Bruno G. Frenguelli

Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein

The cAMP-responsive element-binding protein (CREB) has been implicated in the activation of protein synthesis required for long-term facilitation, a cellular model of memory in Aplysia. Our studies with fear conditioning and with the water maze show that mice with a targeted disruption of the alpha and delta isoforms of CREB are profoundly deficient in long-term memory. In contrast, short-term memory, lasting between 30 and 60 min, is normal. Consistent with models claiming a role for long-term potentiation (LTP) in memory, LTP in hippocampal slices from CREB mutants decayed to baseline 90 min after tetanic stimulation. However, paired-pulse facilitation and posttetanic potentiation are normal. These results implicate CREB-dependent transcription in mammalian long-term memory.

Characterization of Ca2+ signals induced in hippocampal CA1 neurones by the synaptic activation of NMDA receptors.

1. A combination of confocal microscopy, whole‐cell patch‐clamp recording, intracellular dialysis and pharmacological techniques have been employed to study Ca2+ signalling in CA1 pyramidal neurones, within rat hippocampal slices. 2. In the soma of CA1 neurones, depolarizing steps applied through the patch‐pipette resulted in transient increases in the fluorescence emitted by the Ca2+ indicator fluo‐3. The intensity of the fluorescence transients was proportional to the magnitude of the Ca2+ currents recorded through the pipette. Both the somatic fluorescence transients and the voltage‐activated Ca2+ currents ran down in parallel over a period of between approximately 15‐45 min. The fluorescence transients were considered, therefore, to be caused by increases in cytosolic free Ca2+. 3. Under current‐clamp conditions, high‐frequency (tetanic) stimulation (100 Hz, 1 s) of the Schaffer collateral‐commissural pathway led to compound excitatory postsynaptic potentials (EPSPs) and somatic Ca2+ transients. The somatic Ca2+ transients were sensitive to the N‐methyl‐D‐aspartate (NMDA) receptor antagonist D‐2‐amino‐5‐phosphonopentanoate (AP5; 100 microM). These transients, but not the EPSPs, disappeared with a time course similar to that of the run‐down of voltage‐gated Ca2+ currents. Tetanus‐induced somatic Ca2+ transients could not be elicited under voltage‐clamp conditions. 4. Fluorescence images were obtained from the dendrites of CA1 pyramidal neurones starting at least 30 min after obtaining whole‐cell access to the neurone. Measurements were obtained only after voltage‐gated Ca2+ channel activity had run down completely. 5. Tetanic stimulation of the Schaffer collateral‐commissural pathway resulted in compound EPSPs and excitatory postsynaptic currents (EPSCs), under current‐ and voltage‐clamp, respectively. In both cases, these were invariably associated with dendritic Ca2+ transients. In cells voltage‐clamped at ‐35 mV, the fluorescent signal increased on average 2‐fold during the tetanus and decayed to baseline values with a half‐time (t1/2) of approximately 5 s. 6. The alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 10 microM) partially reduced the tetanus‐induced EPSC without affecting the Ca2+ transients. In contrast, AP5, which also depressed the EPSC, substantially reduced or eliminated the Ca2+ transients. 7. In normal (i.e. 1 mM Mg(2+)‐containing) medium, NMDA receptor‐mediated synaptic currents displayed the typical region of negative slope conductance in the peak I‐V relationship (between ‐90 and ‐35 mV). The dendritic tetanus‐induced Ca2+ transients also displayed a similar anomalous voltage dependence, decreasing in size from ‐35 to ‐90 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus

Adenosine is well known to be released during cerebral metabolic stress and is believed to be neuroprotective. ATP release under similar circumstances has been much less studied. We have now used biosensors to measure and compare in real time the release of ATP and adenosine during in vitro ischaemia in hippocampal slices. ATP release only occurred following the anoxic depolarisation, whereas adenosine release was apparent almost immediately after the onset of ischaemia. ATP release required extracellular Ca2+. By contrast adenosine release was enhanced by removal of extracellular Ca2+, whilst TTX had no effect on either ATP release or adenosine release. Blockade of ionotropic glutamate receptors substantially enhanced ATP release, but had only a modest effect on adenosine release. Carbenoxolone, an inhibitor of gap junction hemichannels, also greatly enhanced ischaemic ATP release, but had little effect on adenosine release. The ecto‐ATPase inhibitor ARL 67156, whilst modestly enhancing the ATP signal detected during ischaemia, had no effect on adenosine release. Adenosine release during ischaemia was reduced by pre‐treament with homosysteine thiolactone suggesting an intracellular origin. Adenosine transport inhibitors did not inhibit adenosine release, but instead they caused a twofold increase of release. Our data suggest that ATP and adenosine release during ischaemia are for the most part independent processes with distinct underlying mechanisms. These two purines will consequently confer temporally distinct influences on neuronal and glial function in the ischaemic brain.

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice

1 We have used an enzyme‐based, twin‐barrelled sensor to measure adenosine release during hypoxia in the CA1 region of rat hippocampal slices in conjunction with simultaneous extracellular field recordings of excitatory synaptic transmission. 2 When loaded with a combination of adenosine deaminase, nucleoside phosphorylase and xanthine oxidase, the sensor responded linearly to exogenous adenosine over the concentration range 10 nM to 20 μM. 3 Without enzymes, the sensor when placed on the surface of hippocampal slices recorded a very small net signal during hypoxia of 40 ± 43 pA (mean ±s.e.m.; n= 7). Only when one barrel was loaded with the complete sequence of enzymes and the other with the last two in the cascade did the sensor record a large net difference signal during hypoxia (1226 ± 423 pA; n= 7). 4 This signal increased progressively during the hypoxic episode, scaled with the hypoxic depression of the simultaneously recorded field excitatory postsynaptic potential and was greatly reduced (67 ± 6.5 %; n= 9) by coformycin (0.5‐2 μM), a selective inhibitor of adenosine deaminase, the first enzyme in the enzymic cascade within the sensor. 5 For 5 min hypoxic episodes, the sensor recorded a peak concentration of adenosine of 5.6 ± 1.2 μM (n= 16) with an IC50 for the depression of transmission of approximately 3 μM. 6 In slices pre‐incubated for 3‐6 h in nominally Ca2+‐free artificial cerebrospinal fluid, 5 min of hypoxia resulted in an approximately 9‐fold greater release of adenosine (48.9 ± 17.7 μM; n= 6). 7 High extracellular Ca2+ (4 mM) both reduced the adenosine signal recorded by the sensor during hypoxia (3.5 ± 0.6 μM; n = 4) and delayed the hypoxic depression of excitatory synaptic transmission.

Adenosine and ATP Link PCO2 to Cortical Excitability via pH

In addition to affecting respiration and vascular tone, deviations from normal CO(2) alter pH, consciousness, and seizure propensity. Outside the brainstem, however, the mechanisms by which CO(2) levels modify neuronal function are unknown. In the hippocampal slice preparation, increasing CO(2), and thus decreasing pH, increased the extracellular concentration of the endogenous neuromodulator adenosine and inhibited excitatory synaptic transmission. These effects involve adenosine A(1) and ATP receptors and depend on decreased extracellular pH. In contrast, decreasing CO(2) levels reduced extracellular adenosine concentration and increased neuronal excitability via adenosine A(1) receptors, ATP receptors, and ecto-ATPase. Based on these studies, we propose that CO(2)-induced changes in neuronal function arise from a pH-dependent modulation of adenosine and ATP levels. These findings demonstrate a mechanism for the bidirectional effects of CO(2) on neuronal excitability in the forebrain.

Release of adenosine and ATP during ischemia and epilepsy.

Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions.

The α-Ca2+/calmodulin kinase II: A bidirectional modulator of presynaptic plasticity

The alpha-Ca2+/calmodulin kinase II (alpha CaMKII) is required for long-term potentiation in the CA1 region of the hippocampus. Here, we report that this kinase also has a crucial role in presynaptic plasticity. Paired-pulse facilitation is blunted in the CA1 region of mice heterozygous for a targeted mutation of alpha CaMKII, confirming that this kinase can promote neurotransmitter release. Unexpectedly, field and whole-cell recordings of posttetanic potentiation show that the synaptic responses of mutants are larger than those of controls, indicating that alpha CaMKII can also inhibit transmitter release immediately after tetanic stimulation. Thus, alpha CaMKII has the capacity either to potentiate or to depress excitatory synaptic transmission depending on the pattern of presynaptic activation.

Synaptic Tagging and Capture: Differential Role of Distinct Calcium/Calmodulin Kinases in Protein Synthesis-Dependent Long-Term Potentiation

Weakly tetanized synapses in area CA1 of the hippocampus that ordinarily display long-term potentiation lasting ∼3 h (called early-LTP) will maintain a longer-lasting change in efficacy (late-LTP) if the weak tetanization occurs shortly before or after strong tetanization of an independent, but convergent, set of synapses in CA1. The synaptic tagging and capture hypothesis explains this heterosynaptic influence on persistence in terms of a distinction between local mechanisms of synaptic tagging and cell-wide mechanisms responsible for the synthesis, distribution, and capture of plasticity-related proteins (PRPs). We now present evidence that distinct CaM kinase (CaMK) pathways serve a dissociable role in these mechanisms. Using a hippocampal brain-slice preparation that permits stable long-term recordings in vitro for >10 h and using hippocampal cultures to validate the differential drug effects on distinct CaMK pathways, we show that tag setting is blocked by the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) that, at low concentration, is more selective for CaMKII. In contrast, the CaMK kinase inhibitor STO-609 [7H-benzimidazo(2,1-a)benz(de)isoquinoline-7-one-3-carboxylic acid] specifically limits the synthesis and/or availability of PRPs. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders and test stimulation over long periods of time after LTP induction enable a pharmacological dissociation of these distinct roles of the CaMK pathways in late-LTP and so provide a novel framework for the molecular mechanisms by which synaptic potentiation, and possibly memories, become stabilized.

AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus.

5‐Aminoimidazole‐4‐carboxamide riboside (AICA riboside; Acadesine) activates AMP‐activated protein kinase (AMPK) in intact cells, and is reported to exert protective effects in the mammalian CNS. In rat cerebrocortical brain slices, AMPK was activated by metabolic stress (ischaemia > hypoxia > aglycaemia) and AICA riboside (0.1–10 mm). Activation of AMPK by AICA riboside was greatly attenuated by inhibitors of equilibrative nucleoside transport. AICA riboside also depressed excitatory synaptic transmission in area CA1 of the rat hippocampus, which was prevented by an adenosine A1 receptor antagonist and reversed by application of adenosine deaminase. However, AICA riboside was neither a substrate for adenosine deaminase nor an agonist at adenosine receptors. We conclude that metabolic stress and AICA riboside both stimulate AMPK activity in mammalian brain, but that AICA riboside has an additional effect, i.e. competition with adenosine for uptake by the nucleoside transporter. This results in an increase in extracellular adenosine and subsequent activation of adenosine receptors. Neuroprotection by AICA riboside could be mediated by this mechanism as well as, or instead of, by AMPK activation. Caution should therefore be exercised in ascribing an effect of AICA riboside to AMPK activation, especially in systems where inhibition of adenosine re‐uptake has physiological consequences.

High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices.

We have used improved miniaturized adenosine biosensors to measure adenosine release during hypoxia from within the CA1 region of rat hippocampal slices. These microelectrode biosensors record from the extracellular space in the vicinity of active synapses as they detect the synaptic field potentials evoked in area CA1 by stimulation of the afferent Schaffer collateral‐commissural fibre pathway. Our new measurements demonstrate the rapid production of adenosine during hypoxia that precedes and accompanies depression of excitatory transmission within area CA1. Simultaneous measurement of adenosine release and synaptic transmission gives an estimated IC50 for adenosine on transmission in the low micromolar range. However, on reoxygenation, synaptic transmission recovers in the face of elevated extracellular adenosine and despite a post‐hypoxic surge of adenosine release. This may indicate the occurrence of apparent adenosine A1 receptor desensitization during metabolic stress. In addition, adenosine release is unaffected by pharmacological blockade of glutamate receptors and shows depletion on repeated exposure to hypoxia. Our results thus suggest that adenosine release is not a consequence of excitotoxic glutamate release. The potential for adenosine A1 receptor desensitization during metabolic stress implies that its prevention may be beneficial in extending adenosine‐mediated neuroprotection in a variety of clinically relevant conditions.