SCG10 mRNA localization in the hippocampus: comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs)

SCG10 is a nerve growth factor (NGF)-inducible, neuron-specific protein whose expression is tightly correlated with axonal and/or dendritic growth. We have recently shown that the mRNA encoding SCG10 is expressed at significant levels in certain subsets of neurons in the adult rat brain, while its expression is undetectable or negligible in other non-neuronal tissues. Here we show that regional SCG10 mRNA expression in the adult mouse brain is comparable to that in the rat, however, in the hippocampus its expression profile is distinct. In the mouse, SCG10 mRNA is expressed at high levels in pyramidal cells of CA3–CA4 sub-fields of Ammon's horn and at low levels in the CA1–CA2 sub-fields, while it is found rather uniformly throughout the pyramidal cell layer of the rat hippocampus. SCG10 mRNA is not detectable in the dentate gyrus of the mouse hippocampus, although it is expressed in the rat dentate gyrus. Comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs) such as GAP-43, MAP2, α1-tubulin and stathmin suggests that dentate granule cells express a different repertoire of neuronal growth-associated genes in mouse and rat.     

Temporal profile of adenovirus-mediated E. coli lacZ gene expression in normal and postischemic gerbil hippocampus and ventricle

Abstract

A replication-defective adenoviral vector containing the E. coli lacZ gene was directly injected into normal and post-ischemic gerbil right hippocampus and lateral ventricle, and temporal profiles of the exogenous gene expression were compared. In case of ischemia, common carotid arteries (CCA) were transiently occluded for 5 minI and the adenoviral vector was administered just after the reperfusion. The animals were recovered for 8 h, 7, 3, 7 or 27 days. A small to moderate number of neural cells in the normal hippocampus expressed the gene from 7-3 days except for the cells around dentate gyrus (DG) and the needle route that began to express from 8 h of injection. Some normal hippocampal cells persisted the expression until 7 days. A moderate to large number of ventricular cells expressed the lacZ gene from 8 h to 7 days in the normal brain. On the other hand, no expression of the lacZ gene was observed in the postischemic hippocampus at 8 h including cells at DG and the needle route. Hippocampal CA 7 neurons, that were selectively lost at 7 days of reperfusion, never expressed the gene throughout the post-ischemic course. The other hippocampal cells such as CA3 and dentate granule cells that survived ischemia expressed the gene only transiently at 7 day. A robust expression of the gene persisted in the ventricular cells from 8 h to 7 days. The majority of brain cells in the hippocampus that expressed the lacZ gene was not the pyramidal neurons, but small neurons at around the pyramidal layers of DG. Some astroglial, but no microglial, cells expressed the lacZ gene in the hippocampus. The present study shows that an expression of the lacZ gene was limited in the post-ischemic gerbil hippocampus especially at the vulnerable CA 7 layer in contrast to the strong and persistent expression in the ventricular cells, and that the majority β-gal positive cells were not.the pyramidal neurons but small neurons at around the cell layer both in the control and post-ischemic gerbil hippocampus. [Neural Res 1998; 20: 689-696]     

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.     

Glucocorticoid regulation of glial responses during hippocampal neurodegeneration and regeneration

Glucocorticoids can prevent or accelerate neurodegeneration in the adult rat hippocampus. To investigate these actions of glucocorticoids, we previously cloned genes from the hippocampus. Adrenalectomy specifically increased glial fibrillary acidic protein and transforming growth factor (TGF)-β1 mRNAs in the dentate gyrus and these effects were dependent on induced apoptosis. Corticosterone treatment prevented apoptosis, and decreased glial activation and the influx of activated microglia. Since these effects are opposite to injury and neurodegeneration, we propose that they represent adaptive actions of glucocorticoids, preventing cellular defense mechanisms from overshooting. We used adrenalectomy as a model to investigate how adult granule neurons die in vivo and the effects of neurotrophic factors in protecting against apoptosis. Neurotrophin-4/5 and TGF-β1 protected granule neurons against adrenalectomy-induced apoptosis. Since neurogenesis is also greatly increased in the dentate gyrus following adrenalectomy, we compared the time course of birth and death with glial responses. TGF-β1 mRNA increased before the detection of dying cells in the dentate gyrus, which was coincident with increased proliferation in the neurogenic zone. Glucocorticoids also increased Ndrg2 mRNA in glia in the neurogenic zone; Ndrg2 is a member of a novel gene family involved in neural differentiation and synapse formation. Therefore, studying the effects of glucocorticoid manipulation on the dentate gyrus is increasing our understanding of how mature neurons die by apoptosis and the role of glia in induced apoptosis and neurogenesis. Discovering how endocrine and inflammatory responses regulate neuron birth and survival is important for developing successful neuron replacement strategies to treat neurodegenerative diseases.     

Immunocytochemical mapping of Fos protein following seizures in gerbils indicates the activation of hippocampal neurons

The expression of the proto-oncogene, c-fos, and its protein, Fos, has been shown to be a useful marker for elevated levels of neuronal activity generated in the brain following different stimuli, including seizures. Since previous studies indicated hippocampal involvement in seizure activity in gerbils, Fos immunocytochemistry was used to determine whether hippocampal neurons become activated following environmentally induced seizures in this animal. Gerbils with maximal seizures showed many Fos-immunolabeled neurons in the granule cell layer and hilus of the dentate gyrus, as well as in CA3 and CA1 of the hippocampus. These gerbils had significantly greater numbers of Fos-immunolabeled dentate granule cells than gerbils with less severe seizures or no seizures. The number of dentate granule cells and CA3 pyramidal cells with Fos immunolabeling increased in an exponential manner with increased seizure severity. Many Fos-immunolabeled neurons were found in several regions of the neo- and paleocortex and in other limbic structures including the piriform cortex, cortical amygdaloid nucleus, and arcuate nucleus of the hypothalamus. These results indicate that hippocampal neurons are activated following seizures in a genetic model, and provide further proof that the hippocampal formation is involved in the circuitry for seizures in gerbils.     

Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development.

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.     

Localized expression of BMP and GDF mRNA in the rodent brain.

Expression of BMP- and GDF-related factors within the transforming growth factor-beta (TGF-beta) superfamily was examined in the rat and mouse brain by in situ hybridization. Strong signals were obtained in neurons for GDF-1 and GDF-10. GDF-1 is expressed at postnatal day 6 in the cerebral cortex, hippocampal CA1 through CA3 neurons, while only weakly expressed by cells in the dentate gyrus. Granule cells and neurons in the polymorph layer of the dentate gyrus are GDF-1-positive, as are the majority of neurons in the cortex. GDF-10 shows a distinct pattern of expression: At P6, strong labelling was seen in the superficial layers of cortex, notably in the posterior cingulate cortex, and in CA3 and dentate gyrus. From postnatal day 21, GDF-1 expression is strong in the hippocampus, cortex, and thalamic nuclei, while GDF-10 expression becomes restricted to the granule cell layer in the dentate gyrus. In contrast, OP-1 expression is restricted throughout development to cells of the medial habenular nucleus, choroid plexus, and leptomeninges. The markedly different expression patterns of these BMPs suggest they serve separate functions in the brain.     

Synergistic induction of HSP40 and HSC70 in the mouse hippocampal neurons after cerebral ischemia and ischemic tolerance in gerbil hippocampus.

An ischemia-induced gene was screened using a differential display technique in mouse transient forebrain ischemia. One of the ischemia-responsive clones was found to encode mouse hsp40. HSP40 has a critical regulatory function in the HSC70 ATPase activity. Expression of hsp40 mRNA was low in the nonischemic mouse hippocampus, but it was significantly upregulated 4 hr after ischemia by Northern blot analysis. In situ hybridization analysis revealed hsp40 mRNA induction in the neuron. HSP40 protein expression was also enhanced in the pyramidal and dentate granular neurons from 2 to 4 days after ischemia. The temporal expression and distribution profile of HSC70 protein was similar to that of HSP40, and both proteins were colocalized in ischemic hippocampal neurons. In the gerbil transient forebrain ischemia model, both HSP40 and HSC70 proteins were expressed strongly in ischemia-resistant CA3 neurons and dentate granule cells 1 day after 5 min ischemia, but were not expressed in vulnerable CA1 neurons. However, both proteins were in parallel expressed in the tolerance-acquired CA1 neurons. Based on the current observation that both HSP40 and HSC70 proteins were synergistically expressed in the ischemia-resistant and tolerance-acquired neurons, cochaperone HSP40 may play a significant role against postischemic neuronal response and lead to cell survival through interaction with simultaneously induced HSC70.     

Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia–ischaemia

We have used a porcine model of global hypoxia–ischaemia to examine the mode and extent of cell damage to the newborn brain. Apoptosis and necrosis were observed in neurons and glial cells following transient cerebral hypoxic–ischaemic injury (HII) by haematoxylin and eosin staining and by in situ end labelling (ISEL). Quantitative neuropathological analysis of the cingulate gyrus, the hippocampus and the cerebellum showed that the degree of both apoptosis and necrosis increased with the severity of injury in these brain areas. The hippocampus and cerebellar cortex were particularly sensitive to HII. Furthermore, some cell types were more susceptible to a particular mode of cell death. In the cerebellum, Purkinje cells died by necrosis but never by apoptosis. In contrast, cerebellar granule cells were frequently apoptotic, but never necrotic. In the hippocampus, apoptosis occurred in the inner layer neurons of the dentate fascia and necrosis in the more mature outer layer neurons. This suggests that immature neurons may be more prone to apoptotic death while terminally differentiated neurons die by necrosis. Apoptosis but not necrosis was seen in cerebral white matter. This model may help to elucidate the factors that determine cell fate following HII and aid the development of cerebroprotective strategies.     

Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.     

The intrinsic specification of gamma-aminobutyric acid type A receptor alpha6 subunit gene expression in cerebellar granule cells.

The patterns of gamma-aminobutyric acid type A (GABAA) receptor subunit gene expression in the brain are complex. For example, mouse hippocampal dentate granule cells express many subunit genes, whereas adult cerebellar granule cells, which may share differentiation mechanisms, have a smaller compliment and uniquely express the alpha6 subunit gene. To see how the alpha6 expression component arises, i.e. if intrinsically or environmentally specified, we used a mouse line (Deltaalpha6lacZ) with a beta-galactosidase reporter inserted into the alpha6 gene. Precursor cells from postnatal day 1 Deltaalpha6lacZ cerebellum were transplanted to the adult hippocampus and cerebellum of wild-type mice; 4 weeks after transplantation, Deltaalpha6lacZ cells expressed alpha6-lacZ in the hippocampus, amygdala and cerebellum. Thus, different adult environments support both the development and maintenance of alpha6 gene expression from cerebellar granule cell precursors. Establishing alpha6 gene expression is not likely to require specific patterns of neurotransmitter innervation or other factors present only in the developing brain; instead, alpha6 expression can be timed and maintained autonomously.     

Enhanced neurogenesis in the rodent hippocampus following traumatic brain injury

Recent studies have shown that neurogenesis in the dentate gyrus of the rodent hippocampus continues throughout life. Several physiological and pathological conditions have been reported to alter the rate of progenitor cell division resulting in the increased production of mature granule neurons. Excitotoxic and mechanical lesions of the granule cell layer also stimulate the proliferation of precursor cells suggesting that the death of pre-existing granule neurons may act as a trigger for enhanced neurogenesis. Hippocampal pyramidal neurons, and to a lesser extent granule neurons, have been reported to die as a result of traumatic brain injury in rodents. To determine if the proliferation of precursor cells is enhanced as a result of brain injury in rodents, newly divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). Traumatic brain injury increased the production of BrdU-labeled cells in the dentate gyrus with a maximal rate observed at 3 days post-injury. These cells, a proportion of which co-localize with the immature neuronal marker TOAD-64, implanted themselves into the granule cell layer where they accumulated over time. When examined 1 month post-injury, the majority of BrdU-labeled cells co-labeled with the mature neuronal marker calbindin. These findings show that traumatic brain injury increases neurogenesis in the granule cell layer and suggests that these new cells may contribute to the function of the hippocampus.     

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.     

Distinctive population of Gfap‐expressing neural progenitors arising around the dentate notch migrate and form the granule cell layer in the developing hippocampus

In the adult hippocampus, granule cells continue to be generated from astrocyte‐like progenitors expressing glial fibrillary acidic protein (GFAP) that differ from embryonic neocortical progenitors. However, during the embryonic period, dentate granule neurons and neocortical pyramidal neurons are derived from the ventricular zone (VZ) of the pallium. Our question is when do GFAP+ progenitors of granule neurons appear in the developing hippocampus during the embryonic period, and how do they form the granule cell layer. The present analysis using Gfap‐GFP transgenic mice shows that the GFP+ distinct cell population first appears in the VZ of the medial pallium at the dorsal edge of the fimbria on embryonic day 13.5. During the perinatal period, they form a migratory stream from the VZ to the developing dentate gyrus, and establish the germinal zones in the migratory stream, and the marginal and hilar regions in the developing dentate gyrus. GFP+ cells in these regions were positive for Sox2 and Ki67, but negative for BLBP. GFP+ cells with Neurogenin2 expression were largely distributed in the VZ, whereas GFP+ cells with Tbr2 and NeuroD expressions were seen in the migratory stream and developing dentate gyrus. Prox1‐expressing GFP+ cells were restricted to the developing dentate gyrus. These results suggest that distinctive Gfap‐expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and the developing dentate gyrus where they differentiate into granule neurons, indicating that distinct astrocyte‐like neural progenitors continue to generate granule neurons, from the beginning of dentate development and throughout life. J. Comp. Neurol. 522:261–283, 2014. © 2013 Wiley Periodicals, Inc.     

Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons

New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.