Postsynaptic actions of excitatory amino acids investigated with flash photolysis

Flash photolysis and whole cell patch clamp technique were combined with microspectrofluorimetry of calcium to study the role of inositol trisphosphate as a second messenger, and the role of L-glutamate as a neurotransmitter. Inactive photolabile analogous of inositol trisphosphate, caged InsP3, and fluorescent calcium indicators, Fluo-3 or Furaptra, was introduced into single cells via the whole-cell patch pipette. Astrocytes and neurones from primary cultures of cerebellar granule cells, hippocampal pyramidal cells, striatal neurones, and dorsal root ganglion neurones, and Purkinje cells from acutely prepared thin slices of the cerebellum were voltage clamped (holding potential -70mV). A 1ms pulse of near UV (300-350nm, 100mJ) was used to photolytically release a known concentration of InsP3 in the cytosol, and the changes in the intracellular free calcium concentration, [Ca2+], and conductance monitored. The results may be summarised as follows: (1) Photolytic release of InsP3 (100nM-30μM) in cultured neurones did not alter [Ca2+] or the membrane conductance. In glial cells from the same preparations, however, a rise in [Ca2+] was observed following intracellular release of InsP3 (0.2-5μM). The concentrations of InsP3 required to mobilise calcium in astrocytes is similar to that found in peripheral tissue. (2) Photolytic release of InsP3 (9-76μM) in voltage clamped Purkinje neurones in acutely prepared thick slices of the cerebellum resulted in a large increase in [Ca2+], often reaching 30μM when calibrated with Furaptra, and at the same time an outward current (200-900pA) in the presence or absence of external calcium. The outward current was the result of an increase of the membrane conductance to potassium ions, and under current clamp conditions hyperpolarised the cell to approximately -85mV. The responses occurred with a latency that decreased from a few hundred ms at low (9μM) to 10000X less potent at the NMDA, and >50X at the non-NMDA receptors. When irradiated with a 1ms pulse of near UV light (300-350nm, 100mJ) it cleaved to release L-glutamate, 2-nitrosoacetophenone, and carbon dioxide with a quantum yield of 50%. Neither caged glutamate (1mM) nor its photolysis by-products had any potency as an antagonist at the excitatory amino acid sites. The rate of photolysis was found to have a half time of about 50ms at pH7, too slow to allow for studies of rate of receptor activation. The rate of photolysis, however, is pH dependent increasing 10 fold with one pH unit drop. The ability of the squid giant synapse to withstand acidic conditions was taken advantage of, and L-glutamate was photolytically released from caged glutamate equilibrated with the synapse at pH5.5. This abrupt release of L-glutamate elicited action potentials in the 3rd order fibre of the giant synapse providing good evidence of the role of L-glutamate as a neurotransmitter at this synapse

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Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator

Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.Peer reviewe

Increased susceptibility to cortical spreading depression and epileptiform activity in a mouse model for FHM2

Migraine is a highly prevalent, debilitating, episodic headache disorder affecting roughly 15% of the population. Familial hemiplegic migraine type 2 (FHM2) is a rare subtype of migraine caused by mutations in the ATP1A2 gene, encoding the α2 isoform of the Na+/K+-ATPase, predominantly expressed in astrocytes. Differential comorbidities such as epilepsy and psychiatric disorders manifest in patients. Using a mouse model harboring the G301R disease-mutation in the α2 isoform, we set to unravel whether α2 +/G301R mice show an increased susceptibility for epilepsy and cortical spreading depression (CSD). We performed in vivo experiments involving cortical application of KCl in awake head-restrained male and female mice of different age groups (adult and aged). Interestingly, α2 +/G301R mice indeed showed an increased susceptibility to both CSD and epileptiform activity, closely replicating symptoms in FHM2 patients harboring the G301R and other FHM2-causing mutations. Additionally, this epileptiform activity was superimposed on CSDs. The age-related alteration towards CSD indicates the influence of female sex hormones on migraine pathophysiology. Therefore, the FHM2, α2 +/G301R mouse model can be utilized to broaden our understanding of generalized epilepsy and comorbidity hereof in migraine, and may be utilized toward future selection of possible treatment options for migraine

Restoration of Harmane Induced Memory Consolidation Deficit by Alpha-lipoic Acid in Male Mice

Introduction: there has been a growing number of publications focusing on the effect of beta-carbolines (e.g., harmane) on cognitive behaviors such as different stages of memory formation process. Moreover, several studies have stated that Alpha-lipoic acid (ALA) induces some molecular pathways effects including antioxidant effect and reduction of inflammation process. Thus, in the lines that follow, the question of whether ALA could alter memory consolidation deficit caused by harmane in the male NMRI mice will be addressed. Materials and Methods: The data for this study were collected by step-down inhibitory avoidance task with one trial protocol for evaluation of memory consolidation. The ALA (35 mg/kg) was injected intraperitoneally immediately after training followed by subthreshold and effective doses of harmane (2.5, 5 and 10 mg/kg) with 15-minute interval period. Results: The results show that post-training injection of the highest dose of harmane (10 mg/kg) lowers step-down latency, indicating the amnesia induced by harmane (P&lt;.001). In addition, similar injection of subthreshold dose of ALA (35 mg/kg), 15 minutes before injection of subthreshold and effective doses of harmane, restores step-down latency caused by higher dose of harmane (P&lt;.001) without its effect on the responses induced by subthreshold doses of harmane, indicating benefit effect of ALA on amnesia induced by harmane. Conclusion: An implication of this study is the possibility that ALA can reverse the amnesia induced by harmane. Therefore, future studies on this topic such as molecular mechanisms are recommended. &nbsp

A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1

A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.00