RESUMO
We employed the whole-cell patch-clamp method and ChAT-Cre mice to study the electrophysiological attributes of cholinergic neurons in the external globus pallidus. Most neurons were inactive, although approximately 20% displayed spontaneous firing, including burst firing. The resting membrane potential, the whole neuron input resistance, the membrane time constant and the total neuron membrane capacitance were also characterized. The current-voltage relationship showed time-independent inward rectification without a "sag". Firing induced by current injections had a brief initial fast adaptation followed by tonic firing with minimal accommodation. Intensity-frequency plots exhibited maximal average firing rates of about 10 Hz. These traits are similar to those of some cholinergic neurons in the basal forebrain. Also, we examined their dopamine sensitivity by acutely blocking dopamine receptors. This action demonstrated that the membrane potential, excitability, and firing pattern of pallidal cholinergic neurons rely on the constitutive activity of dopamine receptors, primarily D2-class receptors. The blockade of these receptors induced a resting membrane potential hyperpolarization, a decrease in firing for the same stimulus, the disappearance of fast adaptation, and the emergence of a depolarization block. This shift in physiological characteristics was evident even when the hyperpolarization was corrected with D.C. current. Neither the currents that generate the action potentials nor those from synaptic inputs were responsible. Instead, our findings suggest, that subthreshold slow ion currents, that require further investigation, are the target of this novel dopaminergic signaling.
Assuntos
Dopamina , Globo Pálido , Camundongos , Animais , Dopamina/fisiologia , Potenciais de Ação/fisiologia , Neurônios Colinérgicos , Receptores Dopaminérgicos , ColinérgicosRESUMO
A pipeline is proposed here to describe different features to study brain microcircuits on a histological scale using multi-scale analyses, including the uniform manifold approximation and projection (UMAP) dimensional reduction technique and modularity algorithm to identify neuronal ensembles, Runs tests to show significant ensembles activation, graph theory to show trajectories between ensembles, and recurrence analyses to describe how regular or chaotic ensembles dynamics are. The data set includes ex-vivo NMDA-activated striatal tissue in control conditions as well as experimental models of disease states: decorticated, dopamine depleted, and L-DOPA-induced dyskinetic rodent samples. The goal was to separate neuronal ensembles that have correlated activity patterns. The pipeline allows for the demonstration of differences between disease states in a brain slice. First, the ensembles were projected in distinctive locations in the UMAP space. Second, graphs revealed functional connectivity between neurons comprising neuronal ensembles. Third, the Runs test detected significant peaks of coactivity within neuronal ensembles. Fourth, significant peaks of coactivity were used to show activity transitions between ensembles, revealing recurrent temporal sequences between them. Fifth, recurrence analysis shows how deterministic, chaotic, or recurrent these circuits are. We found that all revealed circuits had recurrent activity except for the decorticated circuits, which tended to be divergent and chaotic. The Parkinsonian circuits exhibit fewer transitions, becoming rigid and deterministic, exhibiting a predominant temporal sequence that disrupts transitions found in the controls, thus resembling the clinical signs of rigidity and paucity of movements. Dyskinetic circuits display a higher recurrence rate between neuronal ensembles transitions, paralleling clinical findings: enhancement in involuntary movements. These findings confirm that looking at neuronal circuits at the histological scale, recording dozens of neurons simultaneously, can show clear differences between control and diseased striatal states: "fingerprints" of the disease states. Therefore, the present analysis is coherent with previous ones of striatal disease states, showing that data obtained from the tissue are robust. At the same time, it adds heuristic ways to interpret circuitry activity in different states.
RESUMO
Differences in the intrinsic properties of intralaminar thalamo-striatal neurons such as expressing low-threshold-spikes (LTS) or after hyperpolarizing potentials (AHPs) of different duration have been attributed to different maturation stages. However, two morphological types: "diffuse" and "bushy" have been described. Therefore, we explored whether electrophysiological differences persist in adult mice using whole cell recordings. Some recorded neurons were identified by intracellular labeling with biocytin and double labeling with retrograde or anterograde tracings using Cre-mice. We classified these neurons by their AHPs during spontaneous firing. Neurons with long duration AHPs, with fast and slow components, were mostly found in the parafascicular (Pf) nucleus. Neurons with brief AHPs were mainly found in the central lateral (CL) nucleus. However, neurons with both AHPs were found in both nuclei in different proportions. Firing frequency adaptation differed between these neuron classes: those with prolonged AHPs exhibited firing frequency adaptation with fast and slow time constants whereas those with brief AHPs were slow adapters. Neurons with more prolonged AHPs had significant higher input resistances than neurons with brief AHPs. Both cell classes could fire in two modes: trains of single action potentials at depolarized potentials or high frequency bursts on top of LTS at more hyperpolarized potentials. LTS were probably generated by Cav3 calcium channels since they were blocked by the selective antagonist TTA-P2. About 11% of neurons with brief AHPs and 55% of neurons with prolonged AHPs do not show LTS and bursts, even when potassium currents are blocked.
Assuntos
Corpo Estriado , Neurônios , Potenciais de Ação , Animais , Canais de Cálcio , CamundongosRESUMO
The striatum is the largest entrance to the basal ganglia. Diverse neuron classes make up striatal microcircuit activity, consisting in the sequential activation of neuronal ensembles. How different neuron classes participate in generating ensemble sequences is unknown. In control mus musculus brain slices in vitro, providing excitatory drive generates ensemble sequences. In Parkinsonian microcircuits captured by a highly recurrent ensemble, a cortical stimulus causes a transitory reconfiguration of neuronal groups alleviating Parkinsonism. Alternation between neuronal ensembles needs interconnectivity, in part due to interneurons, preferentially innervated by incoming afferents. One main class of interneuron expresses parvalbumin (PV+ neurons) and mediates feed-forward inhibition. However, its more global actions within the microcircuit are unknown. Using calcium imaging in ex vivo brain slices simultaneously recording dozens of neurons, we aimed to observe the actions of PV+ neurons within the striatal microcircuit. PV+ neurons in active microcircuits are 5%-11% of the active neurons even if, anatomically, they are <1% of the total neuronal population. In resting microcircuits, optogenetic activation of PV+ neurons turns on circuit activity by activating or disinhibiting, more neurons than those actually inhibited, showing that feed-forward inhibition is not their only function. Optostimulation of PV+ neurons in active microcircuits inhibits and activates different neuron sets, resulting in the reconfiguration of neuronal ensembles by changing their functional connections and ensemble membership, showing that neurons may belong to different ensembles at different situations. Our results show that PV+ neurons participate in the mechanisms that generate alternation of neuronal ensembles, therefore provoking ensemble sequences.
Assuntos
Corpo Estriado , Parvalbuminas , Animais , Gânglios da Base/metabolismo , Corpo Estriado/metabolismo , Interneurônios/metabolismo , Camundongos , Neurônios/metabolismo , Parvalbuminas/metabolismoRESUMO
Striatal projection neurons (SPNs) process motor and cognitive information. Their activity is affected by Parkinson's disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit.
Assuntos
Potenciais de Ação , Neurônios GABAérgicos/fisiologia , Canais de Potássio KCNQ/fisiologia , Neostriado/fisiologia , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Córtex Cerebral/fisiologia , Neurônios Colinérgicos/fisiologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Transgênicos , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Neostriado/citologia , Neostriado/metabolismo , Peptídeos/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismoRESUMO
Neostriatal neurons may undergo events of spontaneous synchronization as those observed in recurrent networks of excitatory neurons, even when cortical afferents are transected. It is necessary to explain these events because the neostriatum is a recurrent network of inhibitory neurons. Synchronization of neuronal activity may be caused by plateau-like depolarizations. Plateau-like orthodromic depolarizations that resemble up-states in medium spiny neostriatal neurons (MSNs) may be induced by a single corticostriatal suprathreshold stimulus. Slow synaptic depolarizations may last hundreds of milliseconds, decay slower than the monosynaptic glutamatergic synaptic potentials that induce them, and sustain repetitive firing. Because inhibitory inputs impinging onto MSNs have a reversal potential above the resting membrane potential but below the threshold for firing, they conform a type of "shunting inhibition". This work asks if shunting GABAergic inputs onto MSNs arrive asynchronously enough as to help in sustaining the plateau-like corticostriatal response after a single cortical stimulus. This may help to begin explaining autonomous processing in the striatal micro-circuitry in the presence of a tonic excitatory drive and independently of spatio-temporally organized inputs. It is shown here that besides synaptic currents from AMPA/KA- and NMDA-receptors, as well as L-type intrinsic Ca(2+)- currents, inhibitory synapses help in maintaining the slow depolarization, although they accomplish the role of depressing firing at the beginning of the response. We then used a NEURON model of spiny cells to show that inhibitory synapses arriving asynchronously on the dendrites can help to simulate a plateau potential similar to that observed experimentally.
Assuntos
Modelos Neurológicos , Neostriado/fisiologia , Inibição Neural/fisiologia , Animais , Sinalização do Cálcio , Rede Nervosa/fisiologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais Sinápticos/fisiologiaRESUMO
Ryanodine-sensitive intracellular Ca2+ channels (RyRs) are present in suprachiasmatic nuclei (SCN) neurons, but the functions served by these channels are not known. Here we addressed whether mobilization of intracellular Ca2+ stores through the RyRs may be a link between the molecular clock and the firing rate in SCN neurons. Activation of the RyRs by administration of either 1 mM caffeine or 100 nM ryanodine increased the firing frequency, whereas inhibition of RyRs by 10 microM dantrolene or 80 microm ryanodine decreased firing rate. Similar results were obtained in experiments conducted at either midday or midnight. Furthermore, these effects were not mediated by synaptic transmission as blockade of GABA A, AMPA and NMDA receptors did not prevent the excitatory or inhibitory effects induced by either dose of ryanodine on SCN firing. We conclude that gating of RyRs is a key element of the intricate output pathway from the circadian clock within SCN neurons in rats.
Assuntos
Relógios Biológicos/fisiologia , Sinalização do Cálcio/fisiologia , Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Núcleo Supraquiasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Relógios Biológicos/efeitos dos fármacos , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Dantroleno/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Relaxantes Musculares Centrais/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Dopamine is a critical modulator of striatal function; its absence produces Parkinson's disease. Most cellular actions of dopamine are still unknown. This work describes the presynaptic actions of dopaminergic receptor agonists on GABAergic transmission between neostriatal projection neurons. Axon collaterals interconnect projection neurons, the main axons of which project to other basal ganglia nuclei. Most if not all of these projecting axons pass through the globus pallidus. Thus, we lesioned the intrinsic neurons of the globus pallidus and stimulated neostriatal efferent axons antidromically with a bipolar electrode located in this nucleus. This maneuver revealed a bicuculline-sensitive synaptic current while recording in spiny cells. D1 receptor agonists facilitated whereas D2 receptor agonists depressed this synaptic current. In contrast, a bicuculline-sensitive synaptic current evoked by field stimulation inside the neostriatum was not consistently modulated, in agreement with previous studies. The data are discussed in light of the most recent experimental and modeling results. The conclusion was that inhibition of spiny cells by axon collaterals of other spiny cells is quantitatively important; however, to be functionally important, this inhibition might be conditioned to the synchronized firing of spiny neurons. Finally, dopamine exerts a potentially important role regulating the extent of lateral inhibition.