Frequency Of Spontaneous Synaptic Events Research Articles

Postsynaptic and Presynaptic NMDARs Have Distinct Roles in Visual Circuit Development

To study contributions of N-methyl-D-aspartate receptors (NMDARs) in presynaptic and postsynaptic neurons of the developing visual system, we microinject antisense Morpholino oligonucleotide (MO) against GluN1 into one cell of two-cell-stage Xenopus laevis embryos. The resulting bilateral segregation of MO induces postsynaptic NMDAR (postNMDAR) knockdown in tectal neurons on one side and presynaptic NMDAR (preNMDAR) knockdown in ganglion cells projecting to the other side. PostNMDAR knockdown reduces evoked NMDAR- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated retinotectal currents. Although the frequency of spontaneous synaptic events is increased, the probability of evoked release is reduced. PreNMDAR knockdown results in larger evoked and unitary synaptic responses. Structurally, postNMDAR and preNMDAR knockdown produce complementary effects. Axonal arbor complexity is reduced by preNMDAR-MO and increased by postNMDAR-MO, whereas tectal dendritic arbors exhibit the inverse. The current study illustrates distinct roles for pre- and postNMDARs in circuit development and reveals extensive transsynaptic regulation of form and function.

Activity-dependent dendritic elaboration requires Pten

Pten, a gene associated with autism spectrum disorder, is an upstream regulator of receptor tyrosine kinase intracellular signaling pathways that mediate extracellular cues to inform cellular development and activity-dependent plasticity. We therefore hypothesized that Pten loss would interfere with activity dependent dendritic growth. We investigated the effects of this interaction on the maturation of retrovirally labeled postnatally generated wild-type and Pten knockout granule neurons in male and female mouse dentate gyrus while using chemogenetics to manipulate the activity of the perforant path afferents. We find that enhancing network activity accelerates the dendritic outgrowth of wild-type, but not Pten knockout, neurons. This was specific to immature neurons during an early developmental window. We also examined synaptic connectivity and physiological measures of neuron maturation. The input resistance, membrane capacitance, dendritic spine morphology, and frequency of spontaneous synaptic events were not differentially altered by activity in wild-type versus Pten knockout neurons. Therefore, Pten and its downstream signaling pathways regulate the activity-dependent sculpting of the dendritic arbor during neuronal maturation.

Dimethylethanolamine Decreases Epileptiform Activity in Acute Human Hippocampal Slices in vitro.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy with about 30% of patients developing pharmacoresistance. These patients continue to suffer from seizures despite polytherapy with antiepileptic drugs (AEDs) and have an increased risk for premature death, thus requiring further efforts for the development of new antiepileptic therapies. The molecule dimethylethanolamine (DMEA) has been tested as a potential treatment in various neurological diseases, albeit the functional mechanism of action was never fully understood. In this study, we investigated the effects of DMEA on neuronal activity in single-cell recordings of primary neuronal cultures. DMEA decreased the frequency of spontaneous synaptic events in a concentration-dependent manner with no apparent effect on resting membrane potential (RMP) or action potential (AP) threshold. We further tested whether DMEA can exert antiepileptic effects in human brain tissue ex vivo. We analyzed the effect of DMEA on epileptiform activity in the CA1 region of the resected hippocampus of TLE patients in vitro by recording extracellular field potentials in the pyramidal cell layer. Epileptiform burst activity in resected hippocampal tissue from TLE patients remained stable over several hours and was pharmacologically suppressed by lacosamide, demonstrating the applicability of our platform to test antiepileptic efficacy. Similar to lacosamide, DMEA also suppressed epileptiform activity in the majority of samples, albeit with variable interindividual effects. In conclusion, DMEA might present a new approach for treatment in pharmacoresistant TLE and further studies will be required to identify its exact mechanism of action and the involved molecular targets.

Copper enhances cellular and network excitabilities, and improves temporal processing in the rat hippocampus.

Copper, an ion with many important metabolic functions, has also been proposed to have a role as modulator on neuronal function, mostly based on its effects on voltage- and neurotransmitter-gated conductance as well as on neurological symptoms of patients with altered copper homeostasis. Nevertheless, the mechanisms by which copper exerts its neuromodulatory effects have not been clearly established in a functional neuronal network. Using rat hippocampus slices as a neuronal network model, the effects of copper in the range of 10-100 nm were tested on the intrinsic, synaptic and network properties of the CA1 region. Most of the previously described effects of this cation were in the micromolar range of copper concentrations. The current results indicate that copper is a multifaceted neuromodulator, having effects that may be grouped into two categories: (i) activity enhancement, by modulating synaptic communication and action potential (AP) conductances; and (ii) temporal processing and correlation extraction, by improving reliability and depressing inhibition. Specifically it was found that copper hyperpolarizes AP firing threshold, enhances neuronal and network excitability, modifies CA3-CA1 pathway gain, enhances the frequency of spontaneous synaptic events, decreases inhibitory network activity, and improves AP timing reliability. Moreover, copper chelation by bathocuproine decreases spontaneous network spiking activity. These results allow the proposal that copper affects the network activity from cellular to circuit levels on a moment-by-moment basis, and should be considered a crucial functional component of hippocampal neuronal circuitry.

Mechanisms underlying rule learning-induced enhancement of excitatory and inhibitory synaptic transmission

Training rats to perform rapidly and efficiently in an olfactory discrimination task results in robust enhancement of excitatory and inhibitory synaptic connectivity in the rat piriform cortex, which is maintained for days after training. To explore the mechanisms by which such synaptic enhancement occurs, we recorded spontaneous miniature excitatory and inhibitory synaptic events in identified piriform cortex neurons from odor-trained, pseudo-trained, and naive rats. We show that olfactory discrimination learning induces profound enhancement in the averaged amplitude of AMPA receptor-mediated miniature synaptic events in piriform cortex pyramidal neurons. Such physiological modifications are apparent at least 4 days after learning completion and outlast learning-induced modifications in the number of spines on these neurons. Also, the averaged amplitude of GABA(A) receptor-mediated miniature inhibitory synaptic events was significantly enhanced following odor discrimination training. For both excitatory and inhibitory transmission, an increase in miniature postsynaptic current amplitude was evident in most of the recorded neurons; however, some neurons showed an exceptionally great increase in the amplitude of miniature events. For both excitatory and inhibitory transmission, the frequency of spontaneous synaptic events was not modified after learning. These results suggest that olfactory discrimination learning-induced enhancement of synaptic transmission in cortical neurons is mediated by postsynaptic modulation of AMPA receptor-dependent currents and balanced by long-lasting modulation of postsynaptic GABA(A) receptor-mediated currents.

Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission

Antibodies against ganglionic acetylcholine receptors (AChR) are implicated as the cause of autoimmune autonomic ganglionopathy (AAG). To characterize ganglionic neurotransmission in an animal model of AAG, evoked and spontaneous excitatory post-synaptic potentials (EPSP) were recorded from neurons in isolated mouse superior cervical ganglia (SCG). In vitro exposure of ganglia to IgG from AAG patients progressively inhibited synaptic transmission. After passive transfer of antibody to mice, evoked EPSP amplitude decreased, and some neurons showed no synaptic responses. EPSP amplitude recovered by day 7 despite persistence of ganglionic AChR antibody in the mouse serum. There was a more persistent (at least 14-day) reduction in miniature EPSP amplitude consistent with antibody-mediated reduction in post-synaptic AChR. Although the quantal size was reduced, a progressive increase in the frequency of spontaneous synaptic events occurred, suggesting a compensatory increase in presynaptic efficacy. The quantal size returned to baseline by 21 days while the frequency remained increased for at least four weeks. Ganglionic AChR antibodies cause an impairment of autonomic ganglionic synaptic transmission. Homeostatic plasticity in autonomic neurotransmission could help explain the spontaneous clinical recovery seen in some AAG patients and may also play an important role in regulating normal autonomic reflexes.

Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.

D2 dopamine modulation of corticoaccumbens synaptic responses changes during adolescence

Dopaminergic afferents from the ventral tegmental area (VTA) modulate information processing in the nucleus accumbens (NA), a brain region critical for motivation and reward mechanisms. In NA medium spiny neurons (MSNs) from young rats, D(2) agonists have been shown to decrease the amplitude of corticoaccumbens synaptic responses. As several dopamine-related functions change during adolescence, we assessed the D(2) modulation of cortical inputs with whole-cell recordings in slices obtained from adult and preadolescent rats. The D(2) agonist quinpirole (5 microM) decreased synaptic response of NA MSNs to electrical cortical stimulation in slices from preadolescent rats. In slices from adult rats, however, quinpirole increased both the amplitude of evoked synaptic responses and the frequency of spontaneous synaptic events. These effects were blocked by the GABA-A antagonist picrotoxin (50 microM), revealing a D(2)-mediated decrease These results suggest that D(2) receptors modulate NA neurons differently in young and adult rats, due to the emergence of a D(2)-facilitated GABA component in corticoaccumbens responses during adolescence.

Reactive Oxygen Species Mediate the Potentiating Effects of ATP on GABAergic Synaptic Transmission in the Immature Hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.

Sex differences in estrogenic regulation of neuronal activity in neonatal cultures of ventromedial nucleus of the hypothalamus

Estrogenic effects have been implicated in sexual differentiation of brain and behavior, in part by affecting neuronal activity in the ventromedial nucleus of the hypothalamus (VMN). We report here a remarkable sex difference in estrogenic regulation of neuronal activity in male vs. female neural networks. Spontaneous synaptic currents originating from a population of neurons were recorded in primary VMN cultures using the whole-cell patch-clamp technique. Treatment with 17beta-estradiol (E2, 10 nM) for 24 h induced opposite effects in the two sexes: the frequency of spontaneous synaptic events decreased significantly in neurons derived from males but increased in those from females. Interestingly, the 24-hour E2 effect was partially reversed by an acute application (5 min) of a second dose of E2 (10 nM), suggesting an interaction between extended (24-hr) and acute (5-min) effects of E2 in VMN neurons. To understand the underlying mechanism of this sexually dimorphic action of E2, we analyzed the E2 effect on GABAergic neurotransmission by recording miniature inhibitory postsynaptic currents. After 24-hour E2 treatment, both the amplitude and frequency of miniature inhibitory postsynaptic currents increased in neurons derived from males but decreased in those from females. These results suggest that E2-induced changes in GABAergic inhibition could at least partially explain E2 effects on neuronal activity. We conclude that E2 may have sexually dimorphic effects on the synaptic output of VMN neurons by modulating GABAergic neurotransmission.

Transplanted CNS stem cells form functional synapses in vivo.

An understanding of developmental mechanisms and new cell therapies can be achieved by transplantation into the nervous system. Multipotential stem cells have been isolated from the foetal and adult central nervous system (CNS). Immortalized and primary precursor cells integrate into the developing brain generating both neurons and glia as defined by immunological and morphological criteria. Here we show for the first time that in vitro-expanded CNS precursors, upon transplantation into the brains of rats, form electrically active and functionally connected neurons. These neurons exhibit spontaneous and evoked postsynaptic events and respond to focal glutamate application. Donor cells were grafted into the foetal hippocampus, and the amplitude and frequency of spontaneous synaptic events were monitored in the grafted cells in area CA1 for the first month of postnatal life. The formation of synapses onto grafted neurons indicates that grafted CNS stem cells can be used to study synaptic development in vivo and has important implications for clinical cell replacement therapies.

Losigamone decreases spontaneous synaptic activity in cultured hippocampal neurons

Losigamone is a new antiepileptic drug with an unknown mechanism of action. Here we report on the effects of losigamone on the synaptic activity in a network of cultured rat hippocampal neurons. Losigamone dose dependently reduced the frequency of spontaneous synaptic events without affecting the mean current amplitude. The drug affected equally the isolated inhibitory as well as excitatory postsynaptic currents. Miniature postsynaptic currents were not altered by losigamone, suggesting that the mechanism of action depends on functional Na + channels. Consistent with these findings, the drug decreased the frequency of spontaneous action potentials and suppressed repetitive firing of neurons. Thus, losigamone generally depresses synaptic activity in a neuronal network without selectively modulating a specific postsynaptic receptor type. We conclude that losigamone acts via a presynaptic mechanism reducing neuronal excitability.

Desensitization of AMPA Receptors Limits the Amplitude of EPSPs and the Excitability of Motoneurons of the Rat Isolated Spinal Cord

Intracellular recording was used to study the effect of cyclothiazide, a selective blocker of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor desensitization, on lumbar motoneurons of the rat isolated spinal cord. Cyclothiazide (25 microM) enhanced the responses to AMPA in a tetrodotoxin-insensitive fashion, without affecting those produced by N-methyl-D-aspartate or gamma-aminobutyric acid. Excitatory postsynaptic potentials (EPSPs) evoked by dorsal root stimulation were strongly potentiated in amplitude while paired-pulse depression (produced by applying pairs of pulses at 2 s interval) of the EPSP was decreased. In the presence of cyclothiazide the frequency of spontaneous synaptic events was greatly increased and network-driven bursting activity developed with eventual loss of electrical excitability. The present results suggest that pharmacological block of AMPA receptor desensitization led to strong excitation of motoneurons and indicate a physiological role of desensitization in protecting these nerve cells from overactivity.

Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses. 5. These results suggest that endogenous release of ATP at developing neuromuscular synapses is responsible for the maintenance of high levels of spontaneous ACh release, which is known to play a crucial role in regulating postsynaptic differentiation.

Properties of synaptic transmission at the neuromuscular junction of the squid, Loligo opalescens

1. 1. Spontaneous and evoked synaptic activity were recorded from the muscles of squid fin and mantle. These spontaneous synaptic potentials were large (up to 30 mV) and pleomorphic. Their amplitudes were not normally distributed, nor did they appear to be clustered in integral multiples of some “unit” event size. 2. 2. Electrical stimulation of the nerve resulted in muscle twitches when the bath calcium concentration was a third normal or higher. The frequency of spontaneous synaptic events was unaffected by low calcium. 3. 3. The large size of spontaneous events may mean that the synchronized release of only a few such “quanta” are sufficient to cause muscle action potentials and contraction. 4. 4. The shapes of spontaneous events correlated poorly with their amplitudes, which is consistent with release from multiple synaptic sites with distinct properties.