Age-related changes in NMDA-induced [3H]acetylcholine release from brain slices of senescence-accelerated mouse.

From the brain slices of normal mice (ddY strain, subcloned from dd strain in National Institute of Health in Japan), N-methyl-D-aspartic acid (NMDA) at 0.01-1 mM evoked [3H]acetylcholine (ACh) release in a concentration dependent manner. [3H]ACh release evoked by 1 mM NMDA was significantly inhibited by 2-amino-5-phosphonovaleric acid (APV), phencyclidine (PCP) and 5-methyl-10,11-dihydroxy-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). The effects of NMDA were not seen in the Ca2+ free medium and were inhibited by physiological concentration (0.83 mM) of Mg2+. NMDA seems to cause ACh release from nerve terminals through the receptor-ion channel mediated mechanism in the mouse brain. Based upon these results, we determined the activity of a high K(+)- or NMDA-evoked [3H]ACh release using prone/8 strain of senescence-accelerated mouse (SAM-P/8) (a murine model of accelerated aging and memory dysfunction) and SAM-resistance/1 strain (SAM-R/1) (normal aging mice as the control) and these release activities were compared between both strains and during aging. [3H]ACh release evoked by 30 mM KCl was significantly lower than that of age-matched SAM-R/1 at 9 and 12 months. NMDA evoked the [3H]ACh release at 2, 6, 10 and 14 months in R/1 mice. In SAM-P/8 mice the activity of NMDA-evoked release was seen at 2 months, but markedly decreased afterwards. Nonsignificant difference was observed on the uptake of [3H]choline and on the spontaneous release of [3H]ACh between SAM-P/8 and SAM-R/1 strains, and during aging.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes of sodium-dependentd-[3H]aspartate and [3H]FK506 binding in rat brain

Summary We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependentd-[³H]aspartate and [³H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependentd-[³H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [³H]FK506 binding was significantly lower only in the hippocampus, thalamus and cerebellum. [³H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependentd-[³H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependentd-[³H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10–25% reduction in sodium-dependentd-[³H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

快速老化模型小鼠学习记忆行为及有关脑区单胺递质水平的增龄性变化

目的 研究快速老化模型小鼠(senescence accelerated mice，SAM)学习记忆能力及其大脑皮层、海马和下丘脑单胺递质含量的增龄性变化及它们之间的关系。方法 分别采用跳台实验和穿梭箱实验测定SAM的被动和主动回避反应能力，采用高效液相色谱电化学检测法测定脑内单胺递质的含量。结果 2月龄快速老化亚系SAM-prone/8(SAMP8)的被动和主动回避反应能力已较同龄抗快速老化亚系SAM-resistance／1(SAMR1)明显降低，且其主动回避反应能力随增龄进一步降低。同时，SAMP8大脑皮层、海马及下丘脑内单胺递质水平多明显高于同龄SAMR1，且随增龄明显增高。结论 SAMP8学习记忆能力的衰退可能与其相关脑区单胺递质的变化密切相关。     

Estimation of endogenous noradrenaline release in rat brain in vivo using [3H]RX 821002

Noradrenaline plays an important role in many normal brain functions, e.g., attention, memory, and emotion. Dysfunction in the noradrenergic system is thought to lead to a number of abnormal brain conditions. The lack of suitable in vivo tracers to monitor noradrenaline release, levels, and regulation has hampered our fully understanding the roles that it plays in the brain. Presented here are data showing that the in vivo binding of the alpha2-adrenoceptor antagonist [3H]RX 821002 is sensitive to endogenous noradrenaline. Elevation of extracellular noradrenaline, using three different pharmacological challenges in rat, led to a reduction in the binding potential (BP) of [3H]RX 821002 when compared with vehicle controls. The challenges used were i.p. administration of D-amphetamine, the imidazoline2 binding site-selective ligand BU224, and L-deprenyl. Of the cortical regions measured, the reduction in BP reached significance in the anterior cingulate cortex for all of these pharmacological challenges. These initial observations in rat indicate that labelling of the alpha2-adrenoceptors with RX 821002 can be used to estimate changes in extracellular noradrenaline concentration in the cortex. This has the potential to enable the investigation of the role that noradrenaline plays both in the normal and abnormal brain and, if the ligand can be radiolabelled with a suitable positron-emitting isotope at high specific radioactivity, it could be an invaluable PET tracer.     

Beta-scorpion toxin induces the release of gamma-[3 H]aminobutyric acid in rat brain slices

The effect of the beta-scorpion toxin, TiTX gamma on the release of [3H]GABA from rat brain cortical slices is described. The stimulatory effect of TiTX gamma on the release of [3H]GABA was dependent on incubation time and TiTX gamma concentration with an EC50 of 0.19 microM. The scorpion toxin effect was calcium dependent and was completely inhibited by tetrodotoxin. beta-Alanine also induced the release of [3H]GABA and this effect was not inhibited by tetrodotoxin but was additive in the presence of TiTX gamma. The data suggest a neuronal origin for the release of [3H]GABA by TiTX gamma.     

The effect of psychostimulants on [3H]dopamine uptake and release in rat brain synaptic vesicles

Summary. Amphetamine and its derivatives are psychostimulants active at the plasma membrane monoamine transporters. In the present study we assessed the interaction of parachloroamphetamine, D-amphetamine, fenfluramine and methylendioxymethamphetamine with brain vesicular monoamine transporter using purified rat striatal synaptic vesicles. All four psychostimulants inhibited vesicular [³H]dopamine uptake in a competitive and dose-dependent manner and had no effect on [³H]dihydrotetrabenazine binding. At higher concentrations the drugs enhanced [³H]dopamine vesicular efflux. Parachloroamphetamine was the most potent agent while methylendioxymethamphetamine was the weakest one. The vesicular activities may be relevant to their neurotoxicity.     

The effect of sevoflurane on the release of [3H]dopamine from rat brain cortical slices

Dopamine is a neurotransmitter that exerts major control on important brain functions and some lines of studies suggest that dopaminergic neurotransmission may be a potential target for volatile anesthetics. In the present study, rat brain cortical slices were labeled with [(3)H]dopamine to investigate the effects of sevoflurane on the release of this neurotransmitter. [(3)H]dopamine release was significantly increased in the presence of sevoflurane (0.46 mM) and this effect was independent of extracellular or intracellular calcium. In addition, [(3)H]dopamine release evoked by sevoflurane was not affected by TTX (blocker of voltage-dependent sodium channels) or reserpine (a blocker of the vesicular monoamine transporter). These data suggest that the dopamine release induced by sevoflurane is non-vesicular, independent of exocytosis and, would be mediated by the dopamine transporter (DAT). GBR12909 and nomifensine, inhibitors of DAT, decreased the release of [(3)H]dopamine evoked by sevoflurane. The same effect was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium. Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce dopamine release through reverse transport, decreased [(3)H]dopamine release induced by sevoflurane. In conclusion, the present study suggests that sevoflurane increases [(3)H]dopamine release in brain cortical slices that is mediated by DAT located at the plasma membrane.     

The antidepressant agents desipramine, fluoxetine, fluvoxamine and norzimelidine inhibit uptake of [H]noradrenaline and [H]5-hydroxytryptamine in slices of human and rat cortical brain tissue

The stability of uptake of [³H]noradrenaline and [³H]5-hydroxytryptamine in rat neocortical brain slices, prepared up to 4–5 h after decapitation, was established. Subsequently, monoamine uptake inhibition by 4 antidepressant agents: desipramine, fluoxetine, fluvoxamine and norzimelidine was compared in human post-mortem neocortical brain slices vs rat neocortical brain slices.In both species, desipramine exhibited considerable selectivity for noradrenaline uptake, while fluoxetine, fluvoxamine and norzimelidine showed varying degrees of selectivity for 5-hydroxytryptamine uptake. These results support the use of rat brain as a model for the study of neurochemical correlates of depression and antidepressant drug action.     

Distribution of N-methyl-D-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain

N-methyl-D-aspartate (NMDA) is an acidic amino acid which depolarizes neurons by selectively interacting with a distinct class of excitatory amino acid receptor. Recent evidence has indicated that this receptor is a neurotransmitter receptor in the spinal cord, cerebral cortex, and hippocampus for which the endogenous ligand is likely to be L-glutamate or a structurally related compound. Using quantitative autoradiography, we have studied the anatomical distribution of the class of L-[3H]glutamate-binding sites displaced by NMDA, which appear to correspond to NMDA receptors. The CA1 region of the hippocampus contains the highest density of sites. In general, telencephalic regions have high levels of binding sites. The cerebral cortex shows significant density variations among the differing layers and regions, with the highest levels found in the frontal cortex layers I to III. Within the basal ganglia, the highest levels are found in the nucleus accumbens, intermediate levels are found in the caudate/putamen, and very low levels are found in the globus pallidus. Thalamic regions have moderate levels with variations among differing regions. Midbrain and brainstem have low levels of binding sites, but within these regions there are structures exhibiting higher levels, e.g., the nucleus of the solitary tract and the inferior olive. The distribution of NMDA sites is consistent with most, but not all, of the regions previously proposed to use glutamate as an excitatory transmitter. Thus, the distribution of NMDA-sensitive L-[3H]glutamate-binding sites suggests that the NMDA receptor represents a major, distinct subset of excitatory amino acid receptors and indicates regions in which neurotransmission may be mediated or modulated by this receptor.     

Sites of D-[3H]aspartate accumulation in mouse cerebellar slices

Slices of mouse cerebellar vermis, cut in the parasagittal plane, were incubated for various times (up to 3 h) in the presence of 1 microM D-[3H]aspartate, a non-metabolized substrate for the glutamate/aspartate carrier in brain tissue. Light microscopic autoradiography indicated that in regions away from the cut edges of the slices the amino acid accumulated in glia and granule cells. Relatively few grains were seen over Purkinje, Golgi, stellate and basket cells or over white matter. Grain counts over the granule cell layers in the middle parts of the slices indicated that after short (15 min) exposures to the labelled substrate, non-granule cell areas (which included glia) contained, on average, slightly more grains than granule cells but with longer exposures (1.5 and 3 h) the relative grain density over granule cells became much higher, possibly because glial uptake prevents D-[3H]aspartate gaining access to neuronal sites in adequate amounts during short incubations and/or because the longer incubations allow time for retrograde migration of the label from parallel fibre terminals to occur. The demonstration of selective uptake of D-[3H]aspartate into granule cells contrasts with previous autoradiographic results (possible reasons for which are discussed) and supports the notion that L-glutamate is the transmitter of granule cells. The results also have a bearing on the importance of the metabolic compartmentation of glutamate in relation to its proposed transmitter role.     

Nitric oxide induces calcium-dependent release of [3H]dopamine release from striatal slices

Hydroxylamine (0.01–30 mM), a nitric oxide (NO) generator, produced a concentration-dependent release of [³H]dopamine ([³H]DA) from rat striatal slices. Hemoglobin (10 μM), a NO scavenger, reduced basal [³H]DA release and blocked hydroxylamine (100 μM)-stimulated [³H]DA efflux. Tetrodotoxin (0.5 μM) had no significant effect. Sodium cyanide was used as a model compound to test the possibility that NO acted through blockade of mitochondrial electron transport. Calcium-free experimental buffer (1 mM EGTA) reduced basal release and the hydroxylamine response, while sodium cyanide-induced release did not change under these experimental conditions. Cadmium (200 μM), a non-selective inhibitor of voltage-dependent calcium channels, reduced the hydroxylamine response by 69%. Methylene blue (10 μM), an inhibitor of guanylate cyclase, produced a 3-fold increase in the basal release but had no significant effect on the hydroxylamine response. These data suggest that NO induces calcium-dependent [³H]DA release from the striatum via a mechanism which is independent of blockade of electron transport or activation of guanylate cyclase. © 1993 Wiley-Liss, Inc.     

The effect of isoflurane on the release of [H]-acetylcholine from rat brain cortical slices

Volatile general anaesthetics are believed to affect synaptic transmission, but their actions in the central nervous system (CNS) remains unclear. Acetylcholine (ACh) is one of the most important neurotransmitter in the CNS and thus, it is possible that its release could be one of the targets for volatile anaesthetic action. However, the effects of these agents on the release of ACh are not yet fully understood. Rat brain cortical slices were loaded with [³H]-choline in order to study the effect of isoflurane on the release of [³H]-ACh from this preparation. Isoflurane (28, 43, 54, 95 and 182 nM) significantly increased the basal release of [³H]-ACh. This effect was independent of the extracellular sodium and calcium concentration but was decreased by tetracaine and dantrolene, inhibitors of Ca^2+-release from intracellular stores. These findings indicate that isoflurane may cause a Ca^2+-release from internal stores that increases [³H]-ACh release in rat brain cortical slices.     

Adenosine uptake and [3H]nitrobenzylthioinosine binding in developing rat brain

The ontogenesis of adenosine transport sites as labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [3H]NBI binding in newborn and adult rats were reflected by changes in Bmax and not Kd values. As a measure of the utility of [3H]NBI as a probe for identifying functional adenosine transport sites, we examined [3H]NBI binding to and [3H]adenosine accumulation by intact brain cells prepared from adult and newborn rats. For [3H]NBI binding to brain cells from adult rats, the values of Kd were 0.092 nM and of Bmax were 274 fmol/mg protein. For newborns, slightly higher Kd and Bmax values were observed; 0.2 nM and 395 fmol/mg protein, respectively. [3H]Adenosine accumulation was higher in brain cells from one-day-old than from adult rat brains. Kinetically this uptake was best described by a two-component model: the Vmax values for the high- and low-affinity uptake, and the Km value for the high-affinity component in one-day-old rats were greater than in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Difference in in vivo receptor binding between [3 H]N-methylspiperone and [3 H]raclopride in reserpine-treated mouse brain

Summary The in vivo binding of [³ H]N-methylspiperone (NMSP) and [³ H]raclopride was compared in mice treated with reserpine (5 mg/kg, 24 hr prior to the tracer injection). With both radioligands, selective accumulation of radioactivity in the striatum following intravenous injection was observed, whereas a relatively low accumulation and a rapid decline in radioactivity in the cerebellum was seen. Reserpine significantly decreased [³ H]NMSP binding in vivo, however it increased [³ H]raclopride binding. By compartment model analysis, it was found that the decrease in [³ H]NMSP binding was primarily due to the decrease in the association rate (K3) and the increase in [³ H]raclopride was due to the decrease in the dissociation rate (K4) in vivo. As both Kd and Bmax of dopamine D2 receptors have been reported to be unaltered by reserpine, these results suggested that some unknown factors except Kd and Bmax which influence on in vivo binding of receptors might be changed by reserpine. These results revealed that it is of importance to measure kinetics of ligand-receptor binding in vivo rather than static analysis. These two different types of radioligands can be combined to reveal functional roles of dopamine receptor in vivo, especially in the study of the human brain with positron emission tomography (PET).     

Regional [H]acetylcholine and [H]nicotine binding in developing mouse brain

The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [³H]acetylcholine ([³H]ACh) and [³H]nicotine ([³H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [³H]NIC increased gradually with age in all brain regions studied. The [³H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [³H]ACh was found to be rather similar to that earlier described for [³H]alpha-bungarotoxin binding sites, whereas that for [³H]NIC differed from that described for other nicotinic ligands.     

Effects of extracellular pH reductions on [3H]d-aspartate and [3H]noradrenaline release by presynaptic nerve terminals isolated from rat cerebral cortex

We analyzed the effects of extracellular pH reductions on the release of [³H]d-aspartate ([³H]d-ASP) and [³H]noradrenaline ([³H]NA) from cerebrocortical synaptosomes isolated from rats. Synaptosomes were superfused with standard medium at a physiologic pH of 7.4 and with acidified medium with a pH of 6.00, 5.50, or 5.0. Medium acidification produced pH-dependent stimulation of [³H]d-ASP release. The increase amounted to 202 ± 12.6% when the pH was reduced to 5.5. The [³H]d-ASP release evoked by low pH (5.50) was still observed in the absence of Ca²⁺ ions, but it was abolished by dl-threo-β-benzyloxyaspartate (dl-TBOA) (100 μM), which inhibits neuronal glutamate/aspartate transport. Exposure to 5-(N,N-hexamethylene)-amiloride (EIPA) (30–100 μM), a selective inhibitor of Na⁺/H⁺ exchange, caused concentration-dependent stimulation of [³H]d-ASP release; the increase observed with EIPA 30 μM was 160 ± 12%. The EIPA-induced release was not dependent on the presence of Ca²⁺ ions in the medium, but it was abolished when synaptosomes were pretreated with 100 μM dl-TBOA. Reduction of the extracellular pH (5.50–5.0) also stimulated the release of [³H]NA from rat cortical synaptosomes. Exposure to medium with a pH of 5.50 increased basal release by 136 ± 9.5%. The release-stimulating effect of this medium was calcium-independent and abolished by 3 μM desipramine (DMI). [³H]NA release was also stimulated by EIPA. The increase induced by a concentration of 30 μM amounted to 136 ± 9.50%, and this effect was calcium-independent and abolished by pretreatment with DMI (3 μM). These findings suggest that reduction of the extracellular pH stimulates release of [³H]D-ASP and [³H]NA by reversing neurotransmitter transport in the nerve terminal. This reversal might be activated by increased cytosolic concentrations of the transmitters caused by reduction of the pH gradient between the cytoplasm and the synaptic vesicles that take up the transmitters. This hypothesis is confirmed by the results of experiments conducted with EIPA. Selective blockade of Na⁺/H⁺ exchange with this compound induces accumulation of H⁺ in the nerve terminals and intracellular acidification, which leads to calcium-independent, transporter-mediated release of [³H]d-ASP and [³H]NA.     

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.     

Ontogeny of influence of clonidine on high potassium-induced release of noradrenaline and specific [3H]clonidine binding in the rat brain cortex

A high (20 mM) K+-induced, Ca2+-dependent release of L-[3H]-noradrenaline (NA) from cerebral cortex slices was detected in rats on day 18 of gestation. Depolarization-evoked release (% of total radioactivity) increased with age between day 18 of gestation and day 70 of postnatal life. Clonidine (0.1 microM) significantly (p less than 0.05) reduced a high K+-induced L-[3H]NA release on days 7 and 70 but not on day 1. Both KD and Bmax of specific [3H]clonidine binding to cerebral cortex membranes rapidly increased between days 1 and 7, followed by a gradual increase to the adult level. Presynaptic alpha 2-adrenoceptors which regulate NA release seems to become sensitive to alpha 2-agonists and to reach functional maturity between days 1 and 7 after birth in the cerebral cortex of rats.