Literature context: (nacalai tesque, cat# 04404-84, RRID:AB_10013361) was raised against His-GFP (fu
The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post-crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post-crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post-crossing dI1 axons in mice, using the Atoh1 enhancer-based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post-crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post-crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.
Literature context: P Nacalai Tesque Cat# 04404-26; RRID:AB_10013361 Chick anti-GFP Aves Labs Cat# G
Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. Here, through a genetic screen in Drosophila, we identify Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. Our data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium.
Literature context: lai, GF090R, RRID:AB_10013361), mouse monoclonal anti-GFP (Th
Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na+ channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Cav1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies.SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.
Literature context: acalai Tesque Cat# 04404-84; RRID:AB_10013361 Guinea pig anti-RBPMS primary a
Many brain regions contain local interneurons of distinct types. How does an interneuron type contribute to the input-output transformations of a given brain region? We addressed this question in the mouse retina by chemogenetically perturbing horizontal cells, an interneuron type providing feedback at the first visual synapse, while monitoring the light-driven spiking activity in thousands of ganglion cells, the retinal output neurons. We uncovered six reversible perturbation-induced effects in the response dynamics and response range of ganglion cells. The effects were enhancing or suppressive, occurred in different response epochs, and depended on the ganglion cell type. A computational model of the retinal circuitry reproduced all perturbation-induced effects and led us to assign specific functions to horizontal cells with respect to different ganglion cell types. Our combined experimental and theoretical work reveals how a single interneuron type can differentially shape the dynamical properties of distinct output channels of a brain region.
Literature context: USA GF090R; RRID:AB_10013361 Chicken polyclonal anti-RFP VRW
The neonatal mammal faces an array of sensory stimuli when diverse neuronal types have yet to form sensory maps. How these inputs interact with intrinsic neuronal activity to facilitate circuit assembly is not well understood. By using longitudinal calcium imaging in unanesthetized mouse pups, we show that layer I (LI) interneurons, delineated by co-expression of the 5HT3a serotonin receptor (5HT3aR) and reelin (Re), display spontaneous calcium transients with the highest degree of synchrony among cell types present in the superficial barrel cortex at postnatal day 6 (P6). 5HT3aR Re interneurons are activated by whisker stimulation during this period, and sensory deprivation induces decorrelation of their activity. Moreover, attenuation of thalamic inputs through knockdown of NMDA receptors (NMDARs) in these interneurons results in expansion of whisker responses, aberrant barrel map formation, and deficits in whisker-dependent behavior. These results indicate that recruitment of specific interneuron types during development is critical for adult somatosensory function.
Literature context: 04404-84; RRID:AB_10013361 Rabbit polyclonal anti-RFP Rock
Little is known about the organizational and functional connectivity of the corticospinal (CS) circuits that are essential for voluntary movement. Here, we map the connectivity between CS neurons in the forelimb motor and sensory cortices and various spinal interneurons, demonstrating that distinct CS-interneuron circuits control specific aspects of skilled movements. CS fibers originating in the mouse motor cortex directly synapse onto premotor interneurons, including those expressing Chx10. Lesions of the motor cortex or silencing of spinal Chx10+ interneurons produces deficits in skilled reaching. In contrast, CS neurons in the sensory cortex do not synapse directly onto premotor interneurons, and they preferentially connect to Vglut3+ spinal interneurons. Lesions to the sensory cortex or inhibition of Vglut3+ interneurons cause deficits in food pellet release movements in goal-oriented tasks. These findings reveal that CS neurons in the motor and sensory cortices differentially control skilled movements through distinct CS-spinal interneuron circuits.
Literature context: Nacalai Tesque 04404-26; RRID:AB_10013361 Mouse anti-beta galactosidase P
The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, we identified S-adenosylmethionine (SAM), a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. Our study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response.
Literature context: Tesque Cat#04404-84; RRID:AB_10013361 Chicken polyclonal anti-Nestin
Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.
Literature context: RRID:AB_10013361
Spontaneous remyelination occurs after spinal cord injury (SCI), but the extent of myelin repair and identity of the cells responsible remain incompletely understood and contentious. We assessed the cellular origin of new myelin by fate mapping platelet-derived growth factor receptor α (PDGFRα), Olig2+, and P0+ cells following contusion SCI in mice. Oligodendrocyte precursor cells (OPCs; PDGFRα+) produced oligodendrocytes responsible for de novo ensheathment of ∼30% of myelinated spinal axons at injury epicenter 3 months after SCI, demonstrating that these resident cells are a major contributor to oligodendrocyte regeneration. OPCs also produced the majority of myelinating Schwann cells in the injured spinal cord; invasion of peripheral myelinating (P0+) Schwann cells made only a limited contribution. These findings reveal that PDGFRα+ cells perform diverse roles in CNS repair, as multipotential progenitors that generate both classes of myelinating cells. This endogenous repair might be exploited as a therapeutic target for CNS trauma and disease.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to profound functional deficits, though substantial numbers of axons often survive. One possible explanation for these deficits is loss of myelin, creating conduction block at the site of injury. SCI leads to oligodendrocyte death and demyelination, and clinical trials have tested glial transplants to promote myelin repair. However, the degree and duration of myelin loss, and the extent and mechanisms of endogenous repair, have been contentious issues. Here, we use genetic fate mapping to demonstrate that spontaneous myelin repair by endogenous oligodendrocyte precursors is much more robust than previously recognized. These findings are relevant to many types of CNS pathology, raising the possibility that CNS precursors could be manipulated to repair myelin in lieu of glial transplantation.
Literature context: alog #04404-84, Nacalai Tesque; RRID:AB_10013361) at room temperature for 16 h.
Temporal lobe epilepsy (TLE) is the most frequent form of focal epilepsies and is generally associated with malfunctioning of the hippocampal formation. Recently, a preferential loss of parvalbumin (PV) neurons has been observed in the subiculum of TLE patients and in animal models of TLE. To demonstrate a possible causative role of defunct PV neurons in the generation of TLE, we permanently inhibited GABA release selectively from PV neurons of the ventral subiculum by injecting a viral vector expressing tetanus toxin light chain in male mice. Subsequently, mice were subjected to telemetric EEG recording and video monitoring. Eighty-eight percent of the mice presented clusters of spike-wave discharges (C-SWDs; 40.0 ± 9.07/month), and 64% showed spontaneous recurrent seizures (SRSs; 5.3 ± 0.83/month). Mice injected with a control vector presented with neither C-SWDs nor SRSs. No neurodegeneration was observed due to vector injection or SRS. Interestingly, mice that presented with only C-SWDs but no SRSs, developed SRSs upon injection of a subconvulsive dose of pentylenetetrazole after 6 weeks. The initial frequency of SRSs declined by ∼30% after 5 weeks. In contrast to permanent silencing of PV neurons, transient inhibition of GABA release from PV neurons through the designer receptor hM4Di selectively expressed in PV-containing neurons transiently reduced the seizure threshold of the mice but induced neither acute nor recurrent seizures. Our data demonstrate a critical role for perisomatic inhibition mediated by PV-containing interneurons, suggesting that their sustained silencing could be causally involved in the development of TLE.SIGNIFICANCE STATEMENT Development of temporal lobe epilepsy (TLE) generally takes years after an initial insult during which maladaptation of hippocampal circuitries takes place. In human TLE and in animal models of TLE, parvalbumin neurons are selectively lost in the subiculum, the major output area of the hippocampus. The present experiments demonstrate that specific and sustained inhibition of GABA release from parvalbumin-expressing interneurons (mostly basket cells) in sector CA1/subiculum is sufficient to induce hyperexcitability and spontaneous recurrent seizures in mice. As in patients with nonlesional TLE, these mice developed epilepsy without signs of neurodegeneration. The experiments highlight the importance of the potent inhibitory action mediated by parvalbumin cells in the hippocampus and identify a potential mechanism in the development of TLE.
Literature context: Â Â RRID:AB_10013361 Â Â
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.
Literature context: 04404-84; RRID:AB_10013361 Rabbit ant
How cell-type-specific physiological properties shape neuronal functions in a circuit remains poorly understood. We addressed this issue in the Drosophila mushroom body (MB), a higher olfactory circuit, where neurons belonging to distinct glomeruli in the antennal lobe feed excitation to three types of intrinsic neurons, α/β, α'/β', and γ Kenyon cells (KCs). Two-photon optogenetics and intracellular recording revealed that whereas glomerular inputs add similarly in all KCs, spikes were generated most readily in α'/β' KCs. This cell type was also the most competent in recruiting GABAergic inhibition fed back by anterior paired lateral neuron, which responded to odors either locally within a lobe or globally across all lobes depending on the strength of stimuli. Notably, as predicted from these physiological properties, α'/β' KCs had the highest odor detection speed, sensitivity, and discriminability. This enhanced discrimination required proper GABAergic inhibition. These results link cell-type-specific mechanisms and functions in the MB circuit.
Literature context: FP expression, (1:1000, Nacalai 04404â€“84, San Diego, CA), goat anti-Foxp
The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.
Literature context: clonal, 04404-84, clone GF090R, AB_100133610.05â€‰Âµg/Âµl (1:1,000; IF)Green fl
The striatum, the largest nucleus of the basal ganglia controlling motor and cognitive functions, can be characterized by a labyrinthine mosaic organization of striosome/matrix compartments. It is unclear how striosome/matrix mosaic formation is spatially and temporally controlled at the cellular level during striatal development. Here, by combining in vivo electroporation and brain slice cultures, we set up a prospective experimental system in which we differentially labeled striosome and matrix cells from the time of birth and followed their distributions and migratory behaviors. Our results showed that, at an initial stage of striosome/matrix mosaic formation, striosome cells were mostly stationary, whereas matrix cells actively migrated in multiple directions regardless of the presence of striosome cells. The mostly stationary striosome cells were still able to associate to form patchy clusters via attractive interactions. Our results suggest that the restricted migratory capability of striosome cells may allow them to cluster together only when they happen to be located in close proximity to each other and are not separated by actively migrating matrix cells. The way in which the mutidirectionally migrating matrix cells intermingle with the mostly stationary striosome cells may therefore determine the topographic features of striosomes. At later stages, the actively migrating matrix cells began to repulse the patchy clusters of striosomes, presumably enhancing the striosome cluster formation and the segregation and eventual formation of dichotomous homogeneous striosome/matrix compartments. Overall, our study reveals temporally distinct migratory behaviors of striosome/matrix cells, which may underlie the sequential steps of mosaic formation in the developing striatum. J. Comp. Neurol. 525:794-817, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: 04404-84 RRID:AB_10013361 GFP Millip
GABAergic interneurons are essential for neural circuit function, and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and, ultimately, for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders.
Literature context: o, Japan; RRID:AB_10013361) antibodie
Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta (il1b). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.
Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.
Nuclei in the central nervous system are 3D aggregates of neurons that have common physiological properties, functionalities, and connectivities. To form specific nuclei, neurons migrate from their birthplace towards the presumptive nuclear region where they change their dynamics to aggregate and rearrange into a distinct 3D structure, a process that we term nucleogenesis. Nuclei, together with the laminar structure, form the basic cytoarchitectonic unit for information processing. However, in contrast to much-studied laminar structures, the neuronal dynamics that contribute to the aggregation process to form nuclei are poorly understood. Here, we analyze nucleogenesis by observing the mouse precerebellar pontine nucleus (PN), and provide the first 4D view of nucleogenesis by tracking neuronal behaviors along the three spatial axes over time. Early- and late-born PN neurons were labeled by in utero electroporation and their behaviors on cultured brain slices were recorded by time-lapse imaging. We find that when PN neurons migrate medially into the nuclear region, many of them switch to migrate radially and laterally, to populate the dorsal and lateral PN regions, respectively. The tendency to switch to radial migration is much less in later-born neurons, whereas that to switch to lateral migration is comparable between the two groups. In contrast to the radial and mediolateral axes, very few PN neurons switch to migrate rostrocaudally. These results could thus provide a framework for understanding the mechanisms that regulate this complex yet important process.
Midbrain dopamine (MbDA) neurons are functionally heterogeneous and modulate complex functions through precisely organized anatomical groups. MbDA neurons are generated from Wnt1-expressing progenitors located in the ventral mesencephalon (vMes) during embryogenesis. However, it is unclear whether the progenitor pool is partitioned into distinct cohorts based on molecular identity and whether the timing of gene expression uniquely identifies subtypes of MbDA neurons. In this study we show that Wnt1-expressing MbDA progenitors from embryonic day (E)8.5-12.5 have dynamic molecular identities that correlate with specific spatial locations in the vMes. We also tested the hypothesis that the timing of Wnt1 expression in progenitors is related to the distribution of anatomically distinct cohorts of adult MbDA neurons using genetic inducible fate mapping (GIFM). We demonstrate that the Wnt1 lineage contributes to specific cohorts of MbDA neurons during a 7-day epoch and that the contribution to MbDA neurons predominates over other ventral Mb domains. In addition, we show that calbindin-, GIRK2-, and calretinin-expressing MbDA neuron subtypes are derived from Wnt1-expressing progenitors marked over a broad temporal window. Through GIFM and quantitative analysis we demonstrate that the Wnt1 lineage does not undergo progressive lineage restriction, which eliminates a restricted competence model of generating MbDA diversity. Interestingly, we uncover that two significant peaks of Wnt1 lineage contribution to MbDA neurons occur at E9.5 and E11.5. Collectively, our findings delineate the temporal window of MbDA neuron generation and show that lineage and timing predicts the terminal distribution pattern of MbDA neurons.
The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using genetic inducible fate mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages' contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the embryonic day 12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains.
Paternal care is rare among mammals, occurring in ≈6% of species. California mice (Peromyscus californicus) are unusual; fathers participate extensively in raising their young and display the same components of parental care as mothers, with the exception of nursing. Parenting is a complex experience, having stressful and enriching aspects. The hippocampus is sensitive to experience and responds to both stress and environmental enrichment with changes in structure and function. In rats, where females care exclusively for offspring, parenting is associated with suppressed hippocampal adult neurogenesis. Since this effect has been causally linked to lactation, it is unlikely that fathers would show a similar change. To investigate this issue, we examined adult neurogenesis in the hippocampus of California mouse fathers compared to males without pups and observed reduced adult neurogenesis. Similar effects were found in California mouse mothers. Next, we investigated whether behaviors linked to the hippocampus, namely, object recognition and novelty-suppressed feeding, were altered in fathers, and observed no substantial changes. During caregiving, suppressed adult neurogenesis does not appear to be related to changes in behaviors associated with the hippocampus, although it is possible that there are other effects on hippocampal function.
The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kgamma. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kgamma-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kgamma has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kgamma isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kgamma may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain.
Inhibitory interneurons in the hippocampus and neocortex are differentiated into several morphological and functional subtypes that innervate distinct subcellular domains of principal neurons. In the olfactory bulb (OB), odor information is processed by local neuronal circuits that include the major inhibitory interneuron, granule cells (GCs). All GCs reported to date target their inhibitory output synapses mainly to dendrites of mitral cells (MCs) and tufted cells (TCs) in the external plexiform layer (EPL). Here we identified a novel type of GC that targets output synapses selectively to the perisomatic region of MCs. In the OB of adult transgenic mice expressing green fluorescent protein (GFP) under the control of nestin gene regulatory regions, we observed cells in the granule cell layer (GCL) that have GC-like morphology and strongly express GFP (referred to as type S cells). Type S cells expressed NeuN and GAD67, molecular markers for GCs. Intracellular labeling of type S cells revealed that their dendrites did not enter the EPL, but formed branches and spines within the GCL, internal plexiform layer, and mitral cell layer. Type S cells typically had huge spines at the ends of the apical dendrites. Some of the terminal spines attached to the perisomatic region of MCs and formed dendrosomatic reciprocal synapses with a presumed granule-to-mitral inhibitory synapse and a mitral-to-granule excitatory synapse. These findings indicate the morphological differentiation of GCs into dendritic-targeting and perisomatic-targeting subsets, and suggest the functional differentiation of the GC subsets in the processing of odor information in the OB.
Tourette syndrome (TS) is an inherited developmental neuropsychiatric disorder characterized by vocal and motor tics. Multiple lines of neurophysiological evidence implicate dysfunction in the corticostriatal-thalamocortical circuits in the etiology of TS. We recently identified rare sequence variants in the Slit and Trk-like family member 1 (SLITRK1) gene associated with TS. SLITRK1, a single-pass transmembrane protein, displays similarities to the SLIT family of secreted ligands, which have roles in axonal repulsion and dendritic patterning, but its function and developmental expression remain largely unknown. Here we provide evidence that SLITRK1 has a developmentally regulated expression pattern in projection neurons of the corticostriatal-thalamocortical circuits. SLITRK1 is further enriched in the somatodendritic compartment and cytoplasmic vesicles of cortical pyramidal neurons in mouse, monkey, and human brain, observations suggestive of an evolutionarily conserved function in mammals. SLITRK1 is transiently expressed in the striosomal/patch compartment of the mammalian striatum and moreover is associated with the direct output pathway; adult striatal expression is confined to cholinergic interneurons. These analyses demonstrate that the expression of SLITRK1 is dynamic and specifically associated with the circuits most commonly implicated in TS and related disorders, suggesting that SLITRK1 contributes to the development of corticostriatal-thalamocortical circuits.
Synapsin III is a synaptic vesicle-associated protein that is expressed in cells of the subgranular layer of the hippocampal dentate gyrus, a brain region known to sustain substantial levels of neurogenesis into adulthood. Here we tested the hypothesis that synapsin III plays a role in adult neurogenesis with synapsin III knockout and wild-type mice. Immunocytochemistry of the adult hippocampal dentate gyrus revealed that synapsin III colocalizes with markers of neural progenitor cell development (nestin, PSA-NCAM, NeuN, and Tuj1) but did not colocalize with markers of mitosis (Ki67 and PCNA). Because neurogenesis consists of a number of stages, the proliferation, survival, and differentiation of neural progenitor cells were systematically quantitated in the hippocampal dentate gyrus of adult synapsin III knockout and wild-type mice. We found a 30% decrease in proliferation and a 55% increase in survival of neural progenitor cells in synapsin III knockout mice. We also observed a 6% increase in the number of neural progenitor cells that differentiated into neurons. No difference in the volume of the dentate gyrus was observed between synapsin III knockout and wild-type mice. Collectively, our results demonstrate a novel role for synapsin III in regulating the proliferation of neural progenitor cells in the adult hippocampal dentate gyrus. These findings suggest a distinct function for this synaptic vesicle protein, in addition to its role in neurotransmission.