Expression of the IP3R1 promoter‐driven nls‐lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the β‐galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5‐trisphosphate receptor (IP₃R1) gene promoter. IP₃R1‐nls‐lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole‐mount X‐gal‐stained cerebellum because of nuclear accumulation of the nls‐lacZ activity. Developing cerebella of 1NM13 mice showed stripe‐like X‐gal staining patterns of parasagittal Purkinje cell subsets. The X‐gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X‐gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X‐gal‐positive (transgene‐positive, Tg⁺) Purkinje cell stripes about the midline as vermal Tg1⁺, Tg2(a, b)⁺ and Tg3(a, b)⁺ stripes and hemispheric Tg4(a, b)⁺, Tg5(a, b)⁺, Tg6(a, b, c)⁺, and Tg7(a, b)⁺ stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c)⁺. The boundaries of X‐gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X‐gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays. © 2010 Wiley‐Liss, Inc.     

Components of the reelin signaling pathway are expressed in the spinal cord

The Reelin signaling pathway in the brain involves the binding of Reelin to very-low-density lipoprotein receptors (VLDLR) and apolipoprotein E receptor 2 (ApoER2). After Reelin binds the lipoprotein receptors on migrating neurons, the intracellular adaptor protein Disabled-1 (Dab1) becomes phosphorylated, ultimately resulting in the proper positioning of cortical neurons. Previous work showed that Reelin also affects the positioning of sympathetic preganglionic neurons (SPN) in the spinal cord (Yip et al. [2000] Proc Natl Acad Sci USA 97:8612-8616). We asked in the present study whether components of the Reelin signaling pathway in the brain also function to control SPN migration in developing spinal cord. Results showed that Reelin and reelin mRNA are found adjacent to migrating SPN. In addition, dab1 mRNA and protein are expressed by migrating SPN, and dab1-null mice show abnormal SPN migration similar to that seen in reeler. Finally, vldlr and apoER2 are also expressed in migrating SPN, and mice lacking both vldlr and apoER2 show aberrant SPN location that is identical to that of reeler and dab1-null mice. Because molecules known to be involved in Reelin signaling in the brain are present in the developing spinal cord, it is likely that the Reelin signaling pathways in the brain and spinal cord function similarly. The relative simplicity of the organization of the spinal cord makes it a potentially useful model system with which to study the molecular and cellular function of the Reelin signaling pathway in control of neuronal migration.     

Migration of sympathetic preganglionic neurons in the spinal cord is regulated by Reelin-dependent Dab1 tyrosine phosphorylation and CrkL

The actions of Reelin in neuronal positioning in the developing cortex and cerebellum are relayed by Src-family kinase (SFK)-mediated phosphorylation of Dab1. Biochemical studies show that after phosphorylation Dab1 binds to an adaptor protein, CrkL. Whether CrkL is important for Reelin signaling in vivo is unknown, because crkl(-/-) embryos die before cortical development is complete. In the developing spinal cord, Reelin and components of its signaling pathway, VLDLR, ApoER2, and Dab1, control the positioning of sympathetic preganglionic neurons (SPN); however, it is not known whether SFKs or Dab1 tyrosine phosphorylation is required. In the present study, we asked whether Reelin-controlled SPN migration depends on tyrosine phosphorylation of Dab1 by SFKs and whether CrkL is involved in SPN migration. To answer these questions, we examined the location of SPN in various mutant mouse embryos. Results showed that, in dab1(5F/5F) embryos, which express a nonphosphorylated mutant of Dab1, and in src(-/-)fyn(-/-) double knockout embryos, the location of SPN is identical to that of reeler. These results show that tyrosine phosphorylation of Dab1 by SFKs is required for Reelin-regulated SPN positioning. In addition, we found that SPN migration in crkl(-/-) showed a partial reeler phenotype, suggesting a partial loss of response of SPN to Reelin signaling. These results suggest a role for CrkL in the Reelin signaling pathway to control neuronal migration.     

Disabled-1 is expressed in type AII amacrine cells in the mouse retina

The organization of several laminated structures in the brain is controlled by a signaling pathway activated by Reelin, a large glycoprotein secreted by pioneer neurons in the developing brain. Reelin binds to transmembrane receptors, including VLDLR and ApoER2, and stimulates tyrosine phosphorylation of Disabled-1 (Dab1), which associates with an NPxY motif present in the cytoplasmic domain of the receptors. Disruption of reelin, dab1, or both the vldr and apoer2 genes results in similar cell positioning defects in laminated brain regions including the cerebellum, hippocampus, and cerebral cortex. Although retinal ganglion cells express reelin during development, there is no obvious disruption of cell positioning in the retina of reeler mice. Here, we examine the expression pattern of Dab1 as a first step toward understanding the function of the Reelin signaling pathway in neural retina. Immunohistochemical analysis of the adult retina revealed that Dab1 is expressed in a specific type of amacrine cell. These cells display a narrow dendritic field and they project to two distinct sublaminae within the inner plexiform layer. Dab1 co-localizes with the high-affinity glycine transporter, indicating that these amacrine cells are glycinergic. Cells that express Dab1 are surrounded by dopaminergic fibers originating from wide-field amacrine cells. These features are characteristic of type AII amacrine cells described in other mammalian species. Analysis of the retina at several stages of development revealed that Dab1 is expressed shortly after birth during the time at which AII amacrine cells extend neurites and form synaptic connections in the inner retina. This raises the possibility that the Reelin/Dab1 signaling pathway contributes to formation of intraretinal circuitry in the neural retina.     

Migration of sympathetic preganglionic neurons in the spinal cord of a C3G‐deficient mouse suggests that C3G acts in the reelin signaling pathway

Proper positioning of sympathetic preganglionic neurons (SPNs) in the spinal cord is regulated by reelin signaling. SPNs in reeler (which lacks reelin), and in mice deficient in components of the reelin signaling pathway (reelin receptors VldlR and ApoER2, the cytoplasmic adaptor protein Dab1, Src and Fyn of the Src‐family of non‐receptor protein tyrosine kinases, and CrkL) are located adjacent to the central canal instead of in the intermediolateral column (IML) of the spinal cord. Events downstream of CrkL in control of SPN migration are unclear. The present study asks whether Rap guanine nucleotide exchange factor (GEF) 1 (C3G/Rapgef1), a Ras family GEF that binds CrkL, could act downstream in the reelin signaling pathway in control of SPN migration. SPN migration was examined in a hypopmorphic C3G mutant mouse (C3G^gt/gt) by using retrograde DiI labeling and NOS immunostaining. The results showed that SPN in the C3G^gt/gt mutant migrate abnormally toward the central canal as in reeler. However, unlike reeler, levels of reelin in the C3G^gt/gt spinal cord are normal, and Dab1 immunostaining is undetectable. These results provide genetic evidence that C3G regulates SPN migration, and suggest that C3G acts downstream in the reelin signaling pathway in control of SPN migration. J. Comp. Neurol., 520:3194–3202, 2012. © 2012 Wiley Periodicals, Inc.     

Beware of your Cre‐Ation: lacZ expression impairs neuronal integrity and hippocampus‐dependent memory

Expression of the lacZ‐sequence is a widely used reporter‐tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß‐galactosidase, an analog to the mammalian senescence‐associated ß‐galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub‐populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus‐dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper‐arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc.     

Integrity of Cajal–Retzius cells in the reeler‐mouse hippocampus

Cajal–Retzius (CR) cells are early‐born glutamatergic neurons that are primarily known as the early main source of the signal protein Reelin. In the reeler mutant, the absence of Reelin causes severe defects in the radial migration of neurons, resulting in abnormal cortical layering. To date, the exact morphological properties of CR‐cells independent of Reelin are unknown. With this in view, we studied the ontogenesis, density, and distribution of CR‐cells in reeler mice that were cross‐bred with a CXCR4‐EGFP reporter mouse line, thus enabling us to clearly identify CR‐cells positions in the disorganized hippocampus of the reeler mouse. As evidenced by morphological analysis, differences were found regarding CR‐cell distribution and density: generally, we found fewer CR‐cells in the developing and adult reeler hippocampus as compared to the hippocampus of wild‐type animals (WT); however, in reeler mice, CR‐cells were much more closely associated to the hippocampal fissure (HF), resulting in relatively higher local CR‐cell densities. This higher local cell density was accompanied by stronger immunoreactivity of the CXCR4 ligand, stroma‐derived factor‐1 (SDF‐1) that is known to regulate CR‐cell positioning. Importantly, confocal microscopy indicates an integration of CR‐cells into the developing and adult hippocampal network in reeler mice, raising evidence that network integration of CR‐cells might be independent of Reelin.     

Dynamic of migration of HSV‐1 from a medullary pronociceptive centre: antinociception by overexpression of the preproenkephalin transgene

Herpes Simplex Virus type 1 (HSV‐1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV‐1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV‐1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of β‐galactosidase (β‐gal) was detected at 2, 4, 7, 10 and 14 days. Numerous β‐gal‐immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV‐1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time‐points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti‐hyperalgesic effects persisted during 4 days, decreasing after that time‐point. The present study demonstrates that selective migration of HSV‐1 should be considered in gene therapy strategies based on HSV‐1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.     

High affinity binding of Dab1 to Reelin receptors promotes normal positioning of upper layer cortical plate neurons

The positions of neurons in the neocortex, hippocampus, cerebellum and various other laminated brain regions are regulated by a signaling pathway initiated by the secreted protein Reelin and requiring the intracellular adaptor protein Dab1. Dab1 and the Reelin receptors VLDLR and ApoER2 are expressed by neurons whose migrations are coordinated by Reelin. In vitro, Dab1 binds with high affinity to the cytoplasmic tails of VLDLR and ApoER2 via its PTB domain. To test the importance of Dab1 binding to VLDLR and ApoER2, we replaced the Dab1 gene with a cDNA cassette encoding a point mutant allele, Dab1(F158V). This mutation strongly decreases Dab1 binding in vitro to peptides containing the ApoER2 or VLDLR cytoplasmic regions. Surprisingly, Dab1(F158V/F158V) homozygotes have no discernable phenotype. However, Dab1(F158V/-) hemizygous animals have a subtle phenotype in which late-generated cortical plate neurons migrate excessively into the marginal zone. Early cortical plate neurons, subplate neurons, hippocampal pyramidal cells and cerebellar Purkinje cells are positioned normally. Thus Dab(F158V) is a weak loss-of-function (hypomorphic) allele that has no detectable effect when homozygous. The phenotype of Dab1(F158V/-) hemizygotes shows that late cortical plate neurons of layers 2-3 require efficient Reelin-Dab1 signaling to prevent them entering the marginal zone. The Dab1(F158V) allele adds to a series of Dab1 alleles that demonstrates cell type-specific variation in the Reelin-Dab1 pathway.     

Both the phosphoinositide and receptor binding activities of Dab1 are required for Reelin-stimulated Dab1 tyrosine phosphorylation

Reelin-stimulated tyrosine phosphorylation of the Dab1 adaptor protein is required during brain development for Reelin-dependent neuron positioning in the cerebral cortex and various other laminated regions. Dab1 contains an amino-terminal PTB/PI domain through which it can bind to Reelin receptors and membrane phosphoinositides. The relative contributions of these binding activities were unknown. Here, we identify a mutation in the PTB domain of Dab1 that inhibits membrane localization without inhibiting receptor binding. In neurons, this mutation reduces both basal and Reelin-stimulated Dab1 tyrosine phosphorylation. In contrast, a mutation that inhibits receptor binding reduces Reelin-stimulated but not basal tyrosine phosphorylation. These results support a model in which phospholipids recruit Dab1 to membranes but do not play a direct role in relaying the Reelin signal, while direct Dab1-receptor interaction is responsible for relaying the Reelin signal but not for membrane recruitment.     

Tyrosine phosphorylation of Disabled-1 is essential for Reelin-stimulated activation of Akt and Src family kinases

Reelin is a large secreted signaling protein that is essential for proper positioning of migratory neurons during mammalian brain development. The Reelin signal is transduced into the cell by the lipoprotein receptors VLDLR and ApoER2, leading to tyrosine phosphorylation of the associated intracellular adaptor protein Disabled-1 (Dab1). Tyrosine phosphorylation of Dab1 is essential for responding to Reelin, as knock-in mice expressing a form of Dab1 that cannot be phosphorylated on tyrosine are indistinguishable from mice lacking Reelin, Reelin-receptors or Dab1. Molecular events dependent on Dab1 tyrosine phosphorylation are unknown. However, Reelin has recently been shown to activate the phosphoinositide-3-kinase (PI 3-K)-dependent kinase, Akt, as well as Src family kinases in wild type but not Dab1-/- primary embryonic neuronal cultures. Using pharmacological inhibitors and mice harboring mutant alleles of Dab1, we show here that tyrosine phosphorylation, but not the carboxyl-terminal region, of Dab1 is required for Reelin-induced activation of Akt and Src family kinases. Additionally, although Fyn is an important regulator of Dab1, Fyn deficiency does not prevent acute Reelin-induced Akt activation. Finally, whereas a number of growth factors propagate signals simultaneously through PI 3-K and mitogen-activated protein kinase (MAPK) cascades, we find Reelin does not engage the canonical MAPK cascade. These results define the first molecular events strictly dependent on Reelin-induced Dab1 tyrosine phosphorylation, and suggest that propagation of the Reelin signal is mediated by Akt, substrates of Src family kinases and/or unidentified molecules that share with these a common molecular link to phosphorylated Dab1.     

5‐HT1A and NMDA receptors interact in the rat medial septum and modulate hippocampal‐dependent spatial learning

Cholinergic and GABAergic neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) projecting to the hippocampus, constitute the septohippocampal projection, which is important for hippocampal‐dependent learning and memory. There is also evidence for an extrinsic as well as an intrinsic glutamatergic network within the MS/vDB. GABAergic and cholinergic septohippocampal neurons express the serotonergic 5‐HT_1A receptor and most likely also glutamatergic NMDA receptors. The aim of the present study was to examine whether septal 5‐HT_1A receptors are important for hippocampal‐dependent long‐term memory and whether these receptors interact with glutamatergic NMDA receptor transmission in a manner important for hippocampal‐dependent spatial memory. Intraseptal infusion of the 5‐HT_1A receptor agonist (R)‐8‐OH‐DPAT (1 or 4 μg/rat) did not affect spatial learning in the water maze task but impaired emotional memory in the passive avoidance task at the higher dose tested (4 μg/rat). While intraseptal administration of (R)‐8‐OH‐DPAT (4 μg) combined with a subthreshold dose of the NMDA receptor antagonist D‐AP5 (1 μg) only marginally affected spatial acquisition, it produced a profound impairment in spatial memory. In conclusion, septal 5‐HT_1A receptors appears to play a more prominent role in emotional than in spatial memory. Importantly, septal 5‐HT_1A and NMDA receptors appear to interact in a manner, which is particularly critical for the expression or retrieval of hippocampal‐dependent long‐term spatial memory. It is proposed that NMDA receptor hypofunction in the septal area may unmask a negative effect of 5‐HT_1A receptor activation on memory, which may be clinically relevant. © 2009 Wiley‐Liss, Inc.     

Heterotrimeric guanosine triphosphate‐binding protein‐coupled modulatory actions of motilin on K+ channels and postsynaptic γ‐aminobutyric acid receptors in mouse medial vestibular nuclear neurons

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice‐patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti‐motilin receptor antibody. Bath application of motilin (0.1 μm ) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose‐dependent manner (K_d, 0.03 μm ). The motilin action on spontaneous action potentials was blocked by apamin (100 nm ), a blocker of small‐conductance Ca²⁺‐activated K⁺ channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate‐γ‐S (1 mm ) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild‐type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ‐aminobutyric acid transporter‐Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small‐conductance Ca²⁺‐activated K⁺ channels and postsynaptic γ‐aminobutyric acid receptors through heterotrimeric guanosine triphosphate‐binding protein‐coupled receptor in a group of glutamatergic MVNs.     

Quantitative study of the developmental changes in calcium‐permeable AMPA receptor‐expressing neurons in the rat somatosensory cortex

The distribution of cells expressing calcium‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (CP‐AMPARs) in the somatosensory cortex of rats at different developmental stages was studied using a kainate‐stimulated Co²⁺‐labeling assay in a quantitative manner. The applicability of this assay for identifying CP‐AMPAR‐expressing cells was first verified using cultured rat cortical neurons by means of fluorescence Ca²⁺ imaging and pharmacological tools. Cells positively identified by the Co²⁺‐labelinig assay resided primarily in the marginal zone and subplate of young fetuses and became more widely distributed throughout the cortex as the fetus matured. The majority, >80%, of these Co²⁺‐positive cells were neurons, exhibiting immunoreactivity with the neuronal marker NeuN. The proportion of neurons that were Co²⁺‐positive increased from ≈25% to ≈60% as the rat fetus grew into adulthood. In contrast, less than 20% of nonneuronal cells were Co²⁺‐positive. Of the Co²⁺‐positive neurons, 15%–31% exhibited GABA immunoreactivity and nonpyramidal‐shaped cell bodies; these were presumably GABAergic neurons. Most of the remaining non‐GABAergic/Co²⁺‐positive neurons had pyramidal‐shaped cell bodies and were presumably excitatory principle neurons. Around 70% of GABAergic neurons in the cortex were Co²⁺‐positive. Furthermore, in the cortex of neonatal rats the Co²⁺‐positive neurons were found to be more susceptible to kainate toxicity than the Co²⁺‐negative cells. The Co²⁺‐positive neurons in the subplate of neonatal rats were more vulnerable to kainate toxicity than their counterparts in the remaining cortical areas. Together, the widespread distribution and distinct susceptibility to excitotoxicity of CP‐AMPAR‐expressing neurons suggest that they play various important roles in the development and physiology of the rat cerebral cortex. J. Comp. Neurol. 518:75–91, 2010. © 2009 Wiley‐Liss, Inc.     

Differential release of β‐NAD+ and ATP upon activation of enteric motor neurons in primate and murine colons

Background The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β‐nicotinamide adenine dinucleotide (β‐NAD⁺) and ADP‐ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. Methods High‐performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5‐HT₃ receptors (5‐HT₃R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. Key Results Nicotinic acetylcholine receptor or 5‐HT₃R agonists increased overflow of ATP and β‐NAD⁺ from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist‐evoked overflow of β‐NAD⁺, but not ATP, was inhibited by tetrodotoxin (0.5 μmol L^?1) or ω‐conotoxin GVIA (50 nmol L^?1), suggesting that β‐NAD⁺ release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. Conclusions & Inferences These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β‐NAD⁺, or its metabolites, serve as the purinergic inhibitory neurotransmitter.     

Effect of neurotrophin‐3 precursor on glutamate‐induced calcium homeostasis deregulation in rat cerebellum granule cells

Neurotrophin‐3 (NT‐3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT‐3) and the precursor (pro‐NT‐3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT‐3 and pro‐NT‐3 in this process remains unclear. The current study explores the effects of NT‐3 and pro‐NT‐3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT‐3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro‐NT‐3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Fura‐2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro‐NT‐3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]_i rise was 1.2‐fold greater in the presence of pro‐NT‐3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu‐induced changes in Са²⁺ homeostasis in the presence of pro‐NT‐3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period. © 2015 Wiley Periodicals, Inc.     

Visualizing corticotropin-releasing hormone receptor type 1 expression and neuronal connectivities in the mouse using a novel multifunctional allele

The corticotropin‐releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1‐expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1‐expressing cells with a tau‐lacZ (tZ) reporter gene. The distribution of neurons expressing β‐galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule‐binding properties of TAU with the Cre‐loxP system allowed to direct the β‐galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1‐expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase‐mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail. J. Comp. Neurol., 520:3150–3180, 2012. © 2012 Wiley Periodicals, Inc.