Neurons In Mature Brain Research Articles

Aberrant cerebellar Purkinje cell function repaired in vivo by fusion with infiltrating bone marrow-derived cells

Bone marrow-derived cells are known to infiltrate the adult brain and fuse with cerebellar Purkinje cells. Histological observations that such heterotypic cell fusion events are substantially more frequent following cerebellar injury suggest they could have a role in the protection of mature brain neurons. To date, the possibility that cell fusion can preserve or restore the structure and function of adult brain neurons has not been directly addressed; indeed, though frequently suggested, the possibility of benefit has always been rather speculative. Here we report, for the first time, that fusion of a bone marrow-derived cell with a neuron in vivo, in the mature brain, results in the formation of a spontaneously firing neuron. Notably, we also provide evidence supporting the concept that heterotypic cell fusion acts as a biological mechanism to repair pathological changes in Purkinje cell structure and electrophysiology. We induced chronic central nervous system inflammation in chimeric mice expressing bone marrow cells tagged with enhanced green fluorescent protein. Subsequent in-depth histological analysis revealed significant Purkinje cell injury. In addition, there was an increased incidence of cell fusion between bone marrow-derived cells and Purkinje cells, revealed as enhanced green fluorescent protein-expressing binucleate heterokaryons. These fused cells resembled healthy Purkinje cells in their morphology, soma size, ability to synthesize the neurotransmitter gamma-aminobutyric acid, and synaptic innervation from neighbouring cells. Extracellular recording of spontaneous firing ex vivo revealed a shift in the predominant mode of firing of non-fused Purkinje cells in the context of cerebellar inflammation. By contrast, the firing patterns of fused Purkinje cells were the same as in healthy control cerebellum, indicating that fusion of bone marrow-derived cells with Purkinje cells mitigated the effects of cell injury on electrical activity. Together, our histological and electrophysiological results provide novel fundamental insights into physiological processes by which nerve cells are protected in adult life.

Biochemical properties of the sensitivity to GABAAergic ligands, Cl-/HCO3--ATPase isolated from fish (Cyprinus carpio) olfactory mucosa and brain.

This paper presents a comparative study of the roles of Cl- and HCO3- in the functioning of the GABAAR-associated Cl-/HCO3--ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl-(15-30mmoll-1), whereas the enzyme from MBNs was not significantly affected by Cl-. By contrast, HCO3-(15-30mmoll-1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl-(15mmoll-1)/HCO3-(27mmoll-1) and these activities were inhibited in the presence of picrotoxin (100μmoll-1), bumetanide (150μmoll-1), and DIDS (1000μmoll-1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~ 56kDa that binds [3H]muscimol and [3H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-32P and Mg2+, as well as Cl-/HCO3- sensitive dephosphorylation, is also associated with this 56kDa peptide. Both preparations also showed one subunit with molecular mass 56kDa that was immunoreactive with GABAAR β3 subunit. The use of a fluorescent dye for Cl- demonstrated that HCO3-(27mmoll-1) causes a twofold increase in Cl- influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO3- had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl- and HCO3- in the regulation of the Cl-/HCO3--ATPases functioning in neurons with different specializations.

Rapid and stable changes in maturation-related phenotypes of the adult hippocampal neurons by electroconvulsive treatment

Electroconvulsive therapy (ECT) is a highly effective and fast-acting treatment for depression. Despite a long history of clinical use, its mechanism of action remains poorly understood. Recently, a novel cellular mechanism of antidepressant action has been proposed: the phenotype of mature brain neurons is transformed to immature-like one by antidepressant drug treatments. We show here that electroconvulsive stimulation (ECS), an animal model of ECT, causes profound changes in maturation-related phenotypes of neurons in the hippocampal dentate gyrus of adult mice. Single ECS immediately reduced expression of mature neuronal markers in almost entire population of dentate granule cells. After ECS treatments, granule cells showed some of physiological properties characteristic of immature granule cells such as higher somatic intrinsic excitability and smaller frequency facilitation at the detate-to-CA3 synapse. The rapid downregulation of maturation markers was suppressed by antagonizing glutamate NMDA receptors, but not by perturbing the serotonergic system. While single ECS caused short-lasting effects, repeated ECS induced stable changes in the maturation-related phenotypes lasting more than 2 weeks along with enhancement of synaptic excitation of granule cells. Augmentation of synaptic inhibition or blockade of NMDA receptors after repeated ECS facilitated regaining the initial mature phenotype, suggesting a role for endogenous neuronal excitation in maintaining the altered maturation-related phenotype probably via NMDA receptor activation. These results suggest that brief neuronal activation by ECS induces “dematuration” of the mature granule cells and that enhanced endogenous excitability is likely to support maintenance of such a demature state. The global increase in neuronal excitability accompanying this process may be relevant to the high efficacy of ECT.

Involvement of PKCα and G‐protein‐coupled receptor kinase 2 in agonist‐selective desensitization of µ‐opioid receptors in mature brain neurons

The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

The Protein Kinase DYRK1A Regulates Caspase-9-Mediated Apoptosis during Retina Development

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome.

Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5

Glioblastoma multifome is the most common and most aggressive primary brain tumor with no current curative therapy. We found expression of the bZip transcription factor ATF5 in all 29 human glioblastomas and eight human and rat glioma cell lines assessed. ATF5 is not detectably expressed by mature brain neurons and astrocytes, but is expressed by reactive astrocytes. Interference with ATF5 function or expression in all glioma cell lines tested causes marked apoptotic cell death. In contrast, such manipulations do not affect survival of ATF5-expressing cultured astrocytes or of several other cell types that express this protein. In a proof-of-principle experiment, retroviral delivery of a function-blocking mutant form of ATF5 into a rat glioma model evokes death of the infected tumor cells, but not of infected brain cells outside the tumors. The widespread expression of ATF5 in glioblastomas and the selective effect of interference with ATF5 function/expression on their survival suggest that ATF5 may be an attractive target for therapeutic intervention in such tumors.

Apoptosis following peripheral sensory deafferentation in the olfactory bulb of adult zebrafish.

Removal of the olfactory organ in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. The current study investigated the potential role of apoptosis in this phenomenon. It was hypothesized that cells in the adult olfactory bulb normally undergo minimal apoptosis and that apoptosis increases upon removal of sensory stimulation. By using both the terminal transferase-mediated deoxyuridine nick-end labeling method and bis-benzimide labeling, the current study showed that, in the normal adult olfactory bulb, cells exhibiting apoptotic profiles were scarce and were localized to the outer layers of the bulb. However, in deafferented animals, there was a significant increase in the number of apoptotic cells. The apoptotic response occurred in two phases and was confined to the rostral half of the bulb. The first phase of cell death peaked at 1 hour postsurgery. These apoptotic profiles appeared to be primarily nonneuronal in nature, in that they exhibited no immunohistochemical labeling to the neuron-specific protein Hu. The second phase of cell death peaked at 24 hours and declined to normal levels by 1 week. At the 24 hour time point, only a fraction of the apoptotic cells was neuronal in nature. Thus, apoptosis of nonneuronal and neuronal elements accounts for at least part of the deafferentation-induced volume decrease in the zebrafish olfactory bulb. This model of anterograde transneuronal degeneration will be useful in elucidating the afferent signals involved in survival and maintenance of mature brain neurons.

Opposing Roles of Elk-1 and Its Brain-specific Isoform, Short Elk-1, in Nerve Growth Factor-induced PC12 Differentiation

The ternary complex factor Elk-1, a major nuclear target of extracellular signal-regulated kinases, is a strong transactivator of serum-responsive element (SRE) driven gene expression. We report here that mature brain neurons and nerve growth factor (NGF)-differentiated PC12 cells also express a second, smaller isoform of Elk-1, short Elk-1 (sElk-1). sElk-1 arises from an internal translation start site in the Elk-1 sequence, which generates a protein lacking the first 54 amino acids of the DNA-binding domain. This deletion severely compromises the ability of sElk-1 to form complexes with serum response factor on the SRE in vitro and to activate SRE reporter genes in the presence of activated Ras. Instead, sElk, but not a mutant that cannot be phosphorylated, inhibits transactivation driven by Elk-1. More pertinent to the neuronal-specific expression of sElk-1, we show it plays an opposite role to Elk-1 in potentiating NGF-driven PC12 neuronal differentiation. Overexpression of sElk-1 but not Elk-1 increases neurite extension, an effect critically linked to its phosphorylation. Interestingly, in the presence of sElk-1, Elk-1 loses its strictly nuclear localization to resemble the nuclear/cytoplasm pattern observed in the mature brain. This is blocked by mutating a normally cryptic nuclear export signal in Elk-1. These data provide new insights into molecular events underlying neuronal differentiation of PC12 cells mediated by the NGF-ERK signaling cascade.

In vivo whole-cell recording from neurons of the superior colliculus in fetal rats

In vivo whole-cell recording was made from neurons of the superior colliculus (SC) in rat fetuses which were connected with the dam by the umbilical cord. Fast action potentials could be generated by changing membrane potentials to depolarizing direction. The firing thresholds of fetal SC neurons appeared to be higher (between −50 and −35 mV) than those of brain neurons in later developmental stages. The action potentials of fetal SC neurons, which were smaller in amplitude and wider in duration as compared to mature brain neurons, revealed a small or no afterhyperpolarization. In addition to these fast action potentials, slow depolarizations of smaller amplitudes were evoked by intracellular injection of long depolarization pulses. Most neurons of the fetal SC (7/11) revealed linear current–voltage ( I– V) relations, while the remaining neurons displayed marked rectification. In some fetal SC neurons, presumed EPSPs and IPSPs occurred spontaneously. These presumed postsynaptic potentials showed temporal summation. These results suggest that rat SC neurons are functionally active even before birth, though the membrane properties remain immature.

Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.

This study examined the effect of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the induction of MHC class I-related genes in functionally mature brain neurons derived from cultures of dissociated rat hippocampal tissue. Patch clamp electrophysiology combined with single cell RT-PCR demonstrated that approximately 50% of the untreated neurons contained mRNA for MHC class I heavy chains, while, with few exceptions, the cells failed to transcribe beta2-microglobulin and TAP1/TAP2 gene transcripts. No constitutive expression of MHC class I protein was detectable by confocal laser microscopy on the surface of neurons. All neurons transcribed the alpha-chain of the interferon-type II receptor (binding IFN-gamma) along with the p55 receptor for TNF-alpha. Sustained exposure to IFN-gamma resulted in transcription of beta2-microglobulin and TAP1/TAP2 genes and MHC class I surface expression in a minor part of the neurons, but did not alter their electrophysiological activities as assessed by whole cell electrophysiology. Suppression of neuronal electric activity by the sodium channel blocker tetrodotoxin drastically increased to almost 100% IFN-gamma-mediated induction of MHC class I chains, of both TAP transporters, and of membrane expression of MHC class I protein. The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate. In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons. Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice

Abstract A major role of DNA-methyltransferase (MTase) is thought to be maintaining the DNA methyl-ation profile through DNA replication. However, previous surveys of mRNA distribution in different tissues by Northern-blot analysis have shown unexpectedly high levels of expression of DNA-MTase mRNA in adult mouse brain, which consists mostly of slowly proliferating glial and nonproliferating neuronal cells. In order to identify cells expressing the gene in the brain, we performed an in situ hybridization analysis of mature brain as well as whole embryos of different ages. In addition to various embryonic tissues with active cell proliferation such as the ventricular neurogenic layer, hair follicle epithelia, thymus and epithelia of the base of intestinal villi, almost all mature neurons in brain of adult and even aged mice expressed DNA-MTase mRNA at substantial levels. No significant expression of the gene was detected in the white matter. These findings suggest some neuron-specific biological function of DNA methylation, unrelated to DNA replication.

Characterization of a novel mRNA expressed by neurons in mature brain.

In previous studies, differential hybridization screening of an activated murine T-lymphocyte cDNA library identified an interleukin 2-responsive mRNA, designated F5, expressed in lymphoid tissues and brain only. We now report characterization of a full-length clone isolated from an adult mouse brain cDNA library. Neither the nucleic acid nor amino acid sequences demonstrated similarity to reported sequences. On Southern blotting, the protein coding sequence hybridized to genomic DNA from a variety of species. On Northern blotting, F5 mRNA was expressed in adult mouse brain, spinal cord, eye, and dorsal root ganglia but not in peripheral nerve. In situ hybridization studies demonstrated prominent expression by neurons in brain. F5 mRNA expression was undetectable in embryonic rat cerebral hemisphere and low until postnatal day 21. F5 is a novel mRNA selectively expressed by proliferating lymphocytes and mature neurons.

Ganglioside-enhanced neurite growth: evidence for a selective induction of high-molecular-weight MAP-2

Neuroblastoma cells maintained in serum-free medium exhibit a significant induction of MAP-1a and Tau proteins, but not MAP-2; the time course of these inductions is highly correlated with an increase in microtubule mass which parallels neurite growth. Bovine brain gangliosides (BBG) enhance the neurite outgrowth response of these cells. We report here that one effect of gangliosides is the selective and dramatic induction of MAP-2 expression. Our results also indicate a strong parallelism between this induction and the increase in microtubule mass which accompanies the appearance of more numerous, longer, and highly branched neurites. These observations suggest that MAP-2 induction in neuroblastoma cells may lead to a further differentiation of neurites equivalent to that observed in mature brain neurons. Finally, our results indicate that gangliosides per se are not neuritogenic factors but rather substances capable of enhancing cell-derived influences which affect the neurite outgrowth response of neuroblastoma cells and the type of MAP that they express.