Effect of cyclothiazide on spontaneous miniature excitatory postsynaptic currents in rat hippocampal pyramidal cells

 Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded from the CA1 region of slices using the whole-cell patch-clamp technique. Cyclothiazide (0.1 mM), a complete blocker of desensitization of (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels, was applied to determine the changes in amplitude and kinetics of mEPSCs occurring with complete suppression of desensitization. The amplitude of mEPSC (A) was not affected significantly by cyclothiazide, but both the rise (τ_r) and the decay time (τ_d) were consistently increased (from 2.3 to 6.5 ms and from 9.9 to 22.2 ms respectively). The amplitude dependence of both τ_d and τ_r became much greater, but there was no upward shift of the best-fitting lines. The slopes of the control best-fitting lines were (±SD; ms/pA; n=5) 0.39±0.05 for τ_d:A and 0.12±0.07 for τ_r:A, but, in the presence of cyclothiazide, the corresponding slopes were much steeper (2.1±0.60 and 0.68±0.21; holding potential was –50 mV and temperature 32°C). These changes, which were slow to develop, suggest that cyclothiazide blocks AMPA receptor channel desensitization, whilst having no effect on the closing rate of AMPA channels. Judging by the extent of change, the speed of diffusion of glutamate in the synaptic cleft is probably similar to that in water. In conclusion, this study provides evidence that: (1) under control conditions, desensitization of AMPA channels plays a major role in shaping the time course of synaptic currents in CA1; and (2) cyclothiazide prolongs their time course solely by abolishing desensitization. Received: 28 May 1998 / Received after revision: 16 July 1998 / Accepted: 3 September 1998     

Changes of spontaneous miniature excitatory postsynaptic currents in rat hippocampal pyramidal cells induced by aniracetam

 Spontaneous miniature excitatory postsynaptic currents (mEPSCs) in rat hippocampal pyramidal neurones in slices (CA1 region) were recorded at 35–37°C using the whole-cell patch-clamp technique before and after addition of aniracetam (1 mM) to determine how a partial blockade of desensitization alters the relationship between the amplitude (A) and kinetics of mEPSCs, and to evaluate the factors that determine their variability. The rise time (τ_r) and the time constant of decay of mEPSCs (τ_d) are essentially amplitude independent in control conditions, but become clearly amplitude dependent in the presence of aniracetam. The slopes of the best fitting lines to τ_d:A and τ_r:A data pairs were (± SD; ms/pA; n = 5): (1) (control) 0.07 ± 0.02 and 0.008 ± 0.003; (2) (aniracetam) 0.40 ± 0.19 and 0.22 ± 0.22. The amplitude-dependent prolongation of τ_d is explained by the concentration dependence of two related processes, the buffering of glutamate molecules by AMPA receptor channels, and the occupancy of the double-bound activatable states. A slower deactivation makes an amplitude-independent contribution. Desensitization reduces the amplitude dependence of τ_d by minimizing repeated openings of α-amino-3-hydroxy-methyl-isoxazole (AMPA) receptor channels. A greater amplitude dependence of τ_r probably involves both pre- and postsynaptic factors. The variability of A and τ_d values did not change significantly, but the factors underlying the variability of τ_d values were much affected. The greater amplitude dependence and the greater scatter about the best fitting lines to τ_d:A data pairs are approximately balanced by the greater mean values. The greater scatter of τ_d about the best fitting lines probably occurs because the saturation of AMPA receptors is not the same at different synapses with different numbers of AMPA receptors. Received: 17 April 1997 1997 / Received after revision: 11 August 1997 / Accepted: 1 September 1997     

Enhancement of N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials in the neostriatum after methamphetamine sensitization. An in vitro slice study

It has been suggested that behavioral methamphetamine sensitization involves changes in cortical excitatory synaptic inputs to neostriatal (Str) projection neurons. To test this, we performed blind whole-cell recording of medium spiny neurons in Str slice preparations. In Str neurons of naive rats, the amplitude of the subcortical white matter stimulation-induced N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials (NMDA-EPSPs) was decreased upon hyperpolarization, owing to the voltage-dependent Mg(2+) blockade of NMDA receptor channels. In contrast, the amplitude of the NMDA-EPSPs in Str neurons of rats undergoing methamphetamine withdrawal (MW) did not show the Mg(2+) blockade and was nearly voltage independent over the membrane potential range of -70 to -110 mV. Application of the specific protein kinase C (PKC) activator, phorbol 12, 13- DL-acetate, blocked the voltage-dependent Mg(2+) blockade of NMDA receptor channels in Str neurons of naive rats. Application of the specific activator of cAMP-dependent protein kinase A (PKA), Sp-cAMPS-triethylamine salt, increased the amplitude of the NMDA receptor-mediated EPSPs at the rest but not at hyperpolarized potentials. Coapplication of the PKC and PKA activators yielded NMDA-EPSPs similar to those seen in Str neurons of MW rats. In Str slices of naive rats, tetanic subcortical white matter stimulation induced long-term depression of field potentials. In Str slices treated with the PKC and/or PKA, the same stimulation induced long-term potentiation of field potentials similar to those observed in slices obtained from MW rats. These results suggest that the enhancement of the NMDA receptor-mediated corticostriatal synaptic transmission plays an important role in behavioral methamphetamine sensitization. This enhancement is probably associated with phosphorylation of NMDA receptors mediated by the simultaneous activation of PKC and PKA.     

Increase of delayed rectifier potassium currents in large aspiny neurons in the neostriatum following transient forebrain ischemia

Large aspiny (LA) neurons in the neostriatum are resistant to cerebral ischemia whereas spiny neurons are highly vulnerable to the same insult. Excitotoxicity has been implicated as the major cause of neuronal damage after ischemia. Voltage-dependent potassium currents play important roles in controlling neuronal excitability and therefore influence the ischemic outcome. To reveal the ionic mechanisms underlying the ischemia-resistance, the delayed rectifier potassium currents (Ik) in LA neurons were studied before and at different intervals after transient forebrain ischemia using brain slices and acute dissociation preparations. The current density of Ik increased significantly 24 h after ischemia and returned to control levels 72 h following reperfusion. Among currents contributing to Ik, the margatoxin-sensitive currents increased 24 h after ischemia while the KCNQ/M current remained unchanged after ischemia. Activation of protein kinase A (PKA) down-regulated Ik in both control and ischemic LA neurons, whereas inhibition of PKA only up-regulated Ik and margatoxin-sensitive currents 72 h after ischemia, indicating an active PKA regulation on Ik at this time. Protein tyrosine kinases had a tonic inhibition on Ik to a similar extent before and after ischemia. Compared with that of control neurons, the spike width was significantly shortened 24 h after ischemia due to facilitated repolarization, which could be reversed by blocking margatoxin-sensitive currents. The increase of Ik in LA neurons might be one of the protective mechanisms against ischemic insult.     

Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons.

Fast application of L-glutamate, AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA (N-methyl-D-aspartate) receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.     

Different physiological properties of spontaneous excitatory postsynaptic currents in nucleus accumbens shell neurons between adult and juvenile rats

Studies have demonstrated the changes of the physiological characteristics of nucleus accumbens (NAc) neurons with the postnatal development of rats. In the present study, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in the slices of NAc shell (NAcS) of adult and juvenile rats. Our results demonstrate that both the average amplitude of sEPSCs and the average frequency of sEPSCs in the NAcS slices of adult rats decreased significantly than that in juvenile rats. The average half width of sEPSCs in the NAcS slices in adult rats increased significantly than that in juvenile rats. The rise time of sEPSCs, the rise 50 time of sEPSCs and the 10–90 rise time of sEPSCs in the NAcS slices increased significantly in adult rats than that in juvenile rats. The decay time of sEPSCs in the NAcS slices also increased significantly in adult rats than that in juvenile rats. The above results strongly indicate that there are marked changes in the electrophysiological properties of single sEPSC in the NAcS slices of juvenile and adult rats.     

Differential contribution of kainate receptors to excitatory postsynaptic currents in superficial layer neurons of the rat medial entorhinal cortex

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.     

Monte Carlo simulation of spontaneous miniature excitatory postsynaptic currents in rat hippocampal synapse in the presence and absence of desensitization

 Using the Monte Carlo method, spontaneous fast excitatory postsynaptic currents (mEPSCs) at a hippocampal synapse were simulated by releasing 150–20,000 glutamate molecules from a point source centred 15 nm above a rectangular grid of 14 × 14 α-amino-3-hydroxy-methyl-isoxazole (AMPA) receptors and assuming the channel kinetics to be as reported by Jonas et al. [J Physiol (Lond) 472:615; 1993]. The relationship between the amplitudes of mEPSCs and their time constants of decay is positive, but not pronounced in physiological conditions (except when the number of molecules released is very high). It increases as desensitization is reduced and becomes highly pronounced when it is eliminated. mEPSCs are prolonged with repeated opening of AMPA channels due to enhancement of two concentration-dependent processes: (1) binding of glutamate molecules by AMPA receptors, and (2) occupancy of both activatable bound states. In contrast, the time constant of decay of the patch currents evoked by a short glutamate pulse is independent of glutamate concentration and current amplitude in control conditions, and only moderately concentration dependent in the absence of desensitization. The fast application protocol thus fails to reproduce synaptic currents reliably when there is repeated binding of glutamate molecules to AMPA receptors. During an mEPSC, the occupancy of desensitized states increases rapidly and it strongly depends on the number of glutamate molecules released. Desensitization reaches its maximum after an mEPSC decays to very low levels, and recovers very slowly (from tens to hundreds of milliseconds), and in a concentration-dependent manner. In conclusion, under physiological conditions the desensitization of AMPA receptors plays a major role in shaping the time course of mEPSCs by minimizing the repeated opening of AMPA channels. Received: 17 April 1997 / Received: after revision: 11 August 1997 / Accepted: 1 September 1997     

Characterization of spontaneous inhibitory postsynaptic currents in cultured rat retinal amacrine cells

Spontaneous postsynaptic current is a reflection of spontaneous neurotransmitter release that plays multiple roles in a variety of neurobiological activities. In the present study, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) by patch-clamp techniques in cultured rat retinal GABAergic amacrine cells (ACs), which provide inhibitory inputs to both bipolar and ganglion cells in the inner retina, and examined if and how Ca²⁺ was involved in the induction of spontaneous GABA release from the terminals of these cells. sIPSCs were completely blocked by application of either 10 μM bicuculline or 10 μM gabazine, and the reversal potential of sIPSCs was close to E_Cl−, indicating that these events were exclusively mediated by GABA_A receptors. Increase of external Ca²⁺ concentrations from 2 to 5 mM significantly enhanced the frequency, but did not change the amplitude of sIPSCs. In contrast, perfusion of Ca²⁺-free external solution greatly reduced the events of sIPSCs and decreased the amplitude of sIPSCs. Consistently, the non-selective voltage-gated calcium channel blocker CdCl₂ (200 μM) considerably suppressed both the frequency and the amplitude of sIPSCs. Furthermore, the ryanodine receptor (RyR) antagonist dantrolene (10 μM) failed to affect sIPSCs, while the inositol 1,4,5-trisphosphate (IP₃) receptor antagonists 2-aminoethyl diphenylborinate (2-APB, 20 μM) and xestospongin C (XeC, 1 μM) significantly decreased the frequency of sIPSCs. In the presence of SKF96365 (10 μM), a non-specific transient receptor potential channel (TRP) blocker, 2-APB persisted to show its effect on sIPSCs. These results suggest that spontaneous GABA release from the terminals of GABAergic ACs is Ca²⁺-dependent, and both extracellular calcium influx through presynaptic calcium channels and Ca²⁺ release through activation of the IP₃-sensitive pathway, but not the ryanodine-sensitive one, from intracellular stores are responsible for the generation of sIPSCs under our experimental conditions.     

Ultrastructure of Golgi-impregnated and gold-toned spiny and aspiny neurons in the monkey neostriatum

Summary Golgi-impregnated, gold-toned spiny and aspiny neurons in the monkey neostriatum were deimpregnated and examined at the electron microscope level.Spiny type I neurons have relatively large nuclei with few indentations and aggregates of chromatin under the nuclear membrane which in some regions give the appearance of a dark rim. The small quantity of surrounding cytoplasm is poor in organelles.Aspiny type I neurons have eccentric, highly indented nuclei. The relatively large proportion of cytoplasm is rich in organelles especially Golgi apparatus and rough endoplasmic reticulum which often appears in stacks.Synapses with symmetric membrane densities are common on the somata of spiny type I neurons. Those on the proximal and distal dendritic shafts are few in number and asymmetric, and those on spines more frequent and primarily asymmetric. Aspiny type I neurons have few synapses on their cell bodies. Proximal and distal dendrites, however, are contacted by numerous profiles which contain small round vesicles and make both symmetric and asymmetric synapses. The same axon terminals also synapse with dendritic spines of spiny neurons, indicating that an input, most likely of afferent origin, is shared by both cell types. Other less frequently occurring profiles forming symmetric membrane densities also contact the dendrites of aspiny and spiny neurons. The axon hillocks and initial segments of both neuronal types receive a synaptic input, which is more common on spiny cells.Results offer unequivocal evidence for the differences in the ultrastructure of these two most common categories of medium-size neostriatal neurons, which may help in their proper identification in standard material, as well as information on the types and distributions of synaptic inputs onto these neurons. Moreover, the findings clarify some controversies in the literature probably originating from observations on a mixed population of cells of medium size.     

Depolarizing IPSPs and depolarization by GABA of rat neostriatum cells in vitro

Summary In neostriatal slices pretreated with sodium pentobarbital (100 M) and 4-aminopyridine (50 M), intrastriatal stimulation elicited EPSPs followed by a slowly decaying depolarization which lasted about 200 ms and was associated with a membrane conductance increase and a suppression of spike potentials. This depolarizing inhibitory synaptic action could be blocked by picrotoxin (50 M) or bicuculline (50 M). The reversal potential for the slowly decaying depolarization was –57 to –62 mV, i.e. it was positive with respect to the resting membrane potential (¯x = –67 mV). GABA, injected into the tissue in the vicinity of the recording electrode by pressure application, or added to the perfusate (10 M–1 mM), depolarized the cells and reduced both the membrane resistance and the amplitude of EPSPs. The reversal potential of GABA depolarization was found in a potential range approximating that of the slowly decaying depolarization. These results are compatible with the assumption that GABA is the transmitter of an intrinsic inhibition in rat neostriatum, but indicate that GABA-mediated IPSPs of neostriatal cells in vitro are depolarizing at the resting membrane potential. The possible reasons for this are discussed.     

Analysis of the slow excitatory postsynaptic potential in bullfrog sympathetic ganglion cells.

The ionic mechanism of the slow excitatory postsynaptic potential (slow EPSP), i.e. the muscarinic action of acetylcholine (ACh), was studied either by stimulating preganglionic nerves or by applying ACh in curarized sympathetic ganglion cells of bullfrogs. There are three different types of cells characterized by the effects of membrane hyperpoliarization on the amplitude of slow EPSP. One group of cells showed an increase in amplitude (type 1 cell) and, in two other groups of cells, it remained unchanged (type 2 cell) or decreased (type 3 cell), when the membrane was hyperpolarized. Under the muscarinic effects of ACh, the slope membrane conductance was increased (type 1 cell), unchanged (type 2 cell) or decreased (type 3 cell) at 10-20 mV hyperpolarized levels, while it was unchanged (type 1 cell) or decreased (types 2 and 3 cells) at resting and depolarized levels. In all cells, the slow ACh potential, corresponding to the slow EPSP, was almost completely suppressed in a high K+, Ca2+-free, Na+-free solution. These results suggest that the slow EPSP is generated by increases in Na+ and Ca2+ conductance and also by a simultaneous decrease in the K+ conductance.     

Adenosine preferentially suppresses serotonin2A receptor-enhanced excitatory postsynaptic currents in layer V neurons of the rat medial prefrontal cortex

Serotonin induces 'spontaneous' (non-electrically evoked) excitatory postsynaptic currents in layer V pyramidal neurons in the prefrontal cortex. This is likely due to a serotonin2A receptor-mediated focal release of glutamate onto apical dendrites. In addition, activation of the serotonin2A receptor selectively enhances late components of electrically evoked excitatory postsynaptic currents. In this study, using in vitro intracellular and whole-cell recording in rat brain slices, we examined the role of adenosine in modulating serotonin2A-enhanced 'spontaneous' and electrically evoked excitatory postsynaptic currents in layer V pyramidal neurons in the medial prefrontal cortex. Adenosine and N6-cyclopentyladenosine, an A1 adenosine agonist, markedly suppressed the serotonin2A-induced ('spontaneous') excitatory postsynaptic currents. However, adenosine had no effect on spontaneous miniature (tetrodotoxin-insensitive) postsynaptic potentials. Adenosine also blocked the late excitatory postsynaptic currents induced by the serotonin2A/2C agonist R(-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. Surprisingly, in contrast to other regions, adenosine had a relatively small effect on electrically evoked fast excitatory postsynaptic currents.These findings represent a novel demonstration of adenosine's ability to preferentially modulate serotonin2A-mediated synaptic events in the medial prefrontal cortex. As the serotonin2A receptor is closely linked with the effects of atypical antipsychotics and hallucinogens, further understanding of the modulators of this receptor such as adenosine may provide useful therapeutic applications.     

A comparison of paired-pulse facilitation of AMPA and NMDA receptor-mediated excitatory postsynaptic currents in the hippocampus

Paired-pulse facilitation of excitatory synaptic transmission was investigated in the CA1 region of rat hippocampal slices using whole-cell patch-clamp recording. To optimise the measurement of excitatory synaptic transmission, -amino-butyric acid (GABA)-mediated synaptic inhibition was eliminated using both GABA_A and GABA_B antagonists. Pure -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) were then isolated pharmacologically. Paired-pulse facilitation of either AMPA or NMDA receptor-mediated EPSCs (EPSC_A and EPSC_N, respectively) was investigated using two stimuli of identical strength delivered at intervals of between 25 and 1000 ms. The paired-pulse facilitation profiles of both EPSC_A and EPSC_N were similar. Pairedpulse facilitation of EPSC_A was independent of holding potential. In contrast paired-pulse facilitation of EPSC_N was markedly voltage-dependent; maximum facilitation was recorded at hyperpolarised membrane potentials. At positive membrane potentials there was little or no paired-pulse facilitation and, in most neurones, pairedpulse depression was observed. Voltage-dependence of paired-pulse facilitation of EPSC_N was similar in the presence or nominal absence of Mg²⁺ in the bathing medium, and was unaffected by extensive dialysis of neurones with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). These data are consistent with a presynaptic locus for paired-pulse facilitation of EPSC_A. However, paired-pulse facilitation of EPSC_N involves postsynaptic factors.     

Modulation of AMPA excitatory postsynaptic currents in the spinal cord dorsal horn neurons by insulin

Glutamate AMPA receptors are critical for sensory transmission at the spinal cord dorsal horn (DH). Plasma membrane AMPA receptor endocytosis that can be induced by insulin may underlie long term modulation of synaptic transmission. Insulin receptors (IRs) are known to be expressed on spinal cord DH neurons, but their possible role in sensory transmission has not been studied. In this work the effect of insulin application on fast excitatory postsynaptic currents (EPSCs) mediated by AMPA receptors evoked in DH neurons was evaluated. Acute spinal cord slices from 6 to 10 day old mice were used to record EPSCs evoked in visually identified superficial DH neurons by dorsal root primary afferent stimulation. AMPA EPSCs could be evoked in all of the tested neurons. In 75% of the neurons the size of the AMPA EPSCs was reduced to 62.1% and to 68.9% of the control values when 0.5 or 10 μM insulin was applied. There was no significant change in the size of the AMPA EPSCs in the remaining 25% of DH neurons. The membrane permeable protein tyrosine kinase inhibitor, lavendustin A (10 μM), prevented the insulin induced AMPA EPSC depression. Our results suggest a possible role of the insulin pathway in modulation of sensory and nociceptive synaptic transmission in the spinal cord.