sEPSC Frequency Research Articles

Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague-Dawley rats without increasing spontaneous glutamate release.

Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non-contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague-Dawley rats that had access to ethanol for 7weeks on alternating days. We found increased basal NAc glutamate at 24h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage-gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol-consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT-1, however, a decrease in slope of the no-net-flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential-dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release.

Effect of interaction between acute administration of morphine and cannabinoid compounds on spontaneous excitatory and inhibitory postsynaptic currents of magnocellular neurons of supraoptic nucleus.

Opioids and cannabinoids are two important compounds that have been shown to influence the activity of magnocellular neurons (MCNs) of supraoptic nucleus (SON). The interaction between opioidergic and cannabinoidergic systems in various structures of the brain and spinal cord is now well established, but not in the MCNs of SON. In this study, whole cell patch clamp recording of neurons in rat brain slice was used to investigate the effect of acute morphine and cannabinoid administration on spontaneous inhibitory and excitatory spostsynaptic currents (sIPSCs and sEPSCs) in MCNs. Bath application of morphine produced an increase in sEPSCs frequency and a decrease in sIPSCs frequency. In contrast, bath application of URB597 (fatty acid amide hydrolase (FAAH) inhibitor) produced a decrease in sEPSCs frequency but an increase in sIPSCs frequency. WIN55212-2 (cannabinoid receptor agonist) decreased both sIPSCs and sEPSCs frequencies of MCNs. Co-application of morphine and URB597 attenuated the effect of morphine on MCNs. Taken together, these data indicated that at the cellular level, pharmacological augmentation of endocannabinoids could attenuate morphine effects on MCNs.

Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures.

The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation. Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus. EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH. These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders.

Calbindin-D-28K like immunoreactivity in superficial dorsal horn neurons and effects of sciatic chronic constriction injury

The neuropathic pain that results from peripheral nerve injury is associated with alterations in the properties of neurons in the superficial spinal laminae. Chronic constriction injury (CCI) of the rat sciatic nerve increases excitatory synaptic drive to excitatory neurons in the substantia gelatinosa while limiting that to inhibitory neurons. Since the calcium-binding protein calbindin D-28K has been associated with excitatory neurons, we examined whether CCI altered the properties of neurons expressing calbindin-like immunoreactivity (Cal+). These account for 30% of the neurons in lamina I and II. Calbindin did not co-localize with any particular electrophysiological phenotype of neuron; in substantia gelatinosa, it was found in some tonic, delay, irregular, phasic and transient firing neurons and in some cells that displayed central, radial or vertical morphology. When neuronal phenotype was defined more precisely in terms of both morphology and electrophysiological properties, no strong correlation with calbindin expression was found. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSC) in calbindin negative (Cal−) neurons was greater than that in Cal+ neurons.CCI did not alter the proportion of Cal+ neurons in the dorsal horn. Although CCI promoted a fourfold increase in sEPSC frequency in Cal+ neurons, sEPSC amplitude was reduced by 22% and charge transfer per second was unchanged. Since synaptic drive to Cal+ neurons is weak and there is no firm correlation between neuronal phenotype and calbindin expression, it is doubtful whether these neurons play a major role in the generation of central sensitization.

Systemic inflammation induces anxiety disorder through CXCL12/CXCR4 pathway

It is evidenced that inflammation is involved in the pathogenesis of anxiety disorder, as well as the dysfunction of glutamate neurotransmission in the central nervous system (CNS). Chemokine CXCL12 has been reported taking part in the regulation of neurotransmitter release, however, the roles of CXCL12 in the development of anxiety are still unclear. In this study, we found that intraperitoneal (i.p) injection of lipopolysaccharide (LPS) induced anxiety-like behaviors in adult mice as measured by elevated plus-maze test (EPM) and open field test (OFT). Astrocytes were responsible for CXCL12 induction upon LPS challenge in hippocampus and amygdala, and microinjection of CXCL12 into amygdala induced mice anxiety-like behaviors. AMD3100, which is an antagonist for CXCL12 receptor CXCR4, prevented the anxiety behaviors induced by microinjection of CXCL12 into amygdala as well as injection i.p of LPS. Knockdown of CXCR4 expression in neurons using short hairpin RNAs (shRNAs) significantly blocked anxiety behaviors mediated by CXCL12 i.c injection. Furthermore, AMD3100 or shCXCR4 prevented the impairment of nesting ability induced by CXCL12 in mice. Whole-cell patch-clamp recordings in the neurons of basolateral amygdala (BLA) revealed that CXCL12 enhanced glutamatergic transmission by increasing sEPSC frequency in the amygdala. AMD3100 inhibited the excitatory glutamatergic neural transmission and involved in the development of anxiety through CXCR4. These findings provide direct evidence that alterations of CXCL12 in BLA play critical roles in the development of anxiety induced by systemic inflammation and that CXCR4 may be a potential therapeutic target for inflammation-induced anxiety.

Selective alterations of NMDAR function and plasticity in D1 and D2 medium spiny neurons in the nucleus accumbens shell following chronic intermittent ethanol exposure

A major mouse model widely adopted in recent years to induce pronounced ethanol intake is the ethanol vapor model known as “CIE” or “Chronic Intermittent Ethanol.” One critical question concerning this model is whether the rapid induction of high blood ethanol levels for such short time periods is sufficient to induce alterations in N-methyl-d-aspartate receptor (NMDAR) function which may contribute to excessive ethanol intake. In this study, we determined whether such short term intermittent ethanol exposure modulates NMDAR function as well as other prominent electrophysiological properties and the expression of plasticity in both D1 (D1+) and D2 (D1−) dopamine receptor expressing medium spiny neurons (MSNs) in the nucleus accumbens (NAc) shell. To distinguish between the two subtypes of MSNs in the NAc we treated Drd1a-TdTomato transgenic mice with CIE vapor and electrophysiological recordings were conducted 24 h after the last vapor exposure. To investigate CIE induced alterations in plasticity, long-term depression (LTD) was induced by pairing low frequency stimulation (LFS) with post synaptic depolarization. In ethanol naïve mice, LFS induced synaptic depression (LTD) was apparent exclusively in D1+ MSNs. Whereas in slices prepared from CIE treated mice, LFS induced synaptic potentiation (LTP) in D1+ MSNs. Furthermore, following CIE exposure, LFS now produced LTD in D1− MSNs. We found that CIE exposure induced an increase in excitability in D1+ MSNs with no change in D1− MSNs. After CIE, we found a significant increase in spontaneous EPSCs (sEPSCs) frequency in D1+ but not D1− MSNs suggesting alterations in baseline α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated signaling. CIE induced changes in NMDAR function were measured using the NMDA/AMPA ratio and input-output curves of isolated NMDAR currents. We observed a significant increase in NMDAR function in D1+ MSNs and a decrease in D1− MSNs after ethanol vapor exposure. The reversal of NMDAR function may account for the CIE induced alterations in the expression of plasticity. The cell type specific alterations in excitatory signaling in the NAc shell may constitute an important neuroadaptation necessary for the expression of increased ethanol consumption induced by intermittent ethanol vapor exposure.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

Short-term repeated corticosterone administration enhances glutamatergic but not GABAergic transmission in the rat motor cortex.

It has been demonstrated that stress impairs performance of skilled reaching and walking tasks in rats due to the action of glucocorticoids involved in the stress response. Skilled reaching and walking are controlled by the primary motor cortex (M1); however, it is not known whether stress-related impairments in skilled motor tasks are related to functional and/or structural alterations within the M1. We studied the effects of single and repeated injections of corticosterone (twice daily for 7 days) on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) recorded from layer II/III pyramidal neurons in ex vivo slices of the M1, prepared 2 days after the last administration of the hormone. We also measured the density of dendritic spines on pyramidal cells and the protein levels of selected subunits of AMPA, NMDA, and GABAA receptors after repeated corticosterone administration. Repeatedly administered corticosterone induced an increase in the frequency but not in the amplitude of sEPSCs, while a single administration had no effect on the recorded excitatory currents. The frequency and amplitude of sIPSCs as well as the excitability of pyramidal cells were changed neither after single nor after repeated corticosterone administration. Treatment with corticosterone for 7 days did not modify the density of dendritic spines on pyramidal neurons. Corticosterone influenced neither the protein levels of GluA1, GluA2, GluN1, GluN2A, and GluN2B subunits of glutamate receptors nor those of α1, β2, and γ2 subunits of the GABAA receptor. The increase in sEPSCs frequency induced by repeated corticosterone administration faded out within 7 days. These data indicate that prolonged administration of exogenous corticosterone selectively and reversibly enhances glutamatergic, but not GABAergic transmission in the rat motor cortex. Our results suggest that corticosterone treatment results in an enhancement of spontaneous glutamate release from presynaptic terminals in the M1 and thereby uncovers a potential mechanism underlying stress-induced motor functions impairment.

Role of brain-derived neurotrophic factor in the excitatory-inhibitory imbalance during the critical period of postnatal respiratory development in the rat.

The critical period of respiratory development in rats is a narrow window toward the end of the second postnatal week (P12–13), when abrupt neurochemical, electrophysiological, and ventilatory changes occur, when inhibition dominates over excitation, and when the animals’ response to hypoxia is the weakest. The goal of this study was to further test our hypothesis that a major mechanism underlying the synaptic imbalance during the critical period is a reduced expression of brain-derived neurotrophic factor (BDNF) and its TrkB receptors. Our aims were to determine (1) that the inhibitory dominance observed in hypoglossal motoneurons during the critical period was also demonstrable in a key respiratory chemosensor, NTSVL; (2) if in vivo application of a TrkB agonist, 7,8-DHF, would prevent, but a TrkB antagonist, ANA-12, would accentuate the synaptic imbalance; and (3) if hypoxia would also heighten the imbalance. Our results indicate that (1) the synaptic imbalance was evident in the NTSVL during the critical period; (2) intraperitoneal injections of 7,8-DHF prevented the synaptic imbalance during the critical period, whereas ANA-12 in vivo accentuated such an imbalance; and (3) acute hypoxia induced the weakest response in both the amplitude and frequency of sEPSCs during the critical period, but it increased the frequency of sIPSCs during the critical period. Thus, our findings are consistent with and strengthen our hypothesis that BDNF and TrkB play a significant role in inducing a synaptic imbalance during the critical period of respiratory development in the rat.

Ethanol produces corticotropin-releasing factor receptor-dependent enhancement of spontaneous glutamatergic transmission in the mouse central amygdala.

Ethanol (EtOH) modulation of central amygdala (CeA) neurocircuitry plays a key role in the development of alcoholism via activation of the corticotropin-releasing factor (CRF) receptor (CRFR) system. Previous work has predominantly focused on EtOH×CRF interactions on the CeA GABA circuitry; however, our laboratory recently showed that CRF enhances CeA glutamatergic transmission. Therefore, this study sought to determine whether EtOH modulates CeA glutamate transmission via activation of CRF signaling. The effects of EtOH on spontaneous excitatory postsynaptic currents (sEPSCs) and basal resting membrane potentials were examined via standard electrophysiology methods in adult male C57BL/6J mice. Local ablation of CeA CRF neurons (CRF(CeAhDTR) ) was achieved by targeting the human diphtheria toxin receptor (hDTR) to CeA CRF neurons with an adeno-associated virus. Ablation was quantified post hoc with confocal microscopy. Genetic targeting of the diphtheria toxin active subunit to CRF neurons (CRF(DTA) mice) ablated CRF neurons throughout the central nervous system, as assessed by quantitative reverse transcriptase polymerase chain reaction quantification of CRF mRNA. Acute bath application of EtOH significantly increased sEPSC frequency in a concentration-dependent manner in CeA neurons, and this effect was blocked by pretreatment of co-applied CRFR1 and CRFR2 antagonists. In experiments utilizing a CRF-tomato reporter mouse, EtOH did not significantly alter the basal membrane potential of CeA CRF neurons. The ability of EtOH to enhance CeA sEPSC frequency was not altered in CRF(CeAhDTR) mice despite a ~78% reduction in CeA CRF cell counts. The ability of EtOH to enhance CeA sEPSC frequency was also not altered in the CRF(DTA) mice despite a 3-fold reduction in CRF mRNA levels. These findings demonstrate that EtOH enhances spontaneous glutamatergic transmission in the CeA via a CRFR-dependent mechanism. Surprisingly, our data suggest that this action may not require endogenous CRF.

Increased excitability and excitatory synaptic transmission during in vitro ischemia in the neonatal mouse hippocampus

Objective: The present study tested the hypothesis that exposure to in vitro hypoxia–ischemia alters membrane properties and excitability as well as excitatory synaptic transmission of CA1 pyramidal neurons in the neonatal mouse. Methods: Experiments were conducted in hippocampal slices in P7–P9 C57Bl/6 mice using whole-cell patch clamp in current- and voltage-clamp mode. Passive membrane potential (Vm), input resistance (Rin) and active (action potential (AP) threshold and amplitude) membrane properties of CA1 pyramidal neurons were assessed at baseline, during 10min in vitro ischemia (oxygen-glucose deprivation (OGD)) and during reoxygenation. Spontaneous and miniature excitatory post-synaptic currents (s and mEPSCs) were studied under similar conditions. Results: OGD caused significant depolarization of CA1 pyramidal neurons as well as decrease in AP threshold and increase in AP amplitude. These changes were blocked by the application of tetrodotoxin (TTX), indicating Na+ channels’ involvement. Following 10min of reoxygenation, significant membrane hyperpolarization was noted and it was associated with a decrease in Rin. AP threshold and amplitude returned to baseline during that stage. sEPSC and mEPSC frequency increased during both OGD and reoxygenation but their amplitude remained unchanged. Additionally, we found that OGD decreases Ih (hyperpolarization activated current) in CA1 neurons from neonatal mice and this effect persists during reoxygenation. Significance: These results indicate that in vitro ischemia leads to changes in membrane excitability mediated by sodium and potassium channels. Further, it results in enhanced neurotransmitter release from presynaptic terminals. These changes are likely to represent one of the mechanisms of hypoxia/ischemia-mediated seizures in the neonatal period.

Bulleyaconitine A depresses neuropathic pain and potentiation at C-fiber synapses in spinal dorsal horn induced by paclitaxel in rats

Paclitaxel, a widely used chemotherapeutic agent, often induces painful peripheral neuropathy and at present no effective drug is available for treatment of the serious side effect. Here, we tested if intragastrical application of bulleyaconitine A (BLA), which has been approved for clinical treatment of chronic pain in China since 1985, could relieve the paclitaxel-induced neuropathic pain. A single dose of BLA attenuated the mechanical allodynia, thermal hyperalgesia induced by paclitaxel dose-dependently. Repetitive administration of the drug (0.4 and 0.8mg/kg, t.i.d. for 7 d) during or after paclitaxel treatment produced a long-lasting inhibitory effect on thermal hyperalgesia, but not on mechanical allodynia. In consistency with the behavioral results, in vivo electrophysiological experiments revealed that spinal synaptic transmission mediated by C-fiber but not A fiber was potentiated, and the magnitude of long-term potentiation (LTP) at C-fiber synapses induced by the same high frequency stimulation was ~50% higher in paclitaxel-treated rats, compared to the naïve rats. Spinal or intravenous application of BLA depressed the spinal LTP, dose-dependently. Furthermore, patch clamp recordings in spinal cord slices revealed that the frequency but not amplitude of both spontaneous excitatory postsynaptic current (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) in lamina II neurons was increased in paclitaxel-treated rats, and the superfusion of BLA reduced the frequency of sEPSCs and mEPSCs in paclitaxel-treated rats but not in naïve ones. Taken together, we provide novel evidence that BLA attenuates paclitaxel-induced neuropathic pain and that depression of spinal LTP at C-fiber synapses via inhibiting presynaptic transmitter release may contribute to the effect.

Nitrous oxide directly inhibits action potential-dependent neurotransmission from single presynaptic boutons adhering to rat hippocampal CA3 neurons.

We evaluated the effects of N2O on synaptic transmission using a preparation of mechanically dissociated rat hippocampal CA3 neurons that allowed assays of single bouton responses evoked from native functional nerve endings. We studied the effects of N2O on GABAA, glutamate, AMPA and NMDA receptor-mediated currents (IGABA, IGlu, IAMPA and INMDA) elicited by exogenous application of GABA, glutamate, (S)-AMPA, and NMDA and spontaneous, miniature, and evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic current (sIPSC, mIPSC, eIPSC, sEPSC, mEPSC and eEPSC) in mechanically dissociated CA3 neurons. eIPSC and eEPSC were evoked by focal electrical stimulation of a single bouton. Administration of 70% N2O altered neither IGABA nor the frequency and amplitude of both sIPSCs and mIPSCs. In contrast, N2O decreased the amplitude of eIPSCs, while increasing failure rates (Rf) and paired-pulse ratios (PPR) in a concentration-dependent manner. On the other hand, N2O decreased IGlu, IAMPA and INMDA. Again N2O did not change the frequency and amplitude of either sEPSCs of mEPSCs. N2O also decreased amplitudes of eEPSCs with increased Rf and PPR. The decay phases of all synaptic responses were unchanged. The present results indicated that N2O inhibits the activation of AMPA/KA and NMDA receptors and also that N2O preferentially depress the action potential-dependent GABA and glutamate releases but had little effects on spontaneous and miniature releases.

Effects of prostaglandin E2 on synaptic transmission in the rat spinal trigeminal subnucleus caudalis

The spinal trigeminal subnucleus caudalis (Vc) receives preferentially nociceptive afferent signals from the orofacial area. Nociceptive stimuli to the orofacial area induce cyclooxygenase both peripherally and centrally, which can synthesize a major prostanoid prostaglandin E2 (PGE2) that implicates in diverse physiological functions. To clarify the roles of centrally-synthesized PGE2 in nociception, effects of exogenous PGE2 on synaptic transmission in the Vc neurons were investigated in the rat brainstem slice. Spontaneously occurring excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) were recorded, respectively, under pharmacological blockade of inhibitory and excitatory transmission by whole-cell patch-clamp mode. Perfusion of PGE2 (1–5μM) increased the frequency of sIPSCs in a concentration-dependent manner but had no significant effect on the amplitude. Similarly to the effects on sIPSCs, PGE2 increased the sEPSC frequency without any effect on the amplitude. These facilitatory effects of PGE2 on spontaneous synaptic transmissions were blocked by an EP1 antagonist SC19220 but not by an EP4 antagonist AH23848. Electrical stimulation of the trigeminal tract evoked short latency EPSCs (eEPSCs) in the Vc neurons. PGE2 (5μM) was ineffective on the eEPSCs. The present study demonstrated that PGE2 facilitated spontaneous synaptic transmissions in the Vc neurons through activating the presynaptic EP1 receptors but had no effect on the trigeminal tract-mediated excitatory transmission. These results suggest that centrally-synthesized PGE2 modifies the synaptic transmission in the Vc region, thereby contributing to the processing of nociceptive signals originated from the orofacial area.

Action of thymol on spontaneous excitatory transmission in adult rat spinal substantia gelatinosa neurons

Thymol, which is contained in thyme essential oil, has various actions including antinociception and nerve conduction inhibition. Although thymol activates transient receptor potential (TRP) channels expressed in heterologous cells, it remains to be examined whether this is so in native neurons. It has not yet been examined how thymol affects synaptic transmission. In order to know how thymol modulates excitatory transmission with a focus on TRP activation, we investigated its effect on glutamatergic spontaneous excitatory transmission in lamina II (substantia gelatinosa; SG) neurons with which nerve terminals expressing TRP channels make synaptic contacts. The experiment was performed by using the blind whole-cell patch-clamp technique in adult rat spinal cord slices. Superfusing thymol (1mM) for 3min reversibly increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a minimal increase in its amplitude in all neurons examined. Seventy-seven% of the neurons produced an outward current at a holding potential of −70mV. The sEPSC frequency increase and outward current produced by thymol were concentration-dependent with almost the same half-maximal effective concentration (EC50) values of 0.18 and 0.14mM, respectively. These activities were repeated at a time interval of 30min, although the sEPSC frequency increase but not outward current recovered with a slow time course. Voltage-gated Na+-channel blocker tetrodotoxin did not affect the thymol activities. The sEPSC frequency increase was inhibited by TRPA1 antagonist HC-030031 but not TRPV1 and TRPM8 antagonist (capsazepine and BCTC, respectively), while these antagonists had no effect on the outward current. This was so, albeit the two thymol activities had similar EC50 values. It is concluded that thymol increases the spontaneous release of L-glutamate onto SG neurons by activating TRPA1 channels while producing an outward current without TRP activation. Considering that the SG plays a pivotal role in modulating nociceptive transmission from the periphery, these actions of thymol could contribute to at least a part of its antinociceptive effect.

Direct Facilitatory Role of Paragigantocellularis Neurons in Opiate Withdrawal-Induced Hyperactivity of Rat Locus Coeruleus Neurons: An In Vitro Study

Studies have shown that following opiate withdrawal, the spontaneous discharge rate of locus coeruleus (LC) neurons remarkably increases. Combination of intrinsic mechanisms with extrinsic excitatory modulations mediates the withdrawal-induced hyperactivity of LC neurons. The nucleus paragigantocellularis (PGi) provides the main excitatory inputs to LC and plays a pivotal role in opiate withdrawal. In the present study the direct facilitatory role of PGi on opiate withdrawal-induced hyperactivity of LC neurons was investigated using a newly developed brain slice, containing both LC and PGi. HRP retrograde neuronal tracing was used to verify the existence of both LC and PGi neurons in the developed slice. The spontaneous discharge rate (SDR), resting membrane potential (RMP) and spontaneous excitatory post-synaptic currents (sEPSCs) were recorded in LC neurons using whole cell patch clamp recording. Results showed that the net SDR and the net RMP of LC neurons in slices containing both LC and PGi neurons are significantly higher than slices lacking intact (uncut) PGi inputs. Also, the frequency of sEPSCs in those LC neurons receiving PGi inputs significantly increased compared to the slices containing no intact PGi inputs. Altogether, our results propose that increase in PGi-mediated excitatory transmission might facilitate the opiate withdrawal-induced hyperactivity of LC neurons.

A new potent analgesic agent with reduced liability to produce morphine tolerance

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.

Effects of chronic alcohol consumption on neuronal function in the non-human primate BNST.

Alterations in hypothalamic-pituitary-adrenal axis function contribute to many of the adverse behavioral effects of chronic voluntary alcohol drinking, including alcohol dependence and mood disorders; limbic brain structures such as the bed nucleus of the stria terminalis (BNST) may be key sites for these effects. Here, we measured circulating levels of several steroid hormones and performed whole-cell electrophysiological recordings from acutely prepared BNST slices of male rhesus monkeys allowed to self-administer alcohol for 12 months or a control solution. Initial comparisons revealed that BNST neurons in alcohol-drinking monkeys had decreased membrane resistance, increased frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with no change in spontaneous excitatory postsynaptic currents (sEPSCs). We then used a combined variable cluster analysis and linear mixed model statistical approach to determine whether specific factors including stress and sex hormones, age and measures of alcohol consumption and intoxication are related to these BNST measures. Modeling results showed that specific measures of alcohol consumption and stress-related hormone levels predicted differences in membrane conductance in BNST neurons. Distinct groups of adrenal stress hormones were negatively associated with the frequency of sIPSCs and sEPSCs, and alcohol drinking measures and basal neuronal membrane properties were additional positive predictors of inhibitory, but not excitatory, PSCs. The amplitude of sEPSCs was highly positively correlated with age, independent of other variables. Together, these results suggest that chronic voluntary alcohol consumption strongly influences limbic function in non-human primates, potentially via interactions with or modulation by other physiological variables, including stress steroid hormones and age.

Tenuigenin enhances hippocampal Schaffer collateral-CA1 synaptic transmission through modulating intracellular calcium

BackgroundTenuigenin (TEN), a natural product from the Chinese herb Polygala tenuifolia root, has been reported to improve cognitive function and exhibits neuroprotective effects in pharmacological studies of the central nervous system. Synaptic transmission is the essential process of brain physiological functions such as learning and memory formation, and TEN has been shown to facilitate the basic synaptic transmission. Hypothesis/PurposeAlthough our previous work has demonstrated that TEN is able to potentiate the basic synaptic transmission, the potential mechanism remains unclear. Here we investigated the effect of TEN on the synaptic transmission and analysed the potential mechanism. We hope that these findings will contribute to explain the role of TEN as a nootropic product or neuroprotective drug in the future. MethodsField excitatory postsynaptic potentials (fEPSPs), spontaneous excitatory postsynaptic currents (sEPSCs) and miniature spontaneous excitatory postsynaptic currents (mEPSCs) were recorded, by using in vitro field potential electrophysiology and whole-cell patch clamp techniques in acute hippocampal slices from rats. ResultsTEN perfusion significantly enhanced the slope of fEPSPs and reduced the ratio of paired-pulse facilitation. Moreover, TEN increased the frequency and amplitude of sEPSCs but only improved the frequency of mEPSCs rather than amplitude in hippocampal CA1 pyramidal neurons. With removal of extracellular calcium, TEN treatment also enhanced the mEPSCs frequency without affecting amplitude. Interestingly, the increase of mEPSCs frequency caused by TEN was blocked by chelation of intracellular calcium with BAPTA-AM. ConclusionThese results indicate that TEN enhances the basic synaptic transmission via stimulating presynaptic intracellular calcium.

Leptin Regulates Synaptic Activity of Brown Adipose Tissue‐Related Pre‐Sympathetic Neurons in the Paraventricular Nucleus of the Mice

Brown adipose tissue (BAT) thermogenesis is critical for maintaining body temperature and thus energy expenditure and it is regulated by the sympathetic nervous system. Recent evidence demonstrated the involvement of leptin in the regulation of the sympathetic outflow to the BAT; however, the underlying cellular mechanisms are poorly understood. In this study, we identified BAT‐related neurons in the paraventricular nucleus of hypothalamus (PVN) of mice (BAT‐PVN) by inoculating BAT with pseudorabies virus 152. Whole‐cell patch‐clamp recordings were performed to test the hypothesis that leptin regulates the synaptic activity of pre‐sympathetic BAT‐PVN neurons. The majority of BAT‐PVN neurons responded to leptin application. Leptin (300 nM) caused an increase of spontaneous excitatory post‐synaptic current (sEPSC) frequency in a subset (40%) of BAT‐PVN neurons, whereas sEPSC frequency was decreased in other subset of BAT‐PVN neurons (30%). This effect was observed without alteration of the amplitude. On the other hand, the frequency of spontaneous inhibitory post‐synaptic currents (sIPSC) was decreased in 50% of BAT‐PVN neurons, while a subset of BAT‐PVN neurons (20%) responded with an increase of sIPSC frequency following leptin application. Leptin did not change the resting membrane potential or input resistance of the recorded BAT‐PVN neurons. These data suggest that leptin contributes to the regulation of sympathetic outflow to the BAT via modulating the synaptic activity of PVN neurons.Supported: NIDDK R01‐DK092587 (HM) and R01‐DK099598 (AZs).