Effects of prenatal irradiation on behaviour and hippocampal neurogenesis in adult rats

Prenatal irradiation is known to have aversive effects on the brain development, manifested in changes in some behavioural parameters in adult individuals. The aim of our work was to assess the effect of prenatal irradiation on different forms of behaviour and on hippocampal neurogenesis in rats. Pregnant female rats were irradiated with a dose of 1 Gy of gamma rays on the 16th day of gravidity. The progeny of irradiated and control animals aged 3 months were tested in Morris water maze (MWM), open field (OF) and in elevated plus maze test (PM). The prenatal irradiation negatively influenced the short-term spatial memory in MWM in female rats, although the long-term memory was not impaired. A statistically significant increase of basic locomotor activity in OF was observed in irradiated rats. The comfort behaviour was not altered. The results of PM showed an increase of anxiety in irradiated females. The level of hippocampal neurogenesis, assessed as the number of cells labelled with 5-bromo-2-deoxyuridine in the area of gyrus dentatus, was not statistically different in irradiated rats. Our results indicate, that prenatal irradiation with a low dose of gamma-rays can affect some innate and learned forms of behaviour in adult rats. We did not confirm a relation of behavioural changes to the changes of hippocampal neurogenesis.     

Enhancement of hippocampal neurogenesis by lithium

Increasing evidence suggests that mood disorders are associated with a reduction in regional CNS volume and neuronal and glial cell atrophy or loss. Lithium, a mainstay in the treatment of mood disorders, has recently been demonstrated to robustly increase the levels of the cytoprotective B-cell lymphoma protein-2 (bcl-2) in areas of rodent brain and in cultured cells. In view of bcl-2's antiapoptotic and neurotrophic effects, the present study was undertaken to determine if lithium affects neurogenesis in the adult rodent hippocampus. Mice were chronically treated with lithium, and 5-bromo-2-deoxyuridine (BrdU) labeling of dividing cells was conducted over 12 days. Immunohistochemical analysis was undertaken 1 day after the last injection, and three-dimensional stereological cell counting revealed that lithium produced a significant 25% increase in the BrdU-labeled cells in the dentate gyrus. Double-labeling immunofluorescence studies were undertaken to co-localize BrdU-positive cells with neuron-specific nuclear protein and showed that approximately 65% of the cells were double-labeled. These results add to the growing body of evidence suggesting that mood stabilizers and antidepressants exert neurotrophic effects and may therefore be of use in the long-term treatment of other neuropsychiatric disorders.     

Modification of hippocampal circuitry by adult neurogenesis

The adult hippocampus is one of the primary neural structures involved in memory formation. In addition to synapse-specific modifications thought to encode information at the subcellular level, changes in the intrahippocampal neuro-populational activity and dynamics at the circuit-level may contribute substantively to the functional capacity of this region. Within the hippocampus, the dentate gyrus has the potential to make a preferential contribution to neural circuit modification owing to the continuous addition of new granule cell population. The integration of newborn neurons into pre-existing circuitry is hypothesized to deliver a unique processing capacity, as opposed to merely replacing dying granule cells. Recent studies have begun to assess the impact of hippocampal neurogenesis by examining the extent to which adult-born neurons participate in hippocampal networks, including when newborn neurons become engaged in ongoing network activity and how they modulate circuit dynamics via their unique intrinsic physiological properties. Understanding the contributions of adult neurogenesis to hippocampal function will provide new insight into the fundamental aspects of brain plasticity, which can be used to guide therapeutic interventions to replace neural populations damaged by disease or injury.     

Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner

Background aimsGraft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD.MethodsA GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line.ResultsThe mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 × 10⁶, intermediate, 1 × 10⁶, and high, 2 × 10⁶) with a consistent splenocyte dose (1 × 10⁶) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan–Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition.ConclusionsWe conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.     

Effects of lipopolysaccharide on 56Fe-particle radiation-induced impairment of synaptic plasticity in the mouse hippocampus

Space radiation, including high-mass, high-Z, high-energy particles (HZE; e.g. (56)Fe), represents a significant health risk for astronauts, and the central nervous system (CNS) may be a vulnerable target. HZE-particle radiation may directly affect neuronal function, or during immunological challenge, it may alter immune system-to-CNS communication. To test these hypotheses, we exposed mice to accelerated iron particles ((56)Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only) and 1 month later prepared hippocampal slices to measure the effects of radiation on neurotransmission and synaptic plasticity in CA1 neurons. In a model of immune system-to-CNS communication, these electrophysiological parameters were measured in irradiated mice additionally challenged with the peripheral immunological stressor lipopolysaccharide (LPS) injected intraperitoneally 4 h before the slice preparation. Exposure to (56)Fe particles alone increased dendritic excitability and inhibited plasticity. In control mice (0 Gy), LPS treatment also inhibited synaptic plasticity. Paradoxically, in mice exposed to 2 Gy, the LPS treatment restored synaptic plasticity to levels similar to those found in controls (0 Gy, no LPS). Our results indicate that HZE-particle radiation alters normal electrophysiological properties of the CNS and the hippocampal response to LPS.     

Intracellular magnesium blocks sodium outward currents in a voltage- and dose-dependent manner.

Tail currents through Na+ channels have been measured in inside-out patches from Xenopus laevis oocytes injected with cDNA-derived mRNA coding for the rat brain type II Na+ channel. It is shown that intracellular Mg2+ blocks outward currents in a voltage- and dose-dependent manner with a half blocking concentration between 3 and 4 mM at 0 mV and a voltage dependence of e-fold per 49 mV.     

Induced terpene accumulation in Norway spruce inhibits bark beetle colonization in a dose-dependent manner

Background

Tree-killing bark beetles (Coleoptera, Scolytinae) are among the most economically and ecologically important forest pests in the northern hemisphere. Induction of terpenoid-based oleoresin has long been considered important in conifer defense against bark beetles, but it has been difficult to demonstrate a direct correlation between terpene levels and resistance to bark beetle colonization.

Methods

To test for inhibitory effects of induced terpenes on colonization by the spruce bark beetle Ips typographus (L.) we inoculated 20 mature Norway spruce Picea abies (L.) Karsten trees with a virulent fungus associated with the beetle, Ceratocystis polonica (Siem.) C. Moreau, and investigated induced terpene levels and beetle colonization in the bark.

Results

Fungal inoculation induced very strong and highly variable terpene accumulation 35 days after inoculation. Trees with high induced terpene levels (n = 7) had only 4.9% as many beetle attacks (5.1 vs. 103.5 attacks m⁻²) and 2.6% as much gallery length (0.029 m m⁻² vs. 1.11 m m⁻²) as trees with low terpene levels (n = 6). There was a highly significant rank correlation between terpene levels at day 35 and beetle colonization in individual trees. The relationship between induced terpene levels and beetle colonization was not linear but thresholded: above a low threshold concentration of ∼100 mg terpene g⁻¹ dry phloem trees suffered only moderate beetle colonization, and above a high threshold of ∼200 mg terpene g⁻¹ dry phloem trees were virtually unattacked.

Conclusion/Significance

This is the first study demonstrating a dose-dependent relationship between induced terpenes and tree resistance to bark beetle colonization under field conditions, indicating that terpene induction may be instrumental in tree resistance. This knowledge could be useful for developing management strategies that decrease the impact of tree-killing bark beetles.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the 'sugar phosphate effect' whereas the S. lividans ppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.     

Forced running exercise attenuates hippocampal neurogenesis impairment and the neurocognitive deficits induced by whole-brain irradiation via the BDNF-mediated pathway

Cranial radiotherapy induces progressive and debilitating cognitive deficits, particularly in long-term cancer survivors, which may in part be caused by the reduction of hippocampal neurogenesis. Previous studies suggested that voluntary exercise can reduce the cognitive impairment caused by radiation therapy. However, there is no study on the effect of forced wheel exercise and little is known about the molecular mechanisms mediating the effect of exercise. In the present study, we investigated whether the forced running exercise after irradiation had the protective effects of the radiation-induced cognitive impairment. Sixty-four Male Sprague–Dawley rats received a single dose of 20 Gy or sham whole-brain irradiation (WBI), behavioral test was evaluated using open field test and Morris water maze at 2 months after irradiation. Half of the rats accepted a 3-week forced running exercise before the behavior detection. Immunofluorescence was used to evaluate the changes in hippocampal neurogenesis and Western blotting was used to assess changes in the levels of mature brain-derived neurotrophic factor (BDNF), phosphorylated tyrosine receptor kinase B (TrkB) receptor, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), calcium-calmodulin dependent kinase (CaMKII), cAMP-calcium response element binding protein (CREB) in the BDNF–pCREB signaling. We found forced running exercise significantly prevented radiation-induced cognitive deficits, ameliorated the impairment of hippocampal neurogenesis and attenuated the down-regulation of these proteins. Moreover, exercise also increased behavioral performance, hippocampal neurogenesis and elevated BDNF–pCREB signaling in non-irradiation group. These results suggest that forced running exercise offers a potentially effective treatment for radiation-induced cognitive deficits.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Aluminium interferes with hippocampal calcium signaling in a species-specific manner

Aluminium (Al) has been implicated in a number of neurodegenerative disorders and the disruption of calcium homeostasis has been proposed as a possible mechanism. To investigate ligand- and structure-specific effects of Al species, calcium imaging was used to probe the influence of five Al complexes - in comparison to inorganic Al (Al-S) - on N-methyl d-aspartate receptor (NMDAR) and voltage-dependent calcium channel (VDCC) function in hippocampal neurontos. The Al complexes utilized comprised three Al-citrate species (AlCit1-3), Al-quinate (AlQ) and Al-N-phosphonomethyliminodiacetate (AlNTAP). Our results suggest variable toxicity among the Al compounds tested: Al-S most potently affected neurons, with a full and irreversible inhibition of NMDAR and VDCC signaling at 500 μM. At all concentrations tested (10, 100, and 500 µM), all Al compounds investigated inhibited NMDA responses, however, no dose-dependency was evident. Furthermore, striking differences were noted with respect to calcium responses via VDCC activation. AlCit2 reduced calcium responses at all concentrations tested, AlQ at 10 and 100 µM, and AlNTAP at 500 µM only. In contrast, AlCit1 and AlCit3 had no significant effect. Collectively, diversely structured Al-ligand species selectively affect neuronal membrane channel function. The distinct chemical reactivity of the various Al forms reflects their unique interactions with neuronal structures and is poised to explain the diverse facets of Al toxicity.     

Co-culture with cardiomyocytes enhanced the myogenic conversion of mesenchymal stromal cells in a dose-dependent manner

To increase the accessibility of myogenic cells for cell therapy in the infarcted heart, we identified conditions to improve the reproducible conversion of bone marrow mesenchymal stromal cells (BMSCs) into myogenic cells. Such cells may permit functional regeneration following a myocardial infarction. BMSCs derived from green fluorescent protein (GFP) transgenic rats were co-cultured with neonatal rat cardiomyocytes (1:1, 1:10, 1:20, and 1:40 ratios) for 7 days. Some BMSCs contracted synchronously with the neonatal cardiomyocytes, and exhibited action potentials that were confirmed with current clamp recordings. The myogenic phenotype of the BMSCs was confirmed by immunohistochemical staining and flow cytometry (antibodies against cardiac specific α-sarcomeric actinin, Troponin I, MEF-2C). An increase in the number of BMSCs expressing cardiac markers correlated with increasing numbers of neonatal cardiomyocytes in the culture. When BMSCs were co-cultured with DiI-labeled neonatal cardiomyocytes, a small percentage of GFP/DiI/Troponin I triple-positive cells were observed after 7 days. This type of myogenic conversion increased nearly twofold when BMSCs were co-cultured with apoptotic (TNF-α-treated) cardiomyocytes. BMSCs co-cultured with cardiomyocytes acquired a functional myogenic phenotype in a dose-dependent manner. Myogenic conversion increased when the BMSCs were cultured with apoptotic cells.     

Differential regulation of antigen-specific CD8+ T cell responses by IL-12p40 in a dose-dependent manner

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 x 10(4) PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 x 10(8) PFU) caused significant suppression of CD8(+) T cell responses. In addition, the enhancement and suppression of OVA-specific CD8(+) T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8(+) T cell response by IL-12p40 was still observed in IL-12Rbeta2 knockout (IL-12Rbeta2KO), but not in IL-12Rbeta1 knockout (IL-12Rbeta1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rbeta1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.     

Acetaldehyde alters Ca2+-release channel gating and muscle contraction in a dose-dependent manner

We studied whether acetaldehyde, which is produced by alcohol consumption, impacts ryanodine receptor (RyR) activity and muscle force. Exposure to 50–200 µM acetaldehyde enhanced channel activity of frog RyR and rabbit RyR1 incorporated into lipid bilayers. An increase in acetaldehyde to 1 mM modified channel activity in a time-dependent manner, with a brief activation and then inhibition. Application of 200 µM acetaldehyde to frog fibers increased twitch tension. The maximum rate of rise of tetanus tension was accelerated to 1.5 and 1.74 times the control rate on exposure of fibers to 50 and 200 µM acetaldehyde, respectively. Fluorescence monitoring with fluo 3 demonstrated that 200–400 µM acetaldehyde induced Ca²⁺ release from the sarcoplasmic reticulum (SR) in frog muscles. Acetaldehyde at 1 mM inhibited twitch tension by 12%, with an increased relaxation time after a small, transient twitch potentiation. These results suggest that moderate concentrations of acetaldehyde can elicit Ca²⁺ release from the SR by increasing the open probability of the RyR channel, resulting in increased tension. However, the effects of acetaldehyde at clinical doses (1–30 µM) are unlikely to mediate alcohol-induced acute muscle dysfunction. ryanodine receptor; single-channel current; fluo 3 fluorescence; calcium ion release; calcium ion uptake     

Learning the same motor task twice impairs its retention in a time- and dose-dependent manner

Anterograde interference emerges when two differing tasks are learned in close temporal proximity, an effect repeatedly attributed to a competition between differing task memories. However, recent development alternatively suggests that initial learning may trigger a refractory period that occludes neuroplasticity and impairs subsequent learning, consequently mediating interference independently of memory competition. Accordingly, this study tested the hypothesis that interference can emerge when the same motor task is being learned twice, that is when competition between memories is prevented. In a first experiment, the inter-session interval (ISI) between two identical motor learning sessions was manipulated to be 2 min, 1 h or 24 h. Results revealed that retention of the second session was impaired as compared to the first one when the ISI was 2 min but not when it was 1 h or 24 h, indicating a time-dependent process. Results from a second experiment replicated those of the first one and revealed that adding a third motor learning session with a 2 min ISI further impaired retention, indicating a dose-dependent process. Results from a third experiment revealed that the retention impairments did not take place when a learning session was preceded by simple rehearsal of the motor task without concurrent learning, thus ruling out fatigue and confirming that retention is impaired specifically when preceded by a learning session. Altogether, the present results suggest that competing memories is not the sole mechanism mediating anterograde interference and introduce the possibility that a time- and dose-dependent refractory period—independent of fatigue—also contributes to its emergence. One possibility is that learning transiently perturbs the homeostasis of learning-related neuronal substrates. Introducing additional learning when homeostasis is still perturbed may not only impair performance improvements, but also memory formation.