RESUMEN
Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer's diseases. This is best exemplified by clinical observations with xanomeline, the efficacy of which is thought to be mediated through co-activation of the M1 and M4 muscarinic acetylcholine receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to the actions of selective M1 and M4 mAChR activators on in vivo intracellular signaling cascades in mice, including 3'-5'-cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation in the striatum, hippocampus, and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electrophysiological signatures in rats using ex vivo recordings from CA1 (Cornu Ammonis 1) as well as in vivo hippocampal theta. As expected, xanomeline's effects across these readouts were consistent with activation of both M1 and M4 mAChRs; however, differences were observed across different brain regions, suggesting non-uniform activation of these receptor subtypes in the central nervous system. Interestingly, despite having nearly equal in vitro potency at the M1 and the M4 mAChRs, during in vivo assays xanomeline produced M4-like effects at significantly lower brain exposures than those at which M1-like effects were observed. Our results raise the possibility that clinical efficacy observed with xanomeline was driven, in part, through its non-uniform activation of mAChR subtypes in the central nervous system and, at lower doses, through preferential agonism of the M4 mAChR.
Asunto(s)
Hipocampo/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Piridinas/farmacología , Receptor Muscarínico M1/metabolismo , Tiadiazoles/farmacología , Acetilcolina/metabolismo , Acetilcolina/farmacología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Hipocampo/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismoRESUMEN
Recent data demonstrated that activation of the muscarinic M1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M2 and M3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M1-selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M1 is sufficient to elicit cholinergic AEs.
Asunto(s)
Isoindoles/farmacología , Lactamas/farmacología , Oxazoles/farmacología , Receptor Muscarínico M1/agonistas , Convulsiones/inducido químicamente , Regulación Alostérica , Anfetamina/farmacología , Animales , Ataxia/inducido químicamente , Diarrea/inducido químicamente , Perros , Donepezilo , Diseño de Fármacos , Femenino , Humanos , Indanos/farmacología , Isoindoles/administración & dosificación , Isoindoles/síntesis química , Isoindoles/toxicidad , Lactamas/administración & dosificación , Lactamas/síntesis química , Lactamas/toxicidad , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Oxazoles/administración & dosificación , Oxazoles/síntesis química , Oxazoles/toxicidad , Piperidinas/farmacología , Ratas Wistar , Receptor Muscarínico M1/antagonistas & inhibidores , Escopolamina/farmacología , Relación Estructura-Actividad , Sulfonamidas/farmacología , Tiadiazoles/farmacología , Vómitos/inducido químicamenteRESUMEN
Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.
Asunto(s)
Región CA1 Hipocampal/fisiología , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/metabolismo , Transmisión Sináptica/fisiología , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The rationale for using M1 selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1 activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1 muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.
Asunto(s)
Encéfalo/efectos de los fármacos , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M1/agonistas , Anfetamina/farmacología , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Células CHO , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiología , Cricetinae , Cricetulus , Dopaminérgicos/farmacología , Relación Dosis-Respuesta a Droga , Fenómenos Electrofisiológicos/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Humanos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Agonistas Muscarínicos/clasificación , Ratas Sprague-Dawley , Receptor Muscarínico M1/metabolismoRESUMEN
Selective activation of the M1 muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. Herein, we describe the characterization of an M1 PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([(3)H]PT-1284), as a tool for characterizing the M1 allosteric binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 ( 1: )) was shown to potentiate acetylcholine (ACh) in an M1 fluorometric imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC50, 165 nM). PT-1284 ( 1: ) also reduced the concentration of ACh required to inhibit [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 µM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 µM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]PT-1284 binding assay and the EC50 values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M1 PAM binding affinity and functional activity, and that [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs.
Asunto(s)
Isoindoles/metabolismo , Piridinas/metabolismo , Ensayo de Unión Radioligante , Receptor Muscarínico M1/metabolismo , Acetilcolina , Regulación Alostérica , Animales , Autorradiografía , Células CHO , Cricetinae , Cricetulus , Fenómenos Electrofisiológicos , Fluorometría , Células HEK293 , Hipocampo/fisiología , Humanos , Cinética , Masculino , Membranas/metabolismo , N-Metilescopolamina/metabolismo , Tomografía de Emisión de Positrones , Ratas Sprague-DawleyRESUMEN
It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer's disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.
Asunto(s)
Descubrimiento de Drogas , Ácidos Picolínicos/farmacología , Receptor Muscarínico M1/agonistas , Tiazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Ácidos Picolínicos/síntesis química , Ácidos Picolínicos/química , Ratas , Receptor Muscarínico M1/metabolismo , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Amidas/química , Amidas/farmacología , Indoles/química , Indoles/farmacología , Receptor Muscarínico M1/metabolismo , Amidas/farmacocinética , Animales , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Indoles/farmacocinética , Ratones , Simulación del Acoplamiento Molecular , Receptor Muscarínico M1/agonistasRESUMEN
The inhibitory output from the internal pallidum and substantia nigra to the thalamus forms an important link in the transmission of basal ganglia processing to cortex. Two hypotheses consider either inhibition of thalamic activity or thalamic excitation via post-inhibitory rebound burst firing as the functional mode of this link. We used optogenetics to characterize the synaptic properties of nigral input to motor thalamus in adult mouse brain slices, and to determine in what conditions the nigral inhibition of motor thalamus is transmitted via inhibition or rebound firing. Our results are more consistent with graded inhibition of spiking for conditions expected in normal awake animals, because inhibitory potentials from nigral input were generally not sufficient to elicit rebound spikes when the thalamic neurons were actively firing. However, with bursty or fast trains of nigral input low-threshold rebound spike bursts could be triggered for low levels of excitation. This may form the basis of pathological burst generation and transmission in parkinsonian conditions.
RESUMEN
The unmet need for the treatment of disorders of the nervous system is growing, and as highlighted in the media and elsewhere, the results of an aging population will ensure this continues with an upward trajectory. Incredibly, the efforts within industry to identify new drugs to treat these conditions have seemingly disappeared despite the growing need. There has been a run of extraordinary failure in the later stages of the drug discovery process for neurological and psychiatric disorders, which has many causes. We believe, though, that we have to confront this dire situation, both by using learnings from the post hoc analysis of our historical failure, as well as harnessing the bewildering array of new technologies and data now available to us, to ensure we are making the right decisions along the very complicated path of drug discovery to registration.
Asunto(s)
Antipsicóticos/química , Descubrimiento de Drogas/métodos , Trastornos Mentales/tratamiento farmacológico , Animales , Descubrimiento de Drogas/tendencias , Humanos , Modelos Animales , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendenciasRESUMEN
Correlated firing among populations of neurons is present throughout the brain and is often rhythmic in nature, observable as an oscillatory fluctuation in the local field potential. Although rhythmic population activity is believed to be critical for normal function in many brain areas, synchronized neural oscillations are associated with disease states in other cases. In the globus pallidus (GP in rodents, homolog of the primate GPe), pairs of neurons generally have uncorrelated firing in normal animals despite an anatomical organization suggesting that they should receive substantial common input. In contrast, correlated and rhythmic GP firing is observed in animal models of Parkinson's disease (PD). Based in part on these findings, it has been proposed that an important part of basal ganglia function is active decorrelation, whereby redundant information is compressed. Mechanisms that implement active decorrelation, and changes that cause it to fail in PD, are subjects of great interest. Rat GP neurons express fast, transient voltage-dependent sodium channels (NaF channels) in their dendrites, with the expression level being highest near asymmetric synapses. We recently showed that the dendritic NaF density strongly influences the responsiveness of model GP neurons to synchronous excitatory inputs. In the present study, we use rat GP neuron models to show that dendritic NaF channel expression is a potential cellular mechanism of active decorrelation. We further show that model neurons with lower dendritic NaF channel expression have a greater tendency to phase lock with oscillatory synaptic input patterns like those observed in PD.
Asunto(s)
Dendritas/fisiología , Globo Pálido/citología , Modelos Neurológicos , Neuronas/citología , Enfermedad de Parkinson/patología , Canales de Sodio/fisiología , Potenciales de Acción/fisiología , Animales , Relojes Biológicos/fisiología , Simulación por Computador , Modelos Animales de Enfermedad , Estimulación Eléctrica , Expresión Génica/fisiología , Neuronas/fisiología , Ratas , Sinapsis/fisiologíaRESUMEN
The voltage and time dependence of ion channels can be regulated, notably by phosphorylation, interaction with phospholipids, and binding to auxiliary subunits. Many parameter variation studies have set conductance densities free while leaving kinetic channel properties fixed as the experimental constraints on the latter are usually better than on the former. Because individual cells can tightly regulate their ion channel properties, we suggest that kinetic parameters may be profitably set free during model optimization in order to both improve matches to data and refine kinetic parameters. To this end, we analyzed the parameter optimization of reduced models of three electrophysiologically characterized and morphologically reconstructed globus pallidus neurons. We performed two automated searches with different types of free parameters. First, conductance density parameters were set free. Even the best resulting models exhibited unavoidable problems which were due to limitations in our channel kinetics. We next set channel kinetics free for the optimized density matches and obtained significantly improved model performance. Some kinetic parameters consistently shifted to similar new values in multiple runs across three models, suggesting the possibility for tailored improvements to channel models. These results suggest that optimized channel kinetics can improve model matches to experimental voltage traces, particularly for channels characterized under different experimental conditions than recorded data to be matched by a model. The resulting shifts in channel kinetics from the original template provide valuable guidance for future experimental efforts to determine the detailed kinetics of channel isoforms and possible modulated states in particular types of neurons.
Asunto(s)
Algoritmos , Membrana Celular/fisiología , Globo Pálido/fisiología , Canales Iónicos/fisiología , Modelos Neurológicos , Neuronas/fisiología , Potenciales de Acción/fisiología , Animales , Globo Pálido/citología , Citometría de Imagen/métodos , Activación del Canal Iónico/fisiología , Canales Iónicos/farmacocinética , Cinética , Neuronas/citología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp/métodos , Fosforilación/fisiología , Ratas , Programas Informáticos , Potenciales Sinápticos/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Conductance-based neuron models are frequently employed to study the dynamics of biological neural networks. For speed and ease of use, these models are often reduced in morphological complexity. Simplified dendritic branching structures may process inputs differently than full branching structures, however, and could thereby fail to reproduce important aspects of biological neural processing. It is not yet well understood which processing capabilities require detailed branching structures. Therefore, we analyzed the processing capabilities of full or partially branched reduced models. These models were created by collapsing the dendritic tree of a full morphological model of a globus pallidus (GP) neuron while preserving its total surface area and electrotonic length, as well as its passive and active parameters. Dendritic trees were either collapsed into single cables (unbranched models) or the full complement of branch points was preserved (branched models). Both reduction strategies allowed us to compare dynamics between all models using the same channel density settings. Full model responses to somatic inputs were generally preserved by both types of reduced model while dendritic input responses could be more closely preserved by branched than unbranched reduced models. However, features strongly influenced by local dendritic input resistance, such as active dendritic sodium spike generation and propagation, could not be accurately reproduced by any reduced model. Based on our analyses, we suggest that there are intrinsic differences in processing capabilities between unbranched and branched models. We also indicate suitable applications for different levels of reduction, including fast searches of full model parameter space.
Asunto(s)
Dendritas/fisiología , Modelos Neurológicos , Neuronas/citología , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Simulación por Computador , Dendritas/efectos de los fármacos , Estimulación Eléctrica , Aminoácidos Excitadores/farmacología , Globo Pálido/citología , Conducción Nerviosa/fisiología , Estadísticas no Paramétricas , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
The globus pallidus (GP) predominantly contains GABAergic projection neurons that occupy a central position in the indirect pathway of the basal ganglia. They have long dendrites that can extend through one-half the diameter of the GP in rats, potentially enabling convergence and interaction between segregated basal ganglia circuits. Because of the length and fine diameter of GP dendrites, however, it is unclear how much influence distal synapses have on spiking activity. Dendritic expression of fast voltage-dependent Na(+) channels (NaF channels) can enhance the importance of distal excitatory synapses by allowing for dendritic spike initiation and by subthreshold boosting of EPSPs. Antibody labeling has demonstrated the presence of NaF channel proteins in GP dendrites, but the quantitative expression density of the channels remains unknown. We built a series of nine GP neuron models that differed only in their dendritic NaF channel expression level to assess the functional impact of this parameter. The models were all similar in their basic electrophysiological features; however, higher expression levels of dendritic NaF channels increased the relative effectiveness of distal inputs for both excitatory and inhibitory synapses, broadening the effective extent of the dendritic tree. Higher dendritic NaF channel expression also made the neurons more resistant to tonic inhibition and highly sensitive to clustered synchronous excitation. The dendritic NaF channel expression pattern may therefore be a critical determinant of convergence for both the striatopallidal and subthalamopallidal projections, while also dictating which spatiotemporal input patterns are most effective at driving GP neuron output.
Asunto(s)
Dendritas/fisiología , Globo Pálido/fisiología , Modelos Neurológicos , Red Nerviosa/fisiología , Neuronas/fisiología , Canales de Sodio/fisiología , Potenciales de Acción/fisiología , Análisis de Varianza , Animales , Simulación por Computador , Electrofisiología , Masculino , Ratas , Ratas Sprague-Dawley , Estadísticas no Paramétricas , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Synchronization of globus pallidus (GP) neurons and cortically entrained oscillations between GP and other basal ganglia nuclei are key features of the pathophysiology of Parkinson's disease. Phase response curves (PRCs), which tabulate the effects of phasic inputs within a neuron's spike cycle on output spike timing, are efficient tools for predicting the emergence of synchronization in neuronal networks and entrainment to periodic input. In this study we apply physiologically realistic synaptic conductance inputs to a full morphological GP neuron model to determine the phase response properties of the soma and different regions of the dendritic tree. We find that perisomatic excitatory inputs delivered throughout the interspike interval advance the phase of the spontaneous spike cycle yielding a type I PRC. In contrast, we demonstrate that distal dendritic excitatory inputs can either delay or advance the next spike depending on whether they occur early or late in the spike cycle. We find this latter pattern of responses, summarized by a biphasic (type II) PRC, was a consequence of dendritic activation of the small conductance calcium-activated potassium current, SK. We also evaluate the spike-frequency dependence of somatic and dendritic PRC shapes, and we demonstrate the robustness of our results to variations of conductance densities, distributions, and kinetic parameters. We conclude that the distal dendrite of GP neurons embodies a distinct dynamical subsystem that could promote synchronization of pallidal networks to excitatory inputs. These results highlight the need to consider different effects of perisomatic and dendritic inputs in the control of network behavior.
Asunto(s)
Dendritas/fisiología , Globo Pálido/citología , Modelos Neurológicos , Neuronas/citología , Sinapsis/fisiología , Potenciales de Acción/fisiología , Animales , Fenómenos Biofísicos/fisiología , Biofisica/métodos , Simulación por Computador , Estimulación Eléctrica/métodos , Conducción Nerviosa/fisiología , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Neuronal recordings and computer simulations produce ever growing amounts of data, impeding conventional analysis methods from keeping pace. Such large datasets can be automatically analyzed by taking advantage of the well-established relational database paradigm. Raw electrophysiology data can be entered into a database by extracting its interesting characteristics (e.g., firing rate). Compared to storing the raw data directly, this database representation is several orders of magnitude higher efficient in storage space and processing time. Using two large electrophysiology recording and simulation datasets, we demonstrate that the database can be queried, transformed and analyzed. This process is relatively simple and easy to learn because it takes place entirely in Matlab, using our database analysis toolbox, PANDORA. It is capable of acquiring data from common recording and simulation platforms and exchanging data with external database engines and other analysis toolboxes, which make analysis simpler and highly interoperable. PANDORA is available to be freely used and modified because it is open-source (http://software.incf.org/software/pandora/home).
Asunto(s)
Simulación por Computador , Sistemas de Administración de Bases de Datos , Bases de Datos Factuales , Fenómenos Electrofisiológicos , Neuronas/fisiología , Programas Informáticos , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica , Ganglios de Invertebrados/fisiología , Globo Pálido/fisiología , Potenciales de la Membrana/efectos de los fármacos , Modelos Neurológicos , Análisis Multivariante , Nephropidae , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Factores de TiempoRESUMEN
Globus pallidus (GP) neurons recorded in brain slices show significant variability in intrinsic electrophysiological properties. To investigate how this variability arises, we manipulated the biophysical properties of GP neurons using computer simulations. Specifically, we created a GP neuron model database with 100,602 models that had varying densities of nine membrane conductances centered on a hand-tuned model that replicated typical physiological data. To test the hypothesis that the experimentally observed variability can be attributed to variations in conductance densities, we compared our model database results to a physiology database of 146 slice recordings. The electrophysiological properties of generated models and recordings were assessed with identical current injection protocols and analyzed with a uniform set of measures, allowing a systematic analysis of the effects of varying voltage-gated and calcium-gated conductance densities on the measured properties and a detailed comparison between models and recordings. Our results indicated that most of the experimental variability could be matched by varying conductance densities, which we confirmed with additional partial block experiments. Further analysis resulted in two key observations: (1) each voltage-gated conductance had effects on multiple measures such as action potential waveform and spontaneous or stimulated spike rates; and (2) the effect of each conductance was highly dependent on the background context of other conductances present. In some cases, such interactions could reverse the effect of the density of one conductance on important excitability measures. This context dependence of conductance density effects is important to understand drug and neuromodulator effects that work by affecting ion channels.
Asunto(s)
Potenciales de Acción/fisiología , Simulación por Computador , Globo Pálido/citología , Canales Iónicos/metabolismo , Modelos Neurológicos , Neuronas/fisiología , Animales , Animales Recién Nacidos , Conductividad Eléctrica , Estimulación Eléctrica/métodos , Técnicas In Vitro , Activación del Canal Iónico/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.