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1.
Methods Mol Biol ; 2551: 147-161, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36310202

RESUMO

Aggregated tau protein plays a key role in the pathogenesis of neurodegenerative tauopathies including Alzheimer's disease. Soluble, low-molecular-weight tau oligomers are formed early in disease processes and are thought to have toxic functions that disrupt neuronal function. The dynamic and transient nature of tau oligomers complicates in vitro functional studies to explore the mechanistic links between oligomer formation and neurodegeneration. We have previously described a method of producing stable and structurally characterized oligomers that maintain their oligomeric conformation and prevent further aggregation. This method allows for the flexibility of stabilizing tau oligomers by specifically labelling cysteine residues with fluorescent or colorless maleimide conjugates. Here, we describe the functional applications of these preformed stable tau oligomers in cell biology and electrophysiological studies. These investigations allow real-time insights into the cellular uptake of exogenous tau oligomers and their functional effects in the recipient cells.


Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Proteínas tau/metabolismo , Tauopatias/metabolismo , Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Eletrofisiologia
2.
Methods Mol Biol ; 2551: 203-224, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36310205

RESUMO

There is growing evidence that tau oligomers are a major pathological species in a number of tauopathies including Alzheimer's disease. However, it is still unclear what exact mechanisms underlie tau oligomer-mediated dysfunction. Studies of tau oligomers in vitro are limited by the high propensity for aggregation and consequent changes in the aggregation state of the produced tau samples over time. In this protocol, we provide a step-by-step description of a validated method for producing stable and structurally characterized oligomers of tau that can be used in biochemical, cellular, and animal model studies to evaluate mechanisms of action of tau in tauopathies.


Assuntos
Doença de Alzheimer , Tauopatias , Animais , Proteínas tau/química , Modelos Animais de Doenças
3.
Aging Cell ; 21(10): e13717, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36135933

RESUMO

A key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Plasticidade Neuronal/fisiologia , Aprendizagem Espacial , Sinapses/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno
4.
Neuropharmacology ; 216: 109172, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35780977

RESUMO

Physiological oscillations in the cortico-thalamo-cortical loop occur during processes such as sleep, but these can become dysfunctional in pathological conditions such as absence epilepsy. The purine neuromodulator adenosine can act as an endogenous anticonvulsant: it is released into the extracellular space during convulsive seizures to activate A1 receptors suppressing on-going activity and delaying the occurrence of the next seizure. However, the role of adenosine in thalamic physiological and epileptiform oscillations is less clear. Here we have combined immunohistochemistry, electrophysiology, and fixed potential amperometry (FPA) biosensor measurements to characterise the release and actions of adenosine in thalamic oscillations measured in rodent slices. In the thalamus, A1 receptors are highly expressed particularly in the ventral basal (VB) thalamus and reticular thalamic nucleus (nRT) supporting a role for adenosine signalling in controlling oscillations. In agreement with previous studies, both adenosine and adenosine A1 receptor agonists inhibited thalamic oscillations in control (spindle-like) and in epileptic conditions. Here we have shown for the first time that both control and epileptiform oscillations are enhanced (i.e., increased number of oscillatory cycles) by blocking A1 receptors consistent with adenosine release occurring during oscillations. Although increases in extracellular adenosine could not be directly detected during control oscillations, clear increases in adenosine concentration could be detected with a biosensor during epileptiform oscillation activity. Thus, adenosine is released during thalamic oscillations and acts via A1 receptors to feedback and reduce thalamic oscillatory activity.


Assuntos
Adenosina , Epilepsia Tipo Ausência , Adenosina/farmacologia , Retroalimentação , Humanos , Convulsões , Tálamo
5.
Front Aging Neurosci ; 14: 861344, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35847678

RESUMO

Sleep apnoea is a highly prevalent disease that often goes undetected and is associated with poor clinical prognosis, especially as it exacerbates many different disease states. However, most animal models of sleep apnoea (e.g., intermittent hypoxia) have recently been dispelled as physiologically unrealistic and are often unduly severe. Owing to a lack of appropriate models, little is known about the causative link between sleep apnoea and its comorbidities. To overcome these problems, we have created a more realistic animal model of moderate sleep apnoea by reducing the excitability of the respiratory network. This has been achieved through controlled genetically mediated lesions of the preBötzinger complex (preBötC), the inspiratory oscillator. This novel model shows increases in sleep disordered breathing with alterations in breathing during wakefulness (decreased frequency and increased tidal volume) as observed clinically. The increase in dyspnoeic episodes leads to reduction in REM sleep, with all lost active sleep being spent in the awake state. The increase in hypoxic and hypercapnic insults induces both systemic and neural inflammation. Alterations in neurophysiology, an inhibition of hippocampal long-term potentiation (LTP), is reflected in deficits in both long- and short-term spatial memory. This improved model of moderate sleep apnoea may be the key to understanding why this disorder has such far-reaching and often fatal effects on end-organ function.

6.
Nat Commun ; 13(1): 4150, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35851064

RESUMO

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.


Assuntos
Analgesia , Depressão , Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P1
7.
Neuropharmacology ; 208: 108982, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35151699

RESUMO

In schizophrenia, mGlu5 receptor hypofunction has been linked with neuropathology and cognitive deficits, making it an attractive therapeutic target. The cognitive impairment associated with schizophrenia remains an unmet clinical need, with existing antipsychotics primarily targeting positive symptoms, with weaker and more variable effects on cognitive deficits. Using the sub-chronic phencyclidine rat model, widely shown to mimic the cognitive impairment and neuropathology of schizophrenia, we have investigated two mGlu5 receptor positive allosteric modulators (PAMs), VU0409551 and VU0360172. We compared the efficacy of these compounds in restoring cognitive deficits and, since these two PAMs have reportedly distinct signalling mechanisms, changes in mGlu5 receptor signalling molecules AKT and MAPK in the PFC. Although not effective at 0.05 and 1 mg/kg, cognitive deficits were significantly alleviated by both PAMs at 10 and 20 mg/kg. The compounds appeared to have differential effects on the scPCP-induced increases in AKT and MAPK phosphorylation: VU0409551 induced a significant decrease in expression of p-AKT, whereas VU0360172 had this effect on p-MAPK levels. Thus, the beneficial effects of PAMs on scPCP-induced cognitive impairment are accompanied by at least partial reversal of scPCP-induced elevated levels of p-MAPK and p-AKT, whose dysfunction is strongly implicated in schizophrenia pathology. These promising data imply an important role for mGlu5 receptor signalling pathways in improving cognition in the scPCP model and provide support for mGlu5 receptor PAMs as a possible therapeutic intervention for schizophrenia.


Assuntos
Receptor de Glutamato Metabotrópico 5 , Esquizofrenia , Regulação Alostérica , Animais , Cognição , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Oxazóis , Fenciclidina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismo , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo
8.
Commun Biol ; 4(1): 1265, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34737403

RESUMO

Tau protein is involved in maintaining neuronal structure. In Alzheimer's disease, small numbers of tau molecules can aggregate to form oligomers. However, how these oligomers produce changes in neuronal function remains unclear. Previously, oligomers made from full-length human tau were found to have multiple effects on neuronal properties. Here we have cut the tau molecule into two parts: the first 123 amino acids and the remaining 124-441 amino acids. These truncated tau molecules had specific effects on neuronal properties, allowing us to assign the actions of full-length tau to different regions of the molecule. We identified one key target for the effects of tau, the voltage gated sodium channel, which could account for the effects of tau on the action potential. By truncating the tau molecule, we have probed the mechanisms that underlie tau dysfunction, and this increased understanding of tau's pathological actions will build towards developing future tau-targeting therapies.


Assuntos
Neurônios/metabolismo , Proteínas tau/química , Humanos , Proteínas tau/metabolismo
9.
Neuropharmacology ; 196: 108686, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34197893

RESUMO

Absence epilepsy is frequently associated with cognitive dysfunction, although the underlying mechanisms are not well understood. Here we report that some forms of hippocampal synaptic plasticity are abnormal in symptomatic Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Metabotropic Glu 1/5 receptor-mediated long term depression (LTD) at Schaffer collateral CA1 synapses is significantly reduced in symptomatic, 5-6 months old WAG/Rij rats compared to age-matched non epileptic control rats. There were no significant changes in mGlu1/5-dependent LTD in pre-symptomatic, 4-6 weeks old WAG/Rij rats compared to age matched controls. The changes in LTD found in symptomatic WAG/Rij forms are not indicative of general deficits in all forms of synaptic plasticity as long term potentiation (LTP) was unchanged. Immunoblot analysis of hippocampal tissue showed a significant reduction in mGlu5 receptor expression, a trend to an increase in pan Homer protein levels and a decrease in GluA1 receptor expression in the hippocampus of symptomatic WAG/Rij rats vs non-epileptic control rats. There were no changes in mGlu1α receptor or GluA2 protein levels. These findings suggest that abnormalities in hippocampal mGlu5 receptor-dependent synaptic plasticity are associated with the pathological phenotype of WAG/Rij rats. This lays the groundwork for the study of mGlu5 receptors as a candidate drug target for the treatment of cognitive dysfunction linked to absence epilepsy.


Assuntos
Epilepsia Tipo Ausência/metabolismo , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Epilepsia Tipo Ausência/fisiopatologia , Proteínas de Arcabouço Homer/metabolismo , Plasticidade Neuronal/fisiologia , Ratos
10.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298872

RESUMO

Connexins can assemble into either gap junctions (between two cells) or hemichannels (from one cell to the extracellular space) and mediate cell-to-cell signalling. A subset of connexins (Cx26, Cx30, Cx32) are directly sensitive to CO2 and fluctuations in the level within a physiological range affect their open probability, and thus, change cell conductance. These connexins are primarily found on astrocytes or oligodendrocytes, where increased CO2 leads to ATP release, which acts on P2X and P2Y receptors of neighbouring neurons and changes excitability. CO2-sensitive hemichannels are also found on developing cortical neurons, where they play a role in producing spontaneous neuronal activity. It is plausible that the transient opening of hemichannels allows cation influx, leading to depolarisation. Recently, we have shown that dopaminergic neurons in the substantia nigra and GABAergic neurons in the VTA also express Cx26 hemichannels. An increase in the level of CO2 results in hemichannel opening, increasing whole-cell conductance, and decreasing neuronal excitability. We found that the expression of Cx26 in the dopaminergic neurons in the substantia nigra at P7-10 is transferred to glial cells by P17-21, displaying a shift from being inhibitory (to neuronal activity) in young mice, to potentially excitatory (via ATP release). Thus, Cx26 hemichannels could have three modes of signalling (release of ATP, excitatory flickering open and shut and inhibitory shunting) depending on where they are expressed (neurons or glia) and the stage of development.


Assuntos
Dióxido de Carbono/metabolismo , Conexinas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Humanos
11.
ACS Pharmacol Transl Sci ; 4(1): 314-326, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33615181

RESUMO

Despite being among the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, they suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and they highlight the dramatic effect that chemical modifications can have on the overall binding mechanism, paving the way for the rational development of a structure-kinetics relationship of A1R agonists.

12.
Methods Mol Biol ; 2346: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32661915

RESUMO

Cell-to-cell communication is an essential process for the efficient function of cells and tissues. Central to this is the purinergic transmission of purines, with ligands such as adenosine triphosphate (ATP). Altered cell-to-cell communication, and in particular changes in the paracrine release of extracellular ATP, plays crucial roles in pathophysiological conditions, such as diabetes. ATP biosensing provides a reliable, real-time measurement of local extracellular ATP concentrations. This allows the detection of altered ATP release, which underlies the progression of inflammation and fibrosis and is a potential therapeutic target. Here we describe in a step-by-step basis how to utilize sensitive microelectrode biosensors to detect low, real-time concentrations of ATP, in vitro.


Assuntos
Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais , Comunicação Celular , Rim/metabolismo , Células Cultivadas , Humanos , Rim/citologia , Transdução de Sinais
13.
STAR Protoc ; 1(3): 100139, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33377033

RESUMO

This protocol provides two independent methods to functionally detect the neuronal expression of CO2-sensitive hemichannels. These hemichannels (consisting of connexins 26 or 30) are directly gated by CO2, independent of pH changes and until recently were thought to be only expressed by glia. This protocol outlines a method to change the concentration of CO2 without changing pH, using isohydric solutions and then utilizing this to detect opening and closing of functional hemichannels using whole-cell patch clamp recording and dye loading. For complete details on the use and execution of this protocol, please refer to Hill et al. (2020).


Assuntos
Neurônios/metabolismo , Proteínas de Transporte de Neurotransmissores/análise , Técnicas de Patch-Clamp/métodos , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Dióxido de Carbono/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Microtomia/métodos , Neuroglia/metabolismo , Neurônios/química , Neurônios/citologia , Proteínas de Transporte de Neurotransmissores/metabolismo
14.
Front Mol Neurosci ; 13: 155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32973448

RESUMO

Tau is a predominantly neuronal protein that is normally bound to microtubules, where it acts to modulate neuronal and axonal stability. In humans, pathological forms of tau are implicated in a range of diseases that are collectively known as tauopathies. Kinases and phosphatases are responsible for maintaining the correct balance of tau phosphorylation to enable axons to be both stable and labile enough to function properly. In the early stages of tauopathies, this balance is interrupted leading to dissociation of tau from microtubules. This leaves microtubules prone to damage and phosphorylated tau prone to aggregation. Initially, phosphorylated tau forms oligomers, then fibrils, and ultimately neurofibrillary tangles (NFTs). It is widely accepted that the initial soluble oligomeric forms of tau are probably the most pathologically relevant species but there is relatively little quantitative information to explain exactly what their toxic effects are at the individual neuron level. Electrophysiology provides a valuable tool to help uncover the mechanisms of action of tau oligomers on synaptic transmission within single neurons. Understanding the concentration-, time-, and neuronal compartment-dependent actions of soluble tau oligomers on neuronal and synaptic properties are essential to understanding how best to counteract its effects and to develop effective treatment strategies. Here, we briefly discuss the standard approaches used to elucidate these actions, focusing on the advantages and shortcomings of the experimental procedures. Subsequently, we will describe a new approach that addresses specific challenges with the current methods, thus allowing real-time toxicity evaluation at the single-neuron level.

15.
Neuropharmacology ; 178: 108240, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32768418

RESUMO

Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABAA receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 h and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models.


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Niacinamida/análogos & derivados , Receptor de Glutamato Metabotrópico 5/agonistas , Receptor de Glutamato Metabotrópico 5/metabolismo , Tálamo/metabolismo , Ácido gama-Aminobutírico/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Relação Dose-Resposta a Droga , Masculino , Niacinamida/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Ratos Wistar , Receptores de GABA-A/metabolismo , Tálamo/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia
16.
iScience ; 23(7): 101343, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32683315

RESUMO

The substantia nigra (SN) and ventral tegmental area (VTA) are vital for the control of movement, goal-directed behavior, and encoding reward. Here we show that the firing of specific neuronal subtypes in these nuclei can be modulated by physiological changes in the partial pressure of carbon dioxide (PCO2). The resting conductance of substantia nigra dopaminergic neurons in young animals (postnatal days 7-10) and GABAergic neurons in the VTA is modulated by changes in the level of CO2. We provide several lines of evidence that this CO2-sensitive conductance results from connexin 26 (Cx26) hemichannel expression. Since the levels of PCO2 in the blood will vary depending on physiological activity and pathology, this suggests that changes in PCO2 could potentially modulate motor activity, reward behavior, and wakefulness.

17.
Neuropharmacology ; 164: 107904, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31812775

RESUMO

The neuromodulator adenosine is released during seizure activity to provide negative feedback suppression of ongoing activity and to delay the occurrence of the next burst of activity. Adenosine acts via multiple G-protein-coupled receptors including the A1 receptor (A1R) which inhibits neurotransmitter release and hyperpolarises neuronal membrane potential. The hyperpolarisation is produced, at least in part, by the activation of G-protein-activated inwardly rectifying K+ (GIRK) channels. We have used tertiapin-Q (TQ), a potent and selective inhibitor of GIRK channels, to assess the role of GIRK channels in controlling seizure activity in areas CA1 and CA2 of mouse hippocampal slices. TQ (100-300 nM) blocked ~50% of the adenosine-mediated membrane potential hyperpolarisation of hippocampal CA1 and CA2 neurons. TQ (100 nM) had no significant effect on synaptic transmission in area CA1 of the hippocampus but enhanced transmission in CA2, an effect prevented by blocking A1Rs. TQ (100 nM) increased the frequency of spontaneous activity (induced by removing Mg2+ and increasing K+) and blunted the effects of exogenous adenosine on the suppression of activity. TQ had a significantly greater effect on electrically-stimulated seizure activity induced in CA2 versus that in CA1, producing a greater increase in both the duration and amplitude of the stimulated bursts. This is consistent with the greater A1R density and A1R activation tone in CA2. Thus GIRK channels play a role in the supressing effects of adenosine on seizure activity.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hipocampo/fisiopatologia , Receptor A1 de Adenosina/genética , Convulsões/genética , Convulsões/fisiopatologia , Adenosina/farmacologia , Animais , Anticonvulsivantes/farmacologia , Venenos de Abelha/farmacologia , Região CA1 Hipocampal/metabolismo , Região CA2 Hipocampal/metabolismo , Estimulação Elétrica , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp , Transmissão Sináptica/efeitos dos fármacos
18.
eNeuro ; 6(5)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31554666

RESUMO

Tau is a highly soluble microtubule-associated protein that acts within neurons to modify microtubule stability. However, abnormally phosphorylated tau dissociates from microtubules to form oligomers and fibrils which associate in the somatodendritic compartment. Although tau can form neurofibrillary tangles (NFTs), it is the soluble oligomers that appear to be the toxic species. There is, however, relatively little quantitative information on the concentration-dependent and time-dependent actions of soluble tau oligomers (oTau) on the electrophysiological and synaptic properties of neurons. Here, whole-cell patch clamp recording was used to introduce known concentrations of oligomeric full-length tau-441 into mouse hippocampal CA1 pyramidal and neocortical Layer V thick-tufted pyramidal cells. oTau increased input resistance, reduced action potential amplitude and slowed action potential rise and decay kinetics. oTau injected into presynaptic neurons induced the run-down of unitary EPSPs which was associated with increased short-term depression. In contrast, introduction of oTau into postsynaptic neurons had no effect on basal synaptic transmission, but markedly impaired the induction of long-term potentiation (LTP). Consistent with its effects on synaptic transmission and plasticity, oTau puncta could be observed in the soma, axon and in the distal dendrites of injected neurons.


Assuntos
Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Transmissão Sináptica/fisiologia , Proteínas tau/toxicidade , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
19.
Neuropharmacology ; 155: 121-130, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31129151

RESUMO

The ability to either erase or update the memories of a previously learned spatial task is an essential process that is required to modify behaviour in a changing environment. Current evidence suggests that the neural representation of such cognitive flexibility involves the balancing of synaptic potentiation (acquisition of memories) with synaptic depression (modulation and updating previously acquired memories). Here we demonstrate that the p38 MAPK/MAPK-activated protein kinase 2 (MK2) cascade is required to maintain the precise tuning of long-term potentiation and long-term depression at CA1 synapses of the hippocampus which is correlated with efficient reversal learning. Using the MK2 knockout (KO) mouse, we show that mGluR-LTD, but not NMDAR-LTD, is markedly impaired in mice aged between 4 and 5 weeks (juvenile) to 7 months (mature adult). Although the amplitude of LTP was the same as in wildtype mice, priming of LTP by the activation of group I metabotropic receptors was impaired in MK2 KO mice. Consistent with unaltered LTP amplitude and compromised mGluR-LTD, MK2 KO mice had intact spatial learning when performing the Barnes maze task, but showed specific deficits in selecting the most efficient combination of search strategies to perform the task reversal. Findings from this study suggest that the mGluR-p38-MK2 cascade is important for cognitive flexibility by regulating LTD amplitude and the priming of LTP.


Assuntos
Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Plasticidade Neuronal/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Reversão de Aprendizagem/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Depressão Sináptica de Longo Prazo/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , /genética
20.
Bioorg Med Chem Lett ; 28(23-24): 3780-3783, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30337231

RESUMO

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.


Assuntos
Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/metabolismo , Neuralgia/tratamento farmacológico , Pirazóis/química , Pirazóis/uso terapêutico , Analgésicos/química , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Descoberta de Drogas , Células HEK293 , Humanos , Masculino , Neuralgia/metabolismo , Técnicas de Patch-Clamp , Piranos/química , Piranos/farmacologia , Piranos/uso terapêutico , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley
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