胞外钙对豚鼠耳蜗Deiters细胞钾电流的调制

为观察胞外钙对豚鼠耳蜗单个离体Deiters细胞钾电流的调控作用并探讨其机制,实验记录了Deiters细胞在正常细胞外液和无钙外液中的全细胞钾电流(whole cell K^ currents,IK),并分析了其电生理学特性的改变。结果观察到,Deiters细胞与在正常细胞外液中相比,在祛除细胞外液中的Ca^2 后Ik电流幅值明显增加,弦电导值亦明显增加,但其平衡电位未明显改变。在无钙外液中Ik电流的反转电位向超极化方向明显移位,更接近于按照Ner-nst方程得出的K^ 理论平衡电位;而且其稳态激活曲线亦向超极化方向明显移位,但其激活趋势与正常相比无明显改变。此外,观察了Deiters细胞中钙抑制性钾电流的电流-电压关系和电导-电压关系,发现两者均呈“S”形,提示此钙抑制性钾电流可能存在2种不同的钾电导成分。由此,推测可能有两种机制参与胞外钙对Deiters细胞钾电流的调控：(1)Deiters细胞中的Ik通道可能存在一个Ca^2 敏感结构域,胞外Ca^2 可能通过改变此结构域而对Ik电流产生调制;(2)Deiters细胞中可能存在一种新型的双相门控性钾通道或钾通道耦联型受体或是一种新型的钾通道亚型,祛除胞外Ca^2 可激活此新型钾电导而对L电流产生调制。由此推测,在听觉形成过程中,胞外钙浓度下降可以对Deiters细胞的全细胞钾电流产生调制,从而更有利于Deiters细胞内K^ 外流,进而有效地缓冲外毛细胞周围的K^ 浓度：而且还可以使Deiters细胞产生更快的复极化并有利于维持其静息状态。     

Anoxia-induced dopamine release from rat striatal slices: involvement of reverse transport mechanism

Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4-dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia-induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia-induced DA depletion, aglycemia- and ischemia-induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK-801 and also by comparing the glutamate-induced DA release with that during anoxia, aglycemia, or ischemia. MK-801 decreased the anoxia-induced DA depletion in a dose-dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia-induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK-801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.     

On the significance of perfusion rate in the study of glutamate release from superfused synaptosomes

The effect of perfusion rate on the apparent release of [³H]glutamate from prelabelled and superfused rat cortical synaptosomes was examined. The proportion of tissue [³H]glutamate released in response to a 4 ml depolarizing pulse of 15 mM K⁺ increased almost linearly with perfusion rates from 1 ml min⁻¹ to 10 ml min⁻¹. Release did not increase markedly between 10 ml min⁻¹ and 20 ml min⁻¹. The basal efflux of [³H]glutamate also increased with perfusion rate. The increase in both basal efflux and K⁺-induced release is interpreted as being due to a greater amount of released transmitter avoiding recapture by uptake processes as perfusion rate increases. This is supported by the observation that increasing the potential number of uptake sites in the tissue decreases both the basal and K⁺-evoked release of the transmitter. The significance of this with respect to optimal perfusion rates for studies on the regulation of glutamate release is discussed.     

Biotransformation of l-DOPA to Dopamine in the Substantia Nigra of Freely Moving Rats: Effect of Dopamine Receptor Agonists and Antagonists

Abstract: We investigated the effects of continuous intranigral perfusion of dopamine D1 and D2 receptor agonists and antagonists on the biotransformation of locally applied l -DOPA to dopamine in the substantia nigra of freely moving rats by means of in vivo microdialysis. The "dual-probe" mode was used to monitor simultaneously changes in extracellular dopamine levels in the substantia nigra and the ipsilateral striatum. Intranigral perfusion of 10 µ M l -DOPA for 20 min induced a significant 180-fold increase in extracellular nigral dopamine level. No effect of the intranigral l -DOPA administration was observed on dopamine levels in the ipsilateral striatum, suggesting a tight control of extracellular dopamine in the striatum after enhanced nigral dopamine levels. Continuous nigral infusion with the D1 receptor agonist CY 208243 (10 µ M ) and with the D2 receptor agonist quinpirole at 10 µ M (a nonselective concentration) attenuated the l -DOPA-induced increase in dopamine in the substantia nigra by 85 and 75%, respectively. However, perfusion of the substantia nigra with a lower concentration of quinpirole (1 µ M ) and the D1 antagonist SCH 23390 (10 µ M ) did not affect the nigral l -DOPA biotransformation. The D2 antagonist (−)-sulpiride (10 µ M ) also attenuated the l -DOPA-induced dopamine release in the substantia nigra to ∼10% of that of the control experiments. We confirm that there is an important biotransformation of l -DOPA to dopamine in the substantia nigra. The high concentrations of dopamine formed after l -DOPA administration may be the cause of dyskinesias or further oxidative stress in Parkinson's disease. Simultaneous administration of D1 receptor agonists with l -DOPA attenuates the biotransformation of l -DOPA to dopamine in the substantia nigra. The observed effects could occur via changes in nigral GABA release that in turn influence the firing rate of the nigral dopaminergic neurons.     

Effects of short-term hypoxia on [3H]glutamate release from preloaded hippocampal and cortical synaptosomes

The effect of short-term hypoxia on the release of [³H]glutamate from preloaded hippocampal and cortical synaptosomes was studied in a rapid superfusion system. The technique minimised the loss of released glutamate by reuptake. The results indicated that the effects of short term hypoxia were qualitatively similar to those reported in previous studies using more long-term hypoxia, but were significantly smaller. The non-Ca²⁺-dependent efflux of glutamate from cortical synaptosomes was increased by hypoxia as was the Ca²⁺-dependent release from hippocampal tissue. Possible mechanisms for these findings were discussed. The small amplitude of these changes in comparison to the effects seen in slowly perfused tissue in vitro and in vivo indicated that the contribution made by changes in neuronal efflux to the overall increase in extracellular glutamate seen in hypoxia is relatively minor.     

Simultaneous determination of cholecystokinin,dopamine, glutamate and aspartate in cortex and striatum of the rat using in vivo microdialysis

Summary Extracellular levels of cholecystokinin (CCK), dopamine (DA), glutamate (Glu) and aspartate (Asp) were simultaneously monitored in the frontoparietal cortex and the striatum of halothane-anaesthetized rats using in vivo microdialysis. Under basal conditions, cortical and striatal CCK levels were 3.11 ± 0.39 pM and 2.76 ± 0.15 pM, respectively. Local KCl (10^–1 M) and bicuculline (10^–4 M) co-application in cortex or striatum increased the CCK levels 18-fold and 26-fold, respectively. The DA level in striatum was 3.78 ± 0.28 nM and the local perfusion with KCl + bicuculline led to a 45-fold increase. The cortical and striatal outputs of Glu were of the order of 2 · 10^–6 M and Asp levels were around 6 · 10^–7 M. Local stimulation with KCl (10^–1 M) and bicuculline (10^–4 M) caused a small increase (2 fold) in cortical and striatal levels of Glu and Asp. The addition of KCl (10^–1 M) and bicuculline (10^–4 M) to the cortical perfusion medium did not modify CCK, DA or Glu concentrations in striatum. These results demonstrate that CCK, DA, Glu and Asp may be simultaneously monitored in vivo and support the idea that their extracellular levels recovered in the microdialysis perfusates could be derived from neuronal pools.     

Bidirectional regulations for glutamate and GABA release in the hippocampus by alpha7 and non-alpha7 ACh receptors

In the assay of glutamate and gamma-aminobutyric acid (GABA) with a high-performance liquid chromatography, spontaneous release of glutamate and GABA from rat hippocampal slices was significantly enhanced by mecamylamine, an inhibitor of non-alpha7 ACh receptors, or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors in the absence of tetrodotoxin (TTX), but not in the presence of TTX. Nicotine significantly enhanced glutamate and GABA release in the absence of TTX, that is abolished by mecamylamine or alpha-bungarotoxin, while it had no effect on the release in the presence of TTX. In the recording of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (AMPA-EPSCs) and GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) from CA1 pyramidal neurons of rat hippocampal slices, nicotine did not affect the rate and amplitude of AMPA-EPSCs and AMPA-miniature EPSCs. In contrast, nicotine significantly increased the rate of GABA(A)-IPSCs, without affecting the amplitude, but such effect was not obtained with GABA(A)-miniature IPSCs. The collective results suggest that alpha7 and non-alpha7 ACh receptors expressed in the hippocampus, activated under the basal conditions, inhibit release of glutamate and GABA controlled through multi-synaptic relays, but that otherwise, those receptors, highly activated by nicotine, stimulate both the release, with a part of GABA released from interneurons transmitting to CA1 pyramidal neurons. Furthermore, the results also suggest that alpha7 and non-alpha7 ACh receptors do not have potency sufficiently to modulate glutamate and GABA release controlled by single synapses.     

Computational modeling of paroxysmal depolarization shifts in neurons induced by the glutamate release from astrocytes

Recent experimental studies have shown that astrocytes respond to external stimuli with a transient increase of the intracellular calcium concentration or can exhibit self-sustained spontaneous activity. Both evoked and spontaneous astrocytic calcium oscillations are accompanied by exocytosis of glutamate caged in astrocytes leading to paroxysmal depolarization shifts (PDS) in neighboring neurons. Here, we present a simple mathematical model of the interaction between astrocytes and neurons that is able to numerically reproduce the experimental results concerning the initiation of the PDS. The timing of glutamate release from the astrocyte is studied by means of a combined modeling of a vesicle cycle and the dynamics of SNARE-proteins. The neuronal slow inward currents (SICs), induced by the astrocytic glutamate and leading to PDS, are modeled via the activation of presynaptic glutamate receptors. The dependence of the bidirectional communication between neurons and astrocytes on the concentration of glutamate transporters is analyzed, as well. Our numerical results are in line with experimental findings showing that astrocyte can induce synchronous PDSs in neighboring neurons, resulting in a transient synchronous spiking activity.     

Effect of intracerebral administration of NMDA and AMPA on dopamine and glutamate release in the ventral pallidum and on motor behavior

The present study investigates the modulation of the ventral tegmental area (VTA)-ventral pallidum (VP) dopaminergic system by glutamate agonists in rats. The glutamate receptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were infused via reversed microdialysis into the VTA, and dopamine (DA), glutamate, and aspartate levels in the VTA and ipsilateral VP were monitored together with motor behavior screened in an open field. NMDA (750 microM) infusion, as well as AMPA (50 microM) infusion, induced an increase of DA and glutamate levels in the VTA, followed by an increase of DA levels in the ipsilateral VP and by enhanced locomotor activity. The increase of DA in the VP was similar after administration of these two glutamate agonists, although motor activity was more pronounced and showed an earlier onset after NMDA infusion. Glutamate levels in the VP were not increased by the stimulation of DA release. It is concluded that DA is released from mesencephalic DA neurons projecting to the VP and that these neurons are controlled by glutamatergic systems, via NMDA and AMPA receptors. Thus, DA in the VP has to be considered as a substantial modulator. Dysregulation of the mesopallidal DA neurons, as well as their glutamatergic control, may play an additional or distinct role in disorders like schizophrenia and drug addiction.     

Effect of Amygdaloid Kindling on the Content and Release of Amino Acids from the Amygdaloid Complex: In Vivo and In Vitro Studies

Abstract: The tissue content and the interstitial fluid levels of glutamate, aspartate, GABA, glutamine, glycine, and serine were studied in amygdaloid-kindled rat brain. Interstitial levels were studied in vivo before and during stage 5 full limbic seizures using microdialysis. Slices of amygdala from kindled and sham-operated animals were used to study baseline and KCl-evoked release in vitro. The contents of these amino acids were measured in slices of amygdala, hippocampus, and cerebral cortex from kindled and sham-operated animals. Kindled brains showed two- to threefold higher levels of glutamate, aspartate, and GABA and 12-fold higher levels of glutamine than sham-operated controls. Correlating with this, interstitial fluid levels of glutamate were two- to threefold higher from kindled amygdala than from control both in vivo (microdialysis) and in vitro (superfusion). GABA levels in interstitial fluid from kindled amygdala were reduced by 67% compared with control amygdala.     

Synthesis and SAR study of a novel series of dopamine receptor agonists

The synthesis of a novel series of dopamine receptor agonists are described as well as their in vitro potency and efficacy on dopamine D₁ and D₂ receptors. This series was designed from pergolide and (4aR,10aR)-1-propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinolin-6-ol (PHBQ) and resulted in the synthesis of (2R,4aR,10aR)-2-methylsulfanylmethyl-4-propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol (compound 27), which has a D₁ and D₂ receptor profile similar to that of the most recently approved drug for Parkinson’s disease, rotigotine.     

In vivo release of endogenous dopamine, 5-hydroxytryptamine and some of their metabolites from rat caudate nucleus by phenylethylamine

The in vivo release of endogenous 3,4-dihydroxyphenylethylamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT), and of 5-hydroxytryptamine (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), has been measured in the caudate nucleus of the anesthetized rat. A push-pull cannula was implanted into the brain, and the tissue perfused with artificial CSF or artificial CSF containing 5×10^–4 M phenylethylamine. The perfusate was collected and analyzed for DA, 5-HT and their metabolites by high performance liquid chromatography with electrochemical detection (HPLC-ECD). DA was released by phenylethylamine at rates significantly greater than its basal rate. 3-MT and 5-HT were undetectable in perfusates collected under basal conditions, but could be detected readlly during phenylethylamine stimulation. DOPAC, HVA and 5-HIAA concentrations were not significantly affected by phenylethylamine. The results suggest (1) that phenylethylamine may exert its behavioural effects through increased release of both DA and 5-HT, and (2) that in vivo measurements of the acid metabolites alone may not be indicative of the release of the amines.Special Issue Dedicated to Dr. Abel Lajtha.     

Feedback control of mesolimbic somatodendritic dopamine release in rat brain

The objective of this study was to examine the role of dopamine (DA) receptors in the nucleus accumbens (ACB) in controlling feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats using ipsilateral dual-probe in vivo microdialysis. Perfusion of the ACB for 60 min with the DA uptake inhibitor GBR-12909 (10-1,000 microM) or nomifensine (10-1,000 microM) dose-dependently increased the extracellular levels of DA in ACB and concomitantly reduced the extracellular levels of DA in the VTA. Coperfusion of 100 microM nomifensine with either 100 microM SCH-23390 (SCH), a D1 antagonist, or 100 microM sulpiride (SUL), a D2 receptor antagonist, produced either an additive (for SCH) or a synergistic (for SUL) elevation in the extracellular levels of DA in the ACB, whereas the reduction in the extracellular levels of DA in the VTA produced by nomifensine alone was completely prevented by addition of either antagonist. Application of 100 microM SCH or SUL alone through the microdialysis probe in the ACB increased the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA remained unchanged. Overall, the results suggest that (a) increasing the synaptic levels of DA in the ACB activates a long-loop negative feedback pathway to the VTA involving both D1 and D2 postsynaptic receptors and (b) terminal DA release within the ACB is regulated directly by D2 autoreceptors and may be indirectly regulated by D1 receptors, possibly on interneurons and/or through postsynaptic inhibition of the negative feedback loop.     

Stress, glucocorticoids and glutamate release: effects of antidepressant drugs

Stressful life events impact on memory, cognition and emotional responses, and are known to precipitate mood/anxiety disorders. It is increasingly recognized that stress and its neurochemical and endocrine mediators induce changes in glutamate synapses and circuitry, and this in turn modify mental states. Half a century after the monoamine hypothesis, it is widely accepted that maladaptive changes in excitatory/inhibitory circuitry have a primary role in the pathophysiology of mood/anxiety disorders. The neuroplasticity hypothesis posits that volumetric changes consistently found in limbic and cortical areas of depressed subjects are in good part due to remodeling of neuronal dendritic arbors and loss of synaptic spines. A considerable body of work, carried out with in vivo microdialysis as well as alternative methodologies, has shown that both stress and corticosterone treatment induce enhancement of activity-dependent glutamate release. Accordingly, results from preclinical studies suggest that stress- and glucocorticoid-induced enhancement of glutamate release and transmission plays a main role in the induction of maladaptive cellular effects, in turn responsible for dendritic remodeling.Additional recent work has showed that drugs employed for therapy of mood/anxiety disorders (antidepressants) prevent the enhancement of glutamate release induced by stress. Understanding the action of traditional drugs on glutamate transmission could be of great help in developing drugs that may work directly at this level.     

NPY and NPY Y1 receptor effects on noradrenaline overflow from the rat brain in vitro

Neurotransmitters and neuropeptides play important roles in the regulation of various neuroendocrine functions particularly feeding. The aim of this study was to investigate whether a functional interaction occurs among neuropeptide Y (NPY) at NPY Y₁ receptors and noradrenaline overflow, as this may contribute to the regulation of appetite. The release of endogenous noradrenaline and its metabolite 3,4-dihydroxyphenylglycol (DHPG) were examined from hypothalamic and medullary prisms using the technique of in vitro superfusion and high performance liquid chromatography (HPLC) with coulometric detection. Noradrenaline and DHPG overflow was investigated at rest, in response to NPY (0.1 μM) and in response to the NPY Y₁ receptor agonist, [Leu³¹,Pro³⁴]NPY (0.1 μM). Perfusion with NPY and [Leu³¹,Pro³⁴]NPY significantly reduced noradrenaline overflow from the hypothalamus and medulla. Perfusion with NPY and [Leu³¹,Pro³⁴]NPY was without significant effect on hypothalamic DHPG overflow, while medullary DHPG overflow was significantly reduced by NPY and [Leu³¹,Pro³⁴]NPY. Results from this study provide evidence of NPY Y₁ receptor-mediated inhibition of noradrenaline release in the hypothalamus and medulla, further illustrating a complex interaction between neurotransmitters and neuropeptides within the rat brain.     

Protein phosphatase 1 and 2A inhibitors prolong the switch in the control of glutamate release by group I metabotropic glutamate receptors: characterization of the inhibitory pathway

We have addressed the role of protein phosphatases (PPs) in the modulation of the switch in glutamate release observed after repetitive stimulation of group I metabotropic glutamate receptors (mGluRs). In cerebrocortical nerve terminals the agonist (S:)-3, 5-dihydroxyphenylglycine facilitated evoked glutamate release. However, a second stimulation, 5 min later, reduced rather than facilitated this release. This switch in the control of glutamate release was reversed when a 30-min interval was left between stimulations. Inhibition of the endogenous PPs, PP1 and PP2A, with calyculin A and okadaic acid prevented the recovery of the facilitatory response and maintained the receptor permanently coupled to the inhibitory pathway. The inhibitors of PP2B, cyclosporin A and cypermethrine, had no effect. The inhibition of glutamate release was insensitive to pertussis toxin and was the result of the loss of the release component coupled to N-type Ca(2+) channels. This inhibitory action was suppressed by addition of the protein kinase C activator 4beta-phorbol 12,13-dibutyrate. We conclude that the balance between protein kinase and phosphatase activity at the nerve terminal plays a key role in accommodating the modulation of glutamate release by group I mGluRs.     

Bis-azaaromatic quaternary ammonium salts as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release: An investigation of binding conformation

A series of conformationally restricted bis-azaaromatic quaternary ammonium salts (3 and 4) have been designed and synthesized in order to investigate the possible binding conformations of N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; 2), a compound which potently inhibits neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked dopamine release. The preliminary structure–activity relationships of these new analogues suggest that bPiDDB binds in an extended conformation at the nAChR binding site, and that flexibility of the linker may be important for its high potency in inhibiting nAChRs mediating nicotine-evoked dopamine release.     

Modulation of ventral pallidal dopamine and glutamate release by the intravenous anesthetic propofol studied by in vivo microdialysis

Summary. The intravenous anesthetic propofol is reported to have various psychological side effects as hallucinations, sexual disinhibition, or euphoria. Hedonic and rewarding states like these are modulated by the dopaminergic system in the nucleus accumbens, prefrontal cortex and also in the ventral pallidum and by the glutamatergic system in the neocortex and limbic system. In the present study, propofol was administered either alone or in combination with the GABAA receptor antagonist bicuculline via reverse microdialysis into the ventral pallidum of freely moving rats. Dialysis fractions were taken every 20min and analyzed for dopamine and glutamate using high performance liquid chromatography. Application of propofol decreased dopamine levels in the ventral pallidum. This effect seems to be mainly mediated through GABAA receptors, since it was compensated by the GABAA receptor antagonist bicuculline. Propofol and propofol plus bicuculline exerted no effect on glutamate release in this brain region. The reduced dopamine release in ventral pallidum was most probably mediated through a GABAergic feedback loop from the ventral pallidum via the nucleus accumbens to the dopaminergic neurons of the ventral tegmental area or by long loop feedback. As an increase but not a decrease of dopamine release in the ventral pallidum is involved in hedonic and rewarding properties, similar symptoms induced by propofol seem to be unrelated to an action of propofol in the ventral pallidum.