The relative variability of excitatory postsynaptic currents (EPSCs) was studied using whole cell recording in CA1 pyramidal cells of hippocampal slices from 2 to 3-week-old rats. EPSCs were evoked by stimulating the Schaffer collateral-commissural pathway and recorded at holding potentials of -75, -30 or +40 mV. The recordings were either isolated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptor mediated EPSCs, or composite ones. EPSC variability was quantified by coefficient of variation (CV). The inverse variability expressed as 1/CV2 was employed in comparisons. Using early (5-15 ms) and late (40-100 ms) measurements to estimate the AMPA and NMDA components of composite EPSCs showed no difference in the variability of the two components. Comparing isolated AMPA EPSCs at -75 mV with NMDA EPSCs at -30 mV also failed to reveal a difference. However, in accord with previous studies by others, NMDA EPSCs recorded at +40 mV were less variable than AMPA EPSCs at -75 mV, the ratio of 1/CV2 for NMDA vs. AMPA being around 1.7. A comparison between isolated AMPA EPSCs revealed a similar pattern of dependency of CV on membrane potential, the EPSCs at +40 mV being less variable than those at -30 or -75 mV (1/CV2 ratios of 1.5-1.6). In conclusion, our results did not demonstrate any inherent difference in CV between AMPA and NMDA receptor mediated EPSCs and the observed differences in CV could be accounted for by a dependency on membrane potential, the mechanism of which remains to be resolved. The present results have implications for the interpretation of CV changes as observed, for instance, during synaptic plasticity.

1. A new preparation of the in vitro rat hippocampal slice has been developed in which the synaptic input to the distal apical dendrites of CA1 pyramidal neurones is isolated. This has been used to investigate the properties of distally evoked synaptic potentials. 2. Distal paired-pulse stimulation (0.1 Hz) evoked a dendritic response consisting of a pair of EPSPs, which showed facilitation. The first EPSP had a rise time (10-90%) of 2.2 +/- 0.05 ms and a half-width of 9.1 +/- 0.13 ms. The EPSPs were greatly reduced by CNQX (10 microM) and the remaining component could be enhanced in Mg(2+)-free Ringer solution and blocked by AP5 (50 microM). In 70% of the dendrites, the EPSPs were followed by a prolonged after-hyperpolarization (AHP) which could be blocked by a selective and potent GABAB antagonist, CGP55845A (2 microM). These results indicate that the EPSPs are primarily mediated by non-NMDA receptors with a small contribution from NMDA receptors, whereas the AHP is a GABAB receptor-mediated slow IPSP. 3. With intrasomatic recordings, the rise time of proximally generated EPSPs (3.4 +/- 0.1 ms) was half that of distally generated EPSPs (6.7 +/- 0.5 ms), whereas the half-widths were similar (19.6 +/- 0.8 ms and 23.8 +/- 1 ms, respectively). These results indicate that propagation through the proximal apical dendrites slows the time-to-peak of distally generated EPSPs. 4. Distal stimulation evoked spikes in 60% of pyramidal neurones. At threshold, the distally evoked spike always appeared on the decaying phase of the dendritic EPSP, indicating that the spike is initiated at some distance proximal to the dendritic recording site. Furthermore, distally and proximally generated threshold spikes had a similar voltage dependency. These results therefore suggest that distally generated threshold spikes are primarily initiated at the initial segment. 5. At threshold, spikes generated by stimulation of distal synapses arose from the decaying phase of the dendritic EPSPs with a latency determined by the time course of the EPSP at the spike initiation zone. With maximal stimulation, however, the spikes arose directly from the peak of the EPSPs with a time-to-spike similar to the time-to-peak of subthreshold dendritic EPSPs. Functionally, this means that the effect of dendritic propagation can be overcome by recruiting more synapses, thereby ensuring a faster response time to distal synaptic inputs. 6. In 42% of the neurones in which distal EPSPs evoked spikes, the relationship between EPSP amplitude and spike latency could be accounted for by a constant dendritic modulation of the EPSP. In the remaining 58%, the change in latency was greater than can be accounted for by a constant dendritic influence. This additional change in latency is best explained by a sudden shift in the spike initiation zone to the proximal dendrites. This would explain the delay observed between the action of somatic application of TTX (10 microM) on antidromically evoked spikes and distally evoked suprathreshold spikes. 7. The present results indicate that full compensation for the electrotonic properties of the main proximal dendrites is not achieved despite the presence of Na+ and Ca2+ currents. Nevertheless, distal excitatory synapses are capable of initiating spiking in most pyramidal neurones, and changes in EPSP amplitude can modulate the spike latency. Furthermore, even though the primary spike initiation zone is in the initial segment, the results suggest that it can move into the proximal apical dendrites under physiological conditions, which has the effect of further shortening the response time to distal excitatory synaptic inputs.

The variation of individual synaptic transmission impacts the dynamics of complex neural circuits. We performed whole-cell recordings from monosynaptically connected hippocampal neurons in rat organotypic slice cultures using a synapse mapping method. The amplitude of unitary excitatory postsynaptic current (uEPSC) varied from trial to trial and was independent of the physical distance between cell pairs. To investigate the source of the transmission variability, we obtained patch-clamp recordings from intact axons. Axonal action potentials (APs) were reliably transmitted throughout the axonal arbour and showed modest changes in width. In contrast, calcium imaging from presynaptic boutons revealed that the amplitude of AP-evoked calcium transients exhibited large variations both among different boutons at a given trial and among trials in a given bouton. These results suggest that a factor contributing to the uEPSC fluctuations is the variability in calcium dynamics at presynaptic terminals. Finally, we acquired triple whole-cell recordings from divergent circuit motifs with one presynaptic neuron projecting to two postsynaptic neurons. Consistent with the independency of calcium dynamics among axonal boutons, a series of uEPSC fluctuations was not correlated between the two postsynaptic cells, indicating that different synapses even from the same neuron act independently.We conclude that the intra-bouton and inter-bouton variability in AP-induced calcium dynamics determine the heterogeneity and independency of uEPSCs.

1. The effects of changes in extra- and intracellular pH (pHo and pHi, respectively) on depolarization-evoked rises in intracellular free Ca2+ concentration ([Ca2+]i) and the activity of a Ca2+-dependent K+ channel were investigated in cultured fetal rat hippocampal neurones. 2. In neurones loaded with 2', 7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF), changes in pHo evoked changes in pHi. At room temperature, the ratio DeltapHi : DeltapHo (the slope of the regression line relating pHi to pHo) was 0.37 under HCO3-/CO2-buffered conditions and 0.45 under Hepes-buffered conditions; corresponding values at 37 C were 0.71 and 0.79, respectively. The measurements of changes in pHi evoked by changes in pHo were employed in subsequent experiments to correct for the effects of changes in pHi on the Kd of fura-2 for Ca2+. 3. In fura-2-loaded neurones, rises in [Ca2+]i evoked by transient exposure to 50 mM K+ were reduced and enhanced during perfusion with acidic and alkaline media, respectively, compared with control responses at pHo 7.3. Fifty percent inhibition of high-[K+]o-evoked rises in [Ca2+]i corresponded to pHo 7.23. In the presence of 10 microM nifedipine, 50 % inhibition of high-[K+]o-evoked responses corresponded to pHo 7.20, compared with a pHo of 7.31 for 50% inhibition of [Ca2+]i transients evoked by N-methyl-D-aspartate. 4. Changes in pHi at a constant pHo were evoked by exposing neurones to weak acids or bases and quantified in BCECF-loaded cells. Following pH-dependent corrections for the Kd of fura-2 for Ca2+, rises in [Ca2+]i evoked by high-[K+]o in fura-2-loaded cells were found to be affected only marginally by changes in pHi. When changes in pHi similar to those observed during the application of weak acids or bases were elicited by changing pHo, reductions in pH inhibited rises in [Ca2+]i evoked by 50 mM K+ whereas increases in pH enhanced them. 5. The effects of changes in pH on the kinetic properties of a BK-type Ca2+-dependent K+ channel were investigated. In inside-out patches excised from neurones in sister cultures to those used in the microspectrofluorimetric studies, with internal [Ca2+] at 20 microM, channel openings at an internal pH of 6.7 were generally absent whereas at pH 7.3 (or 7.8) the open probability was high. In contrast, channel activity in outside-out patches was not affected by reducing the pH of the bath (external) solution from 7.3 to 6.7. In inside-out patches with internal [Ca2+] at 0.7 microM, a separate protocol was applied to generate transient activation of the channel at a potential of 0 mV following a step from a holding level of -80 mV. In this case open probabilities were 0.81 (at pH 7.8), 0.57 (pH 7.3), 0.19 (pH 7.0) and 0.04 (pH 6.7). Channel conductance was not affected by changes in internal pH. 6. The results indicate that, in fetal rat hippocampal neurones, depolarization-evoked rises in [Ca2+]i mediated by the influx of Ca2+ ions through dihydropyridine-sensitive and -resistant voltage-activated Ca2+ channels are modulated by changes in pHo. The effects of pHo cannot be accounted for by changes in pHi consequent upon changes in pHo. However, changes in pHi affect the unitary properties of a Ca2+-dependent K+ channel. The results support the notion that pHo and/or pHi transients may serve a modulatory role in neuronal function.

Naftopidil is used clinically for the treatment of voiding disorders in benign prostatic hyperplasia. Previous in vivo experiments in which naftopidil was applied intrathecally abolished rhythmic bladder contraction, suggesting that naftopidil might inhibit a voiding reflex through interaction with spinal dorsal horn neurons. Here we aimed to clarify the mechanism of action of naftopidil on dorsal horn neurons.

1. The activity of locus coeruleus (LC) neurones (n = 126) was examined in whole-cell (conventional and amphotericin B-perforated patch) recordings, and the relationship of this activity to the respiratory discharge recorded on the C4 or C5 phrenic nerve roots was determined at different CO2 concentrations (2 and 8 %; bath pH 7. 8 and 7.2) in the in vitro brainstem-spinal cord preparation of the neonatal rat (1-5 days old). 2. In most neurones (n = 105) ongoing activity was modulated at respiratory frequency. Typically, this consisted of a phase of depolarization and increased discharge frequency synchronous with the phrenic burst, followed by a phase of hyperpolarization and inhibition of discharge (n = 94 of 105). The incidence of respiratory modulation decreased from 91 % on P1 to 57 % on P5. 3. Bath application of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 microM) or the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 100 microM) abolished both phases of respiratory modulation. The hyperpolarizing phase alone was abolished by the adrenoceptor antagonists idazoxan (5 microM) or phentolamine (0.8 microM). These results indicate that excitatory amino acid pathways are involved in the transmission of both the excitatory and inhibitory components and that the latter involves in addition an alpha2-adrenoceptor-mediated pathway. 4. Increasing the CO2 concentration from 2 to 8 % resulted in a shortening of expiratory duration and weakening or loss of respiratory-phased inhibition; this was accompanied by depolarization, increased discharge frequency and, in those neurones where they were initially present (60 %), an increase in the frequency of subthreshold membrane potential oscillations. The depolarizing response was retained in the presence of tetrodotoxin (TTX, 0.2-1.0 microM). 5. These results indicate that in this neonatal preparation LC neurones form part of the synaptically connected brainstem respiratory network, and that the LC constitutes a site of CO2- or pH-dependent chemoreception.

Giant depolarizing potentials (GDPs) represent a typical spontaneous activity pattern in the immature hippocampus. GDPs are mediated by GABAergic and glutamatergic synaptic inputs and their initiation requires an excitatory GABAergic action, which is typical for immature neurons due to their elevated intracellular Cl- concentration ([Cl-]i). Because GABAA receptors are ligand-gated Cl- channels, activation of these receptors can potentially influence [Cl-]i. However, whether the GABAergic activity during GDPs influences [Cl-]i is unclear. To address this question we performed whole-cell and gramicidin-perforated patch-clamp recordings from visually identified CA3 pyramidal neurons in immature hippocampal slices of mice at postnatal days 4-7. These experiments revealed that the [Cl-]i of CA3 neurons displays a considerable heterogeneity, ranging from 13 to 70 mM (average 38.1 ± 3.2 mM, n = 36). In accordance with this diverse [Cl-]i, GDPs induced either Cl--effluxes or Cl--influxes. In high [Cl-]i neurons with a negative Cl--driving force (DFCl) the [Cl-]i decreased after a GDP by 12.4 ± 3.4 mM (n = 10), while in low [Cl-]i neurons with a positive DFCl [Cl-]i increased by 4.4 ± 0.9 mM (n = 6). Inhibition of GDP activity by application of the AMPA receptor antagonist CNQX led to a [Cl-]i decrease to 24.7 ± 2.9 mM (n = 8). We conclude from these results, that Cl--fluxes via GABAA receptors during GDPs induced substantial [Cl-]i changes and that this activity-dependent ionic plasticity in neuronal [Cl-]i contributes to the functional consequences of GABAergic responses, emphasizing the concept that [Cl-]i is a state- and compartment-dependent parameter of individual cells.

Neuronal excitation evoked after dorsal-root (DR) stimulation in the spinal dorsal horn (DH) of rats was visualized with a high-resolution optical-imaging method, and the propagation mechanism was studied. Transverse slices of the spinal cord were obtained from 2-4 week-old rats and stained with the voltage-sensitive dye RH-482. Single-pulse stimulation to the primary-afferent A fibers in the DR attached to the slice evoked a weak, brief (<10 ms) excitatory optical response in the laminae I and III-V. When the stimulus intensity and duration were increased to activate both A and C fibers, an additional, much greater, and longer-lasting (>100 ms) excitatory response was generated in the laminae I-III, most intensely in the lamina II. A treatment with excitatory amino acid (EAA) antagonists, dl-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2, 3-dione, significantly reduced the amplitude and duration of the response in the lamina II. The optical response in the antagonists-containing solution was quite similar to that recorded in a Ca2+-free solution that blocked afferent synaptic transmission. The late component (>10 ms) was, however, slightly greater than that in the Ca2+-free solution. Treatment with the ATP-receptor antagonist, suramin, had a minimal effect on the response in the presence of EAA antagonists. These results suggested that the propagation of the DR-stimulus-elicited excitation was contributed largely by EAA receptors, but also by other receptors to a much lesser extent.

Acid-sensing ion channels (ASICs) are widely expressed in the mammalian central nervous system where they play a key role in synaptic transmission and in specific forms of memory. On the other hand, ASICs can be persistently active under pathological conditions contributing to neuronal damage in ischemic stroke, brain trauma, epilepsy and Parkinson's disease. However, to date no experimental evidence has linked ASICs to Alzheimer's disease (AD). Aim of the present work was to investigate, in CA1 pyramidal neurons, the possible involvement of ASIC1a in the Aβ-mediated effect on metabotropic glutamate (mGlu) receptor dependent transmission. We found that, in slices pretreated with Aβ, the pharmacological blockade of ASIC1a restored the increased intrinsic excitability following group I mGlu receptor activation. This suggests that, under certain conditions, ASIC1a might further contribute to the Aβ-related depolarizing response. We have recently demonstrated that ASIC1a is also involved long-term depression (LTD) induced either by low-frequency stimulation or by application of the group I mGlu receptor agonist DHPG. Here, we have shown that psalmotoxin-1, a selective blocker of ASIC1a, rescued the DHPG-LTD facilitation associated with genetic and non-genetic models of AD. Overall, these results suggest that a functional coupling between ASIC1a and mGlu receptors occurs and might contribute to the synaptic alterations associated with AD.

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.

1. The aim was to examine whether long-term potentiation (LTP) had effects on short-term synaptic plasticity outside those predicted from its effect on single volley-induced responses. Field recordings from the CA1 region of guinea-pig hippocampal slices were used, and short- term plasticity was evoked by five-impulse trains of 20 and 50 Hz. 2. The five-impulse trains were evoked in the presence of D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 20-50 microM), picrotoxin (100 microM), and 2-OH-saclofen (200 microM), and care was taken to avoid initiation of postsynaptic spike activation. Field responses were thus considered to reflect non-NMDA receptor-mediated activity only, and demonstrated a net facilitation during the trains. 3. The facilitation was found, on average, to be unaffected by LTP, evoked by strong afferent tetanization. This was true also when release probability had been altered either by the adenosine agonist N-cyclohexyladenosine (CHA; 100 nM) or the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 200 nM). When examined for individual experiments, cases with increases, or decreases, of facilitation following LTP were observed. These deviations showed no relation to initial release probability or to LTP magnitude, and they were also observed in control inputs not subjected to LTP. 4. Impairment of non-NMDA receptor desensitization by cyclothiazide (30 microM) increased facilitation observed during a 50 Hz, but not a 20 Hz, train. LTP had no effect on facilitation, in the presence of this drug, either during 20 or 50 Hz trains. 5. The results suggest that the effect of LTP in the hippocampal CA1 region on non-NMDA receptor-mediated synaptic responses to a brief afferent tetanus does not differ from that on a low-frequency, single volley-induced response. They do not support the notion that LTP is based on changes in release probability of previously active synapses. If LTP is based on recruitment of previously, pre- or postsynaptically, silent synapses, these synapses must have, on average, release characteristics similar to the previously active ones.

Systemic nicotine enhances burst firing of dopamine neurons in the ventral tegmental area and dopamine release in the nucleus accumbens, mainly via stimulation of nicotinic acetylcholine receptors in the ventral tegmental area. Given that both the neuronal activity of mesolimbic dopamine neurons and terminal dopamine release are regulated by excitatory amino acid inputs to the ventral tegmental area and that nicotine facilitates glutamatergic transmission in brain, we investigated the putative role of ionotropic glutamate receptors within the ventral tegmental area for the effects of nicotine on dopamine release in the nucleus accumbens using microdialysis, with one probe implanted in the ventral tegmental area for drug application and another in the ipsilateral nucleus accumbens for measuring dopamine, in awake rats. Systemic nicotine (0.5 mg/kg, s.c.) and infusion of nicotine (1.0 mM) into the ventral tegmental area increased dopamine output in the nucleus accumbens. Intrategmental infusion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (0.1 mM) or N-methyl-D-aspartate (0.3 mM) increased accumbal dopamine release; these effects were antagonized by concomitant infusion of a selective antagonist at N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid (0.3 mM), and non-N-methyl-D-aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (0.3 mM), respectively. Infusion of either antagonist (0.3 or 1.0 mM) into the ventral tegmental area did not affect basal dopamine levels, whereas infusion of 2-amino-5-phosphonopentanoic acid, but not 6-cyano-7-nitroquinoxaline-2,3-dione, starting 40 min before nicotine injection dose-dependently attenuated the nicotine-induced increase in accumbal dopamine release. Concurrent intrategmental infusion of 2-amino-5-phosphonopentanoic acid and nicotine decreased nicotine-induced dopamine release in the nucleus accumbens. These results indicate that the stimulatory action of nicotine on the mesolimbic dopamine system is to a considerable extent mediated via stimulation of N-methyl-D-aspartate receptors within the ventral tegmental area.

In the amygdala (AMG) slices obtained from both the young (4-7 months old) and aged (17-20 months old) groups of Senescence-Accelerated Mouse (SAM) P10, spontaneous bursts were recorded in the medial, central and basolateral nuclei. The spontaneous bursts were also observed in the slices from the young group of SAMR1, whereas the mean frequency was significantly lower than that from the young group of SAMP10. The spontaneous burst was barely detectable in slices from the aged group of SAMR1 during perfusing with the standard solution, while bicuculline methiodide (10 microM), a GABAA receptor antagonist, or Mg2+-free solution induced a similar bursting activity observed in the young group. The burst response was also evoked in the medial, central and basolateral AMG following stimulation of the stria terminalis (ST). Both spontaneous and evoked bursts were completely suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 4 microM), an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, but not by (+)-5-methyl-10, 11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801, 30 microM), an N-methyl-d-aspartate receptor antagonist. The hyperexcitability of the AMG neurons was further substantiated by optical recordings. Following stimulation of the ST, the optical signals reflected postsynaptic responses spread into the medial and central AMG areas at 2-5 ms and faded out at 20-30 ms after stimulation. The intensity of the optical signal recorded in the slice from the young SAMP10 was significantly higher than that from SAMR1 or ddY mice. These observations indicate that bursts mediated by AMPA/kainate receptors were transiently generated in the AMG of SAMR1 at the young age, while the bursts with higher frequency were continuously generated in the AMG of SAMP10. The chronic neuronal hyperactivity in the AMG may be partially responsible for the age-related deterioration of memory and learning abilities observed in SAMP10.

Although GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the brain, intense activation of GABA receptors can cause excitation under certain conditions. In the superficial layers of the guinea-pig superior colliculus (SC) slice the excitatory action of GABA (< or = 3 mM) is dominant and sufficient to induce a robust and novel form of long-term potentiation, termed LTPG, of evoked field excitatory postsynaptic potentials (fEPSPs). This action of GABA could neither be mimicked by GABA-A nor -B agonists which were found to suppress synaptic transmission. Additionally, LTPG was not inhibited by the GABA-A receptor antagonist bicuculline while the GABA-C receptor antagonist imidazol-4-acetic acid prevented LTPG. Glutamatergic synaptic transmission was found to be required, as LTPG was partially use-dependent and did not emerge when glutamate receptors of the non-NMDA type were blocked during GABA application. Moreover, LTPG declined to baseline values in the presence of the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (APV). In addition, the L-type calcium channel blocker nifedipine inhibited the induction of LTPG. It is suggested that activation of excitatory GABA non-A, non-B receptors can lead to LTP in the SC, which may be of major importance for plastic events since the content of GABA and GABA receptors are particularly high in this brain area.

Ionotropic glutamate receptors (iGluRs) mediate excitatory neuronal signaling in the mammalian CNS. These receptors are critically involved in diverse physiological processes; including learning and memory formation, as well as neuronal damage associated with neurological diseases. Based on partial sequence and structural similarities, these complex cation-permeable iGluRs are thought to descend from simple bacterial proteins emerging from a fusion of a substrate binding protein (SBP) and an inverted potassium (K+)-channel. Here, we fuse the pore module of the viral K+-channel KcvATCV-1 to the isolated glutamate-binding domain of the mammalian iGluR subunit GluA1 which is structural homolog to SBPs. The resulting chimera (GluATCV*) is functional and displays the ligand recognition characteristics of GluA1 and the K+-selectivity of KcvATCV-1. These results are consistent with a conserved activation mechanism between a glutamate-binding domain and the pore-module of a K+-channel and support the expected phylogenetic link between the two protein families.

Ibogaine is a natural alkaloid of Voacanga africana that is effective in the treatment of withdrawal symptoms and craving in drug addicts. As the synaptic and cellular basis of ibogaine's actions are not well understood, this study tested the hypothesis that ibogaine and Voacanga africana extract modulate neuronal excitability and synaptic transmission in the parabrachial nucleus using the nystatin perforated patch-recording technique. Ibogaine and Voacanga africana extract dose dependently, reversibly, and consistently attenuate evoked excitatory synaptic currents recorded in parabrachial neurons. The ED50 of ibogaine's effect is 5 microM, while that of Voacanga africana extract is 170 micrograms/ml. At higher concentrations, ibogaine and Voacanga africana extract induce inward currents or depolarization that are accompanied by increases in evoked and spontaneous firing rate. The depolarization or inward current is also accompanied by an increase in input resistance and reverses polarity around 0 mV. The depolarization and synaptic depression were blocked by the dopamine receptor antagonist haloperidol. These results indicate that ibogaine and Voacanga africana extract 1) depolarize parabrachial neurons with increased excitability and firing rate; 2) depress non-NMDA receptor-mediated fast synaptic transmission; 3) involve dopamine receptor activation in their actions. These results further reveal that the Voacanga africana extract has one-hundredth the activity of ibogaine in depressing synaptic responses. Thus, ibogaine and Voacanga africana extract may produce their central effects by altering dopaminergic and glutamatergic processes.

It has been reported that kappa opioid receptor (KOR) is expressed in the paraventricular nucleus of thalamus (PVT), a brain region associated with arousal, drug reward and stress. Although intra-PVT infusion of KOR agonist was found to inhibit drug-seeking behavior, it is still unclear whether endogenous KOR agonists directly regulate PVT neuron activity. Here, we investigated the effect of the endogenous KOR agonist dynorphin-A (Dyn-A) on the excitability of mouse PVT neurons at different developmental ages. We found Dyn-A strongly inhibited PVT neurons through a direct postsynaptic hyperpolarization. Under voltage-clamp configuration, Dyn-A evoked an obvious outward current in majority of neurons tested in anterior PVT (aPVT) but only in minority of neurons in posterior PVT (pPVT). The Dyn-A current was abolished by KOR antagonist nor-BNI, Ba(2+) and non-hydrolyzable GDP analogue GDP-β-s, indicating that Dyn-A activates KOR and opens G-protein-coupled inwardly rectifying potassium channels in PVT neurons. More interestingly, by comparing Dyn-A currents in aPVT neurons of mice at various ages, we found Dyn-A evoked significant larger current in aPVT neurons from mice around prepuberty and early puberty stage. In addition, KOR activation by Dyn-A didn't produce obvious desensitization, while mu opioid receptor (MOR) activation induced obvious desensitization of mu receptor itself and also heterologous desensitization of KOR in PVT neurons. Together, our findings indicate that Dyn-A activates KOR and inhibits aPVT neurons in mice at various ages especially around puberty, suggesting a possible role of KOR in regulating aPVT-related brain function including stress response and drug-seeking behavior during adolescence.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a μ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating μ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Epipial application is one of the approaches for drug delivery into the cortex. However, passive diffusion of epipially applied drugs through the cortical depth may be slow, and different drug concentrations may be achieved at different rates across the cortical depth. Here, we explored the pharmacodynamics of the inhibitory effects of epipially applied ionotropic glutamate receptor antagonists CNQX and dAPV on sensory-evoked and spontaneous activity across layers of the cortical barrel column in urethane-anesthetized rats. The inhibitory effects of CNQX and dAPV were observed at concentrations that were an order higher than in slices in vitro, and they slowly developed from the cortical surface to depth after epipial application. The level of the inhibitory effects also followed the surface-to-depth gradient, with full inhibition of sensory evoked potentials (SEPs) in the supragranular layers and L4 and only partial inhibition in L5 and L6. During epipial CNQX and dAPV application, spontaneous activity and the late component of multiple unit activity (MUA) during sensory-evoked responses were suppressed faster than the short-latency MUA component. Despite complete suppression of SEPs in L4, sensory-evoked short-latency multiunit responses in L4 persisted, and they were suppressed by further addition of lidocaine suggesting that spikes in thalamocortical axons contribute ∼20% to early multiunit responses. Epipial CNQX and dAPV also completely suppressed sensory-evoked very fast (∼500 Hz) oscillations and spontaneous slow wave activity in L2/3 and L4. However, delta oscillations persisted in L5/6. Thus, CNQX and dAPV exert inhibitory actions on cortical activity during epipial application at much higher concentrations than in vitro, and the pharmacodynamics of their inhibitory effects is characterized by the surface-to-depth gradients in the rate of development and the level of inhibition of sensory-evoked and spontaneous cortical activity.

The brain is self-writable; as the brain voluntarily adapts itself to a changing environment, the neural circuitry rearranges its functional connectivity by referring to its own activity. How the internal activity modifies synaptic weights is largely unknown, however. Here we report that spontaneous activity causes complex reorganization of synaptic connectivity without any external (or artificial) stimuli. Under physiologically relevant ionic conditions, CA3 pyramidal cells in hippocampal slices displayed spontaneous spikes with bistable slow oscillations of membrane potential, alternating between the so-called UP and DOWN states. The generation of slow oscillations did not require fast synaptic transmission, but their patterns were coordinated by local circuit activity. In the course of generating spontaneous activity, individual neurons acquired bidirectional long-lasting synaptic modification. The spontaneous synaptic plasticity depended on a rise in intracellular calcium concentrations of postsynaptic cells, but not on NMDA receptor activity. The direction and amount of the plasticity varied depending on slow oscillation patterns and synapse locations, and thus, they were diverse in a network. Once this global synaptic refinement occurred, the same neurons now displayed different patterns of spontaneous activity, which in turn exhibited different levels of synaptic plasticity. Thus, active networks continuously update their internal states through ongoing synaptic plasticity. With computational simulations, we suggest that with this slow oscillation-induced plasticity, a recurrent network converges on a more specific state, compared to that with spike timing-dependent plasticity alone.