Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg²⁺-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 5–7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg²⁺-treated group received MgSO₄ 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg²⁺ concentrations increased from1.6 ± 0.1mg/dl to17.7 ± 3.3mg/dl.³H-MK-801 binding was used as an index of NMDA receptor modification. TheB_max in control, hypoxic and Mg²⁺-treated hypoxic piglets was1.09 ± 0.17, 0.70 ± 0.25and0.96 ± 0.14pmoles/mg protein, respectively. TheK_d for the same groups were10.02 ± 2.04, 4.88 ± 1.43and8.71 ± 2.23nM, respectively. TheB_max andK_d in the hypoxic group were significantly lower compared to the control and Mg²⁺-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg²⁺. The activity of Na⁺, K⁺ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg²⁺-treated hypoxic groups. These findings show that MgSO₄ prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction. We suggest that the administration of Mg²⁺ prior to and during hypoxia may be neuroprotective in vivo, possibly by reducing the NMDA receptor-mediated influx of calcium.     

Dissociation between synaptic depression and block of long-term depression induced by raising the temperature in rat hippocampal slices

The influence of raising the bath temperature (39 degrees C) on synaptic transmission and neuronal plasticity was studied in the CA1 region of the rat hippocampus using an extracellular recording technique. Increasing the bath temperature from 32 to 39 degrees C resulted in a depression of field excitatory postsynaptic potential (fEPSP). Application of the selective A(1) receptor agonist, 2-chloro-adenosine (2-CADO, 1 microM) reduced the fEPSP and subsequently occluded the raised temperature-induced synaptic depression. On the other hand, the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) blocked depression of fEPSP produced by raising the temperature. These results suggest that raising temperature-induced synaptic depression is due to an alteration of extracellular adenosine concentration. Long-term depression (LTD) could be reliably induced by the standard low-frequency stimulation (LFS, 1 Hz for 15 min) protocol at 32 degrees C but not at 39 degrees C. The raised temperature-induced block of LTD was mimicked by 2-CADO. Unexpectedly, despite the presence of DPCPX, LFS still could not elicit LTD. NMDA receptor-mediated synaptic component (fEPSP(NMDA)) was decreased when increasing the temperature to 39 degrees C and DPCPX failed to reverse such a depression. The increase in the NMDA response in 0.1 mM Mg(++) compared with 1 mM Mg(++) was significantly greater at 32 degrees C than at 39 degrees C. These results suggest that, by increasing the sensitivity of Mg(++) block, an increase in temperature modulates NMDA responses and thereby inhibits the induction of LTD.     

Early adenosine release contributes to hypoxia‐induced disruption of stimulus‐induced sharp wave‐ripple complexes in rat hippocampal area CA3

We investigated the effects of hypoxia on sharp wave‐ripple complex (SPW‐R) activity and recurrent epileptiform discharges in rat hippocampal slices, and the mechanisms underlying block of this activity. Oxygen levels were measured using Clark‐style oxygen sensor microelectrodes. In contrast to recurrent epileptiform discharges, oxygen consumption was negligible during SPW‐R activity. These network activities were reversibly blocked when oxygen levels were reduced to 20% or less for 3 min. The prolongation of hypoxic periods to 6 min caused reversible block of SPW‐Rs during 20% oxygen and irreversible block when 0% oxygen (anoxia) was applied. In contrast, recurrent epileptiform discharges were more resistant to prolonged anoxia and almost fully recovered after 6 min of anoxia. SPW‐Rs were unaffected by the application of 1‐butyl‐3‐(4‐methylphenylsulfonyl) urea, a blocker of K_ATP channels, but they were blocked by activation of adenosine A₁ receptors. In support of a modulatory function of adenosine, the amplitude and incidence of SPW‐Rs were increased during application of the A₁ receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Interestingly, hypoxia decreased the frequency of miniature excitatory post‐synaptic currents in CA3 pyramidal cells, an effect that was converted into increased frequency by the adenosine A₁ agonist DPCPX. In addition, DPCPX also delayed the onset of hypoxia‐mediated block of SPW‐Rs. Our data suggest that early adenosine release during hypoxia induces a decrease in pre‐synaptic glutamate release and that both might contribute to transient block of SPW‐Rs during hypoxia/anoxia in area CA3.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

Effect of up-regulation of NMDA receptors on cerebral O2 consumption and blood flow in rat

We tested the hypothesis that cerebrocortical blood flow and O₂ consumption would be proportional to an up-regulated number of functional N-methyl- -aspartate (NMDA) receptors. Previous work had shown a relationship between cerebral metabolism and NMDA receptor activity. We increased the specific binding to NMDA receptors in the cerebral cortex, from 2.2 ± 0.9 to 4.5 ± 0.8 (density units) in male Long-Evans rats by daily giving two intraperitoneal injections (30 mg/kg) of CGS-19755, an NMDA receptor inhibitor, for 7 consecutive days (discontinued for 20 h before experiment). Twelve up-regulated (CGS treated) and 12 control rats were used in this study. Under isoflurane anesthesia and after topical stimulation of the right cerebral cortex with 10⁻² M NMDA, the blood flow (¹⁴C-iodoantipyrine method) increased from 98 ± 11 ml/min/100 g in the unstimulated cortex of the control rats to 161 ± 37 ml/min/100 g in the stimulated cortex. The unstimulated value for blood flow (95 ± 7 ml/min/100 g) did not change in the upregulated group but it doubled (194 ± 69 ml/min/100 g) in the stimulated, upregulated cortex. Similarly, O₂ consumption (cryomicrospectrophotometrically determined) in normal rats increased 46%, from 9.3 ± 1 ml/min/100 g to 13.6 ± 4 after NMDA stimulation. While in the upregulated animals, O₂ consumption increased 103% from 7.9 ± 0.6 to 16 ± 6.5 after NMDA stimulation. In conclusion, NMDA receptor upregulation does not alter basal cerebrocortical blood flow or O₂ consumption but in the NMDA-stimulated cortex, the blood flow and O₂ consumption increase is dependent on the number of NMDA receptors present.     

Characterisation of central adenosine A1 receptors and adenosine transporters in mice lacking the adenosine A2a receptor

The present study was designed to assess whether adenosine A_2a receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A₁ receptors were characterised in brain membranes and on slide-mounted sections. The B_MAX for [³H]nitrobenzylthioinosine ([³H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560±52 fmol/mg protein, n=5, P<0.05, Kruskal–Wallis ANOVA) compared to wildtype (1239±213 fmol/mg protein) and heterozygous mice (1300±558 fmol/mg protein). Quantitative autoradiography data indicated that [³H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) (adenosine A₁ receptor density) binding increased significantly (P<0.05, Kruskal–Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [³H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [³H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [³H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A_2a receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.     

Potential use of DPCPX as probe for in vivo localization of brain A1 adenosine receptors

The suitability of (³H)DPCPX (8-cyclopentyl-1,3-dipropylxanthine), a xanthine derivative, as an vivo probe for labelling adenosine A₁ receptors was studied in rats. [³H]DPCPX (nM) penetrated largely into the brain (0.8% of the injected dose per gram of brain tissue 5 min after injection). Brain concentrations stayed at a plateau level from 5 to 15 min after the injection. The distribution in the different brain regions was heterogeneous with the highest amount of [³H]DPCPX in cerebellum and hippocampus and the lowest concentrations in hypothalamus and brain stem. Displacement (45–70% of total radioactivity) was obtained by the injection of 250 nM of cold DPCPX or cyclopentylxanthine, an analog of DPCPX. The ex vivo autoradiographic distribution of [³H]DPCPX was similar to the in vitro autoradiographic distribution of tritiated A₁ adenosine receptor ligand as [³H]CHA. These results suggest the potential use of DPCPX for further in vivo investigation of A₁ adenosine receptors with techniques such as positron emission tomography.     

Endogenous adenosine facilitates neurotransmission via A2A adenosine receptors in the rat superior colliculus in vivo

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A₁ adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A_2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A₁ adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A_2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A_2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A₁ adenosine receptors.     

Neuroprotection of mild hypothermia: differential effects

To estimate whether mild hypothermia during repetitive hypoxia provides a neuroprotective effect on brain tissue, hippocampal slice preparations were subjected to repetitive hypoxic episodes under different temperature conditions. Slices of guinea pig hippocampus (n=40) were placed at the interface of artificial cerebrospinal fluid (aCSF) and gas (normoxia: 95% O₂, 5% CO₂; hypoxia: 95% N₂, 5% CO₂). Evoked potentials (EP) and direct current (DC) potentials were recorded from hippocampal CA1 region. Slices were subjected to two repetitive hypoxic episodes under the following temperature conditions: (A) 34°C/34°C, (B) 30°C/30°C and (C) 34°C/30°C. Hypoxic phases lasted until an anoxic terminal negativity (ATN) occurred. The recovery after first hypoxia lasted 30 min. Tissue function was assessed regarding the latency of ATN and the recovery of evoked potentials. The ATN latencies with protocol A (n=25) for the first and second hypoxia were 5.9±1.3 min (mean±S.E.M., 1st hypoxia) and 2.4±0.9 min (2nd hypoxia), with protocol B the latencies (n=7) were significantly longer: 25.2±7.1 min and 15.6±7.7 min. With protocol C (n=8), the latencies were 5.6±1.8 and 3.3±0.5 min. No differences were seen in the recovery of the EPs with protocols A–C. Our results suggest that a mild hypothermia is only neuroprotective if applied from an initial hypoxia onwards.     

Isoflurane preconditioning decreases glutamate receptor overactivation-induced Purkinje neuronal injury in rat cerebellar slices

A brain slice model was used to test the hypothesis that preconditioning with isoflurane, a commonly used volatile anesthetic in clinical practice, reduces neuronal injury caused by overstimulation of glutamate receptors. Glutamate receptors were stimulated by various concentrations of glutamate for 20 min, N-methyl-d-aspartate (NMDA) for 15 min or alpha-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) for 15 min. Morphology of Purkinje neurons in the cerebellar slices of adult male Sprague-Dawley rats was evaluated 5 h after the agonist stimulation. Glutamate, NMDA and AMPA induced a dose-dependent decrease in the percentage of morphologically normal Purkinje neurons. The concentration to induce the maximal neurotoxic effect was 300 microM for glutamate, 300 microM for NMDA and 30 microM for AMPA. Isoflurane preconditioning (2% isoflurane for 30 min and then a 15-min rest period before the agonist stimulation) significantly reduced the neurotoxicity induced by 300 microM glutamate, 300 microM NMDA or 30 microM AMPA. Isoflurane preconditioning-induced protection against glutamate neurotoxicity was abolished by two protein kinase C (PKC) inhibitors, calphostin C (0.5 microM) and chelerythrine (5 microM), or a nitric oxide synthase (NOS) inhibitor, l-nitro(G)-arginine methyl ester (l-NAME, 1.5 mM), but was not affected by an adenosine A1 receptor inhibitor, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM), or a Gi protein inhibitor, pertussis toxin (PTX, 200 ng/ml). Isoflurane preconditioning-induced protection against NMDA neurotoxicity was also abolished by calphostin C, chelerythrine or l-NAME. Thus, isoflurane preconditioning reduced glutamate receptor overstimulation-induced neuronal injury/death. This neuroprotection may be PKC- and NOS-dependent.     

Stimulus duration-dependent contribution of Kca channel activation and cAMP to hypoxic cerebrovasodilation

Activation of calcium sensitive (K_ca) K channels and cAMP contribute to pial artery dilation observed during a 10-min exposure to hypoxia. Recent studies show that pial dilation during a 20- or 40-min hypoxic exposure was less than that observed during a 5- or 10-min exposure indicating that stimulus duration determines the nature of the vascular response to hypoxia. The present study was designed to determine if the stimulus duration modulates the contribution of K_ca channel activation and cAMP-dependent mechanisms to hypoxic pial artery dilation in piglets equipped with a closed cranial window. The K_ca channel antagonist iberiotoxin had no influence on pial dilation during 5 min of hypoxia (pO₂≈25 mmHg), decremented the dilation during 10- and 20-min exposure, but had no effect on the dilation during a 40-min exposure (33±1% vs. 32±3%, 33±1% vs. 25±1%, 23±1% vs. 19±1%, and 21±2% vs. 17±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after iberiotoxin). NS1619, a K_ca channel agonist, induced pial dilation during hypoxia that was attenuated by 20- and 40-min but not by 5- and 10-min exposure durations. Similarly, the cAMP antagonist Rp 8-Bromo cAMPs had no influence on pial dilation during 5 min of hypoxia, decremented the dilation during a 10-min exposure, but had no effect on the dilation during a 20- or 40-min exposure (36±1% vs. 34±2%, 34±1% vs. 22±1%, 24±2% vs. 21±2%, and 21±2% vs. 19±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after Rp 8-Bromo cAMPs). Additionally, CSF cAMP was unchanged during 5 min, elevated during 10 min, but such elevations were attenuated during 20- and 40-min hypoxic exposure. Pial vasodilation to a cAMP analogue during hypoxia was attenuated by 20- and 40-min but not by 5- and 10-min hypoxic exposure durations. These data show that K_ca channel activation and cAMP contribute to hypoxic pial artery dilation in a stimulus duration-dependent manner. These data suggest that diminished pial artery dilation during longer hypoxic exposure results from attenuated K_ca channel and cAMP-dependent mechanisms.     

Electrical responses in hippocampal slices after prolonged global ischemia: effects of neuroprotectors

A simple and reproducible animal model of global ischemia, induced by decapitation in 30-day-old Wistar rats, has been developed. It allows to perform electrophysiological analysis of the postischemic reperfusion period in the brain slices. Periods of ischemia up to 40 min increase population spikes measured in the CA1 area of the hippocampus during 2–5 h of reperfusion. Thus after 30-min decapitation-induced ischemia (at t_ischem=25°C), the mean amplitude of the recorded maximum orthodromic population spikes was 159% of the control obtained in the non-ischemic animals. Longer ischemic episodes result in the depression of the population spikes. After 2 h of ischemia, the amplitude of population spikes was about 89% of control. After 3 h of decapitation ischemia, the neurons could not be reactivated. The duration of ischemic episode needed for the irreversible depression of the electrical activity of the brain neurons drastically depends on the temperature at which the ischemic brain is maintained. Thus, only 2 h were needed at 30°C as compared to nearly 3 h at 25°C. We have found that intraperitoneal injection of neuroprotectors which precedes decapitation enables reactivation of the post-ischemic neurons even after very long periods of global ischemia. Thus, MK-801, a non-competitive NMDA receptors antagonist, or NBQX, a blocker of AMPA receptors, administrated 15 min before the long-term (90 min) decapitation ischemia (30°C), induced dose-dependent recovery of population spike with ED₅₀ values 0.2 mg/kg and 3 mg/kg respectively. Our results demonstrate that, in spite of the high vulnerability of hippocampal neurons to hypoxia and ischemia, their electrical activity can be restored after prolonged (more then 1 h) decapitation ischemia. Administration of NMDA or AMPA antagonists enhances recovery.     

Spermine dependent activation of the N-methyl-D-aspartate receptor and the effect of nitric oxide synthase inhibition during hypoxia in the cerebral cortex of newborn piglets

This study tests the hypothesis that brain tissue hypoxia results in modification of spermine-dependent activation of the cerebral N-methyl-D-aspartate (NMDA) receptor ion-channel in newborn piglet brains and that pretreatment with N(omega)-nitro-L-arginine (NNLA), an inhibitor of nitric oxide synthase, will reduce the hypoxia-induced modification of the spermine-dependent activation of the receptor. Piglets were assigned to one of four groups; normoxia or hypoxia with or without NNLA. The infusion of NNLA or vehicle lasted for 60 min while the animals were ventilated under either hypoxic or normoxic conditions. Cerebral tissue hypoxia was confirmed by measuring ATP and phosphocreatine (PCr) levels. P2 membranes were isolated and 3H-MK-801 binding was measured in the presence of spermine. Steady state 3H-MK-801 binding in the presence of spermine, showed an increase in receptor affinity in both normoxic (47% of control) and hypoxic (42% of control) animals without change in receptor density. During hypoxia, the spermine-dependent increase in the maximal response of the 3H-MK-801 binding correlated inversely with the ATP concentrations. NNLA pretreatment prior to hypoxia, resulted in a decrease in the slope of the regression line describing the relationship between cellular energy state (ATP) and percent change in maximal response to spermine compared with vehicle treated animals indicating attenuation of the response to hypoxia. We conclude that the spermine-dependent modification of the affinity of the NMDA receptor ion-channel as assessed by 3H-MK-801 binding is similar in hypoxic and normoxic cortical tissue. NNLA administration reduces the hypoxia-induced spermine-dependent activation of the receptor indicating that nitric oxide mediates modification of the spermine site activation of the NMDA receptor ion-channel complex.     

Cyclic adenosine-3′,5′-monophosphate potentiates the synaptic potential mediated by NMDA receptors in the amygdala

An in vitro slice preparation of rat amygdala was used to study the actions of forskolin and cyclic adenosine-3′,5′-monophosphate (cAMP) analogues on the N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potential (EPSP_NMDA). Intracellular recordings were made from basolateral amygdala neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM) and picrotoxin (50 μM) to pharmacologically isolate the EPSP_NMDA. Application of forskolin (25 μM) markedly and persistently potentiated the EPSP_NMDA In contrast, the inactive forskolin analogue, 1,9-dideoxy-forskolin, failed to affect the EPSP_NMDA significantly. Superfusion of dibutyryl-cAMP (dbcAMP, 200 μM) for 15 min caused a transient depression of the amplitude of EPSP_NMDA. The EPSP_NMDA amplitude was reduced to 68 ± 3% of control (n = 10) 15 min after the application, restored to its control value within 25 min, and followed by a long-term potentiation (LTP). Pretreating the slices with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 5 μM), a selective A₁ receptor antagonist, blocked the transient depressive phase produced by dbcAMP. This result suggests that the transient depression induced by dbcAMP was likely due to the interaction of dbcAMP or its breakdown products with adenosine A₁ receptors. To determine the site of action, we examined the effect of forskolin on the postsynaptic responses to exogenously applied NMDA. Forskolin potentiated the postsynaptic depolarization induced by NMDA, suggesting that the enhancement is mediated, at least in part, by a persistent upregulation of postsynaptic NMDA receptor-operated conductances. Occlusion experiments were performed to examine whether the sustained enhancements of EPSP_NMDA produced by tetanic stimulation (TS) and forskolin share a common mechanism. Three episodes of TS were delivered to saturate the LTP and, under this condition, forskolin still caused a further potentiation of the EPSP_NMDA. Similarly, TS, delivered after the EPSP_NMDA was enhanced by forskolin or dbcAMP, produced LTP. These results suggest that the long-term enhancements of EPSP_NMDA produced by TS and forskolin are different and thus do not support the hypothesis that activation of protein kinase A triggers LTP. © 1995 Wiley-Liss, Inc.     

Enhancement of NMDA-induced increases in levels of endogenous adenosine by adenosine deaminase and adenosine transport inhibition in rat striatum

Unilateral microinjection of N-methyl-d-aspartate (NMDA) into striatum of rats subsequently killed by high-energy focused microwave irradiation significantly increased in vivo levels of endogenous adenosine. At a dose of 25 nmol NMDA, levels of adenosine in injected striata were 263% of levels in uninjected contralateral striata. An inhibitor of adenosine deaminase (deoxycoformycin, DCF) in combination with an inhibitor of adenosine transport (dilazep, DLZP) at a dose that did not affect levels of endogenous adenosine, potentiated NMDA-induced increases in adenosine levels to 426% of contralateral striata. In the presence of DCF and DLZP, NMDA dose-dependently increased levels of adenosine (% of contralateral striatum) from 166% at 10 nmol to 622% at 100 nmol. NMDA-induced increases in levels of endogenous adenosine were completely blocked by prior administration of the NMDA receptor antagonist MK 801 (dizocilpine). Inhibitors of adenosine metabolism and transport may provide therapeutic benefit by potentiating excitatory amino acid-induced increases in levels of endogenous adenosine in vivo.     

Effects of excitatory amino acids and neuropeptide Y on the discharge activity of suprachiasmatic neurons in rat brain slices

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-glutamate, AMPA, NMDA and NPY on the discharge activity of neurons located in the ventral subdivision of the suprachiasmatic nucleus were examined in submerged coronal slices of the rat hypothalamus. All substances were bath applied. Application of

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-glutamate (14 neurons examined) induced an excitatory response in 8 suprachiasmatic neurons (+248.9±122.24%, mean±S.E.M.; P<0.001). A biphasic response, i.e. an initial transient excitation (+54.3±8.21%; P<0.001) succeeded by an inhibition (−66.2±9.31%; P<0.001), was observed in 6 neurons. Application of AMPA (36 neurons examined) resulted in an excitation of 31 neurons (+209.2±58.58%; P<0.0001). Application of NMDA (57 neurons examined) induced an excitation in 34 neurons (+253.8±91.18%; P<0.0001), but an inhibition in 8 neurons (−75.7±6.52; P<0.0001). Biphasic effects of NMDA with an excitatory component (+58.7±9.94%; P<0.0001) succeeded by an inhibitory component (−62.0±8.07%; P<0.0001) were observed in 13 neurons. In 5 of 13 examined cases, the inhibitory component of neuronal responses to NMDA was significantly attenuated by the simultaneous application of strychnine (attenuation was 56%; P<0.05). The application of NPY (40 neurons examined) induced significant effects on the discharge rate of 29 suprachiasmatic neurons. 18 of these neurons were inhibited (−59.3±6.39%; P<0.0001) whereas 11 neurons were excited (+156.6±107.22%; P<0.001) by NPY. In 8 of 11 neurons examined, the NPY-induced inhibition was significantly attenuated by 92% during simultaneous application of strychnine (P<0.001). In 23 NPY-sensitive neurons, the discharge activity was also affected by NMDA. Neurons excited by NPY were also excited by NMDA (8 cells). In neurons inhibited by NPY, application of NMDA induced either an inhibition (3 cells) an excitation (5 cells) or a biphasic effect (7 cells). Results suggest a direct excitatory effect of AMPA, NMDA and NPY on suprachiasmatic neurons. In contrast, inhibitory actions of NMDA and NPY are considered induced by an activation of inhibitory interneurons. Antagonistic effects of strychnine suggest an involvement of glycinergic interneurons in a subpopulation of neurons inhibited by NMDA and in most neurons inhibited by NPY. The involvement of inhibitory mechanisms in photic entrainment of the circadian system is discussed. An integrative model of excitatory and inhibitory actions of EAA and NPY on suprachiasmatic neurons is proposed.     

Activation of adenosine A1 receptors by drugs to lower plasma glucose in streptozotocin-induced diabetic rats

To examine the role of the adenosine A1 receptor in glucose regulation in the absence of insulin, the present study investigated the changes of plasma glucose in male streptozotocin-induced diabetic rats (STZ-diabetic rats) using dipyridamole to increase endogenous adenosine and N6-cyclopentyladenosine (CPA) to activate the adenosine A1 receptor. Intravenous injections of dipyridamole or CPA induced a dose-dependent decrease of plasma glucose in fasting STZ-diabetic rats. Plasma glucose lowering action of dipyridamole, like that of CPA, was inhibited in a dose-dependent manner by pre-treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 8-(p-sulfophenyl)theophylline (8-SPT) at which block the adenosine A1 receptors. Action of the adenosine A1 receptors can thus be considered. In isolated skeletal muscle, CPA enhanced the glucose uptake in a concentration-dependent manner. Blockade of this action by DPCPX and 8-SPT again supported the mediation of the adenosine A1 receptor. Also, CPA produced an increase of glycogen synthesis in isolated soleus muscle. Moreover, CPA decreased plasma triglyceride and cholesterol levels significantly in STZ-diabetic rats. These results suggest that activation of adenosine A1 receptors can increase glucose utilization in peripheral tissues by increasing tissue uptake and glycogen synthesis to lower plasma glucose in rats lacking insulin.     

Hydroxylamine blocks adenosine A1 receptor-mediated inhibition of synaptic transmission in rat hippocampus

The nitric oxide donor hydroxylamine (NH₂OH) induced a transient depression of the evoked synaptic potential recorded in the rat hippocampal CA1 region. This depression was abolished with an adenosine A₁ antagonist, 8-cyclopentyltheophylline. In addition, hydroxylamine reversed adenosine A₁ receptor-mediated inhibition of the evoked population spike, the fEPSP and the intracellularly recorded EPSP. The inhibitory modulation of adenosine A₁ receptor activation by hydroxylamine suggests the presence of a potent endogenous regulatory site.     

Glutamate‐induced depression of EPSP–spike coupling in rat hippocampal CA1 neurons and modulation by adenosine receptors

The presence of high concentrations of glutamate in the extracellular fluid following brain trauma or ischaemia may contribute substantially to subsequent impairments of neuronal function. In this study, glutamate was applied to hippocampal slices for several minutes, producing over‐depolarization, which was reflected in an initial loss of evoked population potential size in the CA1 region. Orthodromic population spikes recovered only partially over the following 60 min, whereas antidromic spikes and excitatory postsynaptic potentials (EPSPs) showed greater recovery, implying a change in EPSP–spike coupling (E–S coupling), which was confirmed by intracellular recording from CA1 pyramidal cells. The recovery of EPSPs was enhanced further by dizocilpine, suggesting that the long‐lasting glutamate‐induced change in E–S coupling involves NMDA receptors. This was supported by experiments showing that when isolated NMDA‐receptor‐mediated EPSPs were studied in isolation, there was only partial recovery following glutamate, unlike the composite EPSPs. The recovery of orthodromic population spikes and NMDA‐receptor‐mediated EPSPs following glutamate was enhanced by the adenosine A1 receptor blocker DPCPX, the A_2A receptor antagonist SCH58261 or adenosine deaminase, associated with a loss of restoration to normal of the glutamate‐induced E–S depression. The results indicate that the long‐lasting depression of neuronal excitability following recovery from glutamate is associated with a depression of E–S coupling. This effect is partly dependent on activation of NMDA receptors, which modify adenosine release or the sensitivity of adenosine receptors. The results may have implications for the use of A₁ and A_2A receptor ligands as cognitive enhancers or neuroprotectants.