The effects of chronic lead exposure on long-term depression in area CA1 and dentate gyrus of rat hippocampus in vitro

Long-term potentiation (LTP) and long-term depression (LTD), two forms of synaptic plasticity, are believed to underlie the mechanisms of learning and memory. Previous studies have demonstrated that low-level lead exposure can impair the induction and maintenance of LTP in vivo and in vitro. The present study was carried out to investigate whether the low-level lead exposure affected the induction and maintenance of LTD. Neonatal Wistar rats were exposed to lead from parturition to weaning via milk of dams drinking 0.2% lead acetate solution. Field excitatory postsynaptic potentials (EPSPs) were recorded in hippocampal slices in adult rats (50–65 days) to study the alterations of LTD in area CA1 and dentate gyrus (DG) of hippocampus following chronic lead exposure. The input–output (I/O) curves before conditioning in both areas showed no evident alterations in basic synaptic transmission between the control and lead exposure groups. In area CA1, the mean amplitude of EPSP slope in control rats (61±11%, n=15) decreased significantly greater than that in lead-exposed rats (78±8%, n=8, P<0.05) following low frequency stimulation (LFS, 1 Hz, 15 min), which lasted at least 45 min. In area DG, with application of the same LFS, the LTD was induced in control rats (72±22%, n=8), while the LFS failed to induce LTD in lead-exposed rats (100±26%, n=8). These results showed that chronic lead exposure affected the induction of LTD in both area CA1 and DG. The effect of lead on synaptic plasticity in area CA1 was also investigated. The alteration of the amplitude of LTP in hippocampal slices caused by lead was reexamined in order to compare with that on LTD (control: 189±23, n=5; lead-exposed: 122±12, n=10). The result demonstrated that low-level lead exposure could reduce the range of synaptic plasticity, which might underlie the dysfunction of learning and memory caused by chronic lead exposure.     

Impairment of long-term potentiation and paired-pulse facilitation in rat hippocampal dentate gyrus following developmental lead exposure in vivo

Neonatal rats were exposed to lead from parturition to weaning via the milk of dams drinking 0.2% lead acetate solution. The alterations of long-term potentiation (LTP) and paired-pulse facilitation (PPF) of hippocampal dentate gyrus in adult rats (90–115 days) following developmental lead exposure were studied in vivo. Input/output (I/O) function, paired-pulse facilitation (PPF), excitatory postsynaptic potential (EPSP) and population spike (PS) amplitude were measured in the dentate gyrus (DG) in response to stimulation applied to the lateral perforant path. The results showed that LTP was induced in control rats with an average PS potentiation of 321.1±50.0% (n=18), which was significantly greater than the increase in PS potentiation (173.5±30.0%, n=17, p<0.001) in lead-exposed rats after tetanizing stimulation. The mean EPSP potentiation increased to 172.4±27.0% (n=18) in control and 138.8±21.4% (n=17) in lead-exposed rats after tetanizing stimulation. The lead-induced impairment of LTP of PS potentiation was more serious than that of EPSP potentiation. Following pairs stimulation of perforant fiber at 250 μA and an interpulse interval (IPI) of 10–1000 ms, the average peak facilitation of PS was 211.3±25.0% (n=13) in control and 187.7±23.0% (n=11) in lead-exposed rats. The average facilitation period duration of PS was 243.0±35.8 ms (n=13) in control and 138.0±24.4 ms (n=11) in lead-exposed rats. These results suggested that developmental lead exposure in neonatal rats caused impairments in LTP and PPF of hippocampal dentate gyrus.     

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.     

Priming of long-term potentiation by prior activation of group I and II metabotropic glutamate receptors in the rat dentate gyrus in vitro

The role of metabotropic glutamate receptors (mGluRs) in long-term potentiation (LTP) has remained controversial. However, it has recently been shown that group I mGluR activation, prior to high frequency stimulation (HFS), can facilitate or ‘prime' LTP in the area CA1 of the hippocampus. Here we report that, in the dentate gyrus in vitro, activation of both group I and group II mGluRs primes LTP. Control LTP, 60 min after HFS was 145.4±3.6% of control. The group I mGluR agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 100 μM), resulted in LTP of 180.1±12.1% of control, which was significantly greater than control LTP (n=4; P<0.05). The group I/II mGluR agonist 1S,3R-1-aminocyclopentate-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), and the group II mGluR agonist (2S,3S,4S)-α-(carboxy-cyclopropyl)-glycine (L-CCG-1, 20 μM) also produced LTP that was significantly greater than control LTP (177.7±11.5% and 183.2±9.1% of control respectively; n=5; P<0.05). The group III mGluR agonist -2-amino-4-phosphonobutyric acid (L-AP4, 20 μM), failed to significantly prime LTP (153.8±5.9% of control; n=5). It also proved difficult to depotentiate the primed LTP. Following low frequency stimulation (LFS), control LTP was reduced to 101.1±3.6% of control, and to 145.0±2.1%, 141.2±14.7% and 134.0±8.7% of control for CHPG, ACPD and L-CCG-1 primed LTP respectively. We conclude that LTP may be primed by mGluR activation in the dentate gyrus and that this priming is mediated through group I and II mGluRs.     

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca²⁺ dependent release of the N‐methyl d ‐aspartate receptor (NMDAR) co‐agonist d ‐serine. Previous evidence indicated that d ‐serine release would be regulated by the intracellular Ca²⁺ release channel IP₃ receptor (IP₃R), however, genetic deletion of IP₃R2, the putative astrocytic IP₃R subtype, had no impact on synaptic plasticity or transmission. Although IP₃R2 is widely believed to be the only functional IP₃R in astrocytes, three IP₃R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP₃R and the contribution of the three IP₃R subtypes to Ca²⁺ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP₃R blocker heparin, and rescued by exogenous d ‐serine, indicating that astrocytic IP₃Rs regulate d ‐serine release. To explore which IP₃R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca²⁺ imaging of hippocampal slices from transgenic mice (IP₃R2^?/? and IP₃R2^?/?;3^?/?). This approach revealed that underneath IP₃R2‐mediated global Ca²⁺ events are an overlooked class of IP₃R‐mediated local events, occurring in astroglial processes. Notably, multiple IP₃Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca²⁺ release by astrocytic IP₃Rs. GLIA 2017;65:502–513     

Ammonia-induced depolarization of cultured rat cortical astrocytes

Exposure of cultured rat cortical astrocytes to increased concentrations of ammonia has been shown to induce morphological and biochemical changes similar to those found in hyperammonemic (e.g., hepatic) encephalopathy in vivo. Alterations of electrophysiological properties are not well investigated. In this study, we examined the effect of ammonia on the astrocyte membrane potential by means of perforated patch recordings. Exposure to millimolar concentrations of NH₄Cl induced a slow dose-dependent and reversible depolarization. At steady state, i.e., after several tens of minutes, the cells were significantly depolarized from a resting membrane potential of −96.2±0.6 mV (n=83, S.E.M.) to −89.1±1.6 mV (n=7, S.E.M.) at 5 mM NH₄Cl, −66.3±3.6 mV (n=9, S.E.M.) at 10 mM NH₄Cl and −50.4±2.5 mV (n=12, S.E.M.) at 20 mM NH₄Cl, respectively. In order to examine the underlying depolarizing mechanisms we determined changes in the fractional ion conductances for potassium, chloride and sodium induced by 20 mM NH₄Cl. No significant changes were found in the fractional sodium or chloride conductances, but the dominating fractional potassium conductance decreased slightly from a calculated 0.86±0.04 to 0.77±0.04 (n=9, S.E.M.). Correspondingly, we found a significant fractional ammonium ion (NH₄⁺) conductance of 0.23±0.02 (n=10, S.E.M.) which was blocked by the potassium channel blocker barium and, hence, most likely mediated by barium-sensitive potassium channels. Our data suggest that the sustained depolarization induced by NH₄Cl depended on changes in intracellular ion concentrations rather than changes in ion conductances. Driven by the high membrane potential NH₄⁺ accumulated intracellularly via a barium-sensitive potassium conductance. The concomitant decrease in the intracellular potassium concentration was primarily responsible for the observed slow depolarization.     

Activation of a non-specific cation conductance by intracellular injection of inositol 1,3,4,5-tetrakisphosphate into identified neurons of Aplysia

The ionic mechanism of the effect of intracellulary injected inositol 1,3,4,5-tetrakisphosphate (IP₄) on the membrane of identified neurons (R9–R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure injection, and ion-substitution techniques. Intracellular injection of IP₄ into a neuron voltage-clamped at −45 mV reproducibly induced a slow inward current (20–60 s in duration, 3–5 nA in amplitude) associated with a conductance increase. The current was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential was −21 mV. The IP₄-induced inward current was sensitive to changes in the external Na⁺, Ca²⁺ and K⁺ concentration but not to changes in Cl⁻ concentration, and was resistant to tetrodotoxin (50 μM). When the cell was perfused with tetraethylammonium (5 mM) but not with 4-aminopyridine (5 mM), the IP₄-induced inward current recorded at −45 mV slightly increased. The IP₄-induced inward current was partially reduced by calcium channel blockers (Co²⁺ and Mn²⁺). These results suggest that intracellularly injected IP₄ can activate a non-specific cation conductance.     

Role of cAMP and K channel-dependent mechanisms in piglet hypoxic/ischemic impaired nociceptin/orphanin FQ-induced cerebrovasodilation

This study was designed to determine the role of altered cAMP and K⁺ channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ) following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC/oFQ elicits pial dilation via release of cAMP, which, in turn, activates the calcium sensitive (K_ca) and the ATP-dependent K⁺ (K_ATP) channel. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased pO₂ to 35±3 mmHg with unchanged pCO₂. Topical NOC/oFQ (10⁻⁸, 10⁻⁶ M) induced vasodilation was attenuated by ischemia/reperfusion (I+R) and reversed to vasoconstriction by hypoxia/ischemia/reperfusion (H+I+R) at 1 h of reperfusion (control, 9±1 and 16±1%; I+R, 3±1 and 6±1%; H+I+R, −7±1 and −12±1%). Such altered dilation returned to control values within 4 h in I+R animals and within 12 h in H+I+R animals. NOC/oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I+R animals and reversed to decreases in cAMP concentration in H+I+R animals (control, 1037±58 and 1919±209 fmol/ml; I+R, 1068±33 and 1289±30 fmol/ml; H+I+R, 976±36 and 772±27 fmol/ml for absence and presence of NOC/oFQ 10⁻⁶ M, respectively). Topical 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) pial dilation was unchanged by I+R but blunted by H+I+R (control, 10±1 and 20±1%; I+R, 11±1 and 20±2%; H+I+R, 0±1 and 0±2%). Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K_ATP channel activators, respectively, elicited dilation that was blunted by both I+R and H+I+R while NS1619, a K_ca channel activator, elicited dilation that was unchanged by I+R but blunted by H+I+R. These data indicate that impaired NOC/oFQ dilation following I+R results form altered adenylate cyclase and K_ATP channel-dependent mechanisms. These data further indicate that impaired NOC/oFQ dilation following H+I+R results not only from altered adenylate cyclase and K_ATP channel but also from altered cAMP and K_ca channel-dependent mechanisms.     

Cognitive deficits in interleukin-10-deficient mice after peripheral injection of lipopolysaccharide

Interleukin (IL)-10 is important for regulating inflammation but whether it protects against infection-related deficits in cognitive function is unknown. Therefore, the current study evaluated sickness behavior, hippocampal-dependent matching-to-place performance and several inflammatory cytokines and neurotrophins in wild-type (IL-10^+/+) and IL-10-deficient (IL-10^−/−) mice after i.p. injection of lipopolysaccharide (LPS). Additionally, morphology of dendrites of pyramidal neurons in the dorsal CA1 hippocampus was assessed. Treatment with LPS increased IL-1β, IL-6, and tumor necrosis factor α (TNFα) mRNA in all brain areas examined including the hippocampus, in both IL-10^+/+ and IL-10^−/− mice but the increase was largest in IL-10^−/− mice. Plasma IL-1β, IL-6 and TNFα were also higher in IL-10^−/− mice compared to IL-10^+/+ mice after LPS. Consistent with increased inflammatory cytokines in IL-10^−/− mice after LPS treatment, were a more lengthy sickness behavior syndrome and a more prominent reduction in hippocampal levels of nerve growth factor mRNA; brain-derived neurotrophic factor mRNA was reduced similarly in both genotypes after LPS. In a test of hippocampal-dependent learning and memory that required mice to integrate new information with previously learned information and switch strategies to master a task, IL-10^−/− mice were found to be less efficient after LPS than were similarly treated wild-type mice. LPS did not affect morphology of dendrites of pyramidal neurons in the dorsal CA1 hippocampus in either genotype. Taken together the results are interpreted to suggest that during peripheral infection IL-10 inhibits sickness behavior and tribulations in hippocampal-dependent working memory via its propensity to mitigate inflammation. We conclude that IL-10 is critical for maintaining normal neuro-immune communication during infection.     

Effect of nitroprusside on regional cerebral cyclic GMP, blood flow and O2 consumption in rat

This investigation was conducted to test whether topical nitroprusside (NP), a cytosolic guanylate cyclase activator, would increase the level of cyclic GMP and alter O₂ consumption or blood flow in the cerebral cortex of rats. Male Long-Evans rats were used in a control (n = 9), low dose NP (n = 13, 10⁻³ M) or high dose NP (n = 12, 10⁻² M) group. Nitroprusside or saline was topically applied to the right side of the cerebral cortex and the left side was used as a control. The cyclic GMP level was determined in five rats in each group using a radioimmunoassay. In the o ther rats in each group, regional cerebral blood flow was measured by [¹⁴C]iodoantipyrine and regional arterial and venous O₂ saturations were determined microspectrophotometrically. Nitroprusside significantly increased the cyclic GMP level from 21.4 ± 12.0 pmol/g (contralateral cortex) to 52.2 ± 36.7 pmol/g (NP treated cortex) in low dose nitroprusside group and from 19.9 ± 22.6 pmol/g (contralateral cortex) to 58.5 ± 15.1 pmol/g (NP treated cortex) in high dose nitroprusside group. High dose nitroprusside significantly increased cerebral blood flow from 80 ± 11 ml · min⁻¹ · 100 g (contralateral cortex) to 114 ± 11 ml · min⁻¹ · 100 g (NP treated cortex). However, there was no significant difference in O₂ extraction and O₂ consumption between the NP treated cortex and contralateral cortex in either the low or the high dose NP groups. In the high dose NP group, the O₂ extraction was 8.0 ± 1.3 ml O₂ · 100 ml⁻¹ in the treated cortex and 8.8 ± 1.5 ml O₂ · 100 ml⁻¹ in the contralateral cortex, while the O₂ consumptions in the NP treated cortex and contralateral cortex were 8.1 ± 1.3 ml O₂ · min⁻¹ · 100 g⁻¹ and 7.3 ± 1.2, respectively. Thus, NP increased the cyclic GMP level without a significant change in O₂ consumption in the cerebral cortex. Our data suggested that O₂ consumption in the cerebral cortex was not affected by the increased level of cyclic GMP.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7–12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in 43% of the neurons (53/123); however, in a minority population (6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I_5-HT) in neurons voltage-clamped near the resting potential (-60 mV); the effect was mimicked by the 5-HT₃ receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT₃ receptors. Further, I_5-HT was selectively inhibited by the 5-HT₃ receptor-specific antagonist MDL72222 (1–10 μM), but was unaffected by either 5-HT₁/5-HT₂ receptor antagonist, spiperone, or by 5-HT₂ receptor-specific antagonist, ketanserin (50–100 μM). I_5-HT displayed moderate inward rectification and had a mean reversal potential (±S.E.M.) of −4.3±6.6 mV (n=6). Application of 5-HT (dose range: 0.1–100 μM) produced a dose–response curve that was fitted by the Hill equation with EC₅₀=3.4 μM and Hill coefficient=1.6 (n=8). The activation phase of I_5-HT (10 μM 5-HT at −60 mV) was well fitted by a single exponential with mean (±S.E.M.) time constant of 45±30 ms (n=6). The desensitization phase of I_5-HT was best fitted by a single exponential with mean (±S.E.M.) time constant of 660±167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (±S.E.M) of 2.7±1.5 pS (n=4) at −60 mV. In the minority (6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT₂ receptor antagonist, ketanserin (50 μM). Taken together, these results suggest that 5-HT₃ receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.     

Critical volume of rat cortex and extracellular threshold concentration for a pentylenetetrazol-induced epileptic focus

The initiation of focal interictal epileptiform activity (FIEA) has been shown to depend on the activation of a sufficiently large volume of brain tissue. We estimated the size of this ‘critical volume' for the convulsant pentylenetetrazol (PTZ) by analyzing the diffusion following its microinjection into rat motor cortex. PTZ concentration was monitored 100–200 μm away from the injection site with a PTZ-sensitive microelectrode. Diffusion analysis in 0.3% agar yielded the free diffusion coefficient D (8.50±0.15×10⁻⁶ cm²·s⁻¹ at 37°C, median±S.E.M.). In brain tissue, diffusion was modified by extracellular volume fraction (α), tortuosity (λ=(D/ADC)^1/2; ADC=apparent diffusion coefficient) and non-specific uptake (k′). Using a value of 0.2 for α from previous studies, we found values of λ=1.61±0.01, k′=3.37±0.15×10⁻³ s⁻¹ and an injected volume U of 5.16±0.45×10⁻¹⁰ l for pulses without FIEA, and λ=1.95±0.06, k′=6.24±1.73×10⁻³ s⁻¹ and U=7.40±0.66×10⁻¹⁰ l for pulses with FIEA. From the calculated concentration distribution of PTZ during FIEA we estimated a threshold concentration of about 1.77 mM PTZ and a volume with a radius of about 219 μm in which this concentration had to be exceeded. Since this critical volume was comparable in size to foci elicited by penicillin or electric stimuli in previous studies, it is concluded that it is determined by intrinsic tissue properties rather than by the convulsive agent being used. © 1997 Elsevier Science B.V. All rights reserved.     

Role of impaired cAMP and calcium-sensitive K channel function in altered cerebral hemodynamics following brain injury

Previous studies have shown that pial arteries constricted and responses to dilator opioids were blunted after fluid percussion injury (FPI) in newborn pigs. Membrane potential of vascular muscle is a major determinant of vascular tone and activity of K⁺ channels is a major regulator of membrane potential. Recent data show that opioids elicit dilation via the sequential production of cAMP and subsequent activation of calcium-sensitive K⁺ (

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) channels by this second messenger. The present study was designed to investigate the effect of FPI on cAMP and

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channel function. Chloralose-anesthetized piglets equipped with a closed cranial window were connected to a percussion device consisting of a saline-filled cylindrical reservoir and a metal pendulum. Brain injury of moderate severity (1.9–2.1 atm) was produced by allowing the pendulum to strike a piston on the cylinder. FPI blunted dilation to the cAMP analogs 8-Bromo cAMP and Sp 8-Bromo cAMPs (10⁻⁸, 10⁻⁶ M), (9±1 and 16±1 vs. 2±1 and 3±1% dilations to 8-Bromo cAMP before and after FPI, respectively, n=8). Similarly, FPI attenuated dilation to pituitary adenylate cyclase activating peptide (PACAP), an endogenous activator of adenylate cyclase, and NS 1619, a

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channel agonist (9±1 and 16±1 vs. 3±1 and 5±1% for NS 1619 10⁻⁸, 10⁻⁶ M before and after FPI, respectively, n=8). Moreover, FPI attenuated PACAP, methionine enkephalin, leucine enkephalin, and dynorphin induced elevations in CSF cAMP concentration (940±2, 1457±50, and 2191±53 vs. 810±17, 1033±36, and 1218±49 fmol/ml for control, PACAP 10⁻⁸, 10⁻⁶ M before and after FPI, respectively, n=8). These data show that cAMP and

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channel function is impaired after FPI. Further these data suggest that impaired cAMP and

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channel function contribute to altered cerebral hemodynamics following FPI.     

Kinetic analysis ofl-leucine transport across the blood-brain barrier

Unidirectionall-leucine influx across cerebral capillaries was measured at different concentrations with an in situ rat brain perfusion technique, which has been several advantages over presently-used methods such as the Brain Uptake Index (BUI) technique. The maximal influx rate (V_max) and half-saturation concentration (K_m) equaled1.07 ± 0.02 × 10^3- μmol·s⁻·⁻¹and0.026 *+- 0.002 μmol·ml⁻¹, respectively, for the saturable component, and the constant for non-saturable equaled6.8 ± 1.4 × 10⁻⁵s⁻¹. These values differ by 3–4-fold from respective values obtained with the BUI technique.     

Involvement of peripheral NMDA and non-NMDA receptors in development of persistent firing of spinal wide-dynamic-range neurons induced by subcutaneous bee venom injection in the cat

To study the roles of peripheral excitatory amino acids receptor subtypes N-methyl- -aspartate (NMDA) and non-NMDA receptors in persistent nociception, extracellular single unit recording technique was used to assess the effects of a single dose NMDA and non-NMDA receptor antagonists, AP₅ (5-aminophosphonovaleric acid) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) or DNQX (6,7-dinitroquinoxaline-2,3-dione), on s.c. bee venom-induced increase in firing of wide-dynamic-range (WDR) neurons in the spinal dorsal horn of the urethane–chloralose anesthetized cats. Subcutaneous bee venom injection into the cutaneous receptive field resulted in a single phase of increased firing of WDR neurons over the background activity for more than 1 h. Local pre-administration of AP₅ (200 μg/100 μl) or CNQX (8.3 μg/100 μl) into the bee venom injection site produced 94% (1.01±0.96 spikes/s, n=5) or 76% (2.97±0.58 spikes/s, n=4) suppression of the increased neuronal firing when compared with local saline (16.32±4.55 spikes/s, n=10) or dimethyl sulfoxide (DMSO) (12.37±6.36 spikes/s, n=4) pre-treated group, respectively. Local post-administration of the same dose of AP₅ produced a similar result to the pre-treatment group with a 67% inhibition of the mean firing rate, however, the same treatment with CNQX and even a higher dose of DNQX (100 μg/100 μl) did not produce any inhibition of the neuronal firing induced by s.c. bee venom injection (DNQX vs. DMSO: 23.91±0.25 vs. 22.14±0.04 spikes/s, P=0.0298, n=5). In the control experiments, local pre-administration of the same dose of AP₅ or CNQX into a region on the contralateral hindpaw symmetrical to the bee venom injection site produced no significant influence on the increased firing of the WDR neurons [contralateral AP₅ vs. saline: 14.17±6.27 spikes/s (n=5) vs. 16.32±4.55 spikes/s (n=10), P>0.05; contralateral CNQX vs. DMSO: 12.85±6.38 spikes/s (n=4) vs. 12.37±6.36 spikes/s (n=4), P>0.05], implicating that the suppressive action of local AP₅ or CNQX was not the result of systemic effects. The present results suggest that activation of the peripheral NMDA receptors is involved in both induction and maintenance, while activation of non-NMDA receptors is only involved in induction, but not in the maintenance of persistent firing of the dorsal horn WDR neurons induced by s.c. bee venom injection.     

Neuroprotective effect of the novel glutamate AMPA receptor antagonist YM872 assessed with in vivo MR imaging of rat MCA occlusion

The neuroprotective effect of post-ischemic treatment with the novel, highly water-soluble, glutamate AMPA receptor antagonist YM872 was evaluated by using MR imaging and histopathology of rats subjected to permanent MCA occlusion. Two treatment groups with continuous i.v. infusion of 20 mg kg⁻¹ h⁻¹ YM872 during either the first 4 h or first 24 h after MCA occlusion, called 4 h YM872 treatment group (n=9) and 24 h YM872 treatment group (n=8) respectively, were compared to a control group (n=8). The main end-point was T2 weighted MR imaging and histopathology 24 h after MCA occlusion. Also the time evolution of the ischemic tissue damage was studied by diffusion weighted MR imaging and 24 h after MCA occlusion. The volume of ischemic tissue damage as assessed by diffusion weighted MR imaging h after MCA occlusion was significantly smaller in both YM872 treatment groups (99±52 mm³ and 102±44 mm³ compared to 186±72 mm³ in the control group, ±S.D. and p=0.008). The infarct volume as assessed by T2 weighted MR imaging 24 h after MCA occlusion was significantly smaller only in the 24 h YM872 treatment group (262±57 mm³ compared to 366±49 mm³ in the control group, ±S.D. and p=0.01) while the infarct volume in the 4 h YM872 treatment group (357±88 mm³) was similar to the control group. YM872 treatment significantly reduced the infarct volume 24 h after MCA occlusion when the drug was administered as continuous infusion during the 24-h observation period.     

γ-Hydroxybutyric acid-induced absence seizures in GluR2 null mutant mice

In this electrophysiological study, we examined the susceptibility of GluR2 mutant null mice to absence seizures in comparison with wild-type controls. The prodrug of (GHB), γ-butyrolactone (GBL) was given systemically to induce the absence seizures. We also tested the severity and duration of the seizure activity in this model. The results showed that the latency from GBL administration to onset of seizure was significantly prolonged in GluR2^−/− mice when compared to GluR2^+/+ mice. The duration of spike-and-wave discharges (SWD) was also significantly decreased in the GluR2^−/− mice. Ninety minutes following GBL administration, wild-type animals continued to exhibit intermittent SWD bursts while GluR2^−/− mice had returned to baseline. These data suggest that the GluR2 subunit may be involved in the initiation and maintenance of absence seizures induced by GBL.     

Only ‘de novo’ long-term depression (LTD) in the rat hippocampus in vitro is blocked by the same low concentration of FK506 that blocks LTD in the visual cortex

It has been proposed that the long-term depression (LTD) seen following low frequency stimulation (LFS) in the rat hippocampus involves calcineurin. We have tested this by examining the effect of FK506, a macrolide which blocks calcineurin at nanomolar concentrations, on synaptic transmission in the rat hippocampal slice at a concentration of 1 μM which has been shown to block LTD in the visual cortex. The effect of FK506 on long-term potentiation (LTP) and spontaneous transmitter release was also studied. The magnitude of LTD induced by LFS was 16.7 ± 2.4% in control which was not significantly different from the 22.3 ± 3.0% seen in the same preparations after exposure to FK506 for 25–30 min. In contrast the magnitude of LTD induced ‘de novo’ in preparations exposed to FK506 was significantly reduced. FK506 had no significant effect on LTP, miniature EPSP frequency, miniature EPSP amplitude, resting membrane potential or input resistance. These results, therefore, support the hypothesis that calcineurin is involved in ‘de novo’ LTD but it appears that an event is triggered by LFS whereby FK506-insensitive LTD can subsequently be activated by a second episode of LFS.     

Neuronal plasticity in hippocampal mossy fiber-CA3 synapses of mice lacking the inositol-1,4,5-trisphosphate type 1 receptor

In the present study, we used inositol-1,4,5-trisphosphate (IP3) type 1 receptor (IP3R1) knockout mice to examine the role of this receptor in the induction of LTP, LTD, and DP at mossy fiber-CA3 synapses. No difference in synaptically induced field-EPSPs was seen between the wild-type (IP3R1(+/+)) and IP3R1 knockout mice (IP3R1(-/-)), showing that basic synaptic transmission does not involve IP3R1 activation. Tetanus induced LTP in both wild-type and IP(3)R1(-/-) mice, but the magnitude of LTP was significantly greater in IP3R1(-/-) mice (149.8+/-3.5%, mean+/-S.E.M., n=15) than in wild-type mice (132.4+/-1.5%, n=17), suggesting that the IP3R1 has a suppressive effect on LTP induction. To determine whether this effect involved N-methyl-D-aspartate receptor (NMDAR)-dependent LTP, the effect of tetanus was tested in the present of the NMDAR antagonist, D,L-AP5 (50 microM); under these conditions, the LTP in both IP3R1(-/-) and IP3R1(+/+) mice was not significantly reduced. In addition, group I mGluR activation was shown to be necessary for LTP induction, as the LTP was almost blocked by the group I mGluR antagonist, RS-4CPG (500 microM) in both IP3R1(-/-) (117.6+/-1.7%, n=8) and IP3R1(+/+) (116.9+/-1.8%, n=5) mice. The IP3R1 also plays an essential role in LTD induction, as low-frequency stimulation (LFS) failed to induce LTD in the mutant mice (104.5+/-2.1%, n=10). DP was induced in both IP3R1(-/-) and wild-type mice.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.