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 ADPßS) potentiated memory consolidation and the effect of ADPßS 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 ADPßS 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.

Astrocytic adrenoceptors and learning: alpha1-adrenoceptors.

Noradrenergic receptors are expressed on both on astrocytes and neurons and noradrenergic activation of astrocytic beta(2)- and beta(3)-adrenoceptors are necessary for memory consolidation. In this paper, we marshal evidence for astrocytic alpha(1)-adrenoceptor involvement in memory consolidation. We examine the role of alpha(1)-adrenoceptors in hippocampal and mesopallial (cortical) memory processing using a discriminative avoidance task in the day-old chick. The selective alpha(1)-adrenoceptor agonist, methoxamine, caused the consolidation of weakly-reinforced memory at the time of transition of short-term to intermediate memory and at the time of transition of intermediate to long-term memory. The selective antagonist prazosin prevented memory consolidation at these two times. Blockade of memory by injection of an alpha(2)-adrenoceptor agonist into the LoC could be overcome by mesopallial or hippocampal injection of alpha(1)-, beta(2)- and beta(3)-adrenoceptor agonists. The results of studies where we challenged the ability of methoxamine to promote consolidation by pre-administration of astrocytic metabolic inhibitors of glycogenolysis or oxidative metabolism, suggest that the alpha(1)-adrenoceptor effect is astrocytic. This conclusion is supported by the finding that co-administration of suboptimal doses of methoxamine and thrombin have an additive effect on promoting consolidation. Thrombin causes a calcium response in cultured chick astrocytes but not in neurons. Thrombin, like methoxamine, promotes consolidation at the transition points between short-term, intermediate memory and long-term memory stages. Thrombin enhancement of memory consolidation is blocked by an alpha(1)-adrenoceptor antagonist but not by antagonists of beta(2)- or beta(3)-adrenoceptors. In summary, noradrenaline activation of alpha(1)-adrenoceptors is necessary for consolidation from both short-term and intermediate memory in both the hippocampus and the mesopallium in the chick. Evidence is presented suggesting that the memory consolidating action of alpha(1)-adrenoceptor stimulation results from receptors located on astroctyes and involves an increase in free cytosolic calcium from internal stores.

Astrocytes and interneurons in memory processing in the chick hippocampus: roles for G-coupled protein receptors, GABA(B) and mGluR1.

Glutamate and GABA acting at mGluR1 and GABA(B) receptors, respectively, have roles in memory processing in the hippocampus up to 35 min after bead discrimination learning in the young chick. Activation of mGluR1 receptors is important at 2.5 and 30 min after training, but modulation of these receptors between these two times has no effect on memory. This timing is similar to the action of glutamate on NMDA receptors. The GABA(B) antagonist, phaclofen, and the inhibitor of astrocytic oxidative metabolism, fluoroacetate, inhibited memory when injected between 2.5 and 30 min. Paradoxically, a high dose of the GABA(B) agonist, baclofen, also inhibited memory, but a low dose promoted memory consolidation--an effect possibly caused by too much information and loss of the 'message'. These results are interpreted in terms interactions between interneurons, astrocytes and pyramidal cells and demonstrate the importance of all cell types in memory processing in the hippocampus.

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-Alzheimer's 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.

Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons.

Astrocytes display excitability in the form of intracellular calcium concentration ([Ca(2+)](i)) increases, but the signaling impact of these for neurons remains debated and controversial. A key unresolved issue is whether astrocyte [Ca(2+)](i) elevations impact neurons or not. Here we report that in the CA1 region of the hippocampus, agonists of native P2Y(1) and PAR-1 receptors, which are preferentially expressed in astrocytes, equally elevated [Ca(2+)](i) levels without affecting the passive membrane properties of pyramidal neurons. However, under conditions chosen to isolate NMDA receptor responses, we found that activation of PAR-1 receptors led to the appearance of NMDA receptor-mediated slow inward currents (SICs) in pyramidal neurons. In stark contrast, activation of P2Y(1) receptors was ineffective in this regard. The PAR-1 receptor-mediated increased SICs were abolished by several strategies that selectively impaired astrocyte [Ca(2+)](i) excitability and function. Our studies therefore indicate that evoked astrocyte [Ca(2+)](i) transients are not a binary signal for interactions with neurons, and that astrocytes result in neuronal NMDA receptor-mediated SICs only when appropriately excited. The data thus provide a basis to rationalize recent contradictory data on astrocyte-neuron interactions.

Memory processing in the avian hippocampus involves interactions between beta-adrenoceptors, glutamate receptors, and metabolism.

Noradrenaline is known to modulate memory formation in the mammalian hippocampus. We have examined how noradrenaline and selective beta-adrenoceptor (AR) agonists affect memory consolidation and how antagonists inhibit memory consolidation in the avian hippocampus. Injection of selective beta-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 beta-AR involvement in hippocampal memory processing. Different beta-AR subtypes were recruited in temporal sequence after learning in the order beta(1), beta(3), and beta(2.) We provide evidence that the effect of manipulation of beta(1)-ARs by selective agonists and antagonists within 2.5 min of training parallels the action of NMDA receptor agonists and antagonists. Activation of beta(3)- and beta(2)-ARs facilitated memory but utilized different mechanisms: beta(3)-ARs by stimulating glucose uptake and metabolism, and beta(2)-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 beta-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.

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 approximately 100 and 250 microm, and approximately 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 approximately 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 approximately 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.

An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy.

Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane alpha4beta2 nicotinic and P2X2 channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X2 and alpha4beta2 nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2 and alpha4beta2 channels may form a dimer, with the channels approximately 80 A apart. The measurements also show that P2X2 subunits interact specifically and robustly with the beta2 subunits in alpha4beta2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and alpha4beta2 channels within 100 nm of the plasma membrane of living cells.

ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks.

We investigated the role of extracellular ATP at astrocytes and inhibitory GABAergic interneurons in the stratum radiatum area of the mouse hippocampus. We show that exogenously applied ATP increased astrocyte intracellular Ca2+ levels and depolarized all calbindinand calretinin-positive interneurons in the stratum radiatum region of mouse hippocampus, leading to action potential firing and enhanced synaptic inhibition onto the postsynaptic targets of interneurons. Electrophysiological, pharmacological, and immunostaining studies suggested that the effect of ATP on interneurons was mediated by P2Y1 receptors, and that the depolarization of interneurons was caused by the concomitant reduction and activation of potassium and nonselective cationic conductances, respectively. Electrical stimulation of the Schaffer collaterals and perforant path, as well as local stimulation within the stratum radiatum, evoked increases in intracellular Ca2+ in astrocytes. Facilitation of GABAergic IPSCs onto interneurons also occurred during electrical stimulation. Both the stimulation-evoked increases in astrocyte Ca2+ levels and facilitation of GABAergic IPSCs were sensitive to antagonists of P2Y1 receptors and mimicked by exogenous P2Y1 receptor agonists, suggesting that endogenously released ATP can activate P2Y receptors on both astrocytes and interneurons. Overall, our data are consistent with the hypothesis that ATP released from neurons and astrocytes acts on P2Y1 receptors to excite interneurons, resulting in increased synaptic inhibition within intact hippocampal circuits.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [gamma 2(R43Q)] found in human epilepsy.

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) 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 GABA(A) gamma(2)-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 GABA(A) 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 alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(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 GABA(A) 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.

Pore formation is not associated with macroscopic redistribution of P2X7 receptors.

The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.

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.

Truncation of the GABA(A)-receptor gamma2 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.