Neurons In Slice Cultures Research Articles

All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.

Human Cerebrospinal Fluid Monoclonal LGI1 Autoantibodies Increase Neuronal Excitability.

Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.

Microglia attenuate the kainic acid-induced death of hippocampal neurons in slice cultures.

BackgroundStatus epilepticus‐induced hippocampal neuronal death, astrogliosis, and the activation of microglia are common pathological changes in mesial temporal lobe epilepsy (mTLE) with resistance to antiepileptic drugs. Neuronal death in mTLE gradually progresses and is involved in the aggravation of epilepsy and the impairment of hippocampus‐dependent memory. Thus, clarifying the cellular mechanisms by which neurons are protected in mTLE will significantly contribute to the treatment of epilepsy. Here, mainly using hippocampal slice cultures with or without the pharmacological depletion of microglia, we directly examined whether microglia, the resident immune cells of the brain that can act either neurotoxically or in a neuroprotective manner, accelerate or attenuate kainic acid (KA)‐induced neuronal death in vitro.MethodsHippocampal slice cultures were treated with KA to induce neuronal death in vitro. Clodronate‐containing liposomes or PLX3397 was used to deplete microglia in hippocampal slice cultures, and the effect on KA‐induced neuronal death was immunohistochemically assessed.ResultsThe loss of microglia significantly promoted a decrease in neuronal density in KA‐treated hippocampal slice cultures.ConclusionOur results suggest that microglia are neuroprotective against KA‐induced neuronal death in slice cultures.

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures

Activity can induce structural changes in glutamatergic excitatory synapses, including increase in thickness and curvature of the postsynaptic density (PSD); these structural changes can only be documented by electron microscopy. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, increases in thickness and curvature of PSDs were noticeable within 30 s of stimulation and progressed with time up to 3 min. These structural changes were reversible upon returning the samples to control medium for 5–10 min. Thus, the postsynaptic density is a very dynamic structure that undergoes rapid reorganization of its components upon stimulation, and recovery upon cessation of stimulation. The gradual increase in thickness of PSD could result from a gradual translocation of some PSD proteins to the PSD, and the increase in curvature of the PSD is likely led by postsynaptic elements.

Histone Deacetylase Inhibitor Entinostat (MS-275) Restores Anesthesia-induced Alteration of Inhibitory Synaptic Transmission in the Developing Rat Hippocampus.

Recent evidence strongly supports the idea that common general anesthetics (GAs) such as isoflurane (Iso) and nitrous oxide (N2O; laughing gas), as well as sedative drugs such as midazolam are neurotoxic for the developing mammalian brain having deleterious effects on neural circuits involved in cognition, learning and memory. However, to date, very little is known about epigenetic mechanisms involved in GA-induced plasticity of synaptic transmission in the hippocampus, the main memory-processing region in the brain. Here, we used patch-clamp recordings of miniature inhibitory post-synaptic currents (mIPSCs) from hippocampal neurons in slice cultures exposed to the clinically relevant GA combination. We found that in vitro exposure to a combination of midazolam, 0.75% Iso, and 70% N2O for 6h leads to lasting increase in frequency of mIPSCs, while amplitudes and kinetics of the events were spared. Importantly, co-application of entinostat (MS-275), a selective inhibitor of class I histone deacetylases (HDAC), completely reversed GA-induced synaptic plasticity. Furthermore, when given in vivo to P7 pups exposed to GA with midazolam, Iso and N2O for 6h, MS-275 reversed GA-induced histone-3 hypoacetylation as shown by an increase in Ac-H3 protein expression in the hippocampus. We conclude that exposure to a combination of Iso with N2O and midazolam causes plasticity of mIPSCs in hippocampal neurons by epigenetic mechanisms that target presynaptic sites. We hypothesize that GA-induced epigenetic alterations in inhibitory synaptic transmission in the hippocampus may contribute to altered neuronal excitability and consequently abnormal learning and memory later in life.

Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons.

Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.

Maternal Dexamethasone Exposure Inhibits the Gonadotropin-Releasing Hormone Neuronal Movement in the Preoptic Area of Rat Offspring

Migration and final positioning of gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) is critical for reproduction. It is known that maternal dexamethasone (DEX) exposure impairs reproductive function and behaviour in the offspring. However, it is still not known whether maternal DEX exposure affects the postnatal GnRH neurons in the offspring. This study determined the neuronal movement of enhanced green fluorescent protein (EGFP)-tagged GnRH neurons in slice culture of postnatal day 0 (P0), P5 and P50-60 transgenic male rats. Effect of maternal DEX treatment on EGFP-GnRH neuronal movement and F-actin distribution on GnRH neurons at P0 stage were studied. Time-lapse analysis of P0 and P5 EGFP-GnRH neurons displayed active cellular movement within the POA compared to young adult P50-60 stages, suggesting possible fine-tuning movement for positioning of early postnatal GnRH neurons. The DEX-treated EGFP-GnRH neurons demonstrated decreased motility in the POA and reduced F-actin distribution in the GnRH neurons at 60 h culture compared to the vehicle-treated. These results suggest that the P0 GnRH neuronal movement in the POA is altered by maternal DEX exposure, which possibly disrupts the fine-tuning process for positioning and development of early postnatal GnRH neurons in the brain, potentially linked to reproductive dysfunction in adulthood.

Simultaneous Patch-Clamping and Calcium Imaging in Developing Dendrites

Calcium imaging has been used extensively to explore the role of action potential (AP) firing in the development of neuronal structure and synaptic function because increases in intracellular calcium ([Ca(2+)]i) reliably and, within a certain range, linearly reflect neuronal spiking activity. Patterns of APs in individual cells can be deduced from calcium recordings, which have typically been performed at the level of cell bodies. However, neurons are particularly susceptible to phototoxicity when they are illuminated at the soma. Furthermore, for some imaging experiments (e.g., those that address the interactions between dendrites and axons during synapse formation), the cell body of a given neuron may simply not be in the field of view. In these situations, it would be helpful to determine the spiking patterns of a neuron from the calcium activity in its subcellular compartments such as stretches of dendrites or axons. Here, we describe an approach for determining the relationship between AP firing and dendritic calcium transients by simultaneously imaging calcium transients in small dendritic stretches of hippocampal pyramidal neurons in slice cultures from neonatal rats and recording spiking activity with whole-cell patch-clamp recordings in these neurons. These experiments allow us to correlate the electrophysiological spiking pattern with the accompanying changes in the calcium concentration in individual dendritic segments.

Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences

GABAergic cortical interneurons underlie the complexity of neural circuits and are particularly numerous and diverse in humans. In rodents, cortical interneurons originate in the subpallial ganglionic eminences, but their developmental origins in humans are controversial. We characterized the developing human ganglionic eminences and found that the subventricular zone (SVZ) expanded massively during the early second trimester, becoming densely populated with neural stem cells and intermediate progenitor cells. In contrast with the cortex, most stem cells in the ganglionic eminence SVZ did not maintain radial fibers or orientation. The medial ganglionic eminence exhibited unique patterns of progenitor cell organization and clustering, and markers revealed that the caudal ganglionic eminence generated a greater proportion of cortical interneurons in humans than in rodents. On the basis of labeling of newborn neurons in slice culture and mapping of proliferating interneuron progenitors, we conclude that the vast majority of human cortical interneurons are produced in the ganglionic eminences, including an enormous contribution from non-epithelial SVZ stem cells.

Estrogen and the male hippocampus: Genetic variation in the aromatase gene predicting serum estrogen is associated with hippocampal gray matter volume in men

The neurosteroid 17-beta estradiol (E2) plays an important role in neuronal plasticity, neurogenesis and neuroprotection of hippocampal neurons in slice cultures and the female brain. While some effects of E2 on hippocampal neurons observed in females were also seen in the male hippocampus, others seem to be specific to females. The current study aimed to further explore the effect of E2 on the male hippocampus by investigating the relationship between genetic variations in E2 synthesis and hippocampal gray matter (GM) volume. We chose a single nucleotide polymorphism (rs700158, SNP) in the gene CYP19A1 coding for the final enzyme (aromatase) in E2 synthesis. Men homozygous for the A allele of rs700518 have repeatedly been shown to have higher E2 serum levels than male carriers of the G allele. Two independent cohorts of healthy young men were genotyped for rs700518 and voxel-based morphometry (VBM) was performed on structural magnetic resonance images to determine genotype dependent group differences. Men homozygous for the A allele of rs700518 had greater bilateral posterior hippocampal GM volumes in both cohorts. Thus, the genotype associated with higher E2 serum levels was also associated with greater hippocampal gray matter.

Caffeic acid phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem oxygenase‐1 and brain‐derived neurotrophic factor

Caffeic acid phenethyl ester (CAPE) is a component of honey bee propolis that can induce expression of haem oxygenase-1 (HO-1). Because HO-1 induction has been suggested to protect dopaminergic neurons in the substantia nigra, we examined the effect of CAPE in experimental models of dopaminergic neurodegeneration. Neuroprotective effect of CAPE was investigated in rat organotypic midbrain slice cultures and in vivo, using a mouse model of dopaminergic neurodegeneration induced by intranigral injection of LPS and intrastriatal injection of 6-hydroxydopamine. CAPE protected dopaminergic neurons in slice cultures from IFN-γ/LPS-induced injury. The effect of CAPE was inhibited by zinc protoporphyrin IX, an HO-1 inhibitor, and by neutralizing antibody against brain-derived neurotrophic factor (BDNF). A p38 MAPK inhibitor SB203580 prevented activation of NF-E2-related factor 2, attenuated increased expression of HO-1 and BDNF, and blocked the neuroprotective actions of CAPE. In the LPS-injected mouse model, daily intraperitoneal administration of CAPE protected dopaminergic neurons, up-regulated HO-1 and BDNF, and reduced the increase of activated microglia/macrophages. Neuroprotective effects of CAPE against LPS-induced injury was prevented by zinc protoporphyrin IX or anti-BDNF antibody. CAPE protected dopaminergic neurons and alleviated methamphetamine-induced rotational behaviour also in 6-hydroxydopamine hemiparkinsonian mice. CAPE is a novel type of neuroprotective agent whose actions are mediated by both HO-1 and BDNF. These findings may provide novel clues to develop neuroprotective agents for treatment of neurodegenerative disorders.

Cell surface area regulation in neurons in hippocampal slice cultures is resistant to oxygen-glucose deprivation

Correspondence: Linda r Mills 11-430 Toronto Western Hospital, 399 Bathurst St, Toronto, On, Canada, M5T 2S8 Tel +1 416 603 5420 email linda@uhnres.utoronto.ca or lrmills@gmail.com Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs), to accommodate the decrease in the bilayer. Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D) environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP) targeted to the cytoplasm; and a membrane targeted GFP (lckGFP), targeted to the plasma membrane. Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normoosmotic media changes. Oxygen-glucose deprivation (OGD) caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge. Conclusion: These results suggest cell surface area regulation (SAR) is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

2.P1. Analysis of the morphology of neurites of pilocarpine-treated hippocampal neurons in slice cultures

2.P1. Analysis of the morphology of neurites of pilocarpine-treated hippocampal neurons in slice cultures

An Essential Role for the Integrin-Linked Kinase–Glycogen Synthase Kinase-3β Pathway during Dendrite Initiation and Growth

Multiple cues, including growth factors and circuit activity, signal to regulate the initiation and growth of mammalian dendrites. In this study, we have asked how these environmental cues regulate dendrite formation, and in particular, whether dendrite initiation and growth requires integrin-linked kinase (ILK) or its downstream effector, glycogen synthase kinase-3beta (GSK-3beta). In cultured sympathetic neurons, NGF and neuronal depolarization activated ILK and promoted dendrite initiation and growth, and inhibition of ILK (either pharmacologically, with a dominant-negative form of ILK, or by genetic knockdown) reduced depolarization-induced dendrite formation. In sympathetic neurons, ILK phosphorylated and inhibited GSK-3beta, and inhibition of GSK-3beta (either pharmacologically, with dominant-negative GSK-3beta, or by genetic knockdown) caused robust dendrite initiation. GSK-3beta inhibition also caused dendrite initiation in cultured cortical neurons and growth of hippocampal neurons in slice cultures. GSK-3beta functioned downstream of ILK to regulate dendrite formation, because inhibition of GSK-3beta promoted dendrite initiation even when ILK was simultaneously inhibited. Moreover, GSK-3beta promoted dendrite formation in sympathetic neurons by regulating the activity of a key dendrite formation effector, the MAP (microtubule-associated protein) kinase kinase (MEK)-extracellular signal-regulated protein kinase (ERK) pathway. Specifically, inhibition of GSK-3beta led to increased ERK phosphorylation, and inhibition of MEK completely blocked the effects of GSK-3beta inhibition on dendrite initiation and growth. Thus, the ILK-GSK-3beta pathway plays a key role in regulating dendrite formation in developing mammalian neurons.

Synaptic activity and F-actin coordinately regulate CaMKIIα localization to dendritic postsynaptic sites in developing hippocampal slices

We examined the timing and mechanisms of CaMKIIα recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIα in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIα coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIα is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latrunculin-induced F-actin depolymerization. However, the combination of activity blockade and F-actin depolymerization significantly reduces synaptic CaMKIIα. Conversely, postsynaptic activation induces synaptic recruitment of CaMKIIα even in the presence of F-actin depolymerizing drugs. Thus, synaptic-activity-dependent mechanisms and (synaptic activity-independent) F-actin-based mechanisms are individually sufficient and act in parallel to localize CaMKIIα to the dendritic spine compartment. Moreover, the timing of CaMKIIα recruitment to developing spines suggests a role for CaMKIIα in spine assembly and maintenance.

Calcium Release from Presynaptic Ryanodine-Sensitive Stores Is Required for Long-Term Depression at Hippocampal CA3-CA3 Pyramidal Neuron Synapses

Although Ca2+ release from internal stores has been proposed to be important for the induction of long-term synaptic plasticity, the importance of Ca2+ stores localized in presynaptic terminals remains unclear. Here, we have selectively applied pharmacological antagonists to either the presynaptic or postsynaptic cell in paired whole-cell recordings from hippocampal CA3 pyramidal neurons in slice culture. We demonstrate directly the necessary role of presynaptic, but not postsynaptic, ryanodine-sensitive Ca2+ stores in the induction of NMDA receptor (NMDAR)-dependent long-term depression (LTD). Using two-photon laser scanning microscopy, we further find that release from the ryanodine-sensitive stores during prolonged synaptic stimulation generates a slowly rising Ca2+ signal in the presynaptic terminal that is required for the induction of LTD. Moreover, this form of LTD has a significant presynaptic component of expression because it causes a marked decrease in the rate of release from CA3 neuron presynaptic terminals of FM 1-43, a fluorescent probe of synaptic vesicle cycling. Thus, Ca2+ release from presynaptic ryanodine-sensitive stores is critical in the induction of a presynaptic component of NMDAR-dependent LTD.

New illumination technique for IR-video guided patch-clamp recording from neurons in slice cultures on biomembrane

Slice cultures on biomembrane are the method of choice for studying Ca 2+-dependent plastic changes occurring over several days to weeks. Using IR-differential interference contrast, good visualization of neurons in biomembrane slice cultures has been achieved despite a negative optical effect of the biomembrane, but epifluorescence imaging requires removal of a Wollaston prism and the analyzer. Here, we describe a novel illumination method to overcome this problem. Using optic fiber illumination at a shallow angle from the top of the slice culture, with or without additional illumination from the bottom, we obtained good cellular resolution of neurons in biomembrane slice cultures as well as in acute slices with an infrared-video camera. With this technique, we demonstrate visually guided whole-cell patch-clamp recording of Na +- and K +-currents as well as combination of whole-cell recording with fluorescence imaging of hippocampal and entorhinal cortex neurons in biomembrane slice cultures. Our inexpensive method should prove very useful for studying in vitro effects of long-term manipulations on membrane currents and intracellular Ca 2+-signaling.

Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca 2+ and phospholipase C

The mechanism of the neuroprotective action of the glycolytic pathway intermediate fructose-1,6-bisphosphate (FBP) may involve activation of a phospholipase-C (PLC) dependent MAP kinase signaling pathway. In this study, we determined whether FBP’s capacity to decrease delayed cell death in hippocampal slice cultures is dependent on PLC signaling or activation of the intracellular Ca 2+–MEK/ERK neuroprotective signaling cascade. FBP (3.5 mM) reduced delayed death from oxygen/glucose deprivation in CA1, CA3 and dentate neurons in slice cultures. The phospholipase-C inhibitor U73122 and the MEK1/2 inhibitor U0126 prevented this protection. In hippocampal and cortical neurons, FBP increased phospho-ERK1/2 (p42/44) immunostaining during hypoxic, but not normoxic conditions. Increased phospho-ERK immunostaining was dependent on PLC and also on MEK 1/2, an upstream regulator of ERK. Further, we found that FBP enhancement of phospho-ERK immunostaining depended on [Ca 2+] i: PLC inhibition and the IP 3 receptor blocker xestospongin C prevented FBP from increasing [Ca 2+] i and increasing phospho-ERK levels. However, while FBP-induced increases in [Ca 2+] i were blocked by xestospongin and a PLC inhibitor, [Ca 2+] i increases induced by the neuroprotective growth factor BDNF were not prevented. We conclude that during hypoxia FBP initiates a series of neuroprotective signals which include PLC activation, small increases in [Ca 2+] i, and increased activity of the MEK/ERK signaling pathway.