Frequency Of Excitatory Postsynaptic Potentials Research Articles

Hippocampal mossy cells exhibit some of the earliest signs of increased excitability in the Tg2576 model of Alzheimer’s disease neuropathology

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative illness characterized by progressive accumulation of amyloid beta (Aβ) and neurofibrillary tangles, with cognitive impairment and altered neural activity. Hyperexcitability in the early stages of AD contribute to Aβ accumulation and cognitive impairment, aggravating the progression of AD. However, the hyperexcitability origin is not clear. This study aimed to test whether mossy cells (MCs), an excitatory cell of the hippocampal dentate gyrus, show increased excitability at early stages of AD and contribute to the increased network excitability generation. Indeed, alterations of MCs contribute to hyperexcitability and cognitive impairment in epilepsy. However, the role of MCs in AD has not been substantially explored.MethodsIntrinsic and synaptic properties of MCs and granule cells (GCs) from WT and Tg2576 mice at early ages (1‐2 m.o.) were characterized by whole‐cell patch‐clamp recordings. Synaptic properties included the frequency and amplitude of spontaneous excitatory postsynaptic potentials (EPSPs) and excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). Deterioration in MCs morphology was evaluated using Nissl staining and GluR2/3 labeling by light‐ and confocal microscopy. Aβ deposition was evaluated using the McSA1 antibody.ResultsTg2576 GCs did not have any significant difference in their intrinsic properties, as we shown previously in mice ∼3 m.o. However, an enhanced excitatory and inhibitory input to GCs, depicted by augmented IPSC (7.16 vs 14.04 events/s) and NMDA‐mediated EPSC frequencies (0.81 vs 1.41 events/s) were found. Interestingly, Tg2576 MCs had an augmented EPSP frequency (5.75 vs 9.44 events/s), and their intrinsic properties showed a depolarized RMP (‐72.88 vs ‐58.36 mV), and reduced rheobase (145.56 vs 47.14 pA), AP amplitude (98.14 vs 76.66 mV), time‐to‐peak (552.75 vs 266.16 ms) and maximum rise (171.44 vs 88.68 mV/ms) and decay slopes (‐61.17 vs ‐42.38 mV/ms). The correlation between #APs and current injected showed Tg2576 MCs fired significantly more APs (SEZD = 0.34; z = 2.48). Tg2576 MCs showed robust intracellular Aβ aggregation without any significant morphological change.ConclusionsMCs changes in excitability and early accumulation of Aβ suggest that MCs could be the cause of increased excitability occurring later in GCs. In this manner, MCs could be an important contributor to AD.

Modulation of sympathetic preganglionic neuron activity via adrenergic receptors.

The sympathetic preganglionic neurons (SPNs) play a key role in the sympathetic nervous system. Previous reports have suggested that norepinephrine (NE) directly affects SPNs via both inhibitory hyperpolarization interactions mediated by α2 receptors and excitatory depolarization interactions mediated by α1 receptors. It remains poorly understood, however, whether the excitability of SPNs can be inhibited indirectly (presynaptically) as well as directly (postsynaptically). We intracellularly recorded 41 SPNs using the whole-cell patch-clamp technique in spinal cord slice preparations of neonatal rats. We examined the effects of NE or dexmedetomidine hydrochloride (Dxm) (α2-adrenergic receptor agonist) on SPNs by analyzing the excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs). EPSPs were dominant in 15 SPNs (EPSP-SPNs) and IPSPs were dominant in 7 SPNs (IPSP-SPNs) at baseline. We were unable to analyze the postsynaptic potentials in the other 19 SPNs, due to high frequency of action potential firings (firing-SPNs). At baseline, the membrane potentials and resistances of each type of SPN were similar. NE (1 μM) gradually depolarized the EPSP-SPNs and IPSP-SPNs (P < 0.001) and NE significantly increased the EPSP frequency of the EPSP-SPNs (P < 0.05). Dxm (10 nM) after application of NE decreased the EPSP frequency of the EPSP-SPNs (P < 0.001) and the EPSP voltage and IPSP voltage of the IPSP-SPNs (P < 0.05). In 5 of the 19 firing-SPNs, NE induced membrane hyperpolarization (P < 0.05) and completely inhibited firings. Dxm had no effect in these neurons. The SPNs received inhibitory modulation through α2-adrenergic receptors. Some SPNs can be directly inhibited via effects independent of the α2 receptors.

Melatonin attenuates hLRRK2-induced sleep disturbances and synaptic dysfunction in a Drosophila model of Parkinson's disease.

Sleep problems are the most common non-motor symptoms in Parkinson's disease (PD), and are more difficult to treat than the motor symptoms. In the current study, the role of human leucine-rich repeat kinase 2 (hLRRK2), the most common genetic cause of PD, was investigated with regards to sleep problems, and the therapeutic potential of melatonin in hLRRK2‑associated sleep problems was explored in Drosophila. hLRRK2 was selectively expressed in the mushroom bodies (MBs) in Drosophila and sleep patterns were measured using the Drosophila Activity Monitoring System. MB expression of hLRRK2 resulted in sleep problems, presynaptic dysfunction as evidenced by reduced miniature excitatory postsynaptic current (mEPSC) and excitatory postsynaptic potential (EPSP) frequency, and excessive synaptic plasticity such as increased axon bouton density. Treatment with melatonin at 4mM significantly attenuated the sleep problems and rescued the reduction in mEPSC and EPSP frequency in the hLRRK2 transgenic flies. The present study demonstrates that MB expression of hLRRK2 in flies recapitulates the clinical features of the sleep disturbances in PD, and that melatonin attenuates hLRRK2-induced sleep disorders and synaptic dysfunction, suggesting the therapeutic potential of melatonin in PD patients carrying LRRK2 mutations.

Effects of corticotropin-releasing factor on intermediolateral cell column neurons of newborn rats

Corticotropin-releasing factor (CRF) is a neuropeptide that mediates neuroendocrine, autonomic, and behavioral processes associated with the stress response. CRF-containing fibers and receptors are found in various regions of the central nervous system including the spinal cord. Here, we report excitatory effects of CRF on sympathetic preganglionic neurons in the intermediolateral cell column (IML) of in vitro spinal cord preparations from newborn rats. We also examine the receptor subtypes that are involved in the CRF effects. Application of CRF significantly depolarized the IML neurons and increased the frequency of excitatory postsynaptic potentials (EPSPs) in the IML neurons. These effects were blocked by the CRF receptor 1 antagonist, antalarmin. Menthol, a transient receptor potential channel M8 agonist, depressed EPSPs enhanced by CRF. Our findings suggested that CRF depolarized the IML neurons via direct postsynaptic action and CRF-affected interneurons located in the spinal cord send EPSPs to IML neurons. These excitatory effects of CRF may be caused through CRF1 receptors but not CRF2 receptors.

A novel CaV2.2 channel inhibition by piracetam in peripheral and central neurons

No mechanistic actions for piracetam have been documented to support its nootropic effects. Voltage-gated calcium channels have been proposed as a promising pharmacological target of nootropic drugs. In this study, we investigated the effect of piracetam on Ca(V)2.2 channels in peripheral neurons, using patch-clamp recordings from cultured superior cervical ganglion neurons. In addition, we tested if Ca(V)2.2 channel inhibition could be related with the effects of piracetam on central neurons. We found that piracetam inhibited native Ca(V)2.2 channels in superior cervical ganglion neurons in a dose-dependent manner, with an IC(50) of 3.4 μmol/L and a Hill coefficient of 1.1. GDPβS dialysis did not prevent piracetam-induced inhibition of Ca(V)2.2 channels and G-protein-coupled receptor activation by noradrenaline did not occlude the piracetam effect. Piracetam altered the biophysical characteristics of Ca(V)2.2 channel such as facilitation ratio. In hippocampal slices, piracetam and ω-conotoxin GVIA diminished the frequency of excitatory postsynaptic potentials and action potentials. Our results provide evidence of piracetam's actions on Ca(V)2.2 channels in peripheral neurons, which might explain some of its nootropic effects in central neurons.

Thalamic model of awake alpha oscillations and implications for stimulus processing

We describe a unique conductance-based model of awake thalamic alpha and some of its implications for function. The full model includes a model for a specialized class of high-threshold thalamocortical cells (HTC cells), which burst at the alpha frequency at depolarized membrane potentials (~-56 mV). Our model generates alpha activity when the actions of either muscarinic acetylcholine receptor (mAChR) or metabotropic glutamate receptor 1 (mGluR1) agonists on thalamic reticular (RE), thalamocortical (TC), and HTC cells are mimicked. In our model of mGluR1-induced alpha, TC cells are equally likely to fire during any phase of alpha, consistent with in vitro experiments. By contrast, in our model of mAChR-induced alpha, TC cells tend to fire either at the peak or the trough of alpha, depending on conditions. Our modeling suggests that low levels of mGluR1 activation on a background of mAChR agonists may be able to initiate alpha activity that biases TC cells to fire at certain phases of alpha, offering a pathway for cortical control. If we introduce a strong stimulus by increasing the frequency of excitatory postsynaptic potentials (EPSPs) to TC cells, an increase in alpha power is needed to mimic the level of phasing of TC cells observed in vivo. This increased alpha power reduces the probability that TC cells spike near the trough of alpha. We suggest that mAChR-induced alpha may contribute to grouping TC activity into discrete perceptual units for processing, whereas mGluR1-induced alpha may serve the purpose of blocking unwanted stimuli from reaching the cortex.

284 EFFECTS OF CORTICOTROPIN-RELEASING FACTOR ON INTERMEDIOLATERAL CELL COLUMN NEURONS

Background: CRF receptors are widely distributed in the central nervous system including the spinal cord. Although CRF indirectly participates in control of the blood pressure, it is unknown whether CRF is involved in blood pressure control via direct action on central neurons. Here, we report effects of CRF on sympathetic preganglionic neurons in the intermediolateral cell column (IML) of the spinal cord. Methods: Transverse slices of Th2 segment from newborn rats (0-4 days old) were superfused by artificial cerebrospinal fluid. Membrane potentials of IML neurons were recorded by whole-cell patch-clamp. Preganglionic neurons were confirmed by antidromic action potential induced by Th2 ventral root stimulation and cell staining by Lucifer Yellow. CRF (50 nM) was bath-applied. We measured the membrane potential change and counted the number of excitatory postsynaptic potentials (EPSPs) of the IML neurons. Results: The frequency of EPSPs in the IML neurons was 3.67 ± 3.57 Hz (n = 9) in control solution. Application of CRF increased the number of EPSPs (220%) and induced membrane depolarization by 2.4 ± 1.3 mV. The increase of EPSPs number by CRF was attenuated by pretreatment of a CRF-1 antagonist, antalarmin (124%) but not by pretreatment of a CRF-2 antagonist, astressin2B (268). Conclusion: Our findings suggest that CRF affected directly IML neurons and interneurons in the spinal cord. Moreover, CRF exerted the excitatory effect on the IML neurons through CRF-1 receptors, whereas effect via CRF-2 might be rather inhibitory. Thus, CRF-related drugs are expected as one of the choices of the pharmacologic control of the blood pressure.

AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.

Interaction between hindbrain and spinal networks during the development of locomotion in zebrafish

Little is known about the role of the hindbrain during development of spinal network activity. We set out to identify the activity patterns of reticulospinal (RS) neurons of the hindbrain in fictively swimming (paralyzed) zebrafish larvae. Simultaneous recordings of RS neurons and spinal motoneurons revealed that these were coactive during spontaneous fictive swim episodes. We characterized four types of RS activity patterns during fictive swimming: (i) a spontaneous pattern of discharges resembling evoked high-frequency spiking during startle responses to touch stimuli, (ii) a rhythmic pattern of excitatory postsynaptic potentials (EPSPs) whose frequency was similar to the motoneuron EPSP frequency during swim episodes, (iii) an arrhythmic pattern consisting of tonic firing throughout swim episodes, and (iv) RS cell activity uncorrelated with motoneuron activity. Despite lesions to the rostral spinal cord that prevented ascending spinal axons from entering the hindbrain (normally starting at approximately 20 h), RS neurons continued to display the aforementioned activity patterns at day 3. However, removal of the caudal portion of the hindbrain prior to the descent of RS axons left the spinal cord network unable to generate the rhythmic oscillations normally elicited by application of N-methyl-d-aspartate (NMDA), but in approximately 40% of cases chronic incubation in NMDA maintained rhythmic activity. We conclude that there is an autonomous embryonic hindbrain network that is necessary for proper development of the spinal central pattern generator, and that the hindbrain network can partially develop independently of ascending input.

Novel axonal projection from the caudal end of the ventrolateral medulla to the intermediolateral cell column

We used an optical imaging technique to investigate whether axons of neurons in the caudal end of the ventrolateral medulla (CeVLM), as well as axons of neurons in the rostral ventrolateral medulla (RVLM), project to neurons in the intermediolateral cell column (IML) of the spinal cord. Brain stem-spinal cord preparations from neonatal normotensive Wistar-Kyoto and spontaneously hypertensive rats were stained with a voltage-sensitive dye, and responses to electrical stimulation of the IML at the Th2 level were detected as changes in fluorescence intensity with an optical imaging apparatus (MiCAM-01). The results were as follows: 1) depolarizing responses to IML stimulation during low-Ca high-Mg superfusion were detected on the ventral surface of the medulla at the level of the CeVLM, as well as at the level of the RVLM, 2) depolarizing responses were also detected on cross sections at the level of the CeVLM, and they had a latency of 24.0 +/- 5.5 (SD) ms, 3) antidromic action potentials in response to IML stimulation were demonstrated in the CeVLM neurons where optical images were detected, and 4) glutamate application to the CeVLM increased the frequency of excitatory postsynaptic potentials (EPSPs) and induced depolarization of the IML neurons. The optical imaging findings suggested a novel axonal and functional projection from neurons in the CeVLM to the IML. The increase in EPSPs of the IML neurons in response to glutamate application suggests that the CeVLM participates in the regulation of sympathetic nerve activity and blood pressure and may correspond to the caudal pressor area.

Long-term potentiation of nicotinic synaptic transmission in rat superior cervical ganglia produced by phorbol ester and tetanic stimulation

The long-term potentiation of nicotinic synaptic transmission induced by both active phorbol ester (4β-phorbol-12,13-dibutyrate, PdBu) and tetanic trains of preganglionic stimulation was studied in single neurons of the superior cervical ganglion (SCG) of the rat using intracellular recording techniques. PdBu significantly increased the mean amplitude of both the unitary evoked fast excitatory postsynaptic potentials (EPSPs) and the fast excitatory postsynaptic currents (EPSCs) to 17.0±3.3 mV (control 8.4±1.9 mV, n=5) and 2.8±0.4 nA (control 0.8±0.1 nA, n=10), respectively. There was no significant change in either the resting membrane potential, input resistance, or the threshold for the initiation of an action potential. The response to exogenously applied acetylcholine (ACh) was also not changed following exposure to PdBu. In low-calcium, high-magnesium solutions, PdBu significantly increased the quantal content of EPSPs approximately threefold from a control of 0.9±0.2 ( n=5) to 2.6±0.6 ( n=5). The quantal content of EPSCs was also increased to 1.3±0.2 (control 0.5±0.1, n=10). PdBu increased the frequency of miniature EPSPs (mEPSPs) to 196±47% ( n=6) of control, while the amplitude, rise time, rate of rise, and decay of mEPSPs were not significantly changed. Tetanic stimulation significantly increased the amplitude of the unitary synaptic EPSPs and EPSCs without significantly changing the resting membrane potential, input resistance, threshold for initiation of an action potential, or the response to exogenously applied ACh. Tetanic stimulation significantly increased quantal content of EPSPs and EPSCs threefold. The results obtained with tetanically induced LTP are similar to the results obtained with phorbol ester-induced LTP in these ganglion neurons. These results suggest that both tetanically induced and phorbol ester-induced LTP, in the rat, share similar mechanisms which involve, at least in part, activation of PKC-dependent mechanisms to increase quantal release from sympathetic preganglionic axon terminals.

Structure-activity relationships of 18 endogenous neuropeptides on the motornervous system of the nematode Ascaris suum

Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motornervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motorneurons. These motorneurons are part of the final common output pathway from the motornervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motorneurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motorneuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motorneurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motorneurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motorneurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motorneurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motorneuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motorneuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motorneurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.

Noradrenergic regulation of parvocellular neurons in the rat hypothalamic paraventricular nucleus

Noradrenergic projections to the hypothalamic paraventricular nucleus have been implicated in the secretory regulation of several anterior pituitary hormones, including adrenocorticotropin, thyroid-stimulating hormone, growth hormone and prolactin. In an attempt to elucidate the effects of norepinephrine on the central control of pituitary hormone secretion, we looked at the actions of norepinephrine on the electrical properties of putative parvocellular neurons of the paraventricular nucleus using whole-cell current-clamp recordings in hypothalamic slices. About half (51%) of the putative parvocellular neurons recorded responded to norepinephrine with either a synaptic excitation or a direct inhibition. Norepinephrine (30–300 μM) caused a marked increase in the frequency of excitatory postsynaptic potentials in about 36% of the parvocellular neurons recorded. The increase in excitatory postsynaptic potentials was blocked by prazosin (10 μM), but not by propranolol (10 μM) or timolol (20 μM), indicating that it was mediated by α 1-adrenoreceptor activation. It was also blocked by ionotropic glutamate receptor antagonists, suggesting that the excitatory postsynaptic potentials were caused by glutamate release. The increase in excitatory postsynaptic potentials was completely abolished by tetrodotoxin, indicating the spike dependence of the norepinephrine-induced glutamate release. In a separate group comprising 14% of the parvocellular neurons recorded, norepinephrine elicited a hyperpolarization (6.2±0.69 mV) that was blocked by the β-adrenoreceptor antagonists, propranolol (10 μM) and timolol (20 μM), but not by the α 1-receptor antagonist, prazosin (10 μM). This response was not blocked by tetrodotoxin (1.5–3 μM), suggesting that it was caused by a direct postsynaptic action of norepinephrine. The topographic distribution within the paraventricular nucleus of the norepinephrine-responsive and non-responsive parvocellular neurons was mapped based on intracellular biocytin labeling and neurophysin immunohistochemistry. These data indicate that one parvocellular subpopulation, consisting of about 36% of the paraventricular parvocellular neurons, receives an excitatory input from norepinephrine-sensitive local glutamatergic interneurons, while a second, separate subpopulation, representing about 14% of the parvocellular neurons in the paraventricular nucleus, responds directly to norepinephrine with a β-adrenoreceptor-mediated inhibition. This suggests that excitatory inputs to parvocellular neurons of the paraventricular nucleus are mediated mainly by an intrahypothalamic glutamatergic relay, and that only a relatively small subset of paraventricular parvocellular neurons receives direct noradrenergic inputs, which are primarily inhibitory.

Nonlinear frequency-dependent synchronization in the developing hippocampus.

Synchronous population activity is present both in normal and pathological conditions such as epilepsy. In the immature hippocampus, synchronous bursting is an electrophysiological conspicuous event. These bursts, known as giant depolarizing potentials (GDPs), are generated by the synchronized activation of interneurons and pyramidal cells via GABAA, N-methyl-D-aspartate, and AMPA receptors. Nevertheless the mechanism leading to this synchronization is still controversial. We have investigated the conditions under which synchronization arises in developing hippocampal networks. By means of simultaneous intracellular recordings, we show that GDPs result from local cooperation of active cells within an integration period prior to their onset. During this time interval, an increase in the number of excitatory postsynaptic potentials (EPSPs) takes place building up full synchronization between cells. These EPSPs are correlated with individual action potentials simultaneously occurring in neighboring cells. We have used EPSP frequency as an indicator of the neuronal activity underlying GDP generation. By comparing EPSP frequency with the occurrence of synchronized GDPs between CA3 and the fascia dentata (FD), we found that GDPs are fired in an all-or-none manner, which is characterized by a specific threshold of EPSP frequency from which synchronous GDPs emerge. In FD, the EPSP frequency-threshold for GDP onset is 17 Hz. GDPs are triggered similarly in CA3 by appropriate periodic stimulation of mossy fibers. The frequency threshold for CA3 GDP onset is 12 Hz. These findings clarify the local mechanism of synchronization underlying bursting in the developing hippocampus, indicating that GDPs are fired when background levels of EPSPs or action potentials have built up full synchronization by firing at specific frequencies (>12 Hz). Our results also demonstrate that spontaneous EPSPs and action potentials are important for the initiation of synchronous bursts in the developing hippocampus.

Excitatory amino acids and synaptic transmission in embryonic rat brainstem motoneurons in organotypic culture.

We used brainstem motoneurons recorded in organotypic slice co-cultures maintained for more than 18 days in vitro, together with multibarrel ionophoretic applications of glutamate receptor agonists and bath applications of specific blocking agents, to study the responses of rat brainstem motoneurons to glutamate receptor activation, and the contribution of these receptors to synaptic transmission. Differentiated brainstem motoneurons in vitro are depolarized by glutamate, N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) iontophoresis, and express NMDA, AMPA and also specific kainate receptors, as evidenced by (+/-)2-amino-5-phosphonovaleric acid (APV)- and (-)1-(4-aminophenyl)-3-methyl-carbamoyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzo-diazepine [GYKI 53784 (LY303070)]-resistant depolarizations. Electrical stimulations applied to the dorsal part of the explant trigger excitatory synaptic potentials with latencies distributed in three regularly spaced groups. Excitatory postsynaptic potentials (EPSPs) in the earliest group have a similar latency and time course and correspond to monosynaptic activation. EPSPs in later groups have more scattered latencies and time courses and correspond to polysynaptic activation. Monosynaptic EPSPs are insensitive to the specific NMDA blocker APV, and are completely and reversibly suppressed by the non-competitive AMPA receptor antagonist GYKI 53784 (LY303070). Detailed analysis of the spontaneous excitatory synaptic activity shows that APV decreases the frequency of spontaneous EPSPs without modifying their shape or amplitude. We conclude that excitatory synapses on brainstem motoneurons in vitro are mainly activated through AMPA receptors (AMPA-Rs). NMDA receptors (NMDA-Rs) are present in the membrane, but are located either at extrasynaptic sites or silent synapses, and are not directly involved in synaptic transmission on motoneurons. On the contrary, NMDA receptors contribute to synaptic transmission within the premotor interneuronal network.

Ca 2+-Channels Involved in Neostriatal Glutamatergic Transmission

The actions of peptidic toxins that work as Ca 2+-channel antagonists were investigated on neostriatal glutamatergic transmission. Both intracellularly recorded excitatory postsynaptic potentials (EPSPs) and extracellularly recorded population spikes (PS) evoked by afferent stimulation were evaluated in the presence of 10 μM bicuculline. Percentage of block (mean ± SEM; n = 4) for these events (EPSP and PS, respectively) was: ω-AgTxIVA (100–200 nM): 35 ± 2 and 54 ± 4%; ω-CgTxGVIA (1 μM): 37 ± 3 and 63 ± 6%; ω-CgTxMVIIC (500 nM): 40 ± 4 and 50 ± 2%; and calciseptine (500 nM): 5 ± 4 and 9 ± 6%. When given together, toxins had additive effects. The calciseptine effects were nonsignificant. The toxins were also tested on Ca 2+-dependent random synaptic responses induced by 100 μM 4-AP. Each toxin reduced the frequency of spontaneous EPSPs by more than 60% ( n = 2). The summed actions of individual toxins yields more than 100% block (superadditivity); suggesting that several terminals may possess more than one channel type. The reduction in frequency was not accompanied by a reduction in amplitude confirming that toxins’ actions were presynaptic. It is concluded that at least three different Ca 2+-channel subtypes are involved in glutamate release in neostriatal afferents: N-type, P/Q-type, and a type resistant to the toxins used. The L-type Ca 2+-channel had little, if any, participation.

Synaptic long-term depression alters recovery from, and frequency dependency of, short-term depression in Helix pomatia.

Kinetic properties of synaptic depression were analysed for excitatory postsynaptic potentials in neuron number 3 in the right pallial ganglion of Helix pomatia. It was observed that long-term depression did not only suppress the amplitudes of excitatory postsynaptic potentials but also changed the kinetics of short-term depression. Early during each experiment (before the induction of long-term depression), the degree of short-term depression was inversely related to the frequency of excitatory postsynaptic potentials (contrary to most earlier reports). Later in the experiments, after the formation of long-term depression induced by four spaced excitatory postsynaptic potential series, the inverse frequency dependency had changed into a direct proportionality between short-term depression and frequency. This change was associated with, and could be at least partially explained by, an alternation of recovery from short-term depression induced by long-term depression: early in experiments, recovery showed a transient phase of large recovery between excitatory postsynaptic potentials separated by 3 to 45 s. After the induction of long-term depression, this large and transient recovery was absent. The relationship between the amount of long-term depression formed and the number of preceding excitatory postsynaptic potentials inducing the depression was analysed, and it was observed that a significant long-term depression was present 10 min after only six excitatory postsynaptic potentials. The results point towards the implication that many previous investigations of the kinetics of short-term depression observed in experiments involving a high number of excitatory postsynaptic potentials have been performed under the influence of variable degrees of long-term depression. Since long-term depression (at least in Helix) induced substantial alterations of the kinetic properties of short-term depression, such changes may have significantly influenced the conclusions of earlier reports.

Electrographic Seizures and New Recurrent Excitatory Circuits in the Dentate Gyrus of Hippocampal Slices from Kainate-Treated Epileptic Rats

Mossy fiber sprouting has been proposed to lead to new excitatory connections between dentate granule cells, which in turn cause electrographic seizures. We tested this hypothesis in hippocampal slices from rats made epileptic-by kainate injections. The Timm's histological method revealed intense staining of the inner molecular layer in slices from all kainate-treated rats. In bicuculline (10 microM) and 6 mM [K +]o, antidromic stimulation of the granule cells evoked bursts of population spikes superimposed on long-lasting negative shifts in all slices tested from all kainate-treated rats. Long-duration (2-47 sec), seizure-like bursts with tonic and clonic components occurred spontaneously (53%) or in response to antidromic stimulation (81%). Under identical conditions, prolonged bursts were never seen in slices from controls or from kainate-injected rats 2-4 d after treatment. Glutamate microdrops applied in the granule cell layer evoked abrupt increases in the frequency of excitatory postsynaptic potentials (EPSPs) in two thirds of the cells tested. Glutamate microstimulation was effective at several sites in the granule cell layer but ineffective in the hilus. The proportion of granule cells responding to local application of glutamate by an increase in EPSPs was higher in slices with long bursts (80% with bursts of > 3 sec) than in slices with shorter bursts (33% with bursts of < 3 sec). Glutamate microstimulation did not affect EPSPs in granule cells from control preparations. These results support the hypothesis that kainate-induced mossy fiber sprouting forms new excitatory connections between granule cells and can lead to increased seizure susceptibility in the dentate gyrus.

Synaptic mediation from cutaneous mechanical nociceptors.

1. Responses of dorsal horn neurons to cutaneous mechanical stimulation were studied in an in vitro preparation of hamster spinal cord with partially intact innervation from an isolated patch of hairy skin. Stable extracellular and intracellular recordings were obtained from cells with different mechanoreceptive properties similar to those reported for other species in vivo. Analyses were made of the intracellular responses of 25 dorsal horn neurons activated selectively by mechanical stimulation to the skin patch. 2. Bath application of the broad spectrum, excitatory amino acid (EAA) receptor antagonist, kynurenic acid (1 mM) blocked excitation of 7 of 8 high-threshold mechanoreceptive units by either cutaneous nerve volleys or mechanical stimulation of the skin. This concentration of kynurenic acid suppressed peripherally evoked responses in 8 of 14 neurons responsive to innocuous mechanical stimuli. 3. High-threshold mechanoreceptive neurons of the superficial dorsal horn exhibited one of three distinctive patterns of postsynaptic potentials in response to electrical stimulation of cutaneous afferent fibers: 1) a simple fast excitatory postsynaptic potential (EPSP), 2) a fast EPSP with a prolonged decay phase lasting between 100 and 1,000 ms, and 3) a multiphasic response dissociable on the basis of stimulus strength consisting of a fast EPSP followed by a hyperpolarizing inhibitory postsynaptic potential (IPSP) (duration 80-100 ms). Gentle mechanical stimuli initiated inhibition from areas adjacent to the high-threshold mechanically excitatory field; this suggests that membrane hyperpolarization in these neurons was evoked by input from low-threshold mechanoreceptors. 4. Bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), a competitive EAA antagonist selective for non-N-methyl-D-aspartate (non-NMDA) receptor subtypes, substantially or completely (56-100%) suppressed EPSPs evoked from cutaneous afferent fibers in high-threshold mechanoreceptive neurons. CNQX also decreased the membrane depolarization, the frequency of EPSPs, and the frequency of action potentials evoked by mechanical stimulation of the receptive field. 5. CNQX (10 microM) or kynurenic acid (1 mM) had considerably weaker effects on IPSPs than on EPSPs evoked from the periphery in superficial dorsal horn neurons. IPSP amplitudes were unchanged by these agents in some neurons and decreased by only 20-25% in others. 6. We conclude that L-glutamate acting on non-NMDA receptors mediates fast synaptic excitation of superficial dorsal horn neurons from peripheral mechanical nociceptors with myelinated fibers. Furthermore, the observations imply either an agent other than L-glutamate or one acting at different membrane receptors is a synaptic mediator for other peripheral afferent units including some activated by innocuous mechanical stimuli.

Centrally Generated Rhythmic Activity and Modulatory Function of the Oviductal Dorsal Unpaired Median (Dum) Neurones in Two Orthopteran Species (Calliptamus SP. and Decticus Albifrons)

ABSTRACT The rhythmic firing pattern of the putatively octopaminergic dorsal unpaired median (DUM) neurones supplying the oviductal system of female orthopterans, Calliptamus sp. and Decticus albifrons, was examined. Our data provide evidence that the oviductal DUM neurones in the seventh abdominal ganglion modulate the oviductal motor pattern, both peripherally and centrally, during the inhibition of egg-laying behaviour. In a minimally dissected animal, rhythmic activation of the oviductal DUM and motor neurones can be readily elicited by isolation of the seventh abdominal ganglion from the anterior part of the nerve cord. The bursting activity of the DUM neurones is temporally correlated with the oviductal motor rhythm. Both populations of oviductal neurones retain their rhythmic firing pattern after total isolation of the genital ganglia, indicating the presence of an oviductal central pattern generator. The effects of stimulation of oviductal DUM neurones on the oviductal motor activity were monitored by recording intracellularly from oviductal muscle fibres and extracellularly from motor axons. These effects consist of a reduction in the amplitude and frequency of excitatory postsynaptic potentials (EPSPs) in the muscle fibre and in the firing rate in oviductal motor neurones. We suggest that the change in EPSP amplitude results from peripheral release of octopamine by DUM neurones. The decreased firing rate of motor neurones, however, appears to be a central effect, possibly caused by central release of octopamine by DUM neurones.