K. Krnjević

eIF2α phosphorylation bidirectionally regulates the switch from short to long-term synaptic plasticity and memory

The late phase of long-term potentiation (LTP) and memory (LTM) requires new gene expression, but the molecular mechanisms that underlie these processes are not fully understood. Phosphorylation of eIF2alpha inhibits general translation but selectively stimulates translation of ATF4, a repressor of CREB-mediated late-LTP (L-LTP) and LTM. We used a pharmacogenetic bidirectional approach to examine the role of eIF2alpha phosphorylation in synaptic plasticity and behavioral learning. We show that in eIF2alpha(+/S51A) mice, in which eIF2alpha phosphorylation is reduced, the threshold for eliciting L-LTP in hippocampal slices is lowered, and memory is enhanced. In contrast, only early-LTP is evoked by repeated tetanic stimulation and LTM is impaired, when eIF2alpha phosphorylation is increased by injecting into the hippocampus a small molecule, Sal003, which prevents the dephosphorylation of eIF2alpha. These findings highlight the importance of a single phosphorylation site in eIF2alpha as a key regulator of L-LTP and LTM formation.

Intracellular observations on the disinhibitory action of acetylcholine in the hippocampus

Abstract Intracellular recording (with KCl microelectrodes) from CA1 and CA3 hippocampal neurons in rats under urethane anaesthesia has revealed two kinds of facilitatory actions of acetylcholine (applied microiontophoretically). One was a mild depolarization (mean + 12 mV) accompanied by a rise in input resistance (mean + 14%). The reversal potential for this effect was much more negative than the resting potential, and it differed from the reversal potential of the inhibitory synaptic potential by a mean of 65 mV. It was therefore concluded that one action of acetylcholine tends to reduce K conductance, as in neocortical neurons. The second effect is a reduction in potency of inhibitory synaptic potentials—evoked by fimbrial or entorhinal stimulation—made evident by a 62% average reduction in the conductance increase recorded near the peak of inhibitory potentials. Since acetylcholine did not depress the inhibitory potency of iontophoretic applications of γ-aminobutyrate, it was concluded that acetylcholine must reduce the release of γ-aminobutyrate either by a direct action on inhibitory terminals or by inhibition of inhibitory interneurons. The former appears more likely.

Action of baclofen on mammalian synaptic transmission

Abstract Microiontophoretic and systemic injections were used to investigate the mechanism of baclofens powerful depressant action on transmission at primary afferent synapses in the cat. Iontophoretic applications depressed the spontaneous and evoked activity of cuneate cells and reduced the excitability and input resistance of spinal motoneurones. These effects, which were quick to reverse, resemble those of γ-aminobutyrate and may be due to activation of γ-aminobutyrate receptors by high concentrations of baclofen. Systemic doses of baclofen (0.1–5 mg/kg i.V.), which are known to give only a very low tissue concentration ( These observations are most simply explained if systemic baclofen blocks primary afferent synapses by a presynaptic action, which leads to a depression of transmitter release; this would be in keeping with evidence that, in cortical slices, baclofen selectively inhibits the release of excitatory amino acids.

mTORC2 controls actin polymerization required for consolidation of long-term memory

A major goal of biomedical research is the identification of molecular and cellular mechanisms that underlie memory storage. Here we report a previously unknown signaling pathway that is necessary for the conversion from short- to long-term memory. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which contains the regulatory protein Rictor (rapamycin-insensitive companion of mTOR), was discovered only recently and little is known about its function. We found that conditional deletion of Rictor in the postnatal murine forebrain greatly reduced mTORC2 activity and selectively impaired both long-term memory (LTM) and the late phase of hippocampal long-term potentiation (L-LTP). We also found a comparable impairment of LTM in dTORC2-deficient flies, highlighting the evolutionary conservation of this pathway. Actin polymerization was reduced in the hippocampus of mTORC2-deficient mice and its restoration rescued both L-LTP and LTM. Moreover, a compound that promoted mTORC2 activity converted early LTP into late LTP and enhanced LTM. Thus, mTORC2 could be a therapeutic target for the treatment of cognitive dysfunction.

Electrophysiological and pharmacological characteristics of facilitation of hippocampal population spikes by stimulation of the medial septum

In rats under urethane anesthesia, single shock or tetanic stimulation of the medial septum--which evoked only minimal field potentials--sharply enhanced population spikes evoked in area CA1 by commissural stimulation. An enhancement of population spikes was observed only (a) in areas CA1 and CA2 (adjacent to CA1 in the dorsal hippocampus), but not in the fascia dentata or the deep pyramidal layers CA3 or CA4; (b) in a narrow range of depth, close to the stratum pyramidale; (c) when the intensity of commissural stimulation was of adequate intensity. A comparable facilitation of population spikes was produced at the same sites by microiontophoretic release of acetylcholine. The septal facilitatory action increased in effectiveness with the number of tetanic pulses (up to 10-12) at a given frequency, and it had a maximum at frequencies of 50-100 Hz. It reached a maximum 20-50 ms after the end of septal stimulation, and then decayed slowly, the overall duration being up to 300 ms. The cholinergic nature of the facilitation induced by septal stimulation was confirmed by the parallel potentiation of septal action and that of acetylcholine by physostigmine and their depression by atropine and scopolamine.

Disinhibitory action of acetylcholine in the rat's hippocampus: Extracellular observations

Abstract In rats under urethane anaesthesia, fimbrial-commissural stimulation at a frequency of 1 Hz or less evoked only a positive field in the pyramidal layer of CA1 (and the uppermost region of CA3). Stimulation at intensities 3–5 times threshold and frequencies of 2 or more Hz led to the appearance of large negative population spikes. When acetylcholine was released in the pyramidal layer from a micropipette (10–100 nA), the positive field was reduced and population spikes appeared, even at the lowest frequencies of stimulation (below 1 Hz). Known antagonists of γ-aminobutyrate (bicuculline, picrotoxin and penicillin) had a comparable effect, but with a slower time course—the action of acetylcholine consistently had a very rapid onset and offset (within 5 s). Population spikes were not evoked by acetylcholine when the accompanying fimbrial stimulation was not strong enough to generate spikes at higher frequencies (2 or more Hz), or when acetylcholine was released outside the narrow pyramidal zone where large positive fields are recorded; nor were they evoked by the release of glutamate instead of acetylcholine. On the basis of these observations, it was concluded that acetylcholine probably diminishes the efficiency of synaptic inhibition of pyramidal cells.

EGTA and motoneuronal after-potentials.

1. Intracellular iontophoretic injections of EGTA (5‐‐20 nA) into cat spinal motoneurones consistently greatly reduce the amplitude of the delayed after hyperpolarization (a.h.p.) that follows the spike. 2. This effect is accompanied by a large reduction (on average by 3/4) in the marked increase in input conductance normally associated with the a.h.p. 3. There is also a consistent, though less regular, tendency for the resting input conductance to decrease (on average by 1/5), as well as some depolarization. 4. Recovery of the a.h.p., the associated conductance increase and the resting conductance is ver slow. It is sometimes accelerated by injections of citrate and Cl‐, or CA2+. 5. Other hyperpolarizing phenomena, such as recurrent or othodromically‐evoked i.p.s.p.s, are not depressed by injections of EGTA. 6. When depolarization is minimal EGTA injections that markedly depress the a.h.p. do not affect the rate of rise or fall of the spike. If, as a result of depolarization, an early a.h.p. is visible, it is patently insensitive to EGTA. 7. The post‐spike depolarizing after‐potential (delayed depolarization) is not obviously affected by EGTA, apart from the usual diminution seen during depolarization. 8. Since the main action of EGTA is to bind free Ca2+, the marked depression of the a.h.p. indicates that the sharp increase in K conductance which generates the a.h.p. is probably caused by a influx of Ca2+ accompanying the action potential. It is suggested that this inward Ca2+ current may be manifested in the depolarizing after‐potential.

Suppression of PKR Promotes Network Excitability and Enhanced Cognition by Interferon-γ-Mediated Disinhibition

The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr(-/-) phenotype in WT mice, enhancing long-term memory storage and L-LTP.

Inhibitory conductance changes and action of γ-aminobutyrate in rat hippocampus

Abstract Inhibitory synaptic potentials (IPSPs) and underlying resistance (and conductance) changes were examined in CA1 and CA3 hippocampal neurons in situ in rats. Comparable large conductance increases were evoked by fimbrial-commissural or entorhinal stimulation, and the corresponding inhibitory synaptic potentials had similar reversal potentials. The peak intensity and duration of the inhibitory synaptic potential resistance change were both greater under urethane than under ketamine anaesthesia. A striking rebound of potential and conductance, commonly seen at the end of the inhibitory synaptic potentials, was Cl − sensitive and could be ascribed to a prolonged suppression of ongoing inhibitory input. The time course of inhibitory synaptic potentials was significantly voltage-sensitive, being more prolonged for negative than for positive inhibitory synaptic potentials. Applications of γ-aminobutyrate produced large conductance increases (mean potency 4.2 nS/nA of γ-aminobutyrate iontophoretic current). There was excellent agreement between the reversal potentials for the inhibitory synaptic potential and for the effect of γ-aminobutyrate, recorded in the same cells. As elsewhere the effect of γ-aminobutyrate showed marked fading during applications lasting more than a few seconds. By contrast, glycine seldom caused a definite increase in conductance, but it appeared to depress inhibitory synaptic potentials, probably by an indirect action.

Ethanol Facilitates Glutamatergic Transmission to Dopamine Neurons in the Ventral Tegmental Area

The cellular mechanisms underlying alcohol addiction are poorly understood. In several brain areas, ethanol depresses glutamatergic excitatory transmission, but how it affects excitatory synapses on dopamine neurons of the ventral tegmental area (VTA), a crucial site for the development of drug addiction, is not known. We report here that in midbrain slices from rats, clinically relevant concentrations of ethanol (10–80 mM) increase the amplitude of evoked EPSCs and reduce their paired-pulse ratio in dopamine neurons in the VTA. The EPSCs were mediated by glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. In addition, ethanol increases the frequency but not the amplitude of spontaneous EPSCs. Furthermore, ethanol increases extracellular glutamate levels in the VTA of midbrain slices. The effects of ethanol are mimicked by SKF 38393, a dopamine D1 receptor agonist, and by GBR 12935, a dopamine reuptake inhibitor, and they are blocked by SKF 83566, a D1 antagonist, or by reserpine, which depletes dopamine stores. The enhancement of sEPSC frequency reaches a peak with 40 mM ethanol and declines with concentrations ⩾80 mM ethanol, which is quite likely a result of D2 receptor activation as raclopride, a D2 receptor blocker, significantly enhanced 80 mM ethanol-induced enhancement of sEPSCs. Finally, 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), an AMPA receptor antagonist, attenuates ethanol-induced excitation of VTA DA neurons. We therefore conclude that, acting via presynaptic D1 receptors, ethanol at low concentrations increases glutamate release in the VTA, thus raising somatodendritic dopamine release, which further activates the presynaptic D1 receptors. Enhancement of this positive feedback loop may significantly contribute to the development of alcohol addiction.