Washington Buño

Cholinergic-mediated IP3-receptor activation induces long-lasting synaptic enhancement in CA1 pyramidal neurons

Cholinergic–glutamatergic interactions influence forms of synaptic plasticity that are thought to mediate memory and learning. We tested in vitro the induction of long-lasting synaptic enhancement at Schaffer collaterals by acetylcholine (ACh) at the apical dendrite of CA1 pyramidal neurons and in vivo by stimulation of cholinergic afferents. In vitro ACh induced a Ca2+ wave and synaptic enhancement mediated by insertion of AMPA receptors in spines. Activation of muscarinic ACh receptors (mAChRs) and Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores were required for this synaptic enhancement that was insensitive to blockade of NMDA receptors and also triggered by IP3 uncaging. Activation of cholinergic afferents in vivo induced an analogous atropine-sensitive synaptic enhancement. We describe a novel form of synaptic enhancement (LTPIP3) that is induced in vitro and in vivo by activation of mAChRs. We conclude that Ca2+ released from postsynaptic endoplasmic reticulum stores is the critical event in the induction of this unique form of long-lasting synaptic enhancement.

Selective muscarinic regulation of functional glutamatergic Schaffer collateral synapses in rat CA1 pyramidal neurons

Analysis of the cholinergic regulation of glutamatergic neurotransmission is an essential step in understanding the hippocampus because it can influence forms of synaptic plasticity that are thought to underlie learning and memory. We studied in vitro the cholinergic regulation of excitatory postsynaptic currents (EPSCs) evoked in rat CA1 pyramidal neurons by Schaffer collateral (SC) stimulation. Using ‘minimal’ stimulation, which activates one or very few synapses, the cholinergic agonist carbamylcholine (CCh) increased the failure rate of functional more (36 %) than of silent synapses (7 %), without changes in the EPSC amplitude. These effects of CCh were insensitive to manipulations that increased the probability of release, such as paired pulse facilitation, increases in temperature and increases in the extracellular Ca2+ : Mg2+ ratio. Using ‘conventional’ stimulation, which activates a large number of synapses, CCh inhibited more the pharmacologically isolated non‐NMDA (86 %) than the NMDA (47 %) EPSC. The changes in failure rate, EPSC variance and the increased paired pulse facilitation that paralleled the inhibition imply that CCh decreased release probability. Muscarine had similar effects. The inhibition by both CCh and by muscarine was prevented by atropine. We conclude that CCh reduces the non‐NMDA component of SC EPSCs by selectively inhibiting transmitter release at functional synapses via activation of muscarinic receptors. The results suggest that SCs have two types of terminals, one in functional synapses, selectively sensitive to regulation through activation of muscarinic receptors, and the other in silent synapses less sensitive to that regulation. The specific inhibition of functional synapses would favour activity‐dependent plastic phenomena through NMDA receptors at silent synapses without the activation of non‐NMDA receptors and functional synapses.

Presynaptic inhibition of Schaffer collateral synapses by stimulation of hippocampal cholinergic afferent fibres

It has been known for decades that muscarinic agonists presynaptically inhibit Schaffer collateral synapses contacting hippocampal CA1 pyramidal neurons. However, a demonstration of the inhibition of Schaffer collateral synapses induced by acetylcholine released by cholinergic hippocampal afferents is lacking. We present original results showing that electrical stimulation at the stratum oriens/alveus with brief stimulus trains inhibited excitatory postsynaptic currents evoked by stimulation of Schaffer collaterals in CA1 pyramidal neurons of rat hippocampal slices. The increased paired‐pulse facilitation and the changes in the variance of excitatory postsynaptic current amplitude that paralleled the inhibition suggest that it was mediated presynaptically. The effects of oriens/alveus stimulation were inhibited by atropine, and blocking nicotinic receptors with methyllycaconitine was ineffective, suggesting that the inhibition was mediated via the activation of presynaptic muscarinic receptors. The results provide a novel demonstration of the presynaptic inhibition of glutamatergic neurotransmission by cholinergic fibres in the hippocampus, implying that afferent cholinergic fibres regulate the strength of excitatory synaptic transmission.

The Muscarinic Long-Term Enhancement of NMDA and AMPA Receptor-Mediated Transmission at Schaffer Collateral Synapses Develop through Different Intracellular Mechanisms

We had described a muscarinic-mediated long-term synaptic enhancement at Schaffer collateral synapses caused by the insertion of AMPARs in spines of rat hippocampal CA1 pyramidal neurons that requires Ca2+ release from IP3-sensitive stores (Fernández de Sevilla et al., 2008). We now show that this AMPA-mediated LTPIP3 is precisely matched by an amplification of NMDAR-mediated transmission. The enhanced AMPAR transmission involves SNARE protein activity and CaMKII activation. The amplification of NMDA transmission requires combined CaMKII, PKC, and SRC kinase activity without detectable surface incorporation of NMDARs, suggesting that changes in receptor properties mediate this process. The enhanced AMPAR- and NMDAR-mediated transmission markedly reduce the induction threshold of “Hebbian” LTP. We conclude that both modes of glutamatergic synaptic potentiation may play a critical functional role in the regulation of the learning machinery of the brain by adding flexibility to the demands of the hippocampal network.

Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and fluctuations entrained by sinusoidal injected current

The mechanisms mediating intrinsic and entrained CA1 pyramidal neuron rhythmic membrane potential oscillations were investigated in rat hippocampal slices. Intrinsic oscillations (6–14 Hz, < 10 mV) were evoked by long duration (2 s), depolarizing current pulses in 42% of the cells. Oscillations were also evoked by imposing sinusoidal transmembrane currents at 2, 7, and 14 Hz, adjusted at 7 Hz to imitate the synaptically mediated in vivo “intracellular theta”. Slow all-or-none events (40 mV, 55 ms) — reminiscent of the rhythmic, high threshold slow spikes observed in vivo — were evoked and entrained by the sine wave current cycles with large, imposed depolarization in 35% of the cells. Intrinsic oscillations were insensitive to Ca2+-free, Co2+ (2 mM) and Mn2+ (2 mM) solutions, but were blocked by tetrodotoxin (TTX; 5 μM), illustrating that they were Na+-mediated. Tetraethylammonium (TEA; 15 mM) unmasked slow all-or-none events (40–50 mV, 20–55 ms) and plateau potentials (40–60 mV, 100–700 ms). Plateaus were Co2+ and Mn2+ resistant and were abolished by TTX, hence suggesting that the underlying persistent conductance was Na+-mediated. Plateaus were entrained one-to-one at all sinusoidal current frequencies in Ca2+-free, TEA+Co2+, or TEA+Mn2+ solutions. However, the high threshold Ca2+ spikes uncovered in TEA+TTX could only follow sinusoidal currents of less than 7 Hz. In conclusion, the high threshold Ca2+ and persistent Na+ conductances coexist in CA1 pyramidal cells. The persistent Na+ conductance mediated the intrinsic oscillations, and fluctuated at all the sine wave current frequencies used. The more sluggish high-threshold Ca2+ conductance exclusively oscillated at frequencies of less than 7 Hz and did not support the intrinsic rhythm. Therefore, the findings suggest that the Na+-mediated oscillations may contribute to the high-frequency, type I, hippocampal theta rhythm present in vivo, whereas the high threshold Ca2+ conductance may take part in the low-frequency, type II rhythm.

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

Exploring the principles that govern activity‐dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity‐dependent potentiation of the slow Ca2+‐activated K+ current (sIAHP) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sIAHP using the slice technique and voltage‐clamp recordings with sharp or patch‐electrodes. Using sharp electrodes‐repeated activation with depolarizing pulses evoked a prolonged (8‐min) potentiation of the amplitude (171%) and duration (208%) of the sIAHP. Using patch electrodes, early after entering the whole‐cell configuration (<20 min), responses were as those reported above. However, although the sIAHP remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L‐type Ca2+ current by nifedipine (20 μM) markedly reduced the sIAHP (79%) and its potentiation (55%). Ryanodine (20 μM) that blocks the release of intracellular Ca2+ also reduced sIAHP (29%) and its potentiation (25%). The potentiation of the sIAHP induced a marked and prolonged (>50%; ≈8 min) decrease in excitability. The results suggest that sIAHP is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage‐gated L‐type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+‐binding capacity of the channels mediating the sIAHP. This potentiation of the sIAHP could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long‐term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs. Hippocampus 10:198–206, 2000

Changes of the EPSP Waveform Regulate the Temporal Window for Spike-Timing-Dependent Plasticity

Using spike-timing-dependent plasticity (STDP) protocols that consist of pairing an EPSP and a postsynaptic backpropagating action potential (BAP), we investigated the contribution of the changes in EPSP waveform induced by the slow Ca2+-dependent K+-mediated afterhyperpolarization (sAHP) in the regulation of long-term potentiation (LTP). The “temporal window” between Schaffer collateral EPSPs and BAPs in CA1 pyramidal neurons required to induce LTP was narrowed by a reduction of the amplitude and decay time constant of the EPSP, which could be reversed with cyclothiazide. The EPSP changes were caused by the increased conductance induced by activation of the sAHP. Therefore, the EPSP waveform and its regulation by the sAHP are central in determining the duration of the temporal window for STDP, thus providing a possible dynamic regulatory mechanism for the encoding of cognitive processes.

Insulin-like growth factor I potentiates kainate receptors through a phosphatidylinositol 3-kinase dependent pathway.

Neurotrophic factors modulate synaptic plasticity through mechanisms that include regulation of membrane ion channels and neurotransmitter receptors. Recently, it was shown that insulin-like growth factor I (IGF-I) induces depression of AMPA-mediated currents without affecting NMDA-receptor function in neurons. We now report that IGF-I markedly potentiates the kainate-preferring ionotropic glutamate receptor in young cerebellar granule neurons expressing functional kainate-, but not AMPA-mediated currents. Potentiation of kainate responses by IGF-I is blocked by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating a role for this kinase in the effect of IGF-I. These results reinforce the notion that modulation of ionotropic glutamate receptors are involved in the regulatory actions of IGF-I on neuronal plasticity.

In vivo electrophysiological analysis of lucifer yellow-coupled hippocampal pyramids

Small transient all-or-none depolarizations (also termed in the literature fast prepotentials, spikelets, pseudospikes, d-spikes, or short latency depolarizations) and their association with lucifer yellow (LY) dye-coupling were analyzed in CA1-CA3 hippocampal pyramidal cells in urethane anesthetized rats. It was found that (a) 15 of the 24 LY-injected pyramidal neurons (63%) showed dye-coupling; (b) spontaneous, anti- and orthodromically evoked spikelets (3-7 ms in duration; 3- to 12-mV peak) were recorded in 40 of 95 cells (42%); (c) there was a significantly higher probability of dye-coupled neurons with spikelets and of uncoupled ones without spikelets; (d) spikelet waveform and amplitude were unaffected by spontaneous or imposed polarizations; (e) large hyperpolarizations could reduce the rate and even prevent spikelets; and (f) spikelets could precede or follow spikes, the latter were more frequent with large depolarizations. Electrophysiological findings, and the association of dye-coupling and spikelets, suggest strongly that at least some spikelets are coupling potentials. This implies that pyramidal cells may be electronically coupled under physiological conditions.

Neurotransmitter actions on oral pontine tegmental neurons of the rat: an in vitro study

The actions of neurotransmitters involved in the sleep-wakefulness cycle on neurons located in the ventral part of the oral pontine tegmentum were studied in a rat brain-slice preparation. Results show that glutamate and histamine evoke depolarizations and spike firing while serotonin and gamma-aminobutyric acid evoke hyperpolarizations. The excitatory and inhibitory actions of these neurotransmitters increase pontine neuron activity during the conditions occurring during paradoxical sleep.