Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade

The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.     

Role of presynaptic 5-HT1A and 5-HT3 receptors in modulation of synaptic GABA transmission in dissociated rat basolateral amygdala neurons

Serotonin (5-HT) is considered to play a significant role in anxiety-related behaviors in animals through actions on the amygdaloid complex. To evaluate this role from the point of neurotransmitter release regulation, nystatin-perforated patch recording was employed on mechanically dissociated basolateral amygdala neurons containing functional synaptic boutons. GABAAergic miniature inhibitory postsynaptic currents (mIPSCs) were pharmacologically separated. In subsets of neurons, 8-OH-DPAT (1 microM), a specific 5-HT1A agonist, continuously inhibited mIPSC frequency without effects on mIPSC amplitude. By comparison, mCPBG (1 microM), a specific 5-HT3 agonist, transiently facilitated mIPSC frequency without effects on mIPSC amplitude. Together these results suggest the presynaptic existence of both 5-HT receptor subtypes. In these neurons, application of 8-OH-DPAT and its subsequent removal still suppressed mCPBG-induced responses on mIPSCs. This suppression was not caused by a reduction of presynaptic 5-HT3 receptor affinities to mCPBG and was completely eliminated by pretreatment with N-ethylmaleimide, a pertussis toxin sensitive GTP-binding protein inhibitor. In the neurons exhibiting presynaptic modulation with mCPBG but not 8-OH-DPAT, such suppression by exposure to 8-OH-DPAT was not observed. In conclusion, activation of presynaptic 5-HT1A receptors inhibited mIPSC frequency and at the same time suppressed, via a G-protein-mediated mechanism, the transient facilitation of mIPSC frequency produced by activation of presynaptic 5-HT3 receptors.     

Serotoninergic modulation of GABAergic synaptic transmission in developing rat CA3 pyramidal neurons

Serotoninergic modulation of GABAergic mIPSCs was investigated in immature (postnatal 12–16-days old) rat CA3 pyramidal neurons using a conventional whole-cell patch clamp technique. Serotonin or 5-hydroxytryptamine (5-HT) (10 μmol/L) transiently and explosively increased mIPSC frequency with a small increase in the current amplitude. However, 5-HT did not affect the GABA-induced postsynaptic currents, indicating that 5-HT acts presynaptically to facilitate the probability of spontaneous GABA release. The 5-HT action on GABAergic mIPSC frequency was completely blocked by 100 nmol/L MDL72222, a selective 5-HT₃ receptor antagonist, and mimicked by mCPBG, a selective 5-HT₃ receptor agonist. The 5-HT action on GABAergic mIPSC frequency was completely occluded either in the presence of 200 μmol/L Cd²⁺ or in the Na⁺-free external solution, suggesting that the 5-HT₃ receptor-mediated facilitation of mIPSC frequency requires a Ca²⁺influx passing through voltage-dependent Ca²⁺channels from the extracellular space, and that presynaptic 5-HT₃ receptors are less permeable to Ca²⁺. The 5-HT action on mIPSC frequency in the absence or presence of extracellular Na⁺ gradually increased with postnatal development. Such a developmental change in the 5-HT₃ receptor-mediated facilitation of GABAergic transmission would play important roles in the regulation of excitability as well as development in CA3 pyramidal neurons.     

TRPA1-like channels enhance glycinergic transmission in medullary dorsal horn neurons

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca(2+), suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca(2+) influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na(+) -free external solution or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca(2+), to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.     

The Na(+)/H(+) exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons

The effects of pH(i) on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were studied in mechanically dissociated CA3 pyramidal neurons, by use of ammonium prepulse and whole-cell patch-clamp techniques, under the voltage-clamp condition. NH(4)Cl itself, which is expected to alkalinize pH(i), increased GABAergic mIPSC frequency in a concentration-dependent manner. In contrast, NH(4)Cl decreased mIPSC frequency, either in the presence of 200 microm Cd(2+) or in Ca(2+)-free external solution, suggesting that intraterminal alkalosis decreased GABAergic mIPSC frequency while [NH4(+)] itself may activate Ca(2+) channels by depolarizing the terminal. On the other hand, GABAergic mIPSC frequency was greatly increased immediately after NH(4)Cl removal, a condition expected to acidify pH(i), and recovered to the control level within 2 min after NH(4)Cl removal. This explosive increase in mIPSC frequency observed after NH(4)Cl removal was completely eliminated after depletion of Ca(2+) stores with 1 microm thapsigargin in the Ca(2+)-free external solution, suggesting that acidification increases in intraterminal Ca(2+) concentration via both extracellular Ca(2+) influx and Ca(2+) release from the stores. However, the acidification-induced increase in mIPSC frequency had not recovered by 10 min after NH(4)Cl removal either in the Na(+)-free external solution or in the presence of 10 microm 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific Na(+)/H(+) exchanger (NHE) blocker. The present results suggest that NHEs are major intraterminal pH regulators on GABAergic presynaptic nerve terminals, and that the NHE-mediated regulation of pH(i) under normal physiological or pathological conditions might play an important role in the neuronal excitability by increasing inhibitory tones.     

Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl) glycine (DCGIV; 5 microM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 microM), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 microM) was combined with a cell-permeant PKA inhibitor H-89 (10 microM), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 microM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 microM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 microM), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity.     

Inhibitory effects of 1,4-DHP antagonists on synaptic GABA release modulated by BAY-K 8644 in mechanically dissociated rat substantia innominata

The effects of dihydropyridine (1,4-DHP) agonist and antagonists on miniature inhibitory postsynaptic currents (mIPSCs) were investigated in mechanically dissociated rat substantia innominata neurons attached to native GABAergic presynaptic nerve terminals, namely 'synaptic bouton preparation', using nystatin perforated patch recording mode under voltage-clamp conditions. BAY-K 8644 (BAY-K), an L-type Ca(2+) channel agonist, reversibly and concentration dependently facilitated the GABAergic mIPSC frequency without altering the distribution of current amplitudes. Removal of extracellular Ca(2+) completely suppressed the facilitatory effect of BAY-K on mIPSC frequency. The facilitatory effect of BAY-K on mIPSC frequency was maintained even in the presence of selective N-, P- and Q-type Ca(2+) channel antagonists, such as 3 x 10(-6) M omega-conotoxin-GVIA (omega-CgTX-GVIA), 3 x 10(-8) M omega-agatoxin-IVA (omega-AgTX-IVA) and 3 x 10(-6)M omega-conotoxin-MVIIC (omega-CmTX-MVIIC). However, nicardipine (3 x 10(-6) M) and nimodipine (3 x 10(-6) M), 1,4-DHP antagonists, significantly inhibited the mIPSC frequency enhanced by BAY-K by 37 +/- 5 and 42 +/- 6%, respectively. These results suggest the possible existence of L-type Ca(2+) channels in GABAergic presynaptic nerve terminals.     

BDNF-induced presynaptic facilitation of GABAergic transmission in the hippocampus of young adults is dependent of TrkB and adenosine A<Subscript>2A</Subscript> receptors

Brain-derived neurotrophic factor (BDNF) and adenosine are widely recognized as neuromodulators of glutamatergic transmission in the adult brain. Most BDNF actions upon excitatory plasticity phenomena are under control of adenosine A_2A receptors (A_2ARs). Concerning gamma-aminobutyric acid (GABA)-mediated transmission, the available information refers to the control of GABA transporters. We now focused on the influence of BDNF and the interplay with adenosine on phasic GABAergic transmission. To assess this, we evaluated evoked and spontaneous synaptic currents recorded from CA1 pyramidal cells in acute hippocampal slices from adult rat brains (6 to 10 weeks old). BDNF (10–100 ng/mL) increased miniature inhibitory postsynaptic current (mIPSC) frequency, but not amplitude, as well as increased the amplitude of inhibitory postsynaptic currents (IPSCs) evoked by afferent stimulation. The facilitatory action of BDNF upon GABAergic transmission was lost in the presence of a Trk inhibitor (K252a, 200 nM), but not upon p75^NTR blockade (anti-p75^NTR IgG, 50 μg/mL). Moreover, the facilitatory action of BDNF onto GABAergic transmission was also prevented upon A_2AR antagonism (SCH 58261, 50 nM). We conclude that BDNF facilitates GABAergic signaling at the adult hippocampus via a presynaptic mechanism that depends on TrkB and adenosine A_2AR activation.     

PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.     

Rapid effects of estrogen on G protein-coupled receptor activation of potassium channels in the central nervous system (CNS)

Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding and motivated behaviors. Some of the neurons include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and γ-aminobutyric acid (GABA) neurons. We have elucidated several non-genomic pathways through which the steroid alters synaptic responses in these hypothalamic neurons. We have examined the modulation by estrogen of the coupling of various receptor systems to inwardly-rectifying and small-conductance, Ca²⁺-activated K⁺ (SK) channels using intracellular sharp-electrode and whole-cell recording techniques in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples μ-opioid receptors from G protein-gated inwardly-rectifying K⁺ (GIRK) channels in POMC neurons and GABA_B receptors from GIRK channels in dopamine neurons as manifested by a reduction in the potency of μ-opioid and GABA_B receptor agonists to hyperpolarize their respective cells. This effect is blocked by inhibitors of protein kinase A (PKA) and protein kinase C (PKC). In addition, after 24 h following steroid administration in vivo, the GABA_B/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of ₁-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these preoptic GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of GnRH neurons. These findings indicate a richly complex yet coordinated steroid modulation of K⁺ channel activity in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions.     

Effect of an adenosine A(1) receptor agonist and a novel pyrimidoindole on membrane properties and neurotransmitter release in rat cortical and hippocampal neurons

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.     

Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the cAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]i was diminished by exposing the preparations to zero Ca2+-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2+ chelator (BAPTA-AM) (hypertonic condition), indicate that cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.     

Presynaptic adenosine A1 receptors regulate retinohypothalamic neurotransmission in the hamster suprachiasmatic nucleus

Adenosine has been implicated as a modulator of retinohypothalamic neurotransmission in the suprachiasmatic nucleus (SCN), the seat of the light-entrainable circadian clock in mammals. Intracellular recordings were made from SCN neurons in slices of hamster hypothalamus using the in situ whole-cell patch clamp method. A monosynaptic, glutamatergic, excitatory postsynaptic current (EPSC) was evoked by stimulation of the optic nerve. The EPSC was blocked by bath application of the adenosine A(1) receptor agonist cyclohexyladenosine (CHA) in a dose-dependent manner with a half-maximal concentration of 1.7 microM. The block of EPSC amplitude by CHA was antagonized by concurrent application of the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist CGS21680 was ineffective in attenuating the EPSC at concentrations up to 50 microM. Trains of four consecutive stimuli at 25 ms intervals usually depressed the EPSC amplitude. However, after application of CHA, consecutive responses displayed facilitation of EPSC amplitude. The induction of facilitation by CHA suggested a presynaptic mechanism of action. After application of CHA, the frequency of spontaneous EPSCs declined substantially, while their amplitude distribution was unchanged or slightly reduced, again suggesting a mainly presynaptic site of action for CHA. Application of glutamate by brief pressure ejection evoked a long-lasting inward current that was unaffected by CHA at concentrations sufficient to reduce the evoked EPSC amplitude substantially (1 to 5 microM), suggesting that postsynaptic glutamate receptor-gated currents were unaffected by the drug. Taken together, these observations indicate that CHA inhibits optic nerve-evoked EPSCs in SCN neurons by a predominantly presynaptic mechanism.     

IL-2 receptor signaling through the Shb adapter protein in T and NK cells

We have investigated the effect of hypoxia on the excitatory synaptic transmission in the substantia gelatinosa neurons using perforated-patch-clamp configuration. Brief periods of hypoxia induced a depression in the evoked excitatory postsynaptic current (eEPSC) amplitude. The hypoxia-induced depression of eEPSC was not observed in the presence of theophylline, a nonselective adenosine receptor antagonist, and DPCPX, a selective adenosine receptor A1 antagonist. Application of adenosine (100 microM) also depressed eEPSC in a similar way as with hypoxia. This adenosine-induced depression of eEPSC was inhibited by DPCPX. Hypoxia and exogenous adenosine decreased the frequency of the spontaneous excitatory postsynaptic current (sEPSC) but not the amplitude of sEPSC and increased the paired-pulse ratio. From these results, it is suggested that acute hypoxia depresses the excitatory synaptic transmission by activating the presynaptic adenosine A1 receptor.     

Potentiation of glutamatergic synaptic input to supraoptic neurons by presynaptic nicotinic receptors

The release of vasopressin and oxytocin from the supraoptic nucleus (SON) neurons is tonically regulated by excitatory glutamatergic and inhibitory GABAergic synaptic inputs. Acetylcholine is known to excite SON neurons and to elicit vasopressin release. Cholinergic receptors are located pre- and postsynaptically in the SON, but their functional significance in the regulation of SON neurons is not fully understood. In this study, we determined the role of presynaptic cholinergic receptors in regulation of the excitatory glutamatergic inputs to the SON neurons. The magnocellular neurons in the rat hypothalamic slices were identified microscopically, and the spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole cell voltage-clamp technique. The mEPSCs were abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). Acetylcholine (100 microM) significantly increased the frequency of mEPSCs of 38 SON neurons from 1.87 +/- 0.36 to 3.42 +/- 0.54 Hz but did not alter the amplitude (from 19.61 +/- 0.90 to 19.34 +/- 0.84 pA) and the decay time constant of mEPSCs. Furthermore, the nicotinic receptor antagonist mecamylamine (10 microM, n = 16), but not the muscarinic receptor antagonist atropine (100 microM, n = 12), abolished the excitatory effect of acetylcholine on the frequency of mEPSCs. These data provide new information that the excitatory effect of acetylcholine on the SON neurons is mediated, at least in part, by its effect on presynaptic glutamate release. Activation of presynaptic nicotinic, but not muscarinic, receptors located in the glutamatergic terminals increases the excitatory synaptic input to the SON neurons of the hypothalamus.     

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.     

Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment

Background

Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.

Methodology/Principal Findings

Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.

Conclusions/Significance

These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.     

Allopregnanolone enhancement of GABAergic transmission in rat medial preoptic area neurons

Gamma-aminobutyric acid (GABA)-mediated transmission in the medial preoptic area (MPOA) of the hypothalamus plays an important role in functions such as sex steroid hormone dynamics and control of body temperature. The action of allopregnanolone, the primary metabolite of progesterone, on GABAergic transmission was investigated by employing patch clamp whole cell recording on acutely dissociated rat MPOA neurons with the functional connection of presynaptic terminals. Allopregnanolone enhanced spontaneous GABA release on the MPOA neurons and induced prolonged decay of miniature GABAergic-inhibitory postsynaptic currents (mIPSCs). The facilitation of GABA release from the presynaptic terminals by allopregnanolone disappeared in Ca2+-free extracellular solution. The presynaptic action of this neurosteroid was also blocked by bumetanide, a blocker of cation-Cl- cotransporters, and by removal of extracellular Na+. The results suggest that allopregnanolone enhances GABAergic transmission at the MPOA neurons by pre- and postsynaptic mechanisms. The enhancement of GABA release by allopregnanolone might require a high Cl- concentration in the presynaptic terminal maintained by Na+-dependent, bumetanide-sensitive mechanisms (e.g., Na+-K+-Cl- cotransporter) and might be mediated by Ca2+ influx into presynaptic terminal.     

TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na⁺-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of l-glutamate.