sIPSC Frequency Research Articles

Neuronal excitability and spontaneous synaptic transmission in the entorhinal cortex of BDNF heterozygous mice

Brain Derived Neurotropic Factor (BDNF) is a neutrophic factor that is required for the normal neuronal development and function. BDNF is involved in regulation of synapses as well as neuronal excitability. Entorhinal Cortex (EC) is a key brain area involved in many physiological and pathological processes. In this study we investigated the effects of chronically reduced BDNF levels on layer 3 pyramidal neurons of EC. We aimed to assess the effects of reduced levels of BDNF on firing properties, spontaneous synaptic currents and excitation/inhibition balance from acute brain slices. Patch clamp recordings were obtained from pyramidal neurons of Entorhinal Cortex Layer 3. Findings of BDNF heterozygous (BDNF (+/−)) mice compared to their wild-type littermates at the age of 23–28 days. Action potential threshold was shifted (p = 0,002) to depolarized potentials and spike frequency was smaller in response to somatic current injection steps in BDNF (+/−) mice. Spontaneous synaptic currents were also affected. sEPSC amplitude (p = 0,009), sIPSC frequency (p = 0,001) and sIPSC amplitudes (p = 0,023) were reduced in BDNF (+/−). Decay times of sIPSCs were longer in BDNF (+/−) (p = 0,014). Calculated balance of excitatory/inhibitory balance was shifted in the favor of excitation in BDNF (+/−) mice (p = 0,01). These findings suggest that reductions in concentrations of BDNF results in altered status of excitability and excitation/inhibition imbalance. However, these differences observed in BDNF (+/−) seem to have opposing effects on neuronal activity.

A1609 Microglia regulate sympathetic PVN neuronal activity in both resting and hypertensive states

Objectives: Hypertension is associated with chronic and low-grade neuroinflammation. Our previous studies show that microglia, the resident immune cells in the CNS, are activated in hypertension. Strikingly, loss of naïve or proinflammatory microglia oppositely regulates blood pressure in response to hypertension induction. In this study we are investigating the mechanism by which microglia influence RVLM-projecting PVN neuronal activity in resting and hypertensive state, respectively. Methods: Adult male transgenic CD11-DTR mice were randomly assigned into 4 groups: control, diphtheria toxin (DT; to deplete microglia), L-NAME (hypertensive induction), and L-NAME+DT. Sympathetic neurons in the hypothalamic PVN are retrograde labeled by microinjection of fluorescent microsphere into unilateral RVLM in the brain stem. The electronic profiles of RVLM-PVN neurons are recording in voltage clamp mode. Results: Depletion of naïve or proinflammatory microglia does not alter resting membrane potential or membrane resistance. However, loss of microglia significantly inhibits outward K current. The frequency and amplitude of spontaneous EPSC (sEPSC) and sIPSC are altered by removal of microglia. The overall power of naïve microglial depletion decreases excitatory input to RVLM-PVN neurons. Depletion of proinflammatory microglia in hypertensive mice decreases both excitatory and inhibitory inputs. Conclusion: Our results show that microglia are highly involved in shaping RVLM-PVN neuronal activity in both resting and hypertensive states.

The 5-HT7 receptor antagonist SB 269970 ameliorates corticosterone-induced alterations in 5-HT7 receptor-mediated modulation of GABAergic transmission in the rat dorsal raphe nucleus

RationaleChronic stress and corticosterone have been shown to affect serotonin (5-HT) neurotransmission; however, the influence of stress on the activity of the dorsal raphe nucleus (DRN), the main source of 5-HT in the forebrain, is not well understood. In particular, it is unknown if and how stress modifies DRN 5-HT7 receptors, which are involved in the modulation of the firing of local inhibitory interneurons responsible for regulating the activity of DRN projection cells.ObjectivesOur study aimed to investigate the effect of repeated corticosterone injections on the modulation of the inhibitory transmission within the DRN by 5-HT7 receptors and whether it could be reversed by treatment with a 5-HT7 receptor antagonist.MethodsMale Wistar rats received corticosterone injections repeated twice daily for 14 days. Spontaneous inhibitory postsynaptic currents (sIPSCs) were then recorded from DRN projection cells in ex vivo slice preparations obtained 24 h after the last injection.ResultsRepeated corticosterone administration resulted in decreased frequency, but not amplitude, of sIPSCs in DRN projection cells. There were no changes in the excitability of these cells; however, corticosterone treatment suppressed the 5-HT7 receptor-mediated increase in sIPSC frequency. Administration of the 5-HT7 receptor antagonist SB 269970 for 7 days beginning on the eighth day of corticosterone treatment reversed the detrimental effects of corticosterone on 5-HT7 receptor reactivity and GABAergic transmission in the DRN.ConclusionsElevated corticosterone level reduces DRN 5HT7 receptor reactivity and decreases GABAergic transmission within the DRN, which can be reversed by the 5-HT7 receptor antagonist SB 269970.

Postnatal development of inhibitory synaptic transmission in the anterior piriform cortex

The morphological and functional development of inhibitory circuit in the anterior piriform cortex (aPC) during the first three postnatal weeks may be crucial for the development of odor preference learning in infant rodents. As first step toward testing this hypothesis, we examined the normal development of GABAergic synaptic transmission in the aPC of rat pups during the postnatal days (P) 5–8 and 14-17. Whole cell patch-clamp recordings of layer 2/3 (L2/3) aPC pyramidal cells revealed a significant increase in spontaneous (sIPSC) and miniature (mIPSC) inhibitory postsynaptic current frequencies and a decrease in mIPSC rise and decay-time constant at P14-P17. Moreover, as the development of neocortical inhibitory circuit can be driven by sensory experience, we recorded sIPSC and mIPSC onto L2/3 aPC pyramidal cells from unilateral naris-occluded animals. Early partial olfactory deprivation caused by naris occlusion do not affected the course of age-dependent increase IPSC frequency onto L2/3 aPC pyramidal cell. However, this age-dependent increase of sIPSC and mIPSC frequencies were lower on aPC pyramidal cells ipsilateral to the occlusion side. In addition, the age-dependent increase in sIPSC frequency and amplitude were more pronounced on aPC pyramidal cells contralateral to the occlusion. While mIPSC kinetics were not affected by age or olfactory deprivation, at P5-P8, the sIPSC decay-time constant on aPC pyramidal cells of both hemispheres of naris-occluded animals were significantly higher when compared to sham. These results demonstrated that the GABAergic synaptic transmission on the aPC changed during postnatal development by increasing inhibitory inputs on L2/3 pyramidal cells, with increment in frequency of both sIPSC and mIPSC and faster kinetics of mIPSC. Our data suggested that the maturation of GABAergic synaptic transmission was little affected by early partial olfactory deprivation. These results could contribute to unravel the mechanisms underlying the development of odor processing and olfactory preference learning.

The role of the GLP-1/GLP-1R signaling pathway in regulating seizure susceptibility in rats

ObjectiveThis study aimed to investigate the role of glucagon-like peptide-1 (GLP-1)/GLP-1 receptor(R) signaling in the regulation of seizure susceptibility and to explore the potential mechanism in rats. MethodsHyperthermia-induced seizures in SD rats were generated using hot bath methods, and seizure severity was measured according to Racine scores and electroencephalogram (EEG). Protein levels of GLP-1 and GLP-1R in the brain tissues of rats were evaluated through ELISA, western blot analysis, and immunohistochemistry to explore the possible roles of each in FS. Neuronal excitability, spontaneous inhibitory postsynaptic currents (sIPSCs) and transient receptor potential cation channel subfamily V member 1(TRPV1) currents were tested using the patch-clamp method in cultured hippocampal neurons. ResultSignificant decreases in the levels of GLP-1 and GLP-1R were observed in the hippocampi of rats compared to those in the control group. Furthermore, treatment with the GLP-1R pharmacological inhibitor exendin9-39 increased hyperthermia- induced seizure severity in rats and promoted neuronal firing activity in cultured neurons. Importantly, exendin9-39 and GLP-1R knockdown decreased the amplitude and frequency of sIPSCs in cultured neurons. In addition, GLP-1R knockdown elevated downstream TRPV1 expression and promoted capsaicin-induced TRPV1 function, which may regulate inhibitory neurotransmission to affect seizure susceptibility. ConclusionThe present study suggests that inhibition of GLP-1R signaling promotes seizure activity, which plays a key role in the pathogenesis of FS.

Orexin A presynaptically decreases inhibitory synaptic transmission in rat locus coeruleus neurons

Locus coeruleus nucleus (LC) is a major noradrenergic nucleus in the brain. It receives dense orexinergic projections from lateral hypothalamus. Whilst it is known that orexin A increases firing rate of LC neurons, its effect on spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs, respectively) has not been yet identified. In this research, we investigated the effect of orexin A on eIPSCs and sIPSCs in LC neurons. Whole-cell recordings revealed that orexin A suppresses eIPSCs amplitude in which this effect was blocked by an orexin type-1 receptors antagonist (SB-334867) and cannabinoid type-1 (CB1) receptors antagonist (AM251). Moreover, exposure of neurons to BAPTA (Ca2+ chelator) and U73122 (phospholipase C inhibitor) prevented orexin A-induced eIPSCs depression. On the other hand, orexin A increased pair pulse ratio and sIPSCs frequency but had no effect on sIPSCs amplitude. Our results revealed that eIPSCs suppression in the LC is mediated by CB1 receptor through a presynaptic mechanism.

Developmental nicotine exposure alters glycinergic neurotransmission to hypoglossal motoneurons in neonatal rats.

We tested the hypothesis that nicotine exposure in utero and after birth [developmental nicotine exposure (DNE)] disrupts development of glycinergic synaptic transmission to hypoglossal motoneurons (XIIMNs). Glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brain stem slices from 1- to 5-day-old rat pups of either sex, under baseline conditions and following stimulation of nicotinic acetylcholine (ACh) receptors with nicotine (i.e., an acute nicotine challenge). Under baseline conditions, there were no significant effects of DNE on the amplitude or frequency of either sIPSCs or mIPSCs. In addition, DNE did not alter the magnitude of the whole cell current evoked by bath application of glycine, consistent with an absence of change in postsynaptic glycine-mediated conductance. An acute nicotine challenge (bath application of 0.5 μM nicotine) increased sIPSC frequency in the DNE cells, but not control cells. In contrast, nicotine challenge did not change mIPSC frequency in either control or DNE cells. In addition, there were no significant changes in the amplitude of either sIPSCs or mIPSCs in response to nicotine challenge. The increased frequency of sIPSCs in response to an acute nicotine challenge in DNE cells reflects an enhancement of action potential-mediated input from glycinergic interneurons to hypoglossal motoneurons. This could lead to more intense inhibition of hypoglossal motoneurons in response to exogenous nicotine or endogenous ACh. The former would occur with smoking or e-cigarette use while the latter occurs with changes in sleep state and with hypercapnia. NEW & NOTEWORTHY Here we show that perinatal nicotine exposure does not impact baseline glycinergic neurotransmission to hypoglossal motoneurons but enhances glycinergic inputs to hypoglossal motoneurons in response to activation of nicotinic acetylcholine (ACh) receptors with acute nicotine. Given that ACh is the endogenous ligand for nicotinic ACh receptors, the latter reveals a potential mechanism whereby perinatal nicotine exposure alters motor function under conditions where ACh release increases, such as the transition from non-rapid-eye movement to rapid-eye movement sleep, and during hypercapnia.

Corticosterone-Induced Changes in Inhibitory Neurotransmission in Hippocampal Field CA1 Synapses Depending on the Activity of Inhibitory Synapses Expressing Cannabinoid Receptors

Objectives. To study the effects of corticosterone (100 nM) on spontaneous inhibitory postsynaptic currents(sIPSC) in pyramidal neurons in hippocampal field CA1 and to identify the role of inhibitory synapses expressing cannabinoid receptors in the changes induced by corticosterone. Materials and methods. Experiments were performed on living slices of rat ventral hippocampus using a patch clamp method in the whole cell configuration. Results and conclusions. Addition of corticosterone led to a significant increase in the frequency of sIPSC in the first 10 min, after which the magnitude of this parameter returned to baseline. In addition, the use of corticosterone led to a significant decrease in sIPSC amplitude 40 min after the start of exposure. Activation of cannabinoid CB1 receptors had weak effects on the properties of sIPSC, though addition of corticosterone did not induce the rapid rise in sIPSC frequency occurring at 10 min in controls or the drop in sIPSC amplitude (at 40 min in controls). These data indicate that both the rapid and slow effects of corticosterone developing in the ventral hippocampus are associated with the activity of CB1-expressing inhibitory synapses.

Sex Differences in Oxytocin Effects on CeA GABAergic Signaling in Alcohol‐Dependent and Naive Rats

Alcohol use disorders (AUDs) are significant public health concerns, characterized by compulsive seeking and consumption of alcohol. AUDs have been linked to disruption of normal functioning of brain stress systems, as the transition to dependence recruits these systems leading to the negative affective states seen in withdrawal; the relief of which drives further drinking. The central nucleus of the amygdala (CeA) functions as a neuropeptidergic hub of stress and anxiety processing, and GABAergic signaling within the CeA is involved in the regulation of alcohol consumption. Oxytocin is a stress‐related neuropeptide that has previously been shown to alter GABAergic signaling within the CeA of rodents. Oxytocin also affects alcohol addiction, dependence, and withdrawal, as administration of oxytocin decreases drinking and blocks alcohol withdrawal symptoms in humans and rodents. However, characterization of the electrophysiological effects of oxytocin and alcohol on GABAergic signaling in the CeA have not yet been reported. In this study, we used whole cell patch clamp electrophysiological recordings to examine the effects of oxytocin and alcohol on CeA GABAergic signaling in both alcohol naïve and chronic intermittent alcohol vapor treated (alcohol dependent) Sprague‐Dawley male and female rats. We also used immunohistochemical staining to identify alcohol dependence induced changes in oxytocin expression in male and female rats. Oxytocin decreased sIPSC amplitudes in both naïve and dependent male animals, but blocked alcohol‐induced enhancement of sIPSC frequency in dependents only, indicating both pre‐ and post‐synaptic effects of oxytocin on GABAergic transmission. Pretreatment with an oxytocin antagonist blocked the effects of oxytocin and restored the alcohol enhancement in sIPSC frequency. In female naïve rats, oxytocin has no effects on sIPSCs alone, and does not block the alcohol enhancement of sIPSC frequency, but in alcohol dependent females, oxytocin increases sIPSC frequency alone and additional alcohol increases sIPSC frequency even further. These results provide important insight into the role of oxytocin in CeA neuronal functioning and the sexually dimorphic changes in this system that contributes to the transition to alcohol dependence. This research highlights the potential role of oxytocin in the development of novel targeted therapeutics for the effective treatment of AUDs.Support or Funding InformationNIH/NIAAA F32: AA025262, T32: AA007456, R01: AA017447, AA006420, AA015566This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction of Norepinephrine and Glucocorticoids Modulate Inhibition of Principle Cells of Layer II Medial Entorhinal Cortex in Male Mice.

Spatial memory processing requires functional interaction between the hippocampus and the medial entorhinal cortex (MEC). The grid cells of the MEC are most abundant in layer II and rely on a complex network of local inhibitory interneurons to generate spatial firing properties. Stress can cause spatial memory deficits in males, but the specific underlying mechanisms affecting the known memory pathways remain unclear. Stress activates both the autonomic nervous system and the hypothalamic-pituitary-adrenal axis to release norepinephrine (NE) and glucocorticoids, respectively. Given that adrenergic receptor (AR) and glucocorticoid receptor (GR) expression is abundant in the MEC, both glucocorticoids and NE released in response to stress may have rapid effects on MEC-LII networks. We used whole-cell patch clamp electrophysiology in MEC slice preparations from male mice to test the effects of NE and glucocorticoids on inhibitory synaptic inputs of MEC-LII principal cells. Application of NE (100 μM) increased the frequency and amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) in approximately 75% of the principal cells tested. Unlike NE, bath application of dexamethasone (Dex, 1 μM), a synthetic glucocorticoid, or corticosterone (1 μM) the glucocorticoid in rodents, rapidly decreased the frequency of sIPSCs, but not miniature (mIPSCs) in MEC-LII principal cells. Interestingly, pre-treatment with Dex prior to NE application led to an NE-induced increase in sIPSC frequency in all cells tested. This effect was mediated by the α1-AR, as application of an α1-AR agonist, phenylephrine (PHE) yielded the same results, suggesting that a subset of cells in MEC-LII are unresponsive to α1-AR activation without prior activation of GR. We conclude that activation of GRs primes a subset of principal cells that were previously insensitive to NE to become responsive to α1-AR activation in a transcription-independent manner. These findings demonstrate the ability of stress hormones to markedly alter inhibitory signaling within MEC-LII circuits and suggest the intriguing possibility of modulation of network processing upstream of the hippocampus.

Presynaptic Regulation of Tonic Inhibition by Neuromodulatory Transmitters in the Basal Amygdala.

Tonic inhibition mediated by ambient levels of GABA that activate extrasynaptic GABAA receptors emerges as an essential factor that tunes neuronal network excitability in vitro and shapes behavioral responses in vivo. To address the role of neuromodulatory transmitter systems on this type of inhibition, we employed patch clamp recordings in mouse amygdala slice preparations. Our results show that the current amplitude of tonic inhibition (Itonic) in projection neurons of the basal amygdala (BA) is increased by preincubation with the neurosteroid THDOC, while the benzodiazepine diazepam is ineffective. This suggests involvement of THDOC sensitive δ subunit containing GABAA receptors in mediating tonic inhibition. Moreover, we provide evidence that the neuromodulatory transmitters NE, 5HT, and ACh strongly enhance spontaneous IPSCs as well as Itonic in the BA. As the increase in frequency, amplitude, and charge of sIPSCs by these neuromodulatory transmitters strongly correlated with the amplitude of Itonic, we conclude that spill-over of synaptic GABA leads to activation of Itonic and thereby to dampening of amygdala excitability. Since local injection of THDOC, as a positive modulator of tonic inhibition, into the BA interfered with the expression of contextual fear memory, our results point to a prominent role of Itonic in fear learning.

Developmental plasticity of GABAergic neurotransmission to brainstem motoneurons.

Critical homeostatic behaviours such as suckling, swallowing and breathing depend on the precise control of tongue muscle activity. Perinatal nicotine exposure has multiple effects on baseline inhibitory GABAergic neurotransmission to hypoglossal motoneurons (XIIMNs), consistent with homeostatic compensations directed at maintaining normal motoneuron output. Developmental nicotine exposure (DNE) alters how GABAergic neurotransmission is modulated by acute activation of nicotinic acetylcholine receptors, which may provide insight into mechanisms by which nicotine exposure alters motor function under conditions that result in increased release of GABA, such as hypoxia, or endogenous acetylcholine, as occurs in the transition from NREM to REM sleep, or in response to exogenous nicotine. Nicotinic acetylcholine receptor (nAChR) signalling regulates neuronal differentiation and synaptogenesis. Here we test the hypothesis that developmental nicotine exposure (DNE) disrupts the development of GABAergic synaptic transmission to hypoglossal motoneurons (XIIMNs). GABAergic spontaneous and miniature inhibitory postsynaptic currents (sIPSCs/mIPSCs) were recorded from XIIMNs in brainstem slices from control and DNE rat pups of either sex, 1-5days old, at baseline and following acute stimulation of nAChRs with nicotine. At baseline, sIPSCs were less frequent and smaller in DNE cells (consistent with decreased action potential-mediated GABA release), and mIPSCs were more frequent (consistent with increased vesicular GABA release from presynaptic terminals). Acute nicotine challenge increased sIPSC frequency in both groups, though the increase was greater in DNE cells. Acute nicotine challenge did not change the frequency of mIPSCs in either group, though mIPSC amplitude increased significantly in DNE cells, but not control cells. Stimulation of postsynaptic GABAA receptors with muscimol caused a significantly greater chloride current in DNE cells than in control cells. The increased quantal release of GABA, coupled with the rise in the strength of postsynaptic inhibition may be homeostatic adjustments to the decreased action-potential-mediated input from GABAergic interneurons. However, this will exaggerate synaptic inhibition under conditions where the release of GABA (e.g. hypoxia) or ACh (sleep-wake transitions) is increased. These findings reveal a mechanism that may explain why DNE is associated with deficits in the ability to respond appropriately to chemosensory stimuli or to changes in neuromodulation secondary to changes in central nervous system state.

Neonatal phenobarbital exposure disrupts GABAergic synaptic maturation in rat CA1 neurons.

Phenobarbital is the most commonly utilized drug for the treatment of neonatal seizures. The use of phenobarbital continues despite growing evidence that it exerts suboptimal seizure control and is associated with long-term alterations in brain structure, function, and behavior. Alterations following neonatal phenobarbital exposure include acute induction of neuronal apoptosis, disruption of synaptic development in the striatum, and a host of behavioral deficits. These behavioral deficits include those in learning and memory mediated by the hippocampus. However, the synaptic changes caused by acute exposure to phenobarbital that lead to lasting effects on brain function and behavior remain understudied. Postnatal day (P)7 rat pups were treated with phenobarbital (75mg/kg) or saline. On P13-14 or P29-37, acute slices were prepared and whole-cell patch-clamp recordings were made from CA1 pyramidal neurons. At P14 we found an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in the phenobarbital-exposed as compared to the saline-exposed group. In addition to this change in mIPSC frequency, the phenobarbital group displayed larger bicuculline-sensitive tonic currents, decreased capacitance and membrane time constant, and a surprising persistence of giant depolarizing potentials. At P29+, the frequency of mIPSCs in the saline-exposed group had increased significantly from the frequency at P14, typical of normal synaptic development; at this age the phenobarbital-exposed group displayed a lower mIPSC frequency than did the control group. Spontaneous inhibitory postsynaptic current (sIPSC) frequency was unaffected at either P14 or P29+. These neurophysiological alterations following phenobarbital exposure provide a potential mechanism by which acute phenobarbital exposure can have a long-lasting impact on brain development and behavior.

Role of TLR4 in the Modulation of Central Amygdala GABA Transmission by CRF Following Restraint Stress.

Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) following restraint stress. Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraint stress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRF significantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress. Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response. TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress.

Botulinum toxin type A enhances the inhibitory spontaneous postsynaptic currents on the substantia gelatinosa neurons of the subnucleus caudalis in immature mice.

Botulinum toxin type A (BoNT/A) has been used therapeutically for various conditions including dystonia, cerebral palsy, wrinkle, hyperhidrosis and pain control. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) receive orofacial nociceptive information from primary afferents and transmit the information to higher brain center. Although many studies have shown the analgesic effects of BoNT/A, the effects of BoNT/A at the central nervous system and the action mechanism are not well understood. Therefore, the effects of BoNT/A on the spontaneous postsynaptic currents (sPSCs) in the SG neurons were investigated. In whole cell voltage clamp mode, the frequency of sPSCs was increased in 18 (37.5%) neurons, decreased in 5 (10.4%) neurons and not affected in 25 (52.1%) of 48 neurons tested by BoNT/A (3 nM). Similar proportions of frequency variation of sPSCs were observed in 1 and 10 nM BoNT/A and no significant differences were observed in the relative mean frequencies of sPSCs among 1–10 nM BoNT/A. BoNT/A-induced frequency increase of sPSCs was not affected by pretreated tetrodotoxin (0.5 µM). In addition, the frequency of sIPSCs in the presence of CNQX (10 µM) and AP5 (20 µM) was increased in 10 (53%) neurons, decreased in 1 (5%) neuron and not affected in 8 (42%) of 19 neurons tested by BoNT/A (3 nM). These results demonstrate that BoNT/A increases the frequency of sIPSCs on SG neurons of the Vc at least partly and can provide an evidence for rapid action of BoNT/A at the central nervous system.

Role of Pre-Synaptic NMDA Receptors in the Modulation of Inhibitory Synaptic Transmission in Sensory-Motor and Visual Cortical Pyramidal Neurons in Brain Slices of Young Epileptic Mice.

BackgroundPrevious studies from animal models have shown that pre-synaptic NMDA receptors (preNMDARs) are present in the cortex, but the role of inhibition mediated by preNMDARs during epileptogenesis remains unclear. In this study, we wanted to observe the changes in GABAergic inhibition through preNMDARs in sensory-motor and visual cortical pyramidal neurons after pilocarpine-induced status epilepticus.MethodsUsing a pilocarpine-induced epileptic mouse model, sensory-motor and visual cortical slices were prepared, and the whole-cell patch clamp technique was used to record spontaneous inhibitory post-synaptic currents (sIPSCs).ResultsThe primary finding was that the mean amplitude of sIPSC from the sensory-motor cortex increased significantly in epileptic mice when the recording pipette contained MK-801 compared to control mice, whereas the mean sIPSC frequency was not significantly different, indicating that post-synaptic mechanisms are involved. However, there was no significant pre-synaptic inhibition through preNMDARs in the acute brain slices from pilocarpine-induced epileptic mice.ConclusionIn the acute case of epilepsy, a compensatory mechanism of post-synaptic inhibition, possibly from ambient GABA, was observed through changes in the amplitude without significant changes in the frequency of sIPSC compared to control mice. The role of preNMDAR-mediated inhibition in epileptogenesis during the chronic condition or in the juvenile stage warrants further investigation.

Clenbuterol reduces GABAergic transmission in prefrontal cortex layer 5/6 pyramidal neurons of juvenile rat via reducing action potentials firing frequency of GABAergic interneurons.

Beta-adrenoceptors (β2 -ARs) have beneficial effects on prefrontal cortex (PFC) working memory, however, the cellular and molecular mechanisms are unclear yet. In this study, we probed the effect of β2 -AR-selective agonist clenbuterol (Clen) on synaptic transmission in layer 5/6 pyramidal neurons of PFC. Bath application of Clen reduced spontaneous IPSC (sIPSC) frequency without effects on sEPSCs. Clen did not alter the frequency and amplitude of miniature IPSCs (mIPSCs), but exerted heterogeneous effects on evoked IPSCs (eIPSCs) recorded from PFC layer 5/6 pyramidal neurons. Clen decreased the firing rate of action potentials of fast-spiking GABAergic interneurons. Clen-induced hyperpolarization of fast-spiking GABAergic interneurons required potentiation of an inward rectifier K+ channels. Clen-induced hyperpolarization of fast-spiking interneurons was dependent on Gs protein rather than cAMP and protein kinase A. Our findings demonstrate that Clen (10μM) enhances inward rectifier K+ channels via Gs protein to cause membrane hyperpolarization of fast-spiking GABAergic interneurons resulting in reduction of action potentials firing rate to reduce GABAergic transmission.

Liraglutide modulates GABAergic signaling in rat hippocampal CA3 pyramidal neurons predominantly by presynaptic mechanism

Backgroundγ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain where it regulates activity of neuronal networks. The receptor for glucagon-like peptide-1 (GLP-1) is expressed in the hippocampus, which is the center for memory and learning. In this study we examined effects of liraglutide, a GLP-1 analog, on GABA signaling in CA3 hippocampal pyramidal neurons.MethodsWe used patch-clamp electrophysiology to record synaptic and tonic GABA-activated currents in CA3 pyramidal neurons in rat hippocampal brain slices.ResultsWe examined the effects of liraglutide on the neurons at concentrations ranging from one nM to one μM. Significant changes of the spontaneous inhibitory postsynaptic currents (sIPSCs) were only recorded with 100 nM liraglutide and then in just ≈50% of the neurons tested at this concentration. In neurons affected by liraglutide both the sIPSC frequency and the most probable amplitudes increased. When the action potential firing was inhibited by tetrodotoxin (TTX) the frequency and amplitude of IPSCs in TTX and in TTX plus 100 nM liraglutide were similar.ConclusionsThe results demonstrate that liraglutide regulation of GABA signaling of CA3 pyramidal neurons is predominantly presynaptic and more limited than has been observed for GLP-1 and exendin-4 in hippocampal neurons.

Defective GABAergic neurotransmission in the nucleus tractus solitarius in Mecp2-null mice, a model of Rett syndrome

Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (Mecp2) gene. GABAergic dysfunction has been implicated contributing to the respiratory dysfunction, one major clinical feature of RTT. The nucleus tractus solitarius (NTS) is the first central site integrating respiratory sensory information that can change the nature of the reflex output. We hypothesized that deficiency in Mecp2 gene reduces GABAergic neurotransmission in the NTS. Using whole-cell patch-clamp recordings in NTS slices, we measured spontaneous inhibitory postsynaptic currents (sIPSCs), miniature IPSCs (mIPSCs), NTS-evoked IPSCs (eIPSCs), and GABAA receptor (GABAA-R) agonist-induced responses. Compared to those from wild-type mice, NTS neurons from Mecp2-null mice had significantly (p<0.05) reduced sIPSC amplitude, sIPSC frequency, and mIPSC amplitude but not mIPSC frequency. Mecp2-null mice also had decreased eIPSC amplitude with no change in paired-pulse ratio. The data suggest reduced synaptic receptor-mediated phasic GABA transmission in Mecp2-null mice. In contrast, muscimol (GABAA-R agonist, 0.3–100μM) and THIP (selective extrasynaptic GABAA-R agonist, 5μM) induced significantly greater current response in Mecp2-null mice, suggesting increased extrasynaptic receptors. Using qPCR, we found a 2.5 fold increase in the delta subunit of the GABAA-Rs in the NTS in Mecp2-null mice, consistent with increased extrasynaptic receptors. As the NTS was recently found required for respiratory pathology in RTT, our results provide a mechanism for NTS dysfunction which involves shifting the balance of synaptic/extrasynaptic receptors in favor of extrasynaptic site, providing a target for boosting GABAergic inhibition in RTT.

Spinal analgesic mechanism of minocycline in formalin-induced inflammatory pain

Objective: To investigate the spinal analgesic mechanism of minocycline in formalin-induced inflammatory pain. Methods: Behavioral test: Male Sprague-Dawley rats(3-5-week old) were randomly assigned into four groups: control, model, vehicle-controlled and minocycline group. Ten percent neutral formalin was injected subcutaneously into the right hind paw dorsum of the rats in model, vehicle-controlled and minocycline group. Normal saline was injected subcutaneously into the right hind paw dorsum of the rats in control group. Before 1 h of formalin injection, the rats in vehicle-controlled and minocycline group received intraperitoneal injection of saline and minocycline, respectively. Licking and lifting time was observed as the behavior results of inflammatory pain. Electrophysiologic experiment: In vitro spinal cord parasagittal slices were prepared from the same rats as above. The effect of minocycline on spontaneous inhibitory postsynaptic currents(sIPSCs) of substantia gelatinosa(SG) neurons was observed using whole-cell patch-clamp technique. Results: Compared with the control group, the licking and lifting time in the model group was significantly increased. Compared with the vehicle-controlled group, the licking and lifting time in the minocycline group was significantly decreased. Minocycline significantly increased the frequency(t=9.32, P<0.05)but not the amplitude(t=1.54, P>0.05) of sIPSCs of SG neurons, the frequency of sIPSCs of control and minocycline group were (2.5±0.3)Hz and (5.2±0.6)Hz, respectively. When calcium was removed from the extracellular solution, the frequency before and after minocycline perfusion were (0.9±0.1)Hz and (0.9±0.1)Hz, respectively, the amplitude before and after minocycline perfusion were (18.2±0.7)pA and (18.5± 0.6)pA, respectively, the difference of frequency or amplitude was not statistically significant(t=0.32, 0.82, all P>0.05). However, minocycline still increased the frequency of sIPSCs when glutamate receptor antagonists 6-Cyano-7-nitroquinoxaline-2, 3-dione(CNQX) and D-(-)-2-Amino-5-phosphonopentanoic acid(APV) were included in extracellular solution(t=13.51, P<0.05), the frequency of sIPSCs were (2.0±0.1)Hz and (4.3±0.4)Hz, respectively. Minocycline still increased the frequency of IPSCs when voltage-gated sodium channel blocker tetrodotoxin(TTX) were included in extracellular solution(t=8.67, P<0.05), the frequency of IPSCs were (2.2±0.2)Hz and (5.2±0.5)Hz. Conclusion: Minocycline can attenuate formalin-induced inflammatory pain which may be associated with its increase in the inhibitory synaptic transmission of SG neurons.