David N. Bowser

Mutant GABA A receptor γ2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants. We have found a mutation in a gene encoding a GABAA receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS). There is a recognized genetic relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the γ2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Truncation of the GABAA-Receptor γ2 Subunit in a Family with Generalized Epilepsy with Febrile Seizures Plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes.

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between ∼100 and 250 μm, and ∼15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy

Transmitters such as glutamate and ATP are released from brain astrocytes. Several pathways for their release have been proposed, including exocytosis. In the present study we sought to measure exocytosis from astrocytes with single vesicle imaging methods using synaptopHlourin (SpH) as an optical reporter. We imaged single SpH-laden vesicles with total internal reflection fluorescence (TIRF) microscopy. We observed spontaneous, as well as evoked, single-vesicle exocytosis events. Analysis of the kinetics and spatial spread associated with these events indicated two discernible forms of single vesicle exocytosis. One form, constituting ≈40% of the spontaneous events, was akin to kiss-and-run vesicle fusion and captured a mobile proton buffer from the extracellular medium. The other form seems to represent full vesicle fusion, constitutes ≈60% of the spontaneous events, and is associated with complete mixing of the vesicle and plasma membranes. Activation of calcium-mobilizing receptors on the astrocyte surface selected between the different forms of exocytosis. These data provide evidence for two forms of simultaneously occurring single-vesicle exocytosis events in astrocytes, and also suggest that SpH imaging and TIRF microscopy is useful to study the mechanisms of astrocyte transmitter release.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [γ2(R43Q)] found in human epilepsy

The γ-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABAA receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABAA γ2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α1β2γ2 or α1β2γ2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABAA receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities

Enlarged early endosomes in the neurons of young Down syndrome (DS) and pre-Alzheimers disease (AD) brains suggest that a disturbance in endocytosis is one of the earliest hallmarks of AD pathogenesis in both conditions. We identified a chromosome 21 gene, Intersectin-1 (ITSN1) that is up-regulated in DS brains and has a putative function in endocytosis and vesicle trafficking. To elucidate the function of ITSN1 and assess its contribution to endocytic defects associated with DS and AD, we generated Itsn1 null mice. In knockout mice we found alterations in a number of parameters associated with endocytic and vesicle trafficking events. We found a reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons. Endosome size was increased in neurons and NGF levels were reduced in the septal region of the brain. Our data is the first indication that Itsn1 has a role in endocytosis in an in vivo mammalian model, and that a disruption in Itsn1 expression causes a disturbance in vesicle trafficking and endocytic function in the brain. These results imply a role for ITSN1 in the early endocytic anomalies reported in DS brains which may have ramifications for the onset of AD.

Release of mitochondrial Ca2+ via the permeability transition activates endoplasmic reticulum Ca2+ uptake

Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca2+ concentration ([Ca2+]I), may be of importance in the control of many cell functions, and particularly those involved in initiating cell death. We used targeted Ca2+ sensors (cameleons) to investigate the movement of Ca2+ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca2+ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca2+, perpetuating cellular Ca2+ overload, and cell death. Targeted cameleons (mitochondria and ER) were imaged with confocal microscopy 2–3 days following transient transfection of human embryonic kidney 293 cells. Opening of the MPT resulted in specific loss of mitochondrial Ca2+ (blocked by cyclosporin A), which was sequestered initially by ER. The ER subsequently released this Ca2+ load, leading to a global Ca2+ elevation, a response that was not observed when ER Ca2+‐ATPases were blocked with cyclopiazonic acid. Thus, ER plays an important role in moderating changes in intracellular Ca2+ following MPT and may play a key role in cell death initiated by mitochondrial mechanisms.

Memory Processing in the Avian Hippocampus Involves Interactions between β-Adrenoceptors, Glutamate Receptors, and Metabolism

Noradrenaline is known to modulate memory formation in the mammalian hippocampus. We have examined how noradrenaline and selective β-adrenoceptor (AR) agonists affect memory consolidation and how antagonists inhibit memory consolidation in the avian hippocampus. Injection of selective β-AR agonists and antagonists at specific times within 30 min of a weakly or strongly reinforced, single-trial, bead discrimination learning test in 1-day-old chicks allowed us to determine the pattern of β-AR involvement in hippocampal memory processing. Different β-AR subtypes were recruited in temporal sequence after learning in the order β1, β3, and β2. We provide evidence that the effect of manipulation of β1-ARs by selective agonists and antagonists within 2.5 min of training parallels the action of NMDA receptor agonists and antagonists. Activation of β3- and β2-ARs facilitated memory but utilized different mechanisms: β3-ARs by stimulating glucose uptake and metabolism, and β2-ARs by increasing the breakdown of glycogen—with both metabolic events occurring in astrocytes and affecting intermediate memory. The different receptors are activated at different times within the lifetime of labile memory and within 30 min of learning. We have defined separate roles for the three β-ARs in memory and demonstrated that the avian hippocampus is involved in learning and memory in much the same way as the hippocampus in the mammalian brain.

Confocal Ca2+ imaging of organelles, cells, tissues, and organs.

Publisher Summary The chapter presents a study related to confocal Ca 2+ imaging of organelles, cells, tissues, and organs. The chapter includes two-dimensional (2-D) slices of the cell or by specific regions of interest within the cell. The chapter describes methods to create custom cameleons with any combination of resonance energy transfer (RET) partners, affinities, and desired targeting by using readily available molecular genetic techniques. Cameleons are genetically engineered sensors of free Ca 2+ composed of tandem fusions of blue or cyan mutants of fluorescent protein (GFP), calmodulin, calmodulin-binding protein M13, and an enhanced green or yellow emitting GFP. By monitoring the resonance energy transfer (RET) of the two fused GFPs, the Ca 2+ -dependent conformational change of calmodulin as it binds Ca 2+ can be determined. The ability to “tune” the affinity of cameleons for Ca 2+ according to the targeted location is vital for the successful application of the sensors. The advantages of molecular genetic and RET approaches over chemical synthetic approaches are numerous. The chapter discusses acetoxymethyl (AM) ester loading process, delivery of genetically encoded sensors, targeting of Ca 2+ sensors to intracellular organelles, removal of contaminating cytosolic fluorescence, measuring mitochondrial Ca 2+ with multiple sensors, and several other related concepts.

ATP derived from astrocytes modulates memory in the chick.

Memory consolidation in a discriminative bead pecking task is modulated by endogenous adenosine triphosphate (ATP) acting at purinergic receptors in the hippocampus. Consolidation, from short- to intermediate- to long-term memory during two distinct periods following training, was blocked by the non-selective P2 purinergic receptor antagonist PPADS (pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) tetrasodium salt hydrate and the specific P2Y1 receptor antagonist MRS2179. Direct injections of the ATP agonists (ATPγS and ADPβS) potentiated memory consolidation and the effect of ADPβS was blocked by MRS2179, suggesting an important role of ATP on memory consolidation via the P2Y1 receptor in the chick hippocampus. Incubation of astrocytes with ATPγS and ADPβS resulted in the increase of intracellular calcium ([Ca2+]i), the latter being blocked by MRS2179 suggesting a specific role for P2Y1 receptors in the calcium response. This response was prevented by blocking astrocytic oxidative metabolism with fluoroacetate. We argue that the source of the ATP acting on neuronal P2Y1 receptors is most likely to be astrocytes. Thrombin selectively increases [Ca2+]i in astrocytes but not in neurones. The main findings of the present study are: (a) astrocytic [Ca2+]i plays an important role in the consolidation of short-term to long-term memory; and (b) ATP released from chick astrocytes during learning modulates neuronal activity through astrocytic P2Y1 receptors.