Loss of glycine receptors in the nucleus accumbens and ethanol reward in an Alzheimer´s Disease mouse model.

Alterations in cognitive and non-cognitive cerebral functions characterize Alzheimer's disease (AD). Cortical and hippocampal impairments related to extracellular accumulation of Aß in AD animal models have been extensively investigated. However, recent reports have also implicated intracellular Aß in limbic regions, such as the nucleus accumbens (nAc). Accumbal neurons express high levels of inhibitory glycine receptors (GlyRs) that are allosterically modulated by ethanol and have a role in controlling its intake. In the present study, we investigated how GlyRs in the 2xTg mice (AD model) affect nAc functions and ethanol intake behavior. Using transgenic and control aged-matched litter mates, we found that the GlyRα2 subunit was significantly decreased in AD mice (6-month-old). We also examined intracellular calcium dynamics using the fluorescent calcium protein reporter GCaMP in slice photometry. We also found that the calcium signal mediated by GlyRs, but not GABAAR, was also reduced in AD neurons. Additionally, ethanol potentiation was significantly decreased in accumbal neurons in the AD mice. Finally, we performed drinking in the dark (DID) experiments and found that 2xTg mice consumed less ethanol on the last day of DID, in agreement with a lower blood ethanol concentration. 2xTg mice also showed lower sucrose consumption, indicating that overall food reward was altered. In conclusion, the data support the role of GlyRs in nAc neuron excitability and a decreased glycinergic activity in the 2xTg mice that might lead to impairment in reward processing at an early stage of the disease.

Changes in ethanol effects in knock-in mice expressing ethanol insensitive alpha1 and alpha2 glycine receptor subunits.

AIMS: Glycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. MAIN METHODS: We used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. KEY FINDINGS: Ethanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SIGNIFICANCE: The results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

Overexpression of wild type glycine alpha 1 subunit rescues ethanol sensitivity in accumbal receptors and reduces binge drinking in mice.

The nucleus accumbens (nAc) is a critical region in the brain reward system since it integrates abundant synaptic inputs contributing to the control of neuronal excitability in the circuit. The presence of inhibitory α1 glycine receptor (GlyRs) subunits, sensitive to ethanol, has been recently reported in accumbal neurons suggesting that they are protective against excessive binge consumption. In the present study, we used viral vectors (AAV) to overexpress mutant and WT α1 subunits in accumbal neurons in D1 Cre and α1 KI mice. Injection of a Cre-inducible AAV carrying an ethanol insensitive α1 subunit in D1 Cre neurons was unable to affect sensitivity to ethanol in GlyRs or affect ethanol drinking. On the other hand, using an AAV that transduced WT α1 GlyRs in GABAergic neurons in the nAc of high-ethanol consuming mice caused a reduction in ethanol intake as reflected by lowered drinking in the dark and reduced blood ethanol concentration. As expected, the AAV increased the glycine current density by 5-fold without changing the expression of GABAA receptors. Examination of the ethanol sensitivity in isolated accumbal neurons indicated that the GlyRs phenotype changed from an ethanol resistant to an ethanol sensitive type. These results support the conclusion that increased inhibition in the nAc can control excessive ethanol consumption and that selective targeting of GlyRs by pharmacotherapy might provide a mechanistic procedure to reduce ethanol binge.

GKRP-dependent modulation of feeding behavior by tanycyte-released monocarboxylates.

Objectives: Glucokinase Regulatory Protein (GKRP) is the only known endogenous modulator of glucokinase (GK) localization and activity to date, and both proteins are localized in tanycytes, radial glia-like cells involved in metabolic and endocrine functions in the hypothalamus. However, the role of tanycytic GKRP and its impact on the regulation of feeding behavior has not been investigated. Here, we hypothesize that GKRP regulates feeding behavior by modulating tanycyte-neuron metabolic communication in the arcuate nucleus. Methods: We used primary cultures of tanycytes to evaluate the production of lactate and ß-hydroxybutyrate (ßHB). Similarly, we examined the electrophysiological responses to these metabolites in pro-opiomelanocortin (POMC) neurons in hypothalamic slices. To evaluate the role of GKRP in feeding behavior, we generated tanycyte-selective GKRP-overexpressing and GKRP-knock down mice (GKRPt-OE and GKRPt-KD respectively) using adenovirus-mediated transduction. Results: We demonstrated that lactate release induced by glucose uptake is favored in GKRP-KD tanycytes. Conversely, tanycytes overexpressing GKRP showed an increase in ßHB efflux induced by low glucose concentration. In line with these findings, the excitability of POMC neurons was enhanced by lactate and decreased in the presence of ßHB. In GKRPt-OE rats, we found an increase in post-fasting food avidity, whereas GKRPt-KD caused a significant decrease in feeding and body weight, which is reverted when MCT1 is silenced. Conclusion: Our study highlights the role of tanycytic GKRP in metabolic regulation and positions this regulator of GK as a therapeutic target for boosting satiety in patients with obesity problems.

Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice.

BACKGROUND AND PURPOSE: Glycine receptors composed of α1 and ß subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol. EXPERIMENTAL APPROACH: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice. KEY RESULTS: Similar levels of α and ß receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αß receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol. CONCLUSION AND IMPLICATIONS: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.

Ethanol consumption and sedation are altered in mice lacking the glycine receptor α2 subunit.

BACKGROUND AND PURPOSE: The precise mechanism/s of action of ethanol, although studied for many years, are not well understood. Like other drugs of abuse, ethanol affects dopamine levels in the nucleus accumbens (nAc), an important region of the mesolimbic system, causing a reinforcing effect. It has been shown that glycine receptors (GlyRs) present in the nAc are potentiated by clinically relevant concentrations of ethanol, where α1 and α2 are the predominant subunits expressed. EXPERIMENTAL APPROACH: Using a combination of electrophysiology and behavioural assays, we studied the involvement of GlyR α2 subunits on the effects of low and high doses of ethanol, as well as on consumption using mice lacking the GlyR α2 subunit (male Glra2-/Y and female Glra2-/- ). KEY RESULTS: GlyR α2 subunits exist in accumbal neurons, since the glycine-evoked currents and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in Glra2-/Y mice were drastically decreased. In behavioural studies, differences in ethanol consumption and sedation were observed between wild-type (WT) and Glra2 knockout (KO) mice. Using the drinking in the dark (DID) paradigm, we found that Glra2-/Y mice presented a binge-like drinking behaviour immediately when exposed to ethanol rather than the gradual consumption seen in WT animals. Interestingly, the effect of knocking out Glra2 in female (Glra2-/- ) mice was less evident, since WT female mice already showed higher DID. CONCLUSION AND IMPLICATIONS: The differences in ethanol consumption between WT and KO mice provide additional evidence supporting the conclusion that GlyRs are biologically relevant targets for the sedative and rewarding properties of ethanol.

Influence of nonsynaptic α1 glycine receptors on ethanol consumption and place preference.

Here, we used knock-in (KI) mice that have ethanol-insensitive alpha 1 glycine receptors (GlyRs) (KK385/386AA) to examine how alpha 1 GlyRs might affect binge drinking and conditioned place preference. Data show that tonic alpha 1 GlyR-mediated currents were exclusively sensitive to ethanol only in wild-type mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol. This study suggests that nonsynaptic alpha 1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol.

Alcohol abuse leads to great medical, social, and economic burdens throughout the world. It is believed that the rewarding actions of alcohol are mediated by alterations in the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (NAc). Little is known about the role that ligand-gated ion channels (LGICs), such as glycine receptors (GlyRs), have in regulating levels of ethanol intake and place preference. In this study, we used knock-in (KI) mice that have ethanol-insensitive α1 GlyRs (KK385/386AA) and a combination of electrophysiological and behavioral approaches to examine how expression of ethanol-resistant α1 GlyRs in brain neurons might affect binge drinking and conditioned place preference. Data show that tonic α1 GlyR-mediated currents that modulate accumbal excitability were exclusively sensitive to ethanol only in wild-type (WT) mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol, suggesting that α1 GlyRs in the brain have a protective role against abuse. This study suggests that nonsynaptic α1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol and open a novel opportunity for pharmacotherapy development to treat alcohol use disorders.

Features of alpha-synuclein that could explain the progression and irreversibility of Parkinson's disease.

Alpha-synuclein is a presynaptic protein expressed throughout the central nervous system, and it is the main component of Lewy bodies, one of the histopathological features of Parkinson's disease (PD) which is a progressive and irreversible neurodegenerative disorder. The conformational flexibility of α-synuclein allows it to adopt different conformations, i.e., bound to membranes or form aggregates, the oligomers are believed to be the more toxic species. In this review, we will focus on two major features of α-synuclein, transmission and toxicity, that could help to understand the pathological characteristics of PD. One important feature of α-synuclein is its ability to be transmitted from neuron to neuron using mechanisms such as endocytosis, plasma membrane penetration or through exosomes, thus propagating the Lewy body pathology to different brain regions thereby contributing to the progressiveness of PD. The second feature of α-synuclein is that it confers cytotoxicity to recipient cells, principally when it is in an oligomeric state. This form causes mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, proteasome impairment, disruption of plasma membrane and pore formation that lead to apoptosis pathway activation and consequent cell death. The complexity of α-synuclein oligomerization and formation of toxic species could be a major factor for the irreversibility of PD and could also explain the lack of successful therapies to halt the disease.

Extracellular α-synuclein alters synaptic transmission in brain neurons by perforating the neuronal plasma membrane.

It has been postulated that the accumulation of extracellular α-synuclein (α-syn) might alter the neuronal membrane by formation of 'pore-like structures' that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α-syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α-syn oligomers. In addition, we show here that α-syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α-syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We propose that α-synuclein (α-syn) oligomers form pore-like structures in the plasma membrane of neurons from central nervous system (CNS). We believe that extracellular α-syn oligomers facilitate the formation of α-syn membrane pore-like structures, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We think that alterations in ionic homeostasis and synaptic vesicular depletion are key steps that lead to synaptotoxicity promoted by α -syn membrane pore-like structures.

Alzheimer's Aß interacts with cellular prion protein inducing neuronal membrane damage and synaptotoxicity.

A major feature of Alzheimer's disease is the accumulation of ß-amyloid (Aß) peptide in the brain. Recent studies have indicated that Aß oligomers (Aßo) can interact with the cellular prion protein (PrPc). Therefore, this interaction might be driving some of Aß toxic effects in the synaptic region. In the present study, we report that Aßo binds to PrPc in the neuronal membrane playing a role on toxic effects induced by Aß. Phospholipase C-enzymatic cleavage of PrPc from the plasma membrane attenuated the association of Aßo to the neurons. Furthermore, an anti-PrP antibody (6D11) decreased the association of Aßo to hippocampal neurons with a concomitant reduction in Aßo and PrPc co-localization. Interestingly, this antibody blocked the increase in membrane conductance and intracellular calcium induced by Aßo. Thus, the data indicate that PrPc plays a role on the membrane perforations produced by Aßo, the increase in calcium ions and the release of synaptic vesicles that subsequently leads to synaptic failure. Future studies blocking Aßo interaction with PrPc could be important for the discovery of new therapeutic strategies for Alzheimer's disease.