Neurogenesis in the adult human hippocampus.

The genesis of new cells, including neurons, in the adult human brain has not yet been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the adult human brain, in regions previously identified as neurogenic in adult rodents and monkeys. Human brain tissue was obtained postmortem from patients who had been treated with the thymidine analog, bromodeoxyuridine (BrdU), that labels DNA during the S phase. Using immunofluorescent labeling for BrdU and for one of the neuronal markers, NeuN, calbindin or neuron specific enolase (NSE), we demonstrate that new neurons, as defined by these markers, are generated from dividing progenitor cells in the dentate gyrus of adult humans. Our results further indicate that the human hippocampus retains its ability to generate neurons throughout life.

An automatic system for in vitro cell migration studies.

This paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for cell-type specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia.

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.

Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus.

In several species, including humans, the dentate granule cell layer (GCL) of the hippocampus exhibits neurogenesis throughout adult life. The ability to regulate adult neurogenesis pharmacologically may be of therapeutic value as a mechanism for replacing lost neurons. Insulin-like growth factor-I (IGF-I) is a growth-promoting peptide hormone that has been shown to have neurotrophic properties. The relationship between IGF-I and adult hippocampal neurogenesis is to date unknown. The aim of this study was to investigate the effect of the peripheral administration of IGF-I on cellular proliferation in the dentate subgranular proliferative zone, which contains neuronal progenitor cells, and on the subsequent migration and differentiation of progenitor cells within the GCL. Using bromodeoxyuridine (BrdU) labeling, we found a significant increase of BrdU-immunoreactive progenitors in the GCL after 6 d of peripheral IGF-I administration. To determine the cell fate in progenitor progeny, we characterized the colocalization of BrdU-immunolabeled cells with cell-specific markers. In animals treated with IGF-I for 20 d, BrdU-positive cells increased significantly. Furthermore, the fraction of newly generated neurons in the GCL increased, as evaluated by the neuronal markers Calbindin D(28K), microtubule-associated protein-2, and NeuN. There was no difference in the fraction of newly generated astrocytes. Thus, our results show that peripheral infusion of IGF-I increases progenitor cell proliferation and selectively induces neurogenesis in the progeny of adult neural progenitor cells. This corresponds to a 78 +/- 17% (p < 0.001) increase in the number of new neurons in IGF-I-treated animals compared with controls.

Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes.

In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.

Growth hormone increases connexin-43 expression in the cerebral cortex and hypothalamus.

Several studies indicate that systemic GH influences various brain functions. Connexin-43 forms gap junctions that mediate intercellular communication and establish the astroglial syncytium. We investigated the effects of peripheral administration of bovine GH (bGH) and recombinant human insulin-like growth factor I (rhIGF-I) on the expression of connexin-43 in the rat brain. Hypophysectomized female Sprague Dawley rats were substituted with cortisol (400 microg/kg x day) and L-T4 (10 microg/kg x day) and treated with either bGH (1 mg/kg x day) or rhIGF-I (0.85 mg/kg x day) for 19 days. The abundance of connexin-43 messenger RNA (mRNA) and protein in the brainstem, cerebral cortex, hippocampus, and hypothalamus was quantified by means of ribonuclease protection assays and Western blots. Treatment with bGH increased the amounts of connexin-43 mRNA and protein in the cerebral cortex and hypothalamus. No changes were found in the brainstem or hippocampus. Infusion of rhIGF-I did not affect connexin-43 mRNA or protein levels in any of the brain regions studied. These results show that administration of bGH increases the abundance of cx43 in specific brain regions, suggesting that GH may influence gap junction formation and thereby intercellular communication in the brain.

Gender and strain influence on neurogenesis in dentate gyrus of young rats.

To investigate whether rat hippocampal neurogenesis varies with strain and gender, the authors examined proliferating progenitor cells and their progeny in young male and female Sprague-Dawley (SD) and spontaneously hypertensive rats (SHR) using the thymidine analog bromodeoxyuridine (BrdU) combined with immunohistochemistry for the neuronal marker Calbindin D28k and glial fibrillary acidic protein. Rats were given 7 consecutive daily BrdU injections and were killed 1 day or 4 weeks later to allow for discrimination between proliferation and cell survival. Stereologic analysis of the numbers of BrdU-immunoreactive cells in the dentate gyrus revealed both a strain difference with significantly higher cell proliferation and net neurogenesis in SHR than in SD and a gender difference with males from both strains producing significantly more cells than their female counterparts. Whereas the number of progenitors four weeks after BrdU injections was still significantly greater in male than in female SHRs, resulting in a greater net neurogenesis in the male, the number of BrdU-immunoreactive cells did not differ between male and female SD rats, suggesting a greater survival of newly generated cells in the dentate gyrus in female than in male SD rats. No sex or strain difference was observed in the relative ratio of neurogenesis and gliogenesis.

Regulation of the glial glutamate transporter GLT-1 by glutamate and delta-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 delta-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.

Mu and delta opiate receptors in neuronal and astroglial primary cultures from various regions of the brain--coupling with adenylate cyclase, localisation on the same neurones and association with dopamine (D1) receptor adenylate cyclase.

Primary cultures, enriched in neurones or astroglial cells, from three phylogenetically different regions of the brain of the rat, the cerebral cortex, the striatum and the brain stem, were used to investigate the presence of opiate receptors, coupled to adenylate cyclase. Morphine was used as a mu-receptor agonist and [D-Ala2, D-Leu5]-enkephalin (DADLE) was used as a delta-receptor agonist. In the neuronal cultures, both ligands inhibited the prostaglandin (PG)E1-stimulated intracellular accumulation of cyclic AMP dose-dependently, with the most prominent effects seen in the cultures of striatum and with DADLE being more potent than morphine. The opiate receptor antagonist, naloxone reversed the effects. Morphine and DADLE, added together, inhibited the PGE1-stimulated accumulation of cyclic AMP, less than the sum of the effects of each drug. Therefore, it might be that these opioid receptors are localized together on the same neurone. Striatal neurones contained dopamine receptors coupled to cyclic AMP, as second messenger. It was shown that the D1 (dopamine) receptor-stimulated activity of adenylate cyclase was inhibited by the mu and delta opioid receptor ligands. Thus, interactions at the level of adenylate cyclase seem to exist between D1, mu and delta opiate receptors. In the astroglial enriched cultures, DADLE inhibited the PGE1-induced accumulation of cyclic AMP, however, with a less prominent effect in the brain stem cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Opiate receptors in neuronal primary cultures.

The occurrence and characteristics of mu-, delta- and kappa-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of cAMP in neuronal enriched primary cultures from the cerebral cortex of foetal rats. Morphine or [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) were used as mu-receptor agonists. [D-Ala2,D-Leu5]-Enkephalin (DADLE) or [D-Pen2,D-Pen5]-enkephalin (DPDPE) were used as delta-receptor agonists and dynorphin 1-13 (Dyn) or U-50,488H were used as kappa-receptor agonists. In the presence of 10(-8)-10(-5) M morphine or 10(-8)-10(-5) M DAGO, there was a dose-related inhibition of the 10(-5) M forskolin-stimulated accumulation of cAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10(-9)-10(-6) M DADLE or 10(-9)-10(-6) M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of cAMP and a similar result was obtained in the presence of 10(-9)-10(-5) M Dyn or 10(-9)-10(-5) M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express mu-, delta- and kappa-receptors, that inhibit the forskolin-stimulated accumulation of cAMP. Administration of 10(-5) M morphine and 10(-6) M DADLE or 10(-6) M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of cAMP, indicating the presence of both mu- and delta-receptors on the same population of cells.

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.

GABA induces Ca2+ transients in astrocytes.

By using the Ca(2+)-sensitive indictor Fura-2/AM, the cytosolic Ca2+ levels [Ca2+]i were measured in type 1 astrocytes in rat cortical astroglial primary cultures, after stimulation with GABA, muscimol (GABAA agonist), or baclofen (GABAB agonist). We report the first evidence that stimulation of both GABAA and GABAB receptors evokes Ca2+ transients in type I astrocytes. Two types of Ca2+ responses were seen: the single-phase curve, which was the most common, and the biphasic, which consisted of an initial rise that persisted at the maximal or submaximal level. Both types of Ca2+ responses appeared with some latency. The responses were obtained in astrocytes grown for 12-16 days in culture and the response frequencies for all three agonists were 18% of the total number of examined cells. However, when the astrocytes were grown in a mixed astroglial/neuronal culture the response frequencies for all three agonists increased to 35% of the total number of examined cells. In some cells, the responses after GABA stimulation were blocked to baseline levels after exposure to bicuculline (GABAA antagonist). In other cells, bicuculline only slightly reduced the GABA-evoked responses, and the addition of phaclofen (GABAB antagonist) did not potentiate this partial inhibition. However, the muscimol-evoked rises in [Ca2+]i were completely inhibited after exposure to bicuculline, while the responses after baclofen could only be partly blocked by phaclofen. GABA evoked rises in [Ca2+]i which alternatively were inhibited (mostly) or persisted in Ca(2+)-free buffer. The rises in [Ca2+]i persisted, but were reduced, in Ca(2+)-free buffer after stimulation with muscimol, but were inhibited after baclofen stimulation. The GABA uptake blockers guvacine, 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol and nipecotic acid were also able to reduce the GABA-evoked rises in [Ca2+]i. However, the L-type Ca2+ channel antagonist nifedipine failed to influence on the GABA-evoked Ca2+ transients. The results suggest that type 1 astrocytes in primary culture express GABA receptors which can elevate [Ca2+]i directly or indirectly via Ca2+ channels and/or via release from internal Ca2+ stores. The results also suggest that GABA can have intracellular Ca(2+)-mobilizing sites since the GABA-evoked responses were reduced after incubation with GABA uptake blockers.

Kappa-opioid receptors on astrocytes stimulate L-type Ca2+ channels.

Cultured astrocytes from the cerebral cortex of the rat respond to opioid kappa-receptor stimulation with a substantial elevation of the cytoplasmic free calcium, visualized through the use of the fluorescent calcium indicator Fura-2. The stimulation of kappa-receptors with U-50488H increases the level of calcium through a dose-related stimulatory effect on the transmembrane calcium influx. The kappa-receptor stimulation was completely blocked by the selective kappa-receptor blocker nor-binaltorphimine. Furthermore, the transmembrane calcium influx was completely blocked by nifedipine, indicating the involvement of L-type channels. The presence of L-type channels was verified by stimulation of L-type channels with Bay K8644. The effects of Bay K8644 were completely blocked by nifedipine. L-type channel-coupled kappa-receptors on astrocytes might represent a novel mechanism contributing to the depressant action of opioids on synaptic transmission via decreasing the availability of extracellular calcium necessary for presynaptic transmitter release.

Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions.

Astrocytes are coupled via gap junctions, predominantly formed by connexin-43 proteins, into cellular networks. This coupling is important for the propagation of intercellular calcium waves and for the spatial buffering of K+. Using the scrape-loading/dye transfer technique, we studied gap junction permeability in rat astrocytes cultured from four different brain regions. The cultures were shown to display regional heterogeneity with the following ranking of the gap junction coupling strengths: hippocampus = hypothalamus > cerebral cortex = brain stem. Similar relative patterns were found in connexin-43 messenger RNA and protein levels using solution hybridization/RNase protection assay and western blots, respectively. The percentages of the propagation area of mechanically induced intercellular calcium waves for cortical, brain stem and hypothalamic astrocytes compared with hippocampal astrocytes were approximately 77, 42, and 52, respectively. Thus, the extent of calcium wave propagation was due to more than just gap junctional permeability as highly coupled hypothalamic astrocytes displayed relatively small calcium wave propagation areas. Incubation with 5-hydroxytryptamine decreased and incubation with glutamate increased the calcium wave propagation area in hippocampal (67% and 170% of the control, respectively) and in cortical astrocytes (82% and 163% of the control, respectively). Contrary to hippocampal and cortical astrocytes, the calcium wave propagation in brain stem astrocytes was increased by 5-hydroxytryptamine incubation (158% of control), while in hypothalamic astrocytes, no significant effects were seen. Similar effects from 5-hydroxytryptamine or glutamate treatments were observed on dye transfer, indicating an effect on the junctional coupling strength. These results demonstrate a strong relationship between connexin-43 messenger RNA levels, protein expression, and gap junction permeability among astroglial cells. Furthermore, our results suggest heterogeneity among astroglial cells from different brain regions in intercellular calcium signaling and in its differential modulation by neurotransmitters, probably reflecting functional requirements in various brain regions.

Altered amounts of G-protein mRNA and cAMP accumulation after long-term opioid receptor stimulation of neurons in primary culture from the rat cerebral cortex.

Primary neuronal enriched cultures were incubated with mu (morphine, 10(-5) M), delta (DPDPE, 10(-6) M) and kappa (U-50,488H, 10(-5) M) receptor agonists for 5 days, respectively. Thereafter the acute inhibitory actions of mu, delta or kappa receptor agonists on forskolin stimulated cAMP accumulation was assayed. The effect of long term opioid treatment on the steady-state level of G-protein mRNA (G alpha s, G alpha i-1 and G alpha i-2) was analyzed using an RNAase protection hybridization assay. Incubation for 5 days with kappa receptor agonist resulted in an attenuated ability to decrease the accumulation of cAMP by kappa receptors, as well as mu and delta receptors, which was also observed after 5 days of incubation with the delta receptor agonist. Furthermore, the adenylate cyclase responsiveness to forskolin stimulation was markedly reduced in cultures treated with either delta or kappa receptor agonists. Five days of incubation with kappa receptor agonist resulted in an increase in the levels of G alpha s and G alpha i-2 mRNAs. No effects on the amounts of G alpha s mRNA, G alpha i-1 mRNA or G alpha i-2 mRNA were detected after 5 days of delta receptor stimulation. On the other hand, 5 days of mu receptor stimulation decreased the amounts of G alpha s, G alpha i-1 and G alpha i-2 mRNA. Incubation with kappa receptor agonist for 24 h resulted in a significant decrease in the forskolin-stimulated accumulation of cAMP. The stimulatory effect of forskolin was further decreased after 3 days incubation with kappa receptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of G-PROTEIN mRNA abundancy and cAMP accumulation after long-term opioid incubation in primary cultures of astroglia from the rat cerebral cortex.

Primary astroglial cultures were incubated with delta (10(-6) M DPDPE) or kappa (10(-5) M U-50,488H) receptor agonists for 5 days. Thereafter, the acute inhibitory actions of delta or kappa receptor agonists on forskolin stimulated cAMP accumulation were assayed. The G alpha s, G alpha i-1 and G alpha i-2 mRNA levels were quantified after 5 days of either delta or kappa receptor agonist treatment using a solution hybridization, RNase protection assay. Pronounced effects were observed after 5 days of kappa receptor agonist [10(-5) M U-50,488H] incubation. This treatment resulted in an attenuation in the acute inhibitory action of delta and kappa receptor agonists. Furthermore, a decreased stimulatory action of forskolin was seen. Similar effects were also seen after delta receptor stimulation. We also investigated the effects after 24 h and 3 days of incubation with the kappa receptor agonist (10(-5) M) U-50,488H. The 24 h incubation resulted in a decreased sensitivity to the acute inhibitory action of delta and kappa receptor agonists in the astroglial cultures. This effect was further accentuated after the 3 days of incubation with 10(-5) M U-50,488H. No significant change was seen in the basal accumulation of cAMP after incubation with the kappa agonist U-50,488H. However, after 5 days of incubation with the delta agonist DPDPE, a significantly increased basal accumulation of cAMP was seen in the astroglial cultures. After 5 days of delta or kappa agonist incubation, an increase in G alpha s mRNA level and a decrease in G alpha i-2 mRNA level was seen compared with controls. No statistically significant alterations in the amount of G alpha i-1 mRNA were seen. The data obtained in the present study indicate that the effects of long-term opioid treatment alters the sensitivity of glial cell opioid receptors. Furthermore, long term opioid treatment induces alterations in glial G-protein mRNA levels.

Distribution and development of G alpha i-2 mRNA in the rat cerebral cortex investigated with in situ hybridization and RNAse protection assay.

The distribution of the G alpha i-2 mRNA was investigated with in situ hybridization on tissue sections of the rat cerebral cortex. A non-radioactive method based on digoxygenin labelled cRNA was used for in situ hybridization. The G alpha i-2 mRNA was shown to be present predominantly in large neuronal shaped cells in laminae II-III. The ontogenic development of the expression of G alpha i-2 mRNA in the rat cerebral cortex was quantified using a solution hybridization RNAse protection assay. The abundancy of G alpha i-2 mRNA was shown to increase significantly from embryonic day 16 to adult age. The amount of G alpha i-2 mRNA in adult cerebral cortex of the rat brain was 23 amol/micrograms RNA. The distribution and ontogenic development of G alpha i-2 mRNA are discussed.

Connexin43 mRNA and protein expression during postnatal development of defined brain regions.

Connexin43 (cx43) mRNA and protein were quantified in seven brain regions using RNase protection assays and Western blotting from postnatal day 3, 10, and 17, and adult Sprague-Dawley rats. Increases in cx43 mRNA abundance preceded strong increases in cx43 protein in the cerebral cortex, hippocampus, hypothalamus, and striatum, whereas smaller postnatal increases were seen in the cerebellum, brain stem, and olfactory bulbs.

Volume regulation of single astroglial cells in primary culture.

Relative volume variations in cultured astrocytes were examined by microspectrofluorimetry after loading the cells with the highly fluorescent intracellular probes 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF/AM) or fura-2/AM. At their isosbestic points, 450 nm and 358 nm, respectively, the probes were ion-insensitive and the fluorescent signals emitted related only to the intracellular dye concentration. By varying the excitation wavelengths, changes in intracellular pH or Ca2+ transients could be recorded simultaneously with the relative volume variations of the individual cells. After exposure to a hypotonic buffer, type 1 astrocytes swelled within 30 s and subsequently underwent regulatory volume decrease (RVD). When exposed to a hypertonic buffer, the astrocytes shrunk and exhibited regulatory volume increase (RVI). One mM glutamate induced an increase in astrocyte volume in 60 sec and evoked cytosolic Ca2+ transients but did not change intracellular pH.

[D-Pen2,5]enkephalin and glutamate regulate the expression of delta-opioid receptors in rat cortical astrocytes.

Recent work from our and other laboratories have shown that glial cells in culture express opioid receptors. In the present study we have analyzed the regulation of delta-opioid receptor mRNA and the regulation of delta-opioid receptor activated astroglial Ca2+ responses in primary cultures from the rat cerebral cortex. Astroglial cultures were incubated with glutamate (Glu) or [D-Pen2,5]enkephalin (DPDPE) for 48 h, and delta-opioid receptor mRNA levels were analyzed using a solution hybridization RNase protection assay. Our results suggest that incubation in Glu or DPDPE upregulates the abundance of delta-opioid receptor mRNA in a dose dependent way. Glu incubated cells showed a maximum upregulation at the highest agonist concentration used (10[-5] M), whereas DPDPE was most effective at low concentrations (l0[-9] M). Furthermore, corresponding Ca2+ imaging experiments showed that incubation in Glu or DPDPE upregulated the responding frequency of delta-opioid receptor activated glial calcium fluxes from a control value of 5% to 14% and 17% responding cells, respectively.