RESUMO
PURPOSE: Infections are frequent complications in acute ischemic stroke and may be caused by an altered immune response influencing brain damage. We compared long-term immune responses in stroke patients with or without infections during the recovery period by performing a long-term profiling of clinically relevant inflammatory parameters from stroke onset until day 49. MATERIALS AND METHODS: Thirty-four stroke patients were retrospectively included and divided into two groups depending on infection status. Group 1 had no infections (N = 17) and group 2 had post-admission infection (N = 17). The patients were evaluated carefully for infections and evolution of the peripheral inflammatory response. Neutrophils, monocytes, lymphocytes, total leukocytes and C-reactive protein were evaluated in relation to the occurrence and development of infections. In both patient groups, an acute boost in neutrophils and monocytes were observed whereas the opposite was true for lymphocytes. RESULTS: In Group 1, neutrophils and monocytes approached normal levels after 20-30 days, but remained elevated in Group 2. We found an increase in neutrophils (p = 0.01) and leukocytes (p < 0.01) as well as C-reactive protein (p < 0.01) among infected patients. Lymphocytes remained depressed in Group 2, while Group 1 slowly approached baseline levels. In both groups, CRP levels initially increased with a slow return to baseline levels. From day 0 to 49 after stroke, uninfected patients generally experienced a decline in leukocytes, neutrophils and monocytes (all p < 0.05), while no similar changes happened among infected patients. CONCLUSIONS: Our study provides an overview of general immune cell kinetics after stroke related to infection status. Immune cell numbers were severely disturbed for weeks after the insult, independent of infection status, although infected patients achieved the highest cell counts of neutrophils, leukocytes and for C-reactive protein. The sustained depression of lymphocytes, especially and paradoxically among infected patients, warrants future studies into the mechanisms behind this, with potential for future therapies aimed at restoring normal immunity and thereby improving patient outcome.
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The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4ßδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds.
Assuntos
Adjuvantes Imunológicos/farmacologia , Alcanos/farmacologia , Antagonistas GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Adjuvantes Imunológicos/química , Alcanos/química , Proliferação de Células/efeitos dos fármacos , Antagonistas GABAérgicos/química , Humanos , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Oxidatively generated DNA damage is of paramount importance in a wide range of physiological and pathophysiological processes. Urinary 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is often used as an outcome marker in studies on the role of oxidatively generated DNA damage, but its exact relation to intracellular damage levels and variations in DNA repair have been unclear. Using a new approach of quantitative kinetic modeling inspired by pharmacokinetics, we find evidence that in steady state - i.e. when systemic consequences of given change in damage or cellular removal rates have stabilized - the urinary excretion of 8-oxodG is closely correlated to rates of damage and intracellular 8-oxodG levels, but independent of the rate of cellular removal. Steady state was calculated to occur within approximately 12 h. A similar pattern was observed in a model of the corresponding RNA marker 8-oxo-7,8-dihydroguanosine (8-oxoGuo), but with steady-state occurring slower (up to 5 d). These data have significant implications for the planning of studies and interpretation of data involving urinary 8-oxodG/8-oxoGuo excretion as outcome.HighlightsThe kinetics of 8-oxodG/8-oxoGuo formation, removal and excretion were simulated in silico.The model was based on existing data on 8-oxodG/8-oxoGuo levels and removal/excretion rates.Intracellular 8-oxodG/8-oxoGuo was closely correlated with urinary excretion in steady state.Changes in removal rates did not influence urinary excretion of 8-oxodG/8-oxoGuo.
Assuntos
8-Hidroxi-2'-Desoxiguanosina/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/urina , Simulação por Computador , Guanosina/análogos & derivados , Espaço Intracelular/metabolismo , DNA/metabolismo , Guanosina/metabolismo , Guanosina/urina , Cinética , RNA/metabolismoRESUMO
The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.
Assuntos
Canais Iônicos/metabolismo , Zinco/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Canais Iônicos/genética , Canais Iônicos/fisiologia , Oócitos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Xenopus laevisRESUMO
Brain GABAΑ receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α4ß1δ GABAΑ receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABAΑ receptors. The initial screening hit 2027 (IC50 of 1.03 µM) was used for analogue search resulting in 018 (IC50 of 0.088 µM). 018 was most potent at α3,4,5-subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α4ß1δ receptors and displacement of [3H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-clamp electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABAΑ receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABAΑ receptor-mediated effects of GABA e.g. in the immune system.
Assuntos
Antagonistas de Receptores de GABA-A/isolamento & purificação , Antagonistas de Receptores de GABA-A/farmacologia , Receptores de GABA-A/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/classificação , Células HEK293 , Humanos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Subunidades Proteicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the commonly-used, standard semi-permeable membrane culture methodology which has significantly enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states: the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using immunohistochemistry and electrophysiology, our system was found superior to standard techniques in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as enhance neuronal survival in slice cultures originating from other brain regions.
Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Tegmento Mesencefálico/crescimento & desenvolvimento , Animais , Nível de Alerta/fisiologia , Encéfalo , Meios de Cultura/química , Fenômenos Eletrofisiológicos/fisiologia , Camundongos , Neurônios , Organoides/crescimento & desenvolvimentoRESUMO
Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel NaV1.1 is highly expressed in PV-positive interneurons, but only at low levels in principal cells. Positive modulators of Nav1.1 channels are for this reason considered potential candidates for the treatment of cognitive disorders. Here we examined the effect of the novel positive modulator of voltage-gated sodium channels Lu AE98134. We found that Lu AE98134 facilitated the sodium current mediated by NaV1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6+/- mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found that in the presence of Lu AE98134, the firing frequency was increased while the spike duration and the threshold were decreased. Compounds with similar mode of action appear as promising candidates for restoring cognitive deficits present in schizophrenia.
Assuntos
Compostos Heterocíclicos com 3 Anéis/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Sulfonamidas/farmacologia , Animais , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos MutantesRESUMO
THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a GABAA receptor agonist with varying potencies and efficacies at γ-subunit-containing receptors. More importantly, THIP acts as a selective superagonist at δ-subunit-containing receptors (δ-GABAA Rs) at clinically relevant concentrations. Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated with several animal models of epilepsy, we first investigated the presence of functional δ-GABAA receptors. Both immunohistochemistry and Western blot data demonstrated that δ-GABAA R expression is not only present in the dentate gyrus, but also the expression level was enhanced in the early phase after PTZ kindling. Whole-cell patch-clamp studies in acute hippocampal brain slices revealed that THIP was indeed able to induce a tonic inhibition in dentate gyrus granule cells. However, THIP induced a tonic current of similar magnitude in the PTZ-kindled mice compared to saline-treated animals despite the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties.
RESUMO
Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.
Assuntos
Astrócitos/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Hipocampo/fisiologia , Transmissão Sináptica/fisiologia , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Interneurônios/metabolismo , Modelos Animais , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT3R) agonists sharing little structural similarity with orthosteric 5-HT3R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT3A receptor (5-HT3AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT3AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT3A receptor suggested that the ligand acts through the transmembrane domain of 5-HT3AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT3ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT3ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT3Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT3AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites.
Assuntos
Acrilamidas/farmacologia , Proteínas Mutantes Quiméricas/agonistas , Éteres Fenílicos/farmacologia , Receptores 5-HT3 de Serotonina/genética , Agonistas do Receptor 5-HT3 de Serotonina/farmacologia , Acrilamidas/síntese química , Regulação Alostérica , Sítio Alostérico , Animais , Células COS , Chlorocebus aethiops , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Expressão Gênica , Cinética , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Éteres Fenílicos/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , Receptores 5-HT3 de Serotonina/metabolismo , Serotonina/farmacologia , Agonistas do Receptor 5-HT3 de Serotonina/síntese química , Xenopus laevisRESUMO
A high degree of structural heterogeneity of the GABAA receptors (GABAARs) has been revealed and is reflected in multiple receptor subtypes. The subunit composition of GABAAR subtypes is believed to determine their localization relative to the synapses and adapt their functional properties to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown for only a few orthosteric ligands. Still, these examples show that it is indeed possible to obtain orthosteric subtype selectivity and they serve as models for further development in the orthosteric GABAAR ligand area. This review presents the very few existing structural classes of orthosteric GABAAR antagonists and describes the development of potent antagonists from partial agonists originally derived from the potent GABAAR agonist muscimol. In this process, several heterocyclic aromatic systems have been used in combination with structural models in order to map the orthosteric binding site and to reveal structural details to be used for obtaining potency and subtype selectivity. The challenges connected to functional characterization of orthosteric GABAAR partial agonists and antagonists, especially with regard to GABAAR stoichiometry and alternative binding sites are discussed. GABAAR antagonists have been essential in defining the tonic current but both remaining issues concerning the GABAARs involved and the therapeutic possibilities of modulating tonic inhibition underline the need for GABAAR antagonists with improved selectivity.
Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Sítios de Ligação , Agonismo Parcial de Drogas , Agonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/química , Humanos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismoRESUMO
GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL.
Assuntos
Grânulos Citoplasmáticos/efeitos dos fármacos , Giro Denteado/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Isoxazóis/farmacologia , Neurônios/efeitos dos fármacos , Piperidinas/farmacologia , Animais , Grânulos Citoplasmáticos/metabolismo , Giro Denteado/citologia , Giro Denteado/metabolismo , Antagonistas de Receptores de GABA-A/química , Isoxazóis/química , Camundongos , Neurônios/metabolismo , Piperidinas/química , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/efeitos dos fármacosRESUMO
P300 (P3) event-related potentials (ERPs) have been suggested to be an endogenous marker of cognitive function and auditory oddball paradigms are frequently used to evaluate P3 ERPs in clinical settings. Deficits in P3 amplitude and latency reflect some of the neurological dysfunctions related to several psychiatric and neurological diseases, e.g., Alzheimer's disease (AD). However, only a very limited number of rodent studies have addressed the back-translational validity of the P3-like ERPs as suitable markers of cognition. Thus, the potential of rodent P3-like ERPs to predict pro-cognitive effects in humans remains to be fully validated. The current study characterizes P3-like ERPs in the 192-IgG-SAP (SAP) rat model of the cholinergic degeneration associated with AD. Following training in a combined auditory oddball and lever-press setup, rats were subjected to bilateral intracerebroventricular infusion of 1.25 µg SAP or PBS (sham lesion) and recording electrodes were implanted in hippocampal CA1. Relative to sham-lesioned rats, SAP-lesioned rats had significantly reduced amplitude of P3-like ERPs. P3 amplitude was significantly increased in SAP-treated rats following pre-treatment with 1 mg/kg donepezil. Infusion of SAP reduced the hippocampal choline acetyltransferase activity by 75%. Behaviorally defined cognitive performance was comparable between treatment groups. The present study suggests that AD-like deficits in P3-like ERPs may be mimicked by the basal forebrain cholinergic degeneration induced by SAP. SAP-lesioned rats may constitute a suitable model to test the efficacy of pro-cognitive substances in an applied experimental setup.
Assuntos
Doença de Alzheimer/fisiopatologia , Região CA1 Hipocampal/fisiopatologia , Neurônios Colinérgicos/fisiologia , Potenciais Evocados Auditivos/fisiologia , Degeneração Neural/fisiopatologia , Doença de Alzheimer/tratamento farmacológico , Animais , Anticorpos Monoclonais , Percepção Auditiva/efeitos dos fármacos , Percepção Auditiva/fisiologia , Região CA1 Hipocampal/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Modelos Animais de Doenças , Donepezila , Eletrodos Implantados , Potenciais Evocados Auditivos/efeitos dos fármacos , Indanos/farmacologia , Masculino , Degeneração Neural/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Testes Neuropsicológicos , Piperidinas/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Ratos Sprague-Dawley , Proteínas Inativadoras de Ribossomos Tipo 1 , SaporinasRESUMO
A compromised γ-aminobutyric acid (GABA)ergic system is hypothesized to be part of the underlying pathophysiology of schizophrenia. N-methyl-D-aspartate (NMDA) receptor hypofunction during neurodevelopment is proposed to disrupt maturation of interneurons causing an impaired GABAergic transmission in adulthood. The present study examines prefrontal GABAergic transmission in adult rats administered with the NMDA receptor channel blocker, phencyclidine (PCP), for 3 days during the second postnatal week. Whole-cell patch-clamp recordings from pyramidal cells in PCP-treated rats showed a 22% reduction in the frequency of miniature inhibitory postsynaptic currents in layer II/III, but not in layer V pyramidal neurons of the prefrontal cortex. Furthermore, early postnatal PCP treatment caused insensitivity toward effects of the GABA transporter 1 (GAT-1) inhibitor, 1,2,5,6-tetrahydro-1-[2-[[(diphenyl-methylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid, and also diminished currents passed by δ-subunit-containing GABAA receptors in layer II/III pyramidal neurons. The observed impairments in GABAergic function are compatible with the alteration of GABAergic markers as well as cognitive dysfunction observed in early postnatal PCP-treated rats and support the hypothesis that PCP administration during neurodevelopment affects the functionality of interneurons in later life.
Assuntos
Antagonistas de Aminoácidos Excitatórios/toxicidade , Fenciclidina/toxicidade , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/crescimento & desenvolvimento , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Estimulação Elétrica , Antagonistas GABAérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Ácidos Nipecóticos/farmacologia , Oximas/farmacologia , Técnicas de Patch-Clamp , Córtex Pré-Frontal/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/crescimento & desenvolvimento , Células Piramidais/fisiologia , Piridazinas/farmacologia , Ratos Endogâmicos ACI , Receptores de GABA-A/metabolismo , Técnicas de Cultura de TecidosRESUMO
The Alzheimer's disease (AD) mouse model Tg2576 overexpresses an AD associated mutant variant of human APP and accumulates amyloid beta (Aß) in an age-dependent manner. Using the selective cholinergic immunotoxin mu p75-saporin (SAP), we induced a partial basal forebrain cholinergic degeneration (BFCD) in 3 months old male Tg2576 mice to co-express cholinergic degeneration with Aß overexpression as these characteristics constitutes key hallmarks of AD. At 9 months, SAP lesioned Tg2576 mice were cognitively impaired in two spatial paradigms addressing working memory and mid to long-term memory. Conversely, there was no deterioration of cognitive functioning in sham lesioned Tg2576 mice or wild type littermates (wt) receiving the immunotoxin. At 10 months of age, release of acetylcholine (ACh) was addressed by microdialysis in conscious mice. Scopolamine-induced increases in hippocampal ACh efflux was significantly reduced in SAP lesioned Tg2576 mice compared to sham lesioned Tg2576 mice. Intriguingly, there was no significant difference in ACh efflux between wt treatment groups. Following SAP treatment, choline acetyltransferase activity was reduced in the hippocampus and frontal cortex and the reduction was comparable between groups. Our results suggest that partial BFCD acts collectively with increased levels of Aß to induce cognitive decline and to compromise cholinergic release. Tg2576 mice with BFCD may constitute a new and suitable AD mouse model to study the interrelations between cholinergic deficits and amsyloid deposition.
Assuntos
Acetilcolina/metabolismo , Neurônios Colinérgicos/patologia , Hipocampo/metabolismo , Transtornos da Memória/patologia , Degeneração Neural/etiologia , Prosencéfalo/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Humanos , Imunotoxinas/farmacologia , Aprendizagem em Labirinto , Transtornos da Memória/etiologia , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Reconhecimento Psicológico/fisiologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , SaporinasRESUMO
Stroke is one of the leading causes of death worldwide. At present, the only available treatment is thrombolysis, which should be initiated no later than 4.5 hours after onset of symptoms. Several studies have shown that an attenuation of the inflammatory response in relation to stroke could widen the therapeutic window. However, the immune system has important functions following infarction, such as removal of dead cells and the subsequent astrocytosis as well as prevention of post-ischemic infection. Hence, detailed knowledge concerning the temporal profile of leukocyte infiltration is necessary in order to develop new and effective treatments.The purpose of this review is to determine the temporal profile of leukocyte (neutrophil granulocytes, macrophages and T-cells) infiltration following experimental stroke. We found that the number of neutrophil granulocytes peaks between day 1 and 3 after experimental stroke, with short occlusion times (30 and 60 minutes of middle cerebral artery occlusion (MCAO)) leading to a later peak in response (P <0.001). Macrophages/microglia were found to peak later than day 3 and stay in the infarcted area for longer time periods, whereas duration of occlusion had no influence on the temporal infiltration (P = 0.475). Studies on T-cell infiltration are few; however, a tendency towards infiltration peak at later time points (from day 4 onwards) was seen.This review provides a framework for the instigation of post-stroke anti-inflammatory treatment, which could prove beneficial and widen the therapeutic window compared to current treatment options.
Assuntos
Granulócitos/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Acidente Vascular Cerebral/imunologia , Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Granulócitos/patologia , Macrófagos/patologia , Neutrófilos/patologia , Acidente Vascular Cerebral/patologia , Linfócitos T/patologiaRESUMO
The underlying mechanism of the GABAergic deficits observed in schizophrenia has been proposed to involve NMDA receptor hypofunction. An emerging treatment strategy therefore aims at enhancing GABAergic signalling by increasing the excitatory transmission onto interneurons. We wanted to determine whether behavioural and GABAergic functional deficits induced by the NMDA receptor channel blocker, phencyclidine (PCP), could be reversed by repeated administration of two drugs known to enhance GABAergic transmission: the positive allosteric modulator (PAM) of the metabotropic glutamate receptor 5 (mGluR5), ADX47273, and the partial agonist of the α7 nicotinic acetylcholine receptor (α7 nAChR), SSR180711. Adolescent rats (4-5 weeks) subjected to PCP treatment during the second postnatal week displayed a consistent deficit in prepulse inhibition (PPI), which was reversed by a one-week treatment with ADX47273 or SSR180711. We examined GABAergic transmission by whole cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSC) in pyramidal neurons in layer II/III of prefrontal cortex (PFC) and by activation of extrasynaptic δ-containing GABAA receptors by THIP. Following PCP treatment, pyramidal neurons displayed a reduced mIPSC frequency and up-regulation of extrasynaptic THIP-induced current. ADX47273 treatment restored this up-regulation of THIP-induced current. Reduced receptor function seems to be the underlying cause of the reported changes, since repeated treatment with ADX47273 and SSR180711 decreased the induction of spontaneous inhibitory current caused by acute and direct agonism of mGluR5s and α7 nAChRs in slices. These results show that repeated administration of ADX47273 or SSR180711 reverses certain behavioural and functional deficits induced by PCP, likely through down-regulation or desensitisation of mGluR5s and α7 nAChRs, respectively.
Assuntos
Neurônios GABAérgicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Reflexo de Sobressalto/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colinérgicos/farmacologia , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Feminino , Lobo Frontal/citologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Fenciclidina/efeitos adversos , Piperidinas/metabolismo , Piperidinas/farmacologia , Gravidez , Ratos , Fatores de TempoRESUMO
A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 µM, R(max) 69%) was a moderately potent agonist at human α(1)ß(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)ß(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)ß(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.
Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Muscimol/análogos & derivados , Simulação por Computador , Agonistas de Receptores de GABA-A/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Muscimol/síntese química , Muscimol/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
Cholinergic dysfunction and deposition of plaques containing amyloid ß-peptides (Aß) are two of the characteristics of Alzheimer's disease. Here, we combine APPswe/PS1dE9 (APP/PS1) mice with the cholinergic immunotoxin mu p75-saporin (SAP) to integrate partial basal forebrain cholinergic degeneration and the neuropathology of APP/PS1 mice. By 6 months of age, APP/PS1 mice and wild type littermates (Wt) received intracerebroventricular injection of 0.6 µg SAP (lesion) or PBS (sham). Two months following surgery, APP/PS1 mice treated with SAP were significantly impaired compared to sham treated APP/PS1 mice in a behavioural paradigm addressing working memory. Conversely, the performance of Wt mice was unaffected by SAP treatment. Choline acetyltransferase activity was reduced in the hippocampus and frontal cortex following SAP treatment. The selective effect of a mild SAP lesion in APP/PS1 mice was not due to a more extensive cholinergic degeneration since the reduction in choline acetyltransferase activity was similar following SAP treatment in APP/PS1 mice and Wt. Interestingly, plaque load was significantly increased in SAP treated APP/PS1 mice relative to sham lesioned APP/PS1 mice. Additionally, APP/PS1 mice treated with SAP showed a tendency towards an increased level of soluble and insoluble Aß1-40 and Aß1-42 measured in brain tissue homogenate. Our results suggest that the combination of cholinergic degeneration and Aß overexpression in the APP/PS1 mouse model results in cognitive decline and accelerated plaque burden. SAP treated APP/PS1 mice might thus constitute an improved model of Alzheimer's disease-like neuropathology and cognitive deficits compared to the conventional APP/PS1 model without selective removal of basal forebrain cholinergic neurons.
Assuntos
Doença de Alzheimer/etiologia , Neurônios Colinérgicos/efeitos dos fármacos , Modelos Animais de Doenças , Imunotoxinas/farmacologia , Placa Amiloide/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Colina O-Acetiltransferase/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/patologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Injeções Intraventriculares , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Placa Amiloide/fisiopatologia , Presenilina-1/genética , SaporinasRESUMO
Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) are importantly involved in neurobiological mechanisms governing states of arousal such as sleep and wakefulness as well as other appetitive behaviors, such as drug-seeking. Accordingly, mechanisms controlling their excitability are important to elucidate if we are to understand how these LDT neurons generate arousal states. Glutamate mediates the vast majority of excitatory synaptic transmission in the vertebrate CNS and while presence of glutamate input in the LDT has been shown and ionotropic responses to glutamate have been reported in the LDT, characterization of metabotropic responses is lacking. Therefore, electrophysiological responses and changes in levels of intracellular Ca(2+) in mouse cholinergic LDT neurons following application of specific mGluR agonists and antagonists were examined. Unexpectedly, both the mGluR(5)specific agonist, CHPG, and the group II mGluR (mGlu(2/3)) agonist, LY379268 (LY), induced a TTX-insensitive outward current/hyperpolarization. Both outward currents were significantly reduced by the mGluR antagonist MCPG and the CHPG-induced current was blocked by the specific mGluR(5) antagonist MTEP. Concurrent Ca(2+)imaging revealed that while CHPG actions did include release of Ca(2+) from CPA/thapsigargin-sensitive intracellular stores, actions of LY did not. Both CHPG- and LY-induced outward currents were mediated by a TEA-sensitive potassium conductance. The large-conductance, Ca(2+)-dependent potassium (BK) channel blocker, iberiotoxin, attenuated CHPG actions. Consistent with actions on the BK conductance, CHPG enhanced the amplitude of the fast component of the after hyperpolarizing potential, concurrent with a reduction in the firing rate. We conclude that stimulation of mGluR(5) and group II (mGluR(2/3)) elicits postsynaptically-mediated outward currents/hyperpolarizations in cholinergic LDT neurons. Effects of glutamatergic input would be, thus, expected not only to be excitation via stimulation of ionotropic glutamate receptors and mGluR(1), but also inhibition via actions at mGluR(5) and mGluR(2/3) on these neurons. As these two processes counteract each other, these surprising findings necessitate revision of predictions regarding the net level of excitation generated by glutamate input to cholinergic LDT cells and, by extension, the functional outcome of glutamate transmission on processes which these neurons regulate. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.