Expression of Cytokines and Neurodegeneration in the Rat Hippocampus and Cortex in the Lithium-Pilocarpine Model of Status Epilepticus and the Role of Modulation of Endocannabinoid System.

A significant body of evidence shows that neuroinflammation is one of the key processes in the development of brain pathology in trauma, neurodegenerative disorders, and epilepsy. Various brain insults, including severe and prolonged seizure activity during status epilepticus (SE), trigger proinflammatory cytokine release. We investigated the expression of the proinflammatory cytokines interleukin-1ß (Il1b) and interleukin-6 (Il6), and anti-inflammatory fractalkine (Cx3cl1) in the hippocampus, entorhinal cortex, and neocortex of rats 24 h, 7 days, and 5 months after lithium-pilocarpine SE. We studied the relationship between cytokine expression and neuronal death in the hippocampus and evaluated the effect of modulation of endocannabinoid receptors on neuroinflammation and neurodegeneration after SE. The results of the present study showed that inhibition of endocannabinoid CB1 receptors with AM251 early after SE had a transient neuroprotective effect that was absent in the chronic period and did not affect the development of spontaneous seizures after SE. At the same time, AM251 reduced the expression of Il6 in the chronic period after SE. Higher Cx3cl1 levels were found in rats with more prominent hippocampal neurodegeneration.

The Induction of Long-Term Potentiation by Medial Septum Activation under Urethane Anesthesia Can Alter Gene Expression in the Hippocampus.

We studied changes in the expression of early genes in hippocampal cells in response to stimulation of the dorsal medial septal area (dMSA), leading to long-term potentiation in the hippocampus. Rats under urethane anesthesia were implanted with stimulating electrodes in the ventral hippocampal commissure and dMSA and a recording electrode in the CA1 area of the hippocampus. We found that high-frequency stimulation (HFS) of the dMSA led to the induction of long-term potentiation in the synapses formed by the ventral hippocampal commissure on the hippocampal CA1 neurons. One hour after dMSA HFS, we collected the dorsal and ventral hippocampi on both the ipsilateral (damaged by the implanted electrode) and contralateral (intact) sides and analyzed the expression of genes by qPCR. The dMSA HFS led to an increase in the expression of bdnf and cyr61 in the ipsilateral hippocampi and egr1 in the ventral contralateral hippocampus. Thus, dMSA HFS under the conditions of degeneration of the cholinergic neurons in the medial septal area prevented the described increase in gene expression. The changes in cyr61 expression appeared to be dependent on the muscarinic M1 receptors. Our data suggest that the induction of long-term potentiation by dMSA activation enhances the expression of select early genes in the hippocampus.

Ambiguous Contribution of Glucocorticosteroids to Acute Neuroinflammation in the Hippocampus of Rat.

Effects of modulation of glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) on acute neuroinflammatory response were studied in the dorsal (DH) and ventral (VH) parts of the hippocampus of male Wistar rats. Local neuroinflammatory response was induced by administration of bacterial lipopolysaccharide (LPS) to the DH. The modulation of GR and MR was performed by dexamethasone (GR activation), mifepristone, and spironolactone (GR and MR inhibition, respectively). Experimental drugs were delivered to the dentate gyrus of the DH bilaterally by stereotaxic injections. Dexamethasone, mifepristone, and spironolactone were administered either alone (basal conditions) or in combination with LPS (neuroinflammatory conditions). Changes in expression levels of neuroinflammation-related genes and morphology of microglia 3 days after intrahippocampal administration of above substances were assessed. Dexamethasone alone induced a weak proinflammatory response in the hippocampal tissue, while neither mifepristone nor spironolactone showed significant effects. During LPS-induced neuroinflammation, GR activation suppressed expression of selected inflammatory genes, though it did not prevent appearance of activated forms of microglia. In contrast to GR activation, GR or MR inhibition had virtually no influence on LPS-induced inflammatory response. The results suggest glucocorticosteroids ambiguously modulate specific aspects of neuroinflammatory response in the hippocampus of rats at molecular and cellular levels.

A Single Episode of Cortical Spreading Depolarization Increases mRNA Levels of Proinflammatory Cytokines, Calcitonin Gene-Related Peptide and Pannexin-1 Channels in the Cerebral Cortex.

Cortical spreading depolarization (CSD) is the neuronal correlate of migraine aura and the reliable consequence of acute brain injury. The role of CSD in triggering headaches that follow migraine aura and brain injury remains to be uncertain. We examined whether a single CSD occurring in awake animals modified the expression of proinflammatory cytokines (Il1b, TNF, and Il6) and endogenous mediators of nociception/neuroinflammation-pannexin 1 (Panx1) channel and calcitonin gene-related peptide (CGRP), transforming growth factor beta (TGFb) in the cortex. Unilateral microinjury of the somatosensory cortex triggering a single CSD was produced in awake Wistar rats. Three hours later, tissue samples from the lesioned cortex, intact ipsilesional cortex invaded by CSD, and homologous areas of the contralateral sham-treated cortex were harvested and analyzed using qPCR. Three hours post-injury, intact CSD-exposed cortexes increased TNF, Il1b, Panx1, and CGRP mRNA levels. The strongest upregulation of proinflammatory cytokines was observed at the injury site, while CGRP and Panx1 were upregulated more strongly in the intact cortexes invaded by CSD. A single CSD is sufficient to produce low-grade parenchymal neuroinflammation with simultaneous overexpression of Panx1 and CGRP. The CSD-induced molecular changes may contribute to pathogenic mechanisms of migraine pain and post-injury headache.

Deep Brain Stimulation of the Medial Septal Area Can Modulate Gene Expression in the Hippocampus of Rats under Urethane Anesthesia.

We studied the effects of stimulation of the medial septal area on the gene expression in the dorsal and ventral hippocampus. Rats under urethane anesthesia were implanted with a recording electrode in the right hippocampus and stimulating electrode in the dorsal medial septum (dMS) or medial septal nucleus (MSN). After one-hour-long deep brain stimulation, we collected ipsi- and contralateral dorsal and ventral hippocampi. Quantitative PCR showed that deep brain stimulation did not cause any changes in the intact contralateral dorsal and ventral hippocampi. A comparison of ipsi- and contralateral hippocampi in the control unstimulated animals showed that electrode implantation in the ipsilateral dorsal hippocampus led to a dramatic increase in the expression of immediate early genes (c-fos, arc, egr1, npas4), neurotrophins (ngf, bdnf) and inflammatory cytokines (il1b and tnf, but not il6) not only in the area close to implantation site but also in the ventral hippocampus. Moreover, the stimulation of MSN but not dMS further increased the expression of c-fos, egr1, npas4, bdnf, and tnf in the ipsilateral ventral but not dorsal hippocampus. Our data suggest that the activation of medial septal nucleus can change the gene expression in ventral hippocampal cells after their priming by other stimuli.

Detailed Analysis of Dorsal-Ventral Gradients of Gene Expression in the Hippocampus of Adult Rats.

We performed RNA sequencing of the dorsal and ventral parts of the hippocampus and compared it with previously published data to determine the differences in the dorsoventral gradients of gene expression that may result from biological or technical variability. Our data suggest that the dorsal and ventral parts of the hippocampus differ in the expression of genes related to signaling pathways mediated by classical neurotransmitters (glutamate, GABA, monoamines, etc.) as well as peptide and Wnt ligands. These hippocampal parts also diverge in the expression of axon-guiding molecules (both receptors and ligands) and splice isoforms of genes associated with intercellular signaling and cell adhesion. Furthermore, analysis of differential expressions of genes specific for astrocytes, microglia, oligodendrocytes, and vascular cells suggests that non-neuronal cells may also differ in the characteristics between hippocampal parts. Analysis of expression of transposable elements showed that depletion of ribosomal RNA strongly increased the representation of transposable elements in the RNA libraries and helped to detect a weak predominance of expression of these elements in the ventral hippocampus. Our data revealed new molecular dimensions of functional differences between the dorsal and ventral hippocampus and points to possible cascades that may be involved in the longitudinal organization of the hippocampus.

Glucocorticoids: Dr. Jekyll and Mr. Hyde of Hippocampal Neuroinflammation.

Glucocorticoids (GCs) are an important component of adaptive response of an organism to stressogenic stimuli, a typical stress response being accompanied by elevation of GC levels in blood. Anti-inflammatory effects of GCs are widely used in clinical practice, while pro-inflammatory effects of GCs are believed to underlie neurodegeneration. This is particularly critical for the hippocampus, brain region controlling both cognitive function and emotions/affective behavior, and selectively vulnerable to neuroinflammation and neurodegeneration. The hippocampus is believed to be the main target of GCs since it has the highest density of GC receptors potentially underlying high sensitivity of hippocampal cells to severe stress. In this review, we analyzed the results of studies on pro- and anti-inflammatory effects of GCs in the hippocampus in different models of stress and stress-related pathologies. The available data form a sophisticated, though often quite phenomenological, picture of a modulatory role of GCs in hippocampal neuroinflammation. Understanding the dual nature of GC-mediated effects as well as causes and mechanisms of switching can provide us with effective approaches and tools to avert hippocampal neuroinflammatory events and as a result to prevent and treat brain diseases, both neurological and psychiatric. In the framework of a mechanistic view, we propose a new hypothesis describing how the anti-inflammatory effects of GCs may transform into the pro-inflammatory ones. According to it, long-term elevation of GC level or preliminary treatment with GC triggers accumulation of FKBP51 protein that suppresses activity of GC receptors and activates pro-inflammatory cascades, which, finally, leads to enhanced neuroinflammation.

Neonatal Proinflammatory Stress and Expression of Neuroinflammation-Associated Genes in the Rat Hippocampus.

Differential effect of the neonatal proinflammatory stress (NPS) on the development of neuroinflammation in the hippocampus and induction of the depressive-like behavior in juvenile and adult male and female rats was studied. NPS induction by bacterial lipopolysaccharide in the neonatal period upregulated expression of the Il6 and Tnf mRNAs accompanied by the development of depressive-like behavior in the adult male rats. NPS increased expression of the mRNAs for fractalkine and its receptor in the ventral hippocampus of the juvenile male rats, but did not affect expression of mRNAs for the proinflammatory cytokines and soluble form of fractalkine. NPS downregulated expression of fractalkine mRNA in the dorsal hippocampus of juvenile males. No significant effects of NPS were found in the female rats. Therefore, the NPS induces long-term changes in the expression of neuroinflammation-associated genes in different regions of the hippocampus, which ultimately leads to the induction of neuroinflammation and development of depressive-like behavior in male rats.

Primary culture of mouse embryonic spinal cord neurons: cell composition and suitability for axonal regeneration studies.

Objective: Primary culture is an effective experimental model to study molecular mechanisms that drive axonal regeneration after central nervous system injury. However, the culture of spinal cord (SC) cells remains poorly characterized. Here, we have analyzed the cell composition of a primary SC culture during its maturation. Methods: Primary cell culture was prepared from mouse embryo spinal cords. After 2, 7, and 14 days of cultivation, the cells were fixed and stained with antibodies against ß3-tubulin, nestin, crmp1, SMI-32, DCC or GFAP. We counted percentage of cells positive for the mentioned markers and measured the length of cell processes. Results: We found that ß3-tubulin and nestin were both expressed at day 2 of culture in vitro. Surprisingly (given the use of differentiation-supporting culture medium), the number of nestin+ cells has significantly increased during the first week of cultivation. The GFAP+ cells appeared only at the seventh day in vitro, and their fraction increased during the following cultivation. At 14 day in vitro, SC culture contained cells that expressed the markers typical of commissural and motor neurons. At this age, the neurons had the ability to repair injured neurites after mechanical damage. Conclusion: Primary culture of SC cells is a dynamically developing cell population that contains all main types of SC cells and is capable of self-repair. Therefore, the culture of mouse embryonic SC cells represents an adequate experimental model for studying cellular and molecular processes taking place in SC neurons after axonal damage in the absence of external inhibitors.

Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH.

Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

Neuroinflammatory Cytokine Response, Neuronal Death, and Microglial Proliferation in the Hippocampus of Rats During the Early Period After Lateral Fluid Percussion-Induced Traumatic Injury of the Neocortex.

Time course of changes in neuroinflammatory processes in the dorsal and ventral hippocampus was studied during the early period after lateral fluid percussion-induced neocortical traumatic brain injury (TBI) in the ipsilateral and contralateral hemispheres. In the ipsilateral hippocampus, neuroinflammation (increase in expression of pro-inflammatory cytokines) was evident from day 1 after TBI and ceased by day 14, while in the contralateral hippocampus, it was mainly limited to the dorsal part on day 1. TBI induced an increase in hippocampal corticosterone level on day 3 bilaterally and an accumulation of Il1b on day 1 in the ipsilateral hippocampus. Activation of microglia was observed from day 7 in different hippocampal areas of both hemispheres. Neuronal cell loss was detected in the ipsilateral dentate gyrus on day 3 and extended to the contralateral hippocampus by day 7 after TBI. The data suggest that TBI results in distant hippocampal damage (delayed neurodegeneration in the dentate gyrus and microglia proliferation in both the ipsilateral and contralateral hippocampus), the time course of this damage being different from that of the neuroinflammatory response.

In vivo evidence for the involvement of the carboxy terminal domain in assembling connexin 36 at the electrical synapse.

Connexin 36 (Cx36)-containing electrical synapses contribute to the timing and amplitude of neural responses in many brain regions. A Cx36-EGFP transgenic was previously generated to facilitate their identification and study. In this study we demonstrate that electrical coupling is normal in transgenic mice expressing Cx36 from the genomic locus and suggest that fluorescent puncta present in brain tissue represent distributed electrical synapses. These qualities emphasize the usefulness of the Cx36-EGFP reporter as a tool for the detailed anatomical characterization of electrical synapses in fixed and living tissue. However, though the fusion protein is able to form gap junctions between Xenopus laevis oocytes it is unable to restore electrical coupling to interneurons in the Cx36-deficient mouse. Further experiments in transgenic tissue and non-neural cell lines reveal impaired transport to the plasma membrane as the possible cause. By analyzing the functional deficits exhibited by the fusion protein in vivo and in vitro, we identify a motif within Cx36 that may interact with other trafficking or scaffold proteins and thereby be responsible for its incorporation into electrical synapses.

Intrahippocampal Adeno-Associated Virus-Mediated Overexpression of Nerve Growth Factor Reverses 192IgG-Saporin-Induced Impairments of Hippocampal Plasticity and Behavior.

One of the aspects of Alzheimer disease is loss of cholinergic neurons in the basal forebrain, which leads to development of cognitive impairment. Here, we used a model of cholinergic deficit caused by immunotoxin 192IgG-saporin to study possible beneficial effects of adeno-associated virus (AAV)-mediated overexpression of nerve growth factor (NGF) in the hippocampus of rats with cholinergic deficit. Suspension of recombinant AAV carrying control cassette or cassette with NGF was injected into both hippocampi of control rats or rats with cholinergic deficit induced by intraseptal injection of 192IgG-saporin. Analysis of choline acetyltransferase (ChAT) immunostaining showed that NGF overexpression in the hippocampus did not prevent strong loss of ChAT-positive neurons in the septal area caused by the immunotoxin. Induction of cholinergic deficit in the hippocampus led to impairments in Y-maze and beam-walking test but did not affect behavioral indices in the T-maze, open field test, and inhibitory avoidance training. NGF overexpression in the rats with cholinergic deficit restored normal animal behavior in Y-maze and beam-walking test. Recording of field excitatory postsynaptic potentials in vivo in the hippocampal CA1 area showed that induction of cholinergic deficit decreased magnitude of long-term potentiation (LTP) and prevented a decrease in paired-pulse ratio after LTP induction, and NGF overexpression reversed these negative changes in hippocampal synaptic characteristics. The beneficial effect of NGF was not associated with compensatory changes in the number of cells that express NGF receptors TrkA and NGFR in the hippocampus and medial septal area. NGF overexpression also did not prevent a 192IgG-saporin-induced decrease in the activity of acetylcholine esterase in the hippocampus. We conclude that NGF overexpression in the hippocampus under conditions of cholinergic deficit induces beneficial effects which are not related to maintenance of cholinergic function.

CB2 receptors modulate seizure-induced expression of pro-inflammatory cytokines in the hippocampus but not neocortex.

We compared neuroinflammatory responses induced by nonconvulsive and convulsive seizures and analyzed the role that may be played by cannabinoid CB2 receptors in the neuroinflammatory response induced by generalized tonic-clonic seizures (GTCS). Using quantitative PCR, we analyzed expression of interleukin-1b, CCL2, interleukin-6, tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFb1), fractalkine, and cannabinoid receptor type 2 in the neocortex, dorsal and ventral hippocampus, cortical leptomeninges, dura mater, and spleen in 3 and 6 h after induction of GTCS by a high dose of pentylenetetrazole (PTZ, 70 mg/kg) and absence-like activity by a low dose of PTZ (30 mg/kg). The low dose of PTZ had no effect on the gene expression 3 and 6 h after PTZ injection. In 3 and 6 h after high PTZ dose, the expression of CCL2 and TNF increased in the neocortex. Both ventral and dorsal parts of the hippocampus responded to seizures by elevation of CCL2 expression 3 h after PTZ. Cortical leptomeninges but not dura mater also had elevated CCL2 level and decreased TGFb1 expression 3 h after GTCS. Activation of CB2 receptors by HU308 suppressed an inflammatory response only in the dorsal hippocampus but not neocortex. Suppression of CB2 receptors by AM630 potentiated expression of inflammatory cytokines also in the hippocampus but not in the neocortex. Thus, we showed that GTCS, but not the absence-like activity, provoke inflammatory response in the neocortex, dorsal and ventral hippocampus, and cortical leptomeninges. Modulation of CB2 receptors changes seizure-induced neuroinflammation only in the hippocampus but not neocortex.

Saporin from Saponaria officinalis as a Tool for Experimental Research, Modeling, and Therapy in Neuroscience.

Saporin, which is extracted from Saponaria officinalis, is a protein toxin that inactivates ribosomes. Saporin itself is non-selective toxin but acquires high specificity after conjugation with different ligands such as signaling peptides or antibodies to some surface proteins expressed in a chosen cell subpopulation. The saporin-based conjugated toxins were widely adopted in neuroscience as a convenient tool to induce highly selective degeneration of desired cell subpopulation. Induction of selective cell death is one of approaches used to model neurodegenerative diseases, study functions of certain cell subpopulations in the brain, and therapy. Here, we review studies where saporin-based conjugates were used to analyze cell mechanisms of sleep, general anesthesia, epilepsy, pain, and development of Parkinson's and Alzheimer's diseases. Limitations and future perspectives of use of saporin-based toxins in neuroscience are discussed.

The Ever-Growing Puzzle of Asynchronous Release.

Invasion of an action potential (AP) to presynaptic terminals triggers calcium dependent vesicle fusion in a relatively short time window, about a millisecond, after the onset of the AP. This allows fast and precise information transfer from neuron to neuron by means of synaptic transmission and phasic mediator release. However, at some synapses a single AP or a short burst of APs can generate delayed or asynchronous synaptic release lasting for tens or hundreds of milliseconds. Understanding the mechanisms underlying asynchronous release (AR) is important, since AR can better recruit extrasynaptic metabotropic receptors and maintain a high level of neurotransmitter in the extracellular space for a substantially longer period of time after presynaptic activity. Over the last decade substantial work has been done to identify the presynaptic calcium sensor that may be involved in AR. Several models have been suggested which may explain the long lasting presynaptic calcium elevation a prerequisite for prolonged delayed release. However, the presynaptic mechanisms underlying asynchronous vesicle release are still not well understood. In this review article, we provide an overview of the current state of knowledge on the molecular components involved in delayed vesicle fusion and in the maintenance of sufficient calcium concentration to trigger AR. In addition, we discuss possible alternative models that may explain intraterminal calcium dynamics underlying AR.

Overexpression of Calretinin Enhances Short-Term Synaptic Depression.

Analysis of the effects of various proteins on short-term synaptic plasticity is a difficult task, which may require the use of knockout animals. Here, we propose an alternative experimental approach for studying the roles of desired proteins in synaptic plasticity. We packed the Ca2+-binding protein calretinin and the fluorescent protein Venus into AAV and injected the concentrated viral suspension into the neocortex of newborn rats. The infected layer 2/3 pyramidal cells were identified in rat cortical slices using Venus fluorescence. Analysis of short-term synaptic plasticity using paired patch clamp recordings between layer 2/3 pyramidal cells (presynaptic cell) and fast-spiking (FS) interneurons (post-synaptic cell) showed that calretinin expression in the pyramidal cells did not change the failure rate in this synapse but did decrease synaptic delay. Analysis of the parameters of short-term synaptic plasticity showed that the amplitude of the first EPSP in the train was not affected by calretinin, however, calretinin strongly enhanced short-term depression. In addition, we found that the effect of calretinin depended on the presynaptic firing frequency: an increase in frequency resulted in enhancement of synaptic depression.

Cholinergic Deficit Induced by Central Administration of 192IgG-Saporin Is Associated With Activation of Microglia and Cell Loss in the Dorsal Hippocampus of Rats.

Alzheimer's disease (AD) is associated with degeneration of cholinergic neurons in the basal forebrain. Administration of the immunotoxin 192IgG-saporin to rats, an animal model of AD, leads to degeneration of cholinergic neurons in the medial septal area. In the present study, cholinergic cell death was induced by intracerebroventricular administration of 192IgG-saporin. One and a half months after injection, we studied the histopathology of the hippocampus and the responses of microglia and astrocytes using immunohistochemistry and neuroglial gene expression. We found that treatment with 192IgG-saporin resulted in neuronal loss in the CA3 field of the hippocampus. Microglial proliferation was observed in the dentate gyrus of the dorsal hippocampus and white matter. Massive proliferation and activation of microglia in the white matter was associated with strong activation of astrocytes. However, the expression of microglial marker genes significantly increased only in the dorsal hippocampus, not the ventral hippocampus. These effects were not related to non-specific action of 192IgG-saporin because of the absence of the Nerve growth factor receptor in the hippocampus. Additionally, 192IgG-saporin treatment also induced a decrease in the expression of genes that are associated with transport functions of brain vascular cells (Slc22a8, Ptprb, Sdpr), again in the dorsal hippocampus but not in the ventral hippocampus. Taken together, our data suggest that cholinergic degeneration in the medial septal area induced by intracerebroventricular administration of 192IgG-saporin results in an increase in the number of microglial cells and neuron degeneration in the dorsal hippocampus.

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation.

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH(m) decrease which presumably resulted from MPT opening. This pH(m) decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca(2+) was replaced with antagonist of MPT opening Sr(2+). We found that in Sr(2+)-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH(c) and pH(m) associated with DSD led us to conclude that MD in Sr(2+)-containing medium occurred without involvement of the pore. In contrast, in Ca(2+)-containing medium such "non-pore mechanism" was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range.

BACKGROUND: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). RESULTS: Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. CONCLUSION: We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.