Thorleif Thorlin

Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication

Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K(+) can be buffered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced.

Mu- and delta-opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors

Opioids have previously been shown to affect proliferation and differentiation in various neural cell types. In the present study, cultured rat adult hippocampal progenitors (AHPs) were shown to release β‐endorphin. Membrane preparations of AHPs were found to bind [125I]β‐endorphin, and immunoreactivity for mu‐ and delta‐opioid receptors (MORs and DORs), but not for kappa‐opioid receptors (KORs), was found on cells in culture. Both DNA content and [3H]thymidine incorporation were reduced after a 48‐h incubation with 100 µm naloxone, 10 µm naltrindole or 10 µmβ‐funaltrexamine, but not nor‐binaltorphimine, suggesting proliferative actions of endogenous opioids against MORs and DORs on AHPs. Furthermore, analysis of gene and protein expression after incubation with MOR and DOR antagonists for 48 h using RT‐PCR and Western blotting suggested decreased signalling through the mitogen‐activated protein kinase (MAPK) pathway and lowered levels of genes and proteins that are important in cell cycling. Cultures were incubated with naloxone (10 or 100 µm) for 10 days to study the effects on differentiation. This resulted in an approximately threefold increase in neurogenesis, a threefold decrease in astrogliogenesis and a 50% decrease in oligodendrogenesis. In conclusion, this study suggests that reduced signalling through MORs and DORs decreases proliferation in rat AHPs, increases the number of in vitro‐generated neurons and reduces the number of astrocytes and oligodendrocytes in culture.

Opioid-induced proliferation through the MAPK pathway in cultures of adult hippocampal progenitors

Administration of opioid agonists or antagonists has been reported to regulate proliferation or survival of neural progenitors in vivo. Here we report that beta-endorphin and selective mu-opioid receptor (MOR) and delta-opioid receptor (DOR) agonists stimulate proliferation of isolated rat adult hippocampal progenitors (AHPs). The AHPs were found to express DORs and MORs, but not kappa-opioid receptors. Incubation with beta-endorphin for 48 h increased the number of AHPs found in mitosis, the total DNA content, and the expression of proliferating cell nuclear antigen. This proliferative effect from beta-endorphin on AHPs was antagonized by naloxone. The beta-endorphin-induced proliferation was mediated through phosphorylation of extracellular signal-regulated kinases 1 and 2 and dependent on phosphatidylinositol 3-kinase and both intra- and extracellular calcium. These data suggest a role for the opioid system in the regulation of proliferation in progenitors from the adult hippocampus.

Effects of vagus nerve stimulation on rat hippocampal progenitor proliferation

Vagus nerve stimulation (VNS), used in the treatment of epilepsy, was approved recently for treatment-resistant depression. The mechanisms of action of the VNS anti-depressive effects are not yet fully elucidated. Modulation of hippocampal neurogenesis has been proposed as an important factor in depression pathogenesis. We evaluated the effects of VNS on hippocampal progenitor turnover in the adult rat brain. Rats receiving VNS at the output current of 0.75 mA VNS for 2 days showed a significant 50% increase in dentate gyrus BrdU-incorporation consistent with an increase in progenitor proliferation. Output currents of 0.5 or 1.5 mA yielded non-significant trends for increased BrdU-labeling indicating an inverted U-shaped proliferative dose response to VNS as previously reported for other VNS-induced effects. Specific analysis for progenitor survival revealed no effects by VNS on dentate gyrus BrdU-labeling. These results suggest that VNS induced an increase in the number of available progenitor cells in the adult rat dentate gyrus by a mechanism presumably involving increased progenitor proliferation.

Differential regulation of hippocampal progenitor proliferation by opioid receptor antagonists in running and non-running spontaneously hypertensive rats

Voluntary running in mice and forced treadmill running in rats have been shown to increase the amount of proliferating cells in the hippocampus. Little is known as yet about the mechanisms involved in these processes. It is well known that the endogenous opioid system is affected during running and other forms of physical exercise. In this study, we evaluated the involvement of the endogenous opioids in the regulation of hippocampal proliferation in non‐running and voluntary running rats. Nine days of wheel running was compared with non‐running in spontaneously hypertensive rats (SHR), a rat strain known to run voluntarily. On the last 2 days of the experimental period all rats received two daily injections of the opioid receptor antagonists naltrexone or naltrindole together with injections of bromodeoxyuridine to label dividing cells. Brain sections from the running rats showed approximately a five‐fold increase in newly generated cells in the hippocampus, and this increase was partly reduced by naltrexone but not by naltrindole. By contrast, both naltrexone and naltrindole increased hippocampal proliferation in non‐running rats. In non‐running rats the administration of naltrexone decreased corticosterone levels and adrenal gland weights, whereas no significant effects on these parameters could be detected for naltrindole. However, adrenal gland weights were increased in naltrexone‐ but not in naltrindole‐administered running rats. In addition, in voluntary running rats there was a three‐fold increase in the hippocampal levels of Met‐enkephalin‐Arg‐Phe compared with non‐runners, indicating an increase in opioid activity in the hippocampus during running. These data suggest an involvement of endogenous opioids in the regulation of hippocampal proliferation in non‐running rats, probably through hypothalamic–pituitary–adrenal axis modulation. During voluntary running in SHR naltrexone altered hippocampal proliferation via as yet unknown mechanisms.

5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions.

The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.

Exposure of cultured astroglial and microglial brain cells to 900 MHz microwave radiation

Abstract Thorlin, T., Rouquette, J.-M., Hamnerius, Y., Hansson, E., Persson, M., Björklund, U., Rosengren, L., Rönnbäck, L. and Persson, M. Exposure of Cultured Astroglial and Microglial Brain Cells to 900 MHz Microwave Radiation. Radiat. Res. 166, 409–421 (2006). The rapid rise in the use of mobile communications has raised concerns about health issues related to low-level microwave radiation. The head and brain are usually the most exposed targets in mobile phone users. In the brain, two types of glial cells, the astroglial and the microglial cells, are interesting in the context of biological effects from microwave exposure. These cells are widely distributed in the brain and are directly involved in the response to brain damage as well as in the development of brain cancer. The aim of the present study was to investigate whether 900 MHz radiation could affect these two different glial cell types in culture by studying markers for damage-related processes in the cells. Primary cultures enriched in astroglial cells were exposed to 900 MHz microwave radiation in a temperature-controlled exposure system at specific absorption rates (SARs) of 3 W/kg GSM modulated wave (mw) for 4, 8 and 24 h or 27 W/kg continuous wave (cw) for 24 h, and the release into the extracellular medium of the two pro-inflammatory cytokines interleukin 6 (Il6) and tumor necrosis factor-alpha (Tnfa) was analyzed. In addition, levels of the astroglial cell-specific reactive marker glial fibrillary acidic protein (Gfap), whose expression dynamics is different from that of cytokines, were measured in astroglial cultures and in astroglial cell-conditioned cell culture medium at SARs of 27 and 54 W/kg (cw) for 4 or 24 h. No significant differences could be detected for any of the parameters studied at any time and for any of the radiation characteristics. Total protein levels remained constant during the experiments. Microglial cell cultures were exposed to 900 MHz radiation at an SAR of 3 W/kg (mw) for 8 h, and Il6, Tnfa, total protein and the microglial reactivity marker ED-1 (a macrophage activation antigen) were measured. No significant differences were found. The morphology of the cultured astroglial cells and microglia was studied and appeared to be unaffected by microwave irradiation. Thus this study does not provide evidence for any effect of the microwave radiation used on damage-related factors in glial cells in culture.

Delta-opioid receptors on astroglial cells in primary culture : Mobilization of intracellular free calcium via a pertussis sensitive G protein

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.

Regulation of the glial glutamate transporter GLT-1 by glutamate and δ-opioid receptor stimulation

The excitatory effect of presynaptically released glutamate is tightly regulated and terminated by high affinity sodium‐dependent glutamate transporters. The regulation of the glial glutamate transporter GLT‐1 is potentially important in synaptic modulation. Using astroglial cultures prepared from the rat cerebral cortex, we found that the δ‐opioid receptor agonist [d‐pen2,d‐pen5]‐enkephalin decreases and glutamate increases the expression of the GLT‐1 transporter mRNA. Corresponding changes in the uptake kinetics were found after incubation for 48 h with the respective agonists when glial glutamate uptake was measured in primary astroglial cultures. The data suggest that long‐term receptor activation induces alterations in glial glutamate uptake properties.

Calculating the Current Density and Electric Field in Human Head by Multichannel Transcranial Magnetic Stimulation

In this paper, we present a designed multichannel transcranial magnetic stimulation (mTMS) system with 40 small coils. The induced current density and electric field in realistic human head model by mTMS were calculated by impedance method and the results were compared with figure-of-eight coil. The dependence of stimulation focality on the coil current direction was investigated. Properly adjusting the input current phases can improve the magnitude of the induced fields in the brain.