Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro

Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 microM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42+/-5% and 34+/-6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A(1) receptors in the subthalamic nucleus.     

Spontaneous synaptic activity is primarily GABAergic in vestibular nucleus neurons of the chick embryo

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibular reflexes. In 16-day embryos, the application of glutamate receptor antagonists abolished the postsynaptic responses generated on vestibular-nerve stimulation, but spontaneous synaptic activity was largely unaffected. Here, spontaneous synaptic activity was characterized in principal cells from brain slices at E16 using whole cell voltage-clamp recordings. With KCl electrodes, the frequency of spontaneous inward currents was 3.1 Hz at -60 mV, and the reversal potential was +4 mV. Cs-gluconate pipette solution allowed the discrimination of glycine/GABA(A) versus glutamate receptor-mediated events according to their different reversal potentials. The ratio for spontaneous excitatory to inhibitory events was about 1:4. Seventy-four percent of the outward events were GABA(A), whereas 26% were glycine receptor-mediated events. Both pre- and postsynaptic GABA(B) receptor effects were shown, with presynaptic GABA(B) receptors inhibiting 40% of spontaneous excitatory postsynaptic currents (sEPSCs) and 53% of spontaneous inhibitory postsynaptic currents (sIPSCs). With TTX, the frequency decreased approximately 50% for EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic activity recorded in the principal cells at E16 is primarily inhibitory, action potential-independent, and based on the activation of GABA(A) receptors that can be modulated by presynaptic GABA(B) receptors.     

Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo

We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 microM), kynurenic acid (1 mM), and bicuculline (10 microM) and were found to be sensitive to strychnine (1 microM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of approximately 0.04 Hz, which increased to approximately 0.5 Hz by 30-40 hpf, and was approximately 10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar ( approximately 100 pA), suggesting rapid formation of the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (approximately 0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (tau(off1) and tau(off2)) time courses with a half-width in 26-29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, tau(off1) was approximately 15 ms and tau(off2) was approximately 60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau(off1) was approximately 40 ms and tau(off2) was approximately 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) approximately 9 ms and tau(off2) approximately 40 ms, and in larvae, events declined somewhat further to tau(off1) approximately 4 ms and tau(off2) approximately 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as approximately 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3-10 mM) to outside-out patches obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.     

Activation of metabotropic glutamate receptors inhibits GABAergic transmission in the rat subfornical organ

Glutamate is known to increase neuronal excitability in the subfornical organ, a circumventricular organ devoid of the blood-brain barrier. To understand the synaptic mechanism of neuronal excitation by glutamate in this nucleus, we examined the effects of glutamate on GABAergic spontaneous inhibitory postsynaptic currents recorded from subfornical organ neurons in the rat brain slice. The baseline frequency, amplitude and decay time-constant of such spontaneous synaptic currents were 5.60 Hz, 119 pA and 17.3 ms, respectively. Glutamate (10-1000 microM) selectively inhibited the frequency of spontaneous GABAergic inhibitory postsynaptic currents (half-maximal effective concentration=47 microM) with little effects on their amplitudes and decay time constants. The inhibitory effect of glutamate on the frequency of spontaneous GABAergic postsynaptic currents was not blocked by tetrodotoxin (1 microM), or by the antagonists of ionotropic glutamate receptors. In contrast, such inhibitory effect of glutamate was mimicked by general or group II selective metabotropic glutamate receptor agonists such as DCGIV (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (half-maximal effective concentration=112 nM), but not by the agonists for group I or group III metabotropic glutamate receptors. Under current clamp mode, glutamate reduced the frequencies of spontaneous inhibitory postsynaptic potentials and action potentials in subfornical organ neurons. Our data indicate that glutamate decreases the frequency of spontaneous inhibitory postsynaptic currents by acting on the group II metabotropic glutamate receptors on axonal terminals in the subfornical organ. From these results we suggest that the glutamate-induced modulation of tonic GABAergic inhibitory synaptic activity can influence the excitability of subfornical organ neurons.     

Synaptically released GABA activates both pre- and postsynaptic GABA(B) receptors in the rat globus pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.     

GABA(B) modulation of GABA(A) and glycine receptor-mediated synaptic currents in hypoglossal motoneurons

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.     

High-affinity zinc potentiation of inhibitory postsynaptic glycinergic currents in the zebrafish hindbrain

Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC(50), 0.5-1 microM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR response in control conditions. Using tricine- (10 mM) buffered zinc solution, we then showed that zinc increases the amplitude of outside-out responses evoked by 0.1-0.5 mM glycine with an EC(50) of 15 nM. In contrast zinc had no effect on the amplitude of currents evoked by a saturating concentration (3-10 mM) of glycine. This suggests that zinc enhances GlyR apparent affinity for glycine. The study of the effects of zinc on the kinetics of the response indicates that this increase of apparent affinity is due to a decrease of the glycine dissociation rate constant. We then analyzed the effects of zinc on postsynaptic GlyRs in whole cell recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Chelation of contaminant heavy metals decreased the amplitude and the duration of the mIPSCs; inverse effects were observed by adding zinc in buffered solutions containing nanomolar free zinc concentrations. Zinc plus tricine or tricine alone did not change the coefficient of variation ( approximately 0.85) of the mIPSC amplitude distributions. These results suggest that postsynaptic GlyRs are not saturated after the release of one vesicle.     

Interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons

Whole cell voltage-clamp electrophysiology was used to examine interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons in primary culture. Membrane currents evoked by GABA and glycine were selectively inhibited by low concentrations of bicuculline and strychnine, respectively, suggesting that they activate pharmacologically distinct receptors. However, GABA- and glycine-mediated currents showed cross-inhibition when the two amino acids were applied sequentially. Application of one amino acid inhibited the response to immediate subsequent application of the other. In the majority of neurons, GABA inhibited subsequent glycine-evoked currents and glycine inhibited subsequent GABA-evoked currents. In a small proportion of neurons, however, GABA inhibited glycine-evoked currents but glycine had little effect on GABA-evoked currents. The reverse was true in other neurons, suggesting that alterations in chloride gradients alone did not account for the cross-inhibition. Furthermore, no cross-inhibition was observed between GABA- and glycine-evoked currents in some neurons. The amplitude of the current evoked by the coapplication of saturating concentrations of GABA and glycine in these neurons was nearly the sum of the currents evoked by GABA and glycine alone. In contrast, the currents were not additive in neurons demonstrating cross-inhibition. These results suggest that olfactory bulb neurons heterogeneously express a population of inhibitory amino acid receptors that can bind either GABA or glycine. Interactions between GABA and glycine at inhibitory amino acid receptors may provide a mechanism to modulate inhibitory synaptic transmission.     

The kinetics of inhibition of rat recombinant heteromeric α1β glycine receptors by the low-affinity antagonist SR-95531

The GABA_A antagonist SR-95531 (gabazine) is known to block glycine receptors, albeit with low affinity. We have studied the effect of SR-95531 on rat recombinant α1β glycine receptors expressed in human embryonic kidney (HEK293) cells by recording macroscopic currents elicited by rapid glycine application to outside-out patches. SR-95531 has a fast unbinding rate ( k _offSR, about 3000 s⁻¹) and this means that the time course of its unbinding is comparable to the expected time course of the transmitter in the cleft. We also found that equilibrium applications of SR-95531 reduced the response to brief glycine applications by an amount inversely proportional to the duration of glycine application. The fast unbinding rate of SR-95531 from the glycine receptor will make it useful for establishing the time course of glycine concentration at glycinergic synapses.     

Activity-dependent release of adenosine inhibits the glutamatergic synaptic transmission and plasticity in the hypothalamic hypocretin/orexin neurons

The hypocretin (orexin) neurons in the lateral hypothalamus play a crucial role in the promotion of arousal. Adenosine, an endogenous sleep-promoting factor, modulates both neuronal excitatory and synaptic transmission in the CNS. In this study, the involvement of endogenous adenosine in the regulation of excitatory glutamatergic synaptic transmission to hypocretin neurons was investigated in the hypothalamic slices from transgenic mice by using different frequencies of stimulation. A train of low-frequency stimulation (0.033, 1 Hz) had no effect on the amplitude of evoked excitatory postsynaptic currents (evEPSCs) in hypocretin neurons. Blockade of adenosine A1 receptors with selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT), the amplitude of evEPSCs did not change during 0.033 and 1 Hz stimuli. When the frequency of stimulation was increased upto 2 Hz, a time-dependent depression of amplitude was recorded in hypocretin neurons. Administration of CPT caused no significant change in depressed synaptic response induced by 2 Hz stimulus. While depression induced by 10 and 100 Hz stimuli was partially inhibited by the CPT but not by the selective A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Further findings have demonstrated that high-frequency stimulation could induce long-term potentiation (LTP) of glutamatergic synaptic transmission to hypocretin neurons in acute hypothalamic slices. The experiments with CPT suggested that A1 receptor antagonist could facilitate the induction of LTP, indicating that endogenous adenosine, acting through A1 receptors, may suppress the induction of LTP of excitatory synaptic transmission to hypocretin neurons. These results suggest that in the hypothalamus, endogenous adenosine will be released into extracellular space in an activity-dependent manner inhibiting both basal excitatory synaptic transmission and LTP in hypocretin neurons via A1 receptors. Our data provide further support for the notion that hypocretin neurons in the lateral hypothalamus may be another important target involved in the endogenous adenosine modulating the sleep and wakefulness cycle in the mammalian CNS.     

Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Induction of long-term potentiation without participation of N-methyl-D-aspartate receptors in kitten visual cortex.

1. Intracellular recording was made from layer II-III cells in slice preparations of kitten (30-40 days old) visual cortex. Low-frequency (0.1 Hz) stimulation of white matter (WM) usually evoked an excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP). The postsynaptic potentials (PSPs) showed strong dependence on stimulus frequency. Early component of EPSP and IPSP evoked by weak stimulation both decreased monotonically at frequencies greater than 0.5-1 Hz. Strong stimulation similarly depressed the early EPSP at higher frequencies (greater than 2 Hz) and replaced the IPSP with a late EPSP, which had a maximum amplitude in the stimulus frequency range of 2-5 Hz. 2. Very weak WM stimulation sometimes evoked EPSPs in isolation from IPSPs. The falling phase of the EPSP revealed voltage dependence characteristic to the responses mediated by N-methyl-D-aspartate (NMDA) receptors and was depressed by application of an NMDA antagonist DL-2-amino-5-phosphonovalerate (APV), whereas the rising phase of the EPSP was insensitive to APV. 3. The early EPSPs followed by IPSPs were insensitive to APV but were replaced with a slow depolarizing potential by application of a non-NMDA antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX), indicating that the early EPSP is mediated by non-NMDA receptors. The slow depolarization was mediated by NMDA receptors because it was depressed by membrane hyperpolarization or addition of APV. 4. The late EPSP evoked by higher-frequency stimulation was abolished by APV, indicating that it is mediated by NMDA receptors, which are located either on the recorded cell or on presynaptic cells to the recorded cells. 5. Long-term potentiation (LTP) of EPSPs was examined in cells perfused with solutions containing 1 microM bicuculline methiodide (BIM), a gamma-aminobutyric acid (GABA) antagonist. WM was stimulated at 2 Hz for 15 min as a conditioning stimulus to induce LTP, and the resultant changes were tested by low-frequency (0.1 Hz) stimulation of WM. 6. LTP of early EPSPs occurred in more than one-half of the cells (8/13) after strong conditioning stimulation. The rising slope of the EPSP was increased 1.6 times on average. 7. To test involvement of NMDA receptors in the induction of LTP in the early EPSP, the effect of conditioning stimulation was studied in a solution containing 100 microM APV, which was sufficient to block completely synaptic transmission mediated by NMDA receptors. LTP occurred in the same frequency and magnitude as in control solution.     

Slow inhibitory potentials in the teleost Mauthner cell

In vivo recordings from Mauthner cells in adult zebrafish (Danio rerio) and goldfish (Carassius auratus) preparations with potassium chloride filled electrodes revealed a new class of long-lasting synaptic events in these cells. Their decay time constant ranged from 20 to 80ms, which is about 20 times longer than that of previously identified fast glycinergic inhibitory postsynaptic potentials in this neuron. The average time to peak of these slow events ranged from 1 to 6ms. We demonstrated that they are also inhibitory since (i) they were resistant to antagonists of the excitatory glutamatergic receptors; (ii) their amplitude was increased following chloride loading of the Mauthner cell; (iii) their reversal potential was the same as that of fast, glycinergic inhibitory postsynaptic potentials; and (iv) they produced an inhibitory shunt of the cell's membrane resistance. Furthermore, as with the fast inhibitory postsynaptic potentials, the decay time of the slow events is voltage dependent, increasing when the Mauthner cell is depolarized. However, these inhibitory postsynaptic potentials had a different pharmacological profile to the fast glycinergic ones. That is, they persisted in the presence of strychnine at doses that abolished the fast ones and they were more sensitive to bicuculline. These data are compatible with the notion that these inhibitory postsynaptic potentials are mediated by activation of a different inhibitory receptor type, and may be GABAergic. In addition, the decay time constant of the fast inhibitory postsynaptic current was shorter than the first of the two components that contribute to the bi-exponential decay reported previously for miniature inhibitory postsynaptic currents in Mauthner cells of larval zebrafish. This suggests developmental modifications and/or a switch in the assembly of glycine receptor subtypes. While amplitude distributions of the fast miniature inhibitory postsynaptic potentials recorded in the presence of tetrodotoxin generally could fit with a single Gaussian function, the amplitude histograms of slow miniature events were skewed, often with multiple nearly equally spaced peaks, consistent with the synchronous release of several quantal units. These previously undescribed slow unitary inhibitory postsynaptic potentials contribute to inhibitory synaptic noise recorded in the Mauthner cells. Specifically, autocorrelation analysis revealed gamma-like rhythms (30-80Hz) in each of two phases, characterized as "noisy" and "quiet", and dominated by the fast and slow inhibitory postsynaptic potentials, respectively. The major frequencies of these two states were significantly different (i.e. around 90 and 40Hz, respectively), suggesting that the fast and slow inhibitory postsynaptic potentials are derived from different inhibitory networks. Chloride-filled Mauthner cells gradually hyperpolarized in the presence of tetrodotoxin, reflecting the effect of ongoing activity in the interneurons that produce the slow events. We conclude that this new class of inhibitory postsynaptic potentials contributes to the tonic inhibition which controls the Mauthner cell's excitability. In physiological conditions, this regulatory influence is expressed as a continuous shunt of this neuron's input resistance and responsiveness to sensory inputs.     

Serotonin modulates synaptic transmission in immature rat ventrolateral medulla neurons in vitro.

Patch-clamp recordings in whole-cell configuration were made from ventrolateral medulla neurons of brainstem slices from 8-12-day-old rats. 5-Hydroxytryptamine (3-30 microM) concentration-dependently suppressed excitatory and inhibitory postsynaptic currents evoked by focal stimulation. An augmentation of inhibitory synaptic currents by 5-hydroxytryptamine was noted in a small number of neurons. 5-Hydroxytryptamine depressed synaptic currents with or without causing a significant change in holding currents and membrane conductances; the inward or outward currents induced by exogenously applied glutamate or GABA/glycine were also not significantly changed by 5-hydroxytryptamine. In paired-pulse paradigms designed to evaluate a presynaptic site of action, 5-hydroxytryptamine suppressed synaptic currents but enhanced the paired-pulse facilitation. 5-Hydroxytryptamine reduced the frequency of miniature excitatory postsynaptic currents without significantly affecting the amplitude. 5-Carboxamidotryptamine, 8-hydroxy-2(di-n-propylamino)tetralin, sumatriptan and N-(3-trifluoromethylphenyl)piperazine which exhibit 5-hydroxytryptamine1 receptor agonist activity, depressed synaptic currents with different potencies, with 5-carboxamidotryptamine being the most potent. The non-selective 5-hydroxytryptamine1 receptor antagonist pindolol attenuated the presynaptic effect of 5-hydroxytryptamine, whereas the 5-hydroxytryptamine1A antagonist pindobind-5-hydroxytryptamine1A and 5-hydroxytryptamine2 receptor antagonist ketanserin were ineffective. Our results indicate that 5-hydroxytryptamine suppressed synaptic transmission in ventrolateral medulla neurons by activating presynaptic 5-hydroxytryptamine1 receptors, probably the 5-hydroxytryptamine1B/5-hydroxytryptamine1D subtype. In addition, 5-hydroxytryptamine augmented inhibitory synaptic currents in a small number of neurons the site and mechanism of this potentiating action are not known.     

Theta-frequency facilitation of AMPA receptor-mediated synaptic currents in the principal cells of the medial septum

Recent evidence suggests that Ca(2+)-permeable AMPA receptors display rapid, short-lasting current facilitation. In this study, we investigated the properties of AMPA receptor-mediated synaptic currents in medial septal neurons of the rat in an in vitro slice preparation. Immunocytochemistry with a selective antibody to the GluR2 subunit revealed that both choline acetyltransferase-containing and parvalbumin-containing neurons of the medial septum express no detectable GluR2 subunit immunoreactivity. We used whole cell voltage-clamp recordings to measure synaptically evoked AMPA receptor-mediated currents from medial septal neurons following stimulation of midline afferents. The GYKI 52466 (50 microM)- and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 microM)-sensitive AMPA receptor-mediated component of the synaptic response was isolated by blocking GABA(A)- and N-methyl-D-aspartate receptor-mediated currents with 30 microM bicuculline and 100 microM 2-amino-5-phosphonovaleric acid, respectively. In some cases, patched cells were filled with Lucifer yellow (0.1%) and imaged using 2-photon laser scanning microscopy. AMPA receptor-mediated currents that were observed in large medial septal neurons (20--30 microm) displayed rectification. These currents were sensitive to external application of philanthotoxin-343 (PhTx-343, 50 microM), a potent, high-affinity antagonist of Ca(2+)-permeable, GluR2-lacking AMPA receptors. Rectifying AMPA receptor-mediated currents also displayed a rapid increase in amplitude when evoked five times at low frequency such as 6 Hz. In contrast to currents observed in large medial septal neurons, AMPA-receptor mediated currents evoked in the remaining small (8--11 microm) neurons were nonrectifying and displayed rapid synaptic depression when stimulated five times at 6 Hz. The currents evoked in these cells were unaffected by external application of PhTx-343 and were therefore GluR2-containing AMPA receptors. The results of the present study demonstrate that the principal projection neurons of the medial septum contain PhTx-343-sensitive, GluR2-lacking AMPA receptors that display rapid current facilitation when stimulated at low frequencies.     

Transition from GABAergic to glycinergic synaptic transmission in newly formed spinal networks.

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential-independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABA(A) receptors (GlyR and GABA(A)R) were studied in spinal cord slices of 17- to 18-day-old embryos (E17-18) and 1- to 3-day-old postnatal rats (P1-3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABA(A)R-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABA(A) receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABA(A)R-mediated mIPSCs. The frequency of GABA(A)R-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17-18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABA(A)R-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17-18 to Gaussian mixtures with two to three discrete components at P1-3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABA(A) receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABA(A) receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.     

Presynaptic GABA(B) receptors inhibit synaptic inputs to rat subthalamic neurons.

Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude.These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.     

Muscarinic receptors depress GABAergic synaptic transmission in rat midbrain dopamine neurons

The effects of muscarine and nicotine on evoked and spontaneous release of GABA were studied using intracellular and whole-cell patch-clamp recordings from rat midbrain dopamine neurons in an in vitro slice preparation. Muscarine (30 microM) reversibly depressed the pharmacologically isolated inhibitory postsynaptic potential evoked by local electrical stimulation. The maximal inhibition of the inhibitory postsynaptic potential amplitude was 39.6+/-5%. This depressant effect of muscarine was blocked by the M3/M1 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (100 nM), but was slightly affected by the M1/M3 receptor antagonist pirenzepine (1 microM). In addition, muscarine decreased the frequency of the miniature synaptic currents without any effect on their amplitude. Moreover, muscarine did not change the GABA-induced hyperpolarization, indicating that its effect on the inhibitory postsynaptic potential is mediated by presynaptic receptors. On the contrary, the cholinergic agonist nicotine did not change the frequency or the amplitude of the spontaneous glutamatergic and GABAergic synaptic currents.Our data indicate that a prevalent activation of presynaptic M3 muscarinic receptors inhibits the GABA-mediated synaptic events, while the activation of nicotinic receptors does not affect the release of glutamate and GABA on midbrain dopamine neurons.     

Synaptic activation of kainate receptors on hippocampal interneurons

Although kainate receptor activation has been known to evoke epileptiform activity, little is known about the role of kainate receptors in synaptic transmission. Here we report that kainate (KA) receptors are present on interneurons and, when activated, cause a large increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) driven by action potentials. Stimulation of excitatory afferents generates a pharmacologically identifiable synaptic current mediated by KA receptors in interneurons. This synaptic current is similar to that mediated by AMPA receptors in its response to short stimulus trains, current-voltage relations and coefficient of variation, but it is much smaller in peak amplitude and much slower. KA application also considerably depresses evoked IPSCs. This depression seems to be in large part an indirect consequence of the repetitive firing evoked by the activation of the interneuronal somatic/dendritic KA receptors.