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.

Ethanol drives aversive conditioning through dopamine 1 receptor and glutamate receptor-mediated activation of lateral habenula neurons.

There has been increasing interest in the lateral habenula (LHb) given its potent regulatory role in many aversion-related behaviors. Interestingly, ethanol can be rewarding as well as aversive; we therefore investigated whether ethanol exposure alters pacemaker firing or glutamate receptor signaling in LHb neurons in vitro and also whether LHb activity in vivo might contribute to the acquisition of conditioned place aversion to ethanol. Surprisingly, in epithalamic slices, low doses of ethanol (1.4 mM) strongly accelerated LHb neuron firing (by ~60%), and ethanol's effects were much reduced by blocking glutamate receptors. Ethanol increased presynaptic glutamate release, and about half of this effect was mediated by dopamine subtype 1 receptors (D1Rs) and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In agreement with these findings, c-Fos immunoreactivity in LHb regions was enhanced after a single administration of a low dose of ethanol (0.25 g/kg i.p.). Importantly, the same dose of ethanol in vivo also produced strong conditioned place aversion, and this was prevented by inhibiting D1Rs or neuronal activity within the LHb. By contrast, a higher dose (2 g/kg) led to ethanol conditioned place preference, which was enhanced by inhibiting neuronal activity or D1Rs within the LHb and suppressed by infusing aminomethylphosphonic acid or the D1R agonist SKF38393 within the LHb. Our in vitro and in vivo observations show, for the first time, that ethanol increases LHb excitation, mediated by D1R and glutamate receptors, and may underlie a LHb aversive signal that contributes to ethanol-related aversion.

The Long and Winding Road to Gamma-Amino-Butyric Acid as Neurotransmitter.

This review centers on the discoveries made during more than six decades of neuroscience research on the role of gamma-amino-butyric acid (GABA) as neurotransmitter. In doing so, special emphasis is directed to the significant involvement of Canadian scientists in these advances. Starting with the early studies that established GABA as an inhibitory neurotransmitter at central synapses, we summarize the results pointing at the GABA receptor as a drug target as well as more recent evidence showing that GABAA receptor signaling plays a surprisingly active role in neuronal network synchronization, both during development and in the adult brain. Finally, we briefly address the involvement of GABA in neurological conditions that encompass epileptic disorders and mental retardation.

Salsolinol stimulates dopamine neurons in slices of posterior ventral tegmental area indirectly by activating µ-opioid receptors.

Previous studies in vivo have shown that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse. Although opioid receptors, especially the µ-opioid receptors (MORs), may be involved, the cellular mechanisms mediating the effects of salsolinol have not been fully explored. In the current study, we used whole-cell patch-clamp recordings to examine the effects of salsolinol on dopamine neurons of the ventral tegmental area (VTA) in acute brain slices from Sprague-Dawley rats. Salsolinol (0.01-1 µM) dose-dependently and reversibly increased the ongoing firing of dopamine neurons; this effect was blocked by naltrexone, an antagonist of MORs, and gabazine, an antagonist of GABA(A) receptors. We further showed that salsolinol reduced the frequency without altering the amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents in dopamine neurons. The salsolinol-induced reduction was blocked by both naltrexone and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, an agonist of MORs. Thus, salsolinol excites VTA-dopamine neurons indirectly by activating MORs, which inhibit GABA neurons in the VTA. This form of disinhibition seems to be a novel mechanism underlying the effects of salsolinol.

GABAergic actions mediate opposite ethanol effects on dopaminergic neurons in the anterior and posterior ventral tegmental area.

It is known that the posterior ventral tegmental area (p-VTA) differs from the anterior VTA (a-VTA) in that rats learn to self-administer ethanol into the p-VTA, but not into the a-VTA. Because activation of VTA dopaminergic neurons by ethanol is a cellular mechanism underlying the reinforcement of ethanol consumption, we hypothesized that ethanol may exert different effects on dopaminergic neurons in the p-VTA and a-VTA. In patch-clamp recordings in midbrain slices from young rats (postnatal days 22-32), we detected no significant difference in electrophysiological properties between p-VTA and a-VTA dopaminergic neurons. However, acute exposure to ethanol (21-86 mM) stimulated p-VTA dopaminergic neurons but suppressed a-VTA dopaminergic neurons. Conversely, ethanol (>21 mM) dose-dependently reduced the frequency of the GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) generated by inhibitory neuronal firing but not miniature inhibitory postsynaptic currents (mIPSCs) in p-VTA dopaminergic neurons. By contrast, ethanol increased the frequency and amplitude of both sIPSCs and mIPSCs in a-VTA dopaminergic neurons. All of these effects of ethanol were abolished by a GABA(A) receptor antagonist. There was a strong negative correlation between ethanol-evoked modulation of sIPSCs and neuronal firing in VTA dopaminergic neurons. These results indicate that GABAergic inputs play an important role in ethanol's actions in the VTA. The differential effects of ethanol on sIPSCs and neuronal firing in the p-VTA and a-VTA could be the basis for ethanol reinforcement via the p-VTA.

When and why amino acids?

This article reviews especially the early history of glutamate and GABA as neurotransmitters in vertebrates. The proposal that some amino acids could mediate synaptic transmission in the CNS initially met with much resistance. Both GABA and its parent glutamate are abundant in the brain; but, unlike glutamate, GABA had no obvious metabolic function. By the late 1950s, the switch of interest from electrical to chemical transmission invigorated the search for central transmitters. Its identification with Factor I, a brain extract that inhibited crustacean muscle, focused interest on GABA as a possible inhibitory transmitter. In the first microiontophoretic tests, though GABA strongly inhibited spinal neurons, these effects were considered 'non-specific'. Strong excitation by glutamate (and other acidic amino acids) led to the same conclusion. However, their great potency and rapid actions on cortical neurons convinced other authors that these endogenous amino acids are probably synaptic transmitters. This was partly confirmed by showing that both IPSPs and GABA greatly increased Cl() conductance, their effects having similar reversal potentials. Many anticonvulsants proving to be GABA antagonists, by the 1970s GABA became widely accepted as a mediator of IPSPs. Progress was much slower for glutamate. Being generated on distant dendrites, EPSPs could not be easily compared with glutamate-induced excitation, and the search for specific antagonists was long hampered by the lack of blockers and the variety of glutamate receptors. These difficulties were gradually overcome by the application of powerful techniques, such as single channel recording, cloning receptors, as well as new pharmacological tools.

Propofol facilitates glutamatergic transmission to neurons of the ventrolateral preoptic nucleus.

BACKGROUND: There is much evidence that the sedative component of anesthesia is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors on hypothalamic neurons responsible for arousal, notably in the tuberomammillary nucleus. These GABA(A) receptors are targeted by gamma-aminobutyric acid-mediated (GABAergic) neurons in the ventrolateral preoptic area (VLPO): When these neurons become active, they inhibit the arousal-producing nuclei and induce sleep. According to recent studies, propofol induces sedation by enhancing VLPO-induced synaptic inhibition, making the target cells more responsive to GABA(A). The authors explored the possibility that propofol also promotes sedation less directly by facilitating excitatory inputs to the VLPO GABAergic neurons. METHODS: Spontaneous excitatory postsynaptic currents were recorded from VLPO cells-principally mechanically isolated, but also in slices from rats. RESULTS: In isolated VLPO GABAergic neurons, propofol increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents without affecting their mean amplitude. The action of propofol was mimicked by muscimol and prevented by gabazine, respectively a specific agonist and antagonist at GABA(A) receptors. It was also suppressed by bumetanide, a blocker of Na-K-Cl cotransporter-mediated inward Cl transport. In slices, propofol also increased the frequency of spontaneous excitatory postsynaptic currents and, at low doses, accelerated firing of VLPO cells. CONCLUSION: Propofol induces sedation, at least in part, by increasing firing of GABAergic neurons in the VLPO, indirectly by activation of GABA(A) receptors on glutamatergic afferents: Because these axons/terminals have a relatively high internal Cl concentration, they are depolarized by GABAergic agents such as propofol, which thus enhance glutamate release.

Electrophysiology of cerebral ischemia.

Organized brain activity requires the coordinated firing of vast numbers of nerve cells. To maintain this, all these cells must be adequately polarized, their axons capable of conducting action potentials and releasing transmitters at an even greater numbers of synapses. Hence the often dire consequences of any interruption in the normal supply of O(2) and glucose. Initially, though both cognitive and synaptic functions are soon suppressed, membrane potentials in the brain change little -- indeed, many neurons are hyperpolarized -- and all these effects are fully reversible when glucose and/or O(2) supplies are restored. The early events, suppression of synaptic and cognitive function, sharply reduce the brain's needs of energy, enabling it to maintain the minimal metabolism required for survival. Even this minimum cannot be sustained for more than a few minutes: if ischemia is prolonged, a slowly progressive depolarization (mainly caused by glutamate release) suddenly accelerates owing to the activation of several inward currents. The resulting near-total depolarization and large increase in Ca(2+) influx -- as well as Ca(2+) release from internal stores (including mitochondria) -- leads to a rapid rise in cytoplasmic [Ca(2+)]. As long as this does not reach the critical level that triggers the irreversible processes leading to cell death, restoring energy supplies reactivates the membrane pumps that re-establish normal ionic gradients and membrane potentials, and thus make possible the return of synaptic and cognitive functions, Rapid advances in knowledge suggest a wide spectrum of agents potentially capable of delaying or even preventing irreversible outcomes of brain ischemia.

Ethanol Withdrawal Drives Anxiety-Related Behaviors by Reducing M-type Potassium Channel Activity in the Lateral Habenula.

Alcohol use disorders (AUDs) and anxiety disorders (ADs) are often seen concurrently, but their underlying cellular basis is unclear. For unclear reasons, the lateral habenula (LHb), a key brain region involved in the pathophysiology of ADs, becomes hyperactive after ethanol withdrawal. M-type K+ channels (M-channels), important regulators of neuronal activity, are abundant in the LHb, yet little is known about their role in AUDs and associated ADs. We report here that in rats at 24 h withdrawal from systemic ethanol administration (either by intraperitoneal injection, 2 g/kg, twice/day, for 7 days; or intermittent drinking 20% ethanol in a two-bottle free choice protocol for 8 weeks), the basal firing rate and the excitability of LHb neurons in brain slices was higher, whereas the amplitude of medium afterhyperpolarization and M-type K+ currents were smaller, when compared to ethanol naive rats. Concordantly, M-channel blocker (XE991)-induced increase in the spontaneous firing rate in LHb neurons was smaller. The protein expression of M-channel subunits, KCNQ2/3 in the LHb was also smaller. Moreover, anxiety levels (tested in open field, marble burying, and elevated plus maze) were higher, which were alleviated by LHb inhibition either chemogenetically or by local infusion of the M-channel opener, retigabine. Intra-LHb infusion of retigabine also reduced ethanol consumption and preference. These findings reveal an important role of LHb M-channels in the expression of AUDs and ADs, and suggest that the M-channels could be a potential therapeutic target for alcoholics.

Ethanol potentiates both GABAergic and glutamatergic signaling in the lateral habenula.

Ethanol's aversive property may limit it's use, but the underlying mechanisms are no well-understood. Emerging evidence suggests a critical role for the lateral habenula (LHb) in the aversive response to various drugs, including ethanol. We previously showed that ethanol enhances glutamatergic transmission and stimulates LHb neurons. GABAergic transmission, a major target of ethanol in many brain regions, also tightly regulates LHb activity. This study assessed the action of ethanol on LHb GABAergic transmission in rat brain slices. Application of ethanol accelerated spontaneous action potential firing of LHb neurons, and LHb activity was increased by the GABAA receptor antagonist gabazine, and ethanol-induced acceleration of LHb firing was further increased by gabazine. Additionally, ethanol potentiated GABAergic transmission (inhibitory postsynaptic currents, IPSCs) with an EC50 of 1.5 mM. Ethanol-induced potentiation of IPSCs was increased by a GABAB receptor antagonist; it was mimicked by dopamine, dopamine receptor agonists, and dopamine reuptake blocker, and was completely prevented by reserpine, which depletes store of catecholamine. Moreover, ethanol-induced potentiation of IPSCs involved cAMP signaling. Finally, ethanol enhanced simultaneously glutamatergic and GABAergic transmissions to the majority of LHb neurons: the potentiation of the former being greater than that of the latter, the net effect was increased firing. Since LHb excitation may contribute to aversion, ethanol-induced potentiation of GABAergic inhibition tends to reduce aversion.

Mesencephalic astrocyte-derived neurotrophic factor enhances nigral gamma-aminobutyric acid release.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) - one of a new class of astrocyte-derived human proteins--selectively promotes the survival of dopamine neurons of the ventral midbrain. Using the whole-cell clamp technique, we looked for acute effects of MANF on gamma-aminobutyric acid type A (GABAA) receptor-mediated inhibitory postsynaptic currents (IPSCs) in dopamine neurons of the substantia nigra pars compacta of 6 to 15-day-old rats. In slices, MANF increased the amplitude of evoked IPSCs and decreased the paired pulse ratio. In mechanically dissociated cells, MANF increased the frequency of spontaneous and miniature IPSCs, without changing their mean amplitudes; and in enzymatically dissociated neurons, MANF had no effect on currents induced by exogenous GABA. The presynaptic enhancement of GABAergic inhibition may contribute to MANF's protective action on dopamine cells.

eIF2α-mediated translational control regulates the persistence of cocaine-induced LTP in midbrain dopamine neurons.

Recreational drug use leads to compulsive substance abuse in some individuals. Studies on animal models of drug addiction indicate that persistent long-term potentiation (LTP) of excitatory synaptic transmission onto ventral tegmental area (VTA) dopamine (DA) neurons is a critical component of sustained drug seeking. However, little is known about the mechanism regulating such long-lasting changes in synaptic strength. Previously, we identified that translational control by eIF2α phosphorylation (p-eIF2α) regulates cocaine-induced LTP in the VTA (Huang et al., 2016). Here we report that in mice with reduced p-eIF2α-mediated translation, cocaine induces persistent LTP in VTA DA neurons. Moreover, selectively inhibiting eIF2α-mediated translational control with a small molecule ISRIB, or knocking down oligophrenin-1-an mRNA whose translation is controlled by p-eIF2α-in the VTA also prolongs cocaine-induced LTP. This persistent LTP is mediated by the insertion of GluR2-lacking AMPARs. Collectively, our findings suggest that eIF2α-mediated translational control regulates the progression from transient to persistent cocaine-induced LTP.

Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine.

Adolescents are especially prone to drug addiction, but the underlying biological basis of their increased vulnerability remains unknown. We reveal that translational control by phosphorylation of the translation initiation factor eIF2α (p-eIF2α) accounts for adolescent hypersensitivity to cocaine. In adolescent (but not adult) mice, a low dose of cocaine reduced p-eIF2α in the ventral tegmental area (VTA), potentiated synaptic inputs to VTA dopaminergic neurons, and induced drug-reinforced behavior. Like adolescents, adult mice with reduced p-eIF2α-mediated translational control were more susceptible to cocaine-induced synaptic potentiation and behavior. Conversely, like adults, adolescent mice with increased p-eIF2α became more resistant to cocaine's effects. Accordingly, metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD)-whose disruption is postulated to increase vulnerability to drug addiction-was impaired in both adolescent mice and adult mice with reduced p-eIF2α mediated translation. Thus, during addiction, cocaine hijacks translational control by p-eIF2α, initiating synaptic potentiation and addiction-related behaviors. These insights may hold promise for new treatments for addiction.

Nicotine regulates activity of lateral habenula neurons via presynaptic and postsynaptic mechanisms.

There is much interest in brain regions that drive nicotine intake in smokers. Interestingly, both the rewarding and aversive effects of nicotine are probably critical for sustaining nicotine addiction. The medial and lateral habenular (LHb) nuclei play important roles in processing aversion, and recent work has focused on the critical involvement of the LHb in encoding and responding to aversive stimuli. Several neurotransmitter systems are implicated in nicotine's actions, but very little is known about how nicotinic acetylcholine receptors (nAChRs) regulate LHb activity. Here we report in brain slices that activation of nAChRs depolarizes LHb cells and robustly increases firing, and also potentiates glutamate release in LHb. These effects were blocked by selective antagonists of α6-containing (α6*) nAChRs, and were absent in α6*-nAChR knockout mice. In addition, nicotine activates GABAergic inputs to LHb via α4ß2-nAChRs, at lower concentrations but with more rapid desensitization relative to α6*-nAChRs. These results demonstrate the existence of diverse functional nAChR subtypes at presynaptic and postsynaptic sites in LHb, through which nicotine could facilitate or inhibit LHb neuronal activity and thus contribute to nicotine aversion or reward.

Translational control of nicotine-evoked synaptic potentiation in mice and neuronal responses in human smokers by eIF2α.

Adolescents are particularly vulnerable to nicotine, the principal addictive component driving tobacco smoking. In a companion study, we found that reduced activity of the translation initiation factor eIF2α underlies the hypersensitivity of adolescent mice to the effects of cocaine. Here we report that nicotine potentiates excitatory synaptic transmission in ventral tegmental area dopaminergic neurons more readily in adolescent mice compared to adults. Adult mice with genetic or pharmacological reduction in p-eIF2α-mediated translation are more susceptible to nicotine's synaptic effects, like adolescents. When we investigated the influence of allelic variability of the Eif2s1 gene (encoding eIF2α) on reward-related neuronal responses in human smokers, we found that a single nucleotide polymorphism in the Eif2s1 gene modulates mesolimbic neuronal reward responses in human smokers. These findings suggest that p-eIF2α regulates synaptic actions of nicotine in both mice and humans, and that reduced p-eIF2α may enhance susceptibility to nicotine (and other drugs of abuse) during adolescence.

Presynaptic glycine receptors on GABAergic terminals facilitate discharge of dopaminergic neurons in ventral tegmental area.

GABA-mediated postsynaptic currents (IPSCs) were recorded from dopaminergic (DA) neurons of the ventral tegmental area (VTA) of rats, in acute brain slices, and from enzymatically or mechanically dissociated neurons. In young rats (3-10 d of age), where GABA is excitatory, glycine (1-3 microm) and taurine (10-30 microm) increased the amplitude of evoked IPSCs (eIPSCs) and the frequency of spontaneous IPSCs (sIPSCs) but had minimal postsynaptic effects. Strychnine (1 microm) blocked the action of glycine; when applied alone, it reduced the amplitude of eIPSCs and the frequency of sIPSCs, indicating a tonic facilitation of GABAergic excitation by some endogenous glycine agonist(s). In medium containing no Ca2+, or with Cd2+ or tetrodotoxin added, the amplitude and especially the frequency of sIPSCs greatly diminished. In many cells, glycine had no effect on remaining miniature IPSCs, suggesting a preterminal site of glycine receptors (GlyRs). Fura-2 fluorescent imaging showed a glycine-induced increase of [Ca2+] in nerve terminals (on DA neurons), which was suppressed by strychnine or 3 microm omega-conotoxin MVIIA. Therefore, the presynaptic GlyR-mediated facilitation of GABAergic transmission seems to be mediated by N- and/or P/Q-type Ca2+ channels. In older rats (22-30 d of age), where GABA causes inhibition, the effect of strychnine on GABAergic IPSCs was reversed to facilitation, indicating a tonic glycinergic inhibition of GABA release. Furthermore, glycine (1-3 microm) reduced the amplitude of eIPSCs and the frequency of sIPSCs. Hence, the overall effect of the presynaptic action of glycine is to enhance the firing of DA cells, both in very young and older rats.

From cAMP to adenosine: an illuminating shift of focus.

In a remarkable career, straddling five decades, John Phillis pursued with fierce determination and exceptional energy the main goal of his scientific life, to throw light on the chemical agents that control brain function. Starting in Australia, he settled in North America, first in Canada, then in the USA, where his long tenure at Wayne State brought his career to its culmination.

How does a little acronym become a big transmitter?

After an overview of the early, chequered history of the discovery of GABA and its gradual acceptance as inhibitory synaptic transmitter in the brain, the article lists and discusses some of the more unexpected later developments in studies of GABA, especially its role as excitatory transmitter in the immature brain.

Translational control of mGluR-dependent long-term depression and object-place learning by eIF2α.

At hippocampal synapses, activation of group I metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD), which requires new protein synthesis. However, the underlying mechanism remains elusive. Here we describe the translational program that underlies mGluR-LTD and identify the translation factor eIF2α as its master effector. Genetically reducing eIF2α phosphorylation, or specifically blocking the translation controlled by eIF2α phosphorylation, prevented mGluR-LTD and the internalization of surface AMPA receptors (AMPARs). Conversely, direct phosphorylation of eIF2α, bypassing mGluR activation, triggered a sustained LTD and removal of surface AMPARs. Combining polysome profiling and RNA sequencing, we identified the mRNAs translationally upregulated during mGluR-LTD. Translation of one of these mRNAs, oligophrenin-1, mediates the LTD induced by eIF2α phosphorylation. Mice deficient in phospho-eIF2α-mediated translation are impaired in object-place learning, a behavioral task that induces hippocampal mGluR-LTD in vivo. Our findings identify a new model of mGluR-LTD, which promises to be of value in the treatment of mGluR-LTD-linked cognitive disorders.

Salsolinol modulation of dopamine neurons.

Salsolinol, a tetrahydroisoquinoline present in the human and rat brains, is the condensation product of dopamine and acetaldehyde, the first metabolite of ethanol. Previous evidence obtained in vivo links salsolinol with the mesolimbic dopaminergic (DA) system: salsolinol is self-administered into the posterior of the ventral tegmental area (pVTA) of rats; intra-VTA administration of salsolinol induces a strong conditional place preference and increases dopamine release in the nucleus accumbens (NAc). However, the underlying neuronal mechanisms are unclear. Here we present an overview of some of the recent research on this topic. Electrophysiological studies reveal that DA neurons in the pVTA are a target of salsolinol. In acute brain slices from rats, salsolinol increases the excitability and accelerates the ongoing firing of dopamine neurons in the pVTA. Intriguingly, this action of salsolinol involves multiple pre- and post-synaptic mechanisms, including: (1) depolarizing dopamine neurons; (2) by activating µ opioid receptors on the GABAergic inputs to dopamine neurons - which decreases GABAergic activity - dopamine neurons are disinhibited; and (3) enhancing presynaptic glutamatergic transmission onto dopamine neurons via activation of dopamine type 1 receptors, probably situated on the glutamatergic terminals. These novel mechanisms may contribute to the rewarding/reinforcing properties of salsolinol observed in vivo.