Excitatory and inhibitory control of epileptiform discharges in combined hippocampal/entorhinal cortical slices

We examined whether epileptiform activity can be induced and prevented by mild reduction of GABA_A receptor-mediated inhibition and non-NMDA receptor-mediated excitation, respectively, in different regions of combined hippocampal/entorhinal cortical slices from juvenile rats (P15–21). We used the receptor antagonists bicuculline (GABA_A) and CNQX (non-NMDA) as tools to investigate the sensitivities of the CA1, the subiculum (SUB) and the medial entorhinal cortex (MEC) for generating epileptiform discharges upon extracellular stimulation. We found that low concentrations of bicuculline (<3.5 μM) were enough to induce epileptiform discharges in the three regions. These discharges were similar to those observed under high concentrations of bicuculline (>10 μM) and consisted of stereotyped population bursts, recorded both extra- and intracellularly. Interestingly, the CA1 and SUB were more susceptible to generate discharges compared to the MEC in the same slices. We also found that non-NMDA excitation was critical in controlling discharges, as they were blocked by CNQX in a concentration-dependent manner. The sensitivity of the CA1 region to CNQX was lower than that of the SUB and MEC. Based on these regional differences, we show that epileptiform activity can be pharmacologically isolated within the CA1 region in the hippocampal–entorhinal circuitry in vitro.     

Effects of NMDA and non-NMDA receptor antagonists on the medullary inspiratory neuronal activity during spontaneous augmented breaths in anesthetized rats

To elucidate whether there is a difference between the effects of iontophoretically applied N-methyl-

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-aspartate (NMDA) and non-NMDA receptor antagonists on the activity of inspiratory neurons during spontaneous augmented breaths, extracellular single unit recording of inspiratory neurons (I-augmenting, I-decrementing and I-other) was performed in pentobarbital anesthetized rats. The spontaneous augmented breath was divided into two different phases; the first phase (phase I) resembled a normal inspiration, but the second phase (phase II) consisted of a marked increase in diaphragm electromyogram activity. The mean firing frequency of I-aug type neurons was significantly decreased after 50 nA application of both

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-2-amino-5-phosphonopentanoic acid (AP-5) (NMDA receptor antagonist) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (non-NMDA receptor antagonist). The mean firing frequency of both I-dec and I-other neurons was significantly decreased by both AP-5 and CNQX applications (70 nA). After AP-5 application, relative changes in the discharge rates during inspiratory phases I and II of spontaneous augmented breaths were significantly suppressed in all types of neurons, but CNQX application had no significant effect on the response changes during phase II. In all cell types of neurons, a significant difference between the iontophoretic AP-5 and CNQX applications in the relative mean firing rate was observed. These results suggested that activation of the NMDA receptor-induced neurotransmission can modify the discharge rate of medullary inspiratory neurons, irrespective of the cell types, during the inspiratory phase II of spontaneous augmented breaths, but that non-NMDA receptor blockade may not significantly influence their discharge rate.     

Focal stimulation of specific pathways in the rat hippocampus causes a reduction in radioligand binding to the haloperidol-sensitive sigma receptor

Summary Focal electrical stimulation of selected excitatory pathways in the hippocampal slice caused a decrease in the binding of [³H]-1,3-di(2-tolyl)guanidine (DTG) or [³H]-(+)-3-[hydroxyphenyl]-N-(1-propyl)piperidine ((³H)-(+)3-PPP) to haloperidol-sensitive sigma binding sites in the slice. Activation of the mossy fibers or perforant path by high frequency electrical stimulation caused the reduction in [³H]-DTG binding; whereas activation of fibers in the strata radiatum, lacunosummoleculare, alveus, or oriens did not affect [³H]-DTG binding. The decrease in binding observed was calcium-dependent and tetrodotoxin sensitive and varied with the frequency, intensity, and duration of stimulation. Although haloperidol-sensitive [³H]-DTG binding sites are distributed throughout the hippocampus, stimulation of the perforant path or mossy fibers resulted in a significant reduction in binding only in the dentate region of the slice. The decrease in binding following perforant path stimulation was blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); whereas the decrease in binding caused by mossy fiber stimulation was not affected by CNQX or DL-APV. The results obtained support the hypothesis that activation of the granule cells in the hippocampal slice caused the release of an endogenous ligand which acts at the haloperidol-sensitive sigma binding site in the dentate gyrus.     

The involvement of N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the responsiveness of rat spinal neurons with input from the chronically inflamed ankle

Unilateral adjuvant inflammation was induced at the rat ankle 2 or 20 days before an evaluation of the contribution of N-methyl-d-aspartate (NMDA) and non-NMDA receptors to the processing of nociceptive information by wide dynamic range neurons in the spinal cord. Microionophoretic application of either the NMDA receptor antagonists ketamine and DL-2-amino-5-phosphonovalerate (AP5) or the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the responses to innocuous and noxious mechanical stimulation of the inflamed ankle. The pattern of these effects was comparable to that in rats with acute inflammation suggesting that non-NMDA and NMDA receptors are similarly involved in acute, prolonged acute and chronic inflammation-evoked activity.     

逍遥散对肝郁脾虚证大鼠边缘系统神经元超微结构的影响

目的:探讨慢性束缚应激所致肝郁脾虚证大鼠海马CA1区和杏仁核细胞超微结构的变化,明确逍遥散和6-氰基-7-硝喹啉-2,3-双酮(CNQX)对慢性应激所致肝郁脾虚证的保护作用及其机制。方法:将100只雄性SD大鼠随机分为正常组(A组)、假手术组(B组)、模型组(C组)、逍遥散组(D组)、CNQX组(E组)和逍遥散+CNQX组(F组)。C、D、E、F组大鼠通过连续21d慢性束缚应激建立肝郁脾虚证候模型,D、F组大鼠每天束缚前灌服逍遥散5.32g/kg,E、F组大鼠在第1、4、7、10、13、16、19、21d右侧杏仁核区微量注射α-氨基羟甲基恶唑丙酸(AMPA)受体拮抗剂CNQX0.5μl。第22d处死动物,用透射电镜方法观察海马CA1区和左侧杏仁核神经细胞和突触的形态结构。结果:模型组大鼠海马出现了细胞核变形、线粒体嵴模糊,突触结构不清等,正常组、假手术组和给药各组大鼠CA1区未见明显异常;各组大鼠杏仁核均未见明显异常。结论:逍遥散可改善慢性束缚应激导致的海马CA1区超微结构的损伤,其作用机制可能与CNQX一致。     

Rhythm generation in organotypic medullary cultures of newborn rats

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO₂ and H⁺. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 μM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 μM) and/or hydroxysaclofen (200 μM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 μM) abolished rhythmicity. (3) While atropine sulphate (5 μM) was ineffective in modulating periodic discharges nicotine (100 μM) — like CO₂ — shortened the intervals between the periodic events; hexamethonium (50–100 μM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 μM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10–50 μM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50–100 μM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO₂/acidosis. (6) Dantrolene (10 μM), an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 μM) which blocks endoplasmic Ca²⁺-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)–(7) indicate that transmembrane calcium fluxes as well as intracellular Ca²⁺-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.     

Modulation by divalent cations of the current generated by vasopressin in facial motoneurons

Vasopressin generates a voltage-gated, sodium-dependent current in facial motoneurons in brainstem slices. Reducing the extracellular calcium concentration from 2 to 0.01 mM caused a 30 to 120% increase in the amplitude of this current. Lowering extracellular magnesium also enhanced it, but less efficiently. In the physiological solution, the response of facial neurons to vasopressin is thus partially blocked. Increasing extracellular calcium was without effect. Current-voltage curves indicate that the vasopressin current reversed at around 0 mV and suggest that the low-calcium-induced potentiation was due to an attenuation of the region of negative slope conductance.     

Cerebellar AMPA/KA receptor antagonism by CNQX inhibits vestibuloocular reflex adaptation

Vestibuloocular reflex (VOR) performance and adaptation have been investigated during antagonism of cerebellar AMPA/quisqualate and kainate receptors (AMPA/KA) by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Injection of CNQX into the vestibulo-cerebellum of the goldfish before adaptation significantly inhibited and, at the highest dosage, completely prevented acquisition of adaptive reflex gain increases and decreases during a 3-h training period. Injection of CNQX before initiation of VOR adaptive training did not affect pre-adapted baseline performance of the reflex. Injection of CNQX, 1 to 2 h after the initiation of training did not alter the performance of adaptive gain increases that occurred before the injection. If injection of CNQX occurred at the end of adaptive training, there was an accelerated loss of the previously adapted gain changes during the retention period when the animal remained stationary in the dark. CNQX injection did not produce any permanent or long-term deficits, because goldfish could be retrained 48 h later to produce adaptive VOR gain changes similar to control animals. Thus, this work demonstrates that the AMPA/KA receptors located in the vestibulo-cerebellum of the goldfish are necessary for acquisition of short-term adaptive VOR gain increases and decreases. The deficit in adaptive capability was not the result of a deficit in performance, because CNQX did not inhibit an adaptive change that had already occurred as long as the adapting vestibular and visual stimulation continued. This adaptive performance could possibly be maintained by other glutamatergic (metabotropic) receptors located on the Purkinje cells. The retention of adapted gain increases and decreases after CNQX application was inhibited because AMPA/KA antagonism accelerated VOR gain loss after the completion of training when no vestibular or visual stimulation was present. Because the AMPA/KA receptors are located only in the molecular layer of the goldfish cerebellum, these results are, presumably, the result of AMPA/KA receptor antagonism at synapses located on the Purkinje cell dendrite tree.     

Nanomolar dose of bisphenol A rapidly modulates spinogenesis in adult hippocampal neurons

We demonstrated the rapid effects of 10nM bisphenol A (BPA) on the spinogenesis of adult rat hippocampal slices. The density of spines was analyzed by imaging Lucifer Yellow-injected CA1 neurons in slices. Not only the total spine density but also the head diameter distribution of spine was quantitatively analyzed. Spinogenesis was significantly enhanced by BPA within 2h. In particular, the density of middle-head spine (with head diameter of 0.4-0.5μm) was significantly increased. Hydroxytamoxifen, an antagonist of both estrogen-related receptor gamma (ERRγ) and estrogen receptors (ERα/ERβ), blocked the BPA-induced enhancement of the spine density. However, ICI 182,780, an antagonist of ERα/ERβ, did not suppress the BPA effects. Therefore, ERRγ is deduced to be a high affinity receptor of BPA, responsible for modulation of spinogenesis. The BPA-induced enhancement of spinogenesis was also suppressed by MAP kinase inhibitor, PD98059, and the blocker of NMDA receptors, MK-801. Washout of BPA for additional 2h after 2h BPA treatment abolished the BPA-induced enhancement of spinogenesis, suggesting that the BPA effect was reversible. ERRγ was localized at synapses as well as cell bodies of principal neurons. ERRγ at synapses may contribute to the observed rapid effect. The level of BPA in the hippocampal slices was determined by mass-spectrometric analysis.