window for this dietary flavonoid. © 2015 Wiley Periodicals, Inc.     

Micro‐RNA‐30a regulates ischemia‐induced cell death by targeting heat shock protein HSPA5 in primary cultured cortical neurons and mouse brain after stroke

Micro‐RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR‐30a protects N2A cells against ischemic injury, in part through enhancing beclin 1‐mediated autophagy. The present study explores further the involvement of miR‐30a in ischemia‐induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR‐30a level is significantly decreased in cortical neurons after 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation. Overexpression of miR‐30a aggravated the OGD‐induced neuronal cell death, whereas inhibition of miR‐30a attenuated necrosis and apoptosis as determined by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐di‐phenyl‐2H‐tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase‐3. The amount of HSPA5 protein, which is predicted to be a putative target of miR‐30a by TargetScan, could be reduced by pre‐miR‐30a, whereas it was increased by anti‐miR‐30a. Furthermore, the luciferase reporter assay confirmed that miR‐30a directly binds to the predicted 3′‐UTR target sites of the hspa5 gene. The cell injury regulated by miR‐30a in OGD‐treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase‐12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress‐induced apoptosis. In vivo, reduced miR‐30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia‐stroked mice. Downregulation of miR‐30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress‐induced apoptosis might be one of the mechanisms underlying HSPA5‐mediated neuroprotection. © 2015 Wiley Periodicals, Inc.     

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Synapsin knockdown is associated with decreased neurite outgrowth,functional synaptogenesis impairment,and fast high‐frequency neurotransmitter release

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle‐associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time‐dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn‐silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn‐silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short‐term plasticity paradigms. In addition, Syn‐silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool. © 2015 Wiley Periodicals, Inc.     

5‐hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system

The epigenetic mark 5‐hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5‐methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non‐mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478–497, 2017. © 2016 Wiley Periodicals, Inc.