Detection of cortical laminar architecture using manganese-enhanced MRI

Changes in manganese-enhanced MRI (MEMRI) contrast across the rodent somatosensory cortex were compared to the cortical laminae as identified by tissue histology and administration of an anatomical tracer to cortex and thalamus. Across the cortical thickness, MEMRI signal intensity was low in layer I, increased in layer II, decreased in layer III until mid-layer IV, and increased again, peaking in layer V, before decreasing through layer VI. The reeler mouse mutant was used to confirm that the cortical alternation in MEMRI contrast was related to laminar architecture. Unlike in wild-type mice, the reeler cortex showed no appreciable changes in MEMRI signal, consistent with absence of cortical laminae in histological slides. The tract tracing ability of MEMRI was used to further confirm assignments and demonstrate laminar specificity. Twelve to 16 h after stereotaxic injections of MnCl(2) to the ventroposterior thalamic nuclei, an overall increase in signal intensity was detected in primary somatosensory cortex compared to other brain regions. Maximum intensity projection images revealed a distinctly bright stripe located 600-700 microm below the pial surface, in layer IV. The data show that both systemic and tract tracing forms of MEMRI are useful for studying laminar architecture in the brain.     

Dose response and time course of manganese-enhanced magnetic resonance imaging for visual pathway tracing in vivo

Axonal tracing is useful for detecting optic nerve injury and regeneration,but many commonly used methods cannot be used to observe axoplasmic flow and synaptic transmission in vivo.Manganese(Mn2+)-enhanced magnetic resonance imaging(MEMRI) can be used for in vivo longitudinal tracing of the visual pathway.Here,we explored the dose response and time course of an intravitreal injection of Mn Cl2 for tracing the visual pathway in rabbits in vivo using MEMRI.We found that 2 m M Mn Cl2 enhanced images of the optic nerve but not the lateral geniculate body or superior colliculus,whereas at all other doses tested(5–40 m M),images of the visual pathway from the retina to the contralateral superior colliculus were significantly enhanced.The images were brightest at 24 hours,and then decreased in brightness until the end of the experiment(7 days).No signal enhancement was observed in the visual cortex at any concentration of Mn Cl2.These results suggest that MEMRI is a viable method for temporospatial tracing of the visual pathway in vivo.Signal enhancement in MEMRI depends on the dose of Mn Cl2,and the strongest signals appear 24 hours after intravitreal injection.     

Neuroaxonal ion dyshomeostasis of the normal-appearing corpus callosum in experimental autoimmune encephalomyelitis

Atrophy of the corpus callosum (CC) is a well-documented observation in clinically definite multiple sclerosis (MS) patients. One recent hypothesis for the neurodegeneration that occurs in MS is that ion dyshomeostasis leads to neuroaxonal damage. To examine whether ion dyshomeostasis occurs in the CC during MS onset, experimental autoimmune encephalomyelitis (EAE) was utilized as an animal MS model to induce autoimmunity-mediated responses. To date, in vivo investigations of neuronal ion homeostasis has not been feasible using traditional neuroscience techniques. Therefore, the current study employed an emerging MRI method, called Mn2+-enhanced MRI (MEMRI). Mn2+ dynamics is closely associated with important neuronal activity events, and is also considered to be a Ca2+ surrogate. Furthermore, when injected intracranially, Mn2+ can be used as a multisynaptic tracer. These features enable MEMRI to detect neuronal ion homeostasis within a multisynaptic circuit that is connected to the injection site. Mn2+ was injected into the visual cortex to trace the CC, and T1-weighted imaging was utilized to observe temporal changes in Mn2+-induced signals in the traced pathways. The results showed that neuroaxonal functional changes associated with ion dyshomeostasis occurred in the CC during an acute EAE attack. In addition, the pathway appeared normal, although EAE-induced immune-cell infiltration was visible around the CC. The findings suggest that ion dyshomeostasis is a major neuronal aberration underlying the deterioration of normal-appearing brain tissues in MS, supporting its involvement in neuroaxonal functioning in MS.     

Manganese-enhanced MRI: an exceptional tool in translational neuroimaging

The metal manganese is a potent magnetic resonance imaging (MRI) contrast agent that is essential in cell biology. Manganese-enhanced magnetic resonance imaging (MEMRI) is providing unique information in an ever-growing number of applications aimed at understanding the anatomy, the integration, and the function of neural circuits both in normal brain physiology as well as in translational models of brain disease. A major drawback to the use of manganese as a contrast agent, however, is its cellular toxicity. Therefore, paramount to the successful application of MEMRI is the ability to deliver Mn2+ to the site of interest using as low a dose as possible while preserving detectability by MRI. In the present work, the different approaches to MEMRI in translational neuroimaging are reviewed and challenges for future identified from a practical standpoint.     

Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics

The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca(2+)) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca(2+) dynamics in astrocytes, thereby suppressing Ca(2+) wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca(2+)-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca(2+) wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca(2+) was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca(2+) dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1-10 microM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca(2+) waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca(2+) uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca(2+) levels in cells using the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca(2+) at concentrations as low as 1 muM. Examination of ATP-stimulated changes in mitochondrial Ca(2+) indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca(2+). It is concluded that exposure of astrocytes to low concentrations of Mn(2+) results in sequestration of Ca(2+) within the mitochondria that reduces the available pool of releasable Ca(2+) within the ER, thereby inhibiting calcium wave activity.     

P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia

Overactivation of subtype P2X7 receptors can induce excitotoxic neuronal death by calcium (Ca(2+)) overload. In this study, we characterize the functional properties of P2X7 receptors using electrophysiology and Ca(2+) monitoring in primary cortical neuron cultures and in brain slices. Both electrical responses and Ca(2+) influx induced by ATP and benzoyl-ATP were reduced by Brilliant Blue G (BBG) at concentrations which specifically inhibit P2X7 receptors. In turn, oxygen-glucose deprivation (OGD) caused neuronal death that was reduced with BBG application. OGD in neuron cultures and brain slices generated an inward current, which was delayed and reduced by BBG. To assess the relevance of these in vitro findings, we used middle cerebral artery occlusion in rats as a model of transient focal cerebral ischemia to study the neuroprotective effect of BBG in vivo. Treatment with BBG (twice per day, 30 mg/kg) produced a 60% reduction in the extent of brain damage compared to treatment with vehicle alone. These results show that P2X7 purinergic receptors mediate tissue damage after OGD in neurons and following transient brain ischemia. Therefore, these receptors are a relevant molecular target for the development of new treatments to attenuate brain damage following stroke.     

In vivo characterization of a smart MRI agent that displays an inverse response to calcium concentration

Contrast agents for magnetic resonance imaging (MRI) that exhibit sensitivity toward specific ions or molecules represent a challenging but attractive direction of research. Here a Gd(3+) complex linked to an aminobis(methylenephosphonate) group for chelating Ca(2+) was synthesized and investigated. The longitudinal relaxivity (r(1)) of this complex decreases during the relaxometric titration with Ca(2+) from 5.76 to 3.57 mM(-1) s(-1) upon saturation. The r(1) is modulated by changes in the hydration number, which was confirmed by determination of the luminescence emission lifetimes of the analogous Eu(3+) complex. The initial in vivo characterization of this responsive contrast agent was performed by means of electrophysiology and MRI experiments. The investigated complex is fully biocompatible, having no observable effect on neuronal function after administration into the brain ventricles or parenchyma. Distribution studies demonstrated that the diffusivity of this agent is significantly lower compared with that of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).     

Intracellular calcium chelation with BAPTA-AM modulates ethanol-induced behavioral effects in mice

Calcium (Ca(2+)) has been characterized as one of the most ubiquitous, universal and versatile intracellular signaling molecules responsible for controlling numerous cellular processes. Ethanol-induced effects on Ca(2+) distribution and flux have been widely studied in vitro, showing that acute ethanol administration can modulate intracellular Ca(2+) concentrations in a dose dependent manner. In vivo, the relationship between Ca(2+) manipulation and the corresponding ethanol-induced behavioral effects have focused on Ca(2+) flux through voltage-gated Ca(2+) channels. The present study investigated the role of inward Ca(2+) currents in ethanol-induced psychomotor effects (stimulation and sedation) and ethanol intake. We studied the effects of the fast Ca(2+) chelator, BAPTA-AM, on ethanol-induced locomotor activity and the sedative effects of ethanol. Swiss (RjOrl) mice were pretreated with BAPTA-AM (0-10 mg/kg) 30 min before an ethanol (0-4 g/kg) challenge. Our results revealed that pretreatment with BAPTA-AM prevented locomotor stimulation produced by ethanol without altering basal locomotion. In contrast, BAPTA-AM reversed ethanol-induced hypnotic effects. In a second set of experiments, we investigated the effects of intracellular Ca(2+) chelation on ethanol intake. Following a drinking-in-the-dark methodology, male C57BL/6J mice were offered 20% v/v ethanol, tap water, or 0.1% sweetened water. The results of these experiments revealed that BAPTA-AM pretreatment (0-5 mg/kg) reduced ethanol consumption in a dose-dependent manner while leaving water and sweetened water intake unaffected. Our findings support the role of inward Ca(2+) currents in mediating different behavioral responses induced by ethanol. Our results are discussed together with data indicating that ethanol appears to be more sensitive to intracellular Ca(2+) manipulations than other psychoactive drugs.     

Effects of calmodulin and protein kinase C modulators on transient Ca2+ increase and capacitative Ca2+ entry in human platelets: relevant to pathophysiology of bipolar disorder

Disturbed intracellular calcium (Ca(2+)) homeostasis has been implicated in bipolar disorder, which mechanisms may be involved in the dysregulation of protein kinase C (PKC) and calmodulin systems. In this study, we investigated a transient intracellular Ca(2+) increase induced by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase pump (SERCA), and a capacitative Ca(2+) entry followed by addition of extracellular Ca(2+), in the presence or absence of PKC/calmodulin modulators in the platelets of healthy subjects in order to elucidate the role of SERCA in Ca(2+) homeostasis and to assess how both PKC and calmodulin systems regulate the two Ca(2+) responses. Moreover, we also examined the thapsigargin-elicited transient Ca(2+) increase and capacitative Ca(2+) entry in patients with mood disorders. PKC and calmodulin systems have opposite regulatory effects on the transient Ca(2+) increase and capacitative Ca(2+) entry in the platelets of normal subjects. The inhibitory effect of PKC activation on capacitative Ca(2+) entry is significantly increased and the stimulatory effect of PKC inhibition is significantly decreased in bipolar disorder compared to major depressive disorder and normal controls. These results suggest the possibility that increased PKC activity may activate the inhibitory effect of capacitative Ca(2+) entry in bipolar disorder. However, this is a preliminary study using a small sample, thus further studies are needed to examine the PKC and calmodulin modulators on the capacitative Ca(2+) entry in a larger sample.     

Pixel-based criteria-oriented analysis of time-lapse Ca2+-fluorescence images

Since its inception, the analysis of time-lapse video-images acquired during Ca2+ imaging experiments using fluorescence microscopy has been progressively optimized for achieving a high temporal resolution. In contrast, the spatial resolution of the acquired images is often compromised during analysis to varying degrees by the need to draw regions of interest (ROI). We developed a strategy to analyze images at the acquired spatial resolution-pixel-by-pixel, grouping all pixels based on criteria of interest (COI) in regard to their associated fluorescence values over time and visualizing the distributions of the pixel-groups detected in a pseudo-colored map. We applied this pixel-based COI-strategy to the analysis of relative intracellular free calcium levels (Ca(i)(2+)) in attached cultured embryonic hippocampal cells under baseline and experimental conditions designed to evaluate the contribution of extracellular Ca2+ (Ca(e)(2+)) to baseline Ca(i)(2+) levels. We discovered distinct groups of Ca(e)(2+)-dependent Ca(i)(2+) regulation patterns emergent during the earliest phases of hippocampal cell differentiation, which were not limited to inter-cell differences. Thus, pixel-based COI-analysis of time-lapse images can be used to disclose distinct patterns of Ca(e)(2+)-dependent Ca(i)(2+) levels and their corresponding subcellular distributions in developing hippocampal cells. Such a strategy should be useful in studying the emergence and distribution of Ca(i)(2+) signaling at subcellular levels of resolution using fluorescence microscopy.     

Manganese inhibits NMDA receptor channel function: implications to psychiatric and cognitive effects

Humans exposed to excess levels of manganese (Mn(2+)) express psychiatric problems and deficits in attention and learning and memory. However, there is a paucity of knowledge on molecular mechanisms by which Mn(2+) produces such effects. We now report that Mn(2+) is a potent inhibitor of [(3)H]-MK-801 binding to the NMDA receptor channel in rat neuronal membrane preparations. The inhibition of [(3)H]-MK-801 to the NMDA receptor channel by Mn(2+) was activity-dependent since Mn(2+) was a more potent inhibitor in the presence of the NMDA receptor co-agonists glutamate and glycine (K(i)=35.9+/-3.1 microM) than in their absence (K(i)=157.1+/-6.5 microM). We also show that Mn(2+) is a NMDA receptor channel blocker since its inhibition of [(3)H]-MK-801 binding to the NMDA receptor channel is competitive in nature. That is, Mn(2+) significantly increased the affinity constant (K(d)) with no significant effect on the maximal number of [(3)H]-MK-801 binding sites (B(max)). Under stimulating conditions, Mn(2+) was equipotent in inhibiting [(3)H]-MK-801 binding to NMDA receptors expressed in neuronal membrane preparations from different brain regions. However, under basal, non-stimulated conditions, Mn(2+) was more potent in inhibiting NMDA receptors in the cerebellum than other brain regions. We have previously shown that chronic Mn(2+) exposure in non-human primates increases Cu(2+), but not zinc or iron concentrations in the basal ganglia [Guilarte TR, Chen M-K, McGlothan JL, Verina T, Wong DF, Zhou Y, Alexander M, Rohde CA, Syversen T, Decamp E, Koser AJ, Fritz S, Gonczi H, Anderson DW, Schneider JS. Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates. Exp Neurol 2006a;202:381-90]. Therefore, we also tested the inhibitory effects of Cu(2+) on [(3)H]-MK-801 binding to the NMDA receptor channel. The data shows that Cu(2+) in the presence of glutamate and glycine is a more potent inhibitor of the NMDA receptor than Mn(2+). Our findings suggest that the inhibitory effect of Mn(2+) and/or Cu(2+) on the NMDA receptor may produce a deficit in glutamatergic transmission in the brain of individuals exposed to excess levels of Mn(2+) and produce neurological dysfunction.     

Speciation of manganese in cells and mitochondria: a search for the proximal cause of manganese neurotoxicity

Recent studies of speciation of manganese (Mn) in brain mitochondria, neuron-like cells, and astrocytes are reviewed. No evidence is found for oxidation of Mn(2+) complexes to a Mn(3+) complex. The only evidence for any Mn(3+) complex is found in a spectrum essentially identical to that of mitochondrial manganese superoxide dismutase (MnSOD). While this does not prove that no Mn(3+) is produced in these tissues by oxidation of Mn(2+), it does suggest that formation of an active Mn(3+) complex by oxidation of Mn(2+) probably does not play as important a role in Mn toxicity as has been suggested earlier. Since these results suggest that we should look elsewhere for the proximal causes of Mn neurotoxicity, we consider the possibilities that Mn(3+) may be transported into the cell via transferrin and that Mn(2+) may inhibit Ca(2+)-activation and control of the rate of ATP production by oxidative phosphorylation.     

Inositol 1,4,5 trisphosphate receptor and chromogranin B are concentrated in different regions of the hippocampus

Calcium (Ca(2+)) release from intracellular stores plays a crucial role in many cellular functions in the brain. These intracellular signals have been shown to be transmitted within and between cells. We report a non-uniform distribution of proteins essential for Ca(2+) signaling in acutely prepared brain slice preparations and organotypic slice cultures, both made from rat hippocampus. The Type I inositol-1,4,5 trisphosphate receptor (InsP(3)R1) is the main InsP(3)R subtype in neurons. Immunohistochemistry experiments showed a prominent expression of InsP(3)R1 in the CA1 region of the hippocampus whereas the CA3 region and dentate gyrus (DG) showed only moderate immunoreactivity. In contrast, chromogranin B (CGB), a protein binding to the InsP(3)R1 on the luminal side of the endoplasmic reticular membrane was enriched in the CA3 region whereas DG and the CA1 region showed only faint CGB signals. The neuronal kinases leading to the formation of inositol-1,4,5 trisphosphate (InsP(3)), phosphatidylinositol-4-kinase (PI4K), and phosphatidylinositol-4-phosphate-5-kinase (PIPK), showed strong immunoreactivity throughout all hippocampal cell fields with differences in the subcellular distribution. Moreover, a distinct band of strong CGB and PIPK immunoreactivity was observed in the CA3 region that coincides with the mossy fiber tract (stratum lucidum). These data show differential expression of the components of the signaling toolkit leading to InsP(3)-mediated Ca(2+) release in cells of the hippocampus. The regulation of these differences may play an important role in various neuropathologic conditions such as Alzheimer's disease, epilepsy, or schizophrenia.     

Are elevated admission calcium levels associated with better outcomes after ischemic stroke?

Calcium (Ca(2+)) and magnesium (Mg(2+)) influence the molecular pathways of ischemic neuronal death. The authors evaluated the impact of admission serum Ca(2+) and Mg(2+) levels, on incident stroke severity and discharge functional outcome. After adjusting for covariates, higher admission Ca(2+) was significantly associated with lesser stroke severity and better discharge functional outcome. Admission Mg(2+) was not an independent clinical outcome prognosticator.     

Involvement of K(Ca2+) channels in the local abnormalities and hyperkalemia following the ischemia-reperfusion injury of rat skeletal muscle.

Patch-clamp technique was used to investigate the properties of the muscular Ca2+-activated K+ channel (K(Ca2+)) in the ischemic and ischemic-reperfused rat muscle fibers and the possible involvement of this ion channel in the reperfusion-dependent hyperkalemic state. The properties of the muscular K(Ca2+) channel were unaltered following 4 h of ischemia of the lower limbs and that the serum K+ level did not change following ischemia. In contrast, after 3 h of reperfusion an over-activation of K(Ca2+) channel was observed which was related to the increase in the number of functional channels per patch area. Currents from cation aselective channels were also routinely detected in these muscles and ion channel abnormalities similar to those observed in the ischemic-reperfused muscles were also found in the contralateral muscles. Significant hyperkalemia was observed following 3 h of reperfusion. Administration of L-NAME (10 mg.kg(-1)), a nitric-oxide synthase (NOS) inhibitor, during reperfusion prevented the increase of K(Ca2+) channel activity and the activation of the cation aselective channel. The L-NAME treatment also partially antagonised the characteristic hyperkalemia observed following reperfusion. In contrast, D-NAME (20 mg.kg(-1)), the inactive antipode on NOS enzyme administered to the rats during reperfusion failed to prevent the overactivation of the K(Ca2+) channel or the hyperkalemia. Our results indicate that overactivation of K(Ca2+) channel found in the muscles following reperfusion is either directly or indirectly related to NOS activation, and contributes to the hyperkalemia. Moreover, the discovery of abnormalities similar to those of the ischemic-reperfused muscles in the contralaterals suggests that proinflammatory molecules were released from the ischemic area, accentuating the pathological state.     

Role of intracellular Ca(2+) stores shaping normal activity in brain.

The role of intracellular Ca(2+) stores in the control of brain activity was investigated in microdialysis experiments by monitoring changes in the extracellular concentration of amino acids (AA) in the hippocampus of the rat after intracerebroventricular (icv) administration of the intracellular Ca(2+) release blocker, dantrolene in vivo, as well as in D-aspartate release and transmembrane Ca(2+) flux measurements in dantrolene-treated (50 microM) hippocampal homogenates containing resealed plasmalemma fragments and nerve endings in vitro. Microdialysis data demonstrate that icv injection of 0.6 mM dantrolene significantly decreases ( approximately 20%) the background (Glu) in the hippocampus. Both the (Glu; approximately 300%) and the inhibitory effect of dantrolene thereupon ( approximately 50%) was significantly increased when 0.5 mM of the Glu uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid, was dialysed into the hippocampus. NMDA and (S)-AMPA induced [(3)H]-D-aspartate release in hippocampal homogenates. Preincubation of these homogenates with 50 microM dantrolene was found to reduce the response to NMDA, but not to (S)-AMPA, in a NMDA-dependent manner. Increased rates of transmembrane influx and efflux of Ca(2+) in hippocampal homogenates with half-times of 4 ms and 200 ms, respectively, can be observed by the addition of 100 microM NMDA as recorded using a stopped-flow UV/fluorescence spectrometer in combination with the Ca(2+) indicator dye, bisfura-2. Both the Ca(2+) influx and efflux rates of the NMDA response were reduced (25-fold and >5-fold, respectively) in homogenates preloaded with 50 microM dantrolene. These results suggest a role for NMDA-inducible intracellular Ca(2+) stores in the control of normal brain activity in vivo.     

Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca2+ channels in rat hippocampal neurons

Vascular endothelial growth factor (VEGF) protects neurons against ischemic injury. An overload of intracellular calcium ions (Ca(2+)) caused by the excessive release of glutamate is widely considered to be one of the molecular mechanisms of ischemic neuronal death. In the present study, we investigated whether VEGF could modulate the activity of Ca(2+) channels on the neuronal membrane. We used the Fluo-3 image method assisted by confocal laser scan microscopy to detect any Ca(2+) influx in primary cultured hippocampal neurons. Whole-cell patch-clamp techniques were used to record the activity of the high-voltage-activated (HVA) Ca(2+) currents in the CA1 pyramidal neurons of hippocampal slices that were freshly prepared from neonatal brains of rats. The results obtained from the Fluo-3 image experiments showed that VEGF pretreatment of cultured neurons at a final concentration of 50, 100, or 200 ng/ml acutely and dose dependently attenuated the Ca(2+) influx induced by application of KCl (60 mM) or glutamate (50 microM). This effect was blocked by SU1498, an antagonist of Flk-1 VEGF receptor. The influx of Ca(2+) returned to basal levels after removal of VEGF. Furthermore, electrophysiological recording data showed that VEGF could acutely reduce the amplitudes of the HVA Ca(2+) currents in a dose- and voltage-dependent manner. The HVA Ca(2+) currents also returned to the levels of the control after removal of VEGF from the system. Taken together, the results obtained from the present study demonstrated that VEGF specifically reduced the influx of Ca(2+) via the inhibitory activity of the HVA Ca(2+) channels in hippocampal neurons.     

Role of Ca2+-independent phospholipase A2 and n-3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation.

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A(2) and cyclooxygenases. These enzymes release the prostaglandin precursor, the n-6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H(2). Docosahexaenoic acid, which belongs to the n-3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca(2+) signaling, which results in changes of activity of Ca(2+)-dependent phospholipase A(2), hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A(2), i.e. Ca(2+)-dependent phospholipase A(2) and Ca(2+)-independent phospholipase A(2), respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca(2+)- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.     

Cyclic AMP/protein kinase a signal attenuates Ca(2+)-induced fibroblast growth factor-1 synthesis in rat cortical neurons

Fibroblast growth factor (FGF)-1 is increased in particular brain regions after birth, suggesting an involvement of some regulatory neuronal circuits. To address the neuronal activity responsible for FGF-1 synthesis, effects of various neurotransmitter receptor activation on cellular FGF-1 content were examined using cultured rat cortical neurons. Histamine, glutamate, carbachol, serotonin or gamma-aminobutyric acid (GABA) caused an increase of FGF-1 content. Because this effect was mimicked by (1) N-methyl-D-aspartate, a glutamatergic agonist; (2) Ca(2+) ionophore; (3) depolarization with high concentration of KCl, but was abolished in Ca(2+)-free medium, Ca(2+) influx was thought to trigger FGF-1 synthesis. Such Ca(2+)-mediated enhancement of FGF-1 synthesis, however, did not occur in the presence of norepinephrine (NE), but was restored by KT-5720, an inhibitor of protein kinase A (PKA), suggesting an interplay between Ca(2+)-activated and cAMP/PKA signals for neuronal FGF-1 synthesis. This mechanism was proved to function in vivo by stimulation of FGF-1 expression in neurons of the cerebral cortex after intracerebral administration of propranolol, an antagonist of adrenergic beta receptors. This demonstrates that FGF-1 synthesis is essentially upregulated by Ca(2+) influx through excitatory neuronal activities, but such an effect is abolished by neurotransmission that evokes cAMP/PKA signals. FGF-1 produced is thought to act on establishment and maintenance of particular neuronal circuits in the brain, which may be one of the ways neurotransmitters regulate brain function.     

Magnetic resonance imaging at 9.4 T as a tool for studying neural anatomy in non-vertebrates

This report describes magnetic resonance imaging (MRI) methods we have developed at 9.4 T for observing internal organs and the nervous system of an invertebrate organism, the crayfish, Cherax destructor. We have compared results acquired using two different pulse sequences, and have tested manganese (Mn(2+)) as an agent to enhance contrast of neural tissues in this organism. These techniques serve as a foundation for further development of functional MRI and neural tract-tracing methods in non-vertebrate systems.