Dopamine-containing neurons of the midbrain are required to control voluntary movements, behavior... more Dopamine-containing neurons of the midbrain are required to control voluntary movements, behavior and motivation. Damage of these neurons in the substantia nigra zona compacta (SNc) is considered as the cause of Parkinson's disease. In this work, the firing characteristics of SNc neurons were studied in vitro. Neurons were identified by intracellular labeling tyrosine hydroxylase (TH) immunocytochemistry, and their electrophysiological characteristics. TH-positive neurons displayed three different firing modes. At around -55 mV neurons exhibited a regular single spike firing mode (pacemaking). At around -65 mV, neurons discharged in an irregular single spike firing mode (idling). At around -75 mV, many neurons exhibited a mode of discharge characterized by low threshold spikes (LTS), oscillations and, on some occasions, bursting. When present, bursts were of short duration and commonly made up of incomplete somatodendritic action potentials. TH-negative neurons also exhibited single spikes and bursting modes of firing. Both types of SNc neurons generated LTS oscillations following a hyperpolarization. However, LTS in TH-negative neurons could be evoked from less negative membrane potentials and produced more prolonged bursts. LTS from both types of neurons could be differentiated. LTS were blocked by 100 microM Ni2+ and became prolonged in the presence of TEA.
Adenosine A1 and A2A receptors are expressed in striatal projection neurons (SPNs). A1 receptors ... more Adenosine A1 and A2A receptors are expressed in striatal projection neurons (SPNs). A1 receptors are located in direct (dSPN) and indirect SPNs (iSNP). A2A receptors are only present in iSPNs. Dopamine D2 receptors are also expressed in iSPNs and interactions between D2 and A2A receptors have received attention. iSPNs activity increases during parkinsonism (PD) and A2A receptors may be responsible by enhancing Ca2+ currents (iCa2+). Therefore, A2A receptors blockade is a therapeutic approach. We asked whether A2A receptors need the interaction with D2 receptors (D2R) to exert their actions. By using isolated and identified iSPNs to avoid indirect influences, we show that D2R action habilitates A2A receptors (A2AR) modulation. iCa2+ through voltage gated Ca2+ channels (CaV) was used as a signal to observe this interaction. Voltage-clamp recordings in acutely dissociated iSPNs, current-clamp recordings in slices and calcium imaging in transgenic A2A-Cre mice, showed that D2R reduction...
Amantadine and clozapine have proved to reduce abnormal involuntary movements (AIMs) in preclinic... more Amantadine and clozapine have proved to reduce abnormal involuntary movements (AIMs) in preclinical and clinical studies of L-DOPA-Induced Dyskinesias (LID). Even though both drugs decrease AIMs, they may have different action mechanisms by using different receptors and signaling profiles. Here we asked whether there are differences in how they modulate neuronal activity of multiple striatal neurons within the striatal microcircuit at histological level during the dose-peak of L-DOPA in ex-vivo brain slices obtained from dyskinetic mice. To answer this question, we used calcium imaging to record the activity of dozens of neurons of the dorsolateral striatum before and after drugs administration in vitro. We also developed an analysis framework to extract encoding insights from calcium imaging data by quantifying neuronal activity, identifying neuronal ensembles by linking neurons that coactivate using hierarchical cluster analysis and extracting network parameters using Graph Theory. The results show that while both drugs reduce LIDs scores behaviorally in a similar way, they have several different and specific actions on modulating the dyskinetic striatal microcircuit. The extracted features were highly accurate in separating amantadine and clozapine effects by means of principal components analysis (PCA) and support vector machine (SVM) algorithms. These results predict possible synergistic actions of amantadine and clozapine on the dyskinetic striatal microcircuit establishing a framework for a bioassay to test novel antidyskinetic drugs or treatments in vitro.
In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked lon... more In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked long‐lasting plateau potentials in neostriatal neurons. Plateau potentials were often followed by spontaneous voltage transitions between two preferred membrane potentials. While the induction of plateau potentials was disrupted by non‐NMDA and NMDA glutamate receptor antagonists, the maintenance of spontaneous voltage transitions was only blocked by NMDA receptor and L‐type Ca2+ channel antagonists. The frequency and duration of depolarized events, resembling up‐states described in vivo, were increased by NMDA and L‐type Ca2+ channel agonists as well as by GABAA receptor and K+ channel antagonists. NMDA created a region of negative slope conductance and a positive slope crossing indicative of membrane bistability in the current‐voltage relationship. NMDA‐induced bistability was partially blocked by L‐type Ca2+ channel antagonists. Although evoked by synaptic stimulation, plateau potentials...
A pipeline is proposed here to describe different features to study brain microcircuits on a hist... more 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 includesex-vivoNMDA-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 conne...
This work investigated if diverse properties could be ascribed to evoked inhibitory postsynaptic ... more This work investigated if diverse properties could be ascribed to evoked inhibitory postsynaptic currents (IPSCs) recorded on rat neostriatal neurons when field stimulation was delivered at two different locations: the globus pallidus (GP) and the neostriatum (NS). Previous work stated that stimulation in the GP could antidromically excite projection axons from medium spiny neurons. This maneuver would predominantly activate the inhibitory synapses that interconnect spiny cells. In contrast, intrastriatal stimulation would preferentially activate inhibitory synapses provided by interneurons. This study shows that, in fact, intensity-amplitude experiments are able to reveal different properties for IPSCs evoked from these two locations (GP and NS). In addition, while all IPSCs evoked from the GP were always sensitive to omega-conotoxin GVIA (Ca(V2.2)2.2 or N-channel blocker), one-half of the inhibition evoked from the NS exhibited little sensitivity to omega-conotoxin GVIA. Characteristically, all omega-conotoxin GVIA-insensitive IPSCs exhibited strong paired pulse depression, whereas omega-conotoxin GVIA-sensitive IPSCs evoked from either the GP or the NS could exhibit short-time depression or facilitation. omega-Agatoxin TK (Ca(V2.1)2.1+ or P/Q-channel blocker) blocked IPSCs evoked from both locations. Therefore 1) distinct inhibitory inputs onto projection neostriatal cells can be differentially stimulated with field electrodes; 2) N-type Ca2+ channels are not equally expressed in inhibitory terminals activated in the NS; and 3) synapses that interconnect spiny neurons use both N- and P/Q-type Ca2+ channels.
Dopamine-containing neurons of the midbrain are required to control voluntary movements, behavior... more Dopamine-containing neurons of the midbrain are required to control voluntary movements, behavior and motivation. Damage of these neurons in the substantia nigra zona compacta (SNc) is considered as the cause of Parkinson's disease. In this work, the firing characteristics of SNc neurons were studied in vitro. Neurons were identified by intracellular labeling tyrosine hydroxylase (TH) immunocytochemistry, and their electrophysiological characteristics. TH-positive neurons displayed three different firing modes. At around -55 mV neurons exhibited a regular single spike firing mode (pacemaking). At around -65 mV, neurons discharged in an irregular single spike firing mode (idling). At around -75 mV, many neurons exhibited a mode of discharge characterized by low threshold spikes (LTS), oscillations and, on some occasions, bursting. When present, bursts were of short duration and commonly made up of incomplete somatodendritic action potentials. TH-negative neurons also exhibited single spikes and bursting modes of firing. Both types of SNc neurons generated LTS oscillations following a hyperpolarization. However, LTS in TH-negative neurons could be evoked from less negative membrane potentials and produced more prolonged bursts. LTS from both types of neurons could be differentiated. LTS were blocked by 100 microM Ni2+ and became prolonged in the presence of TEA.
Adenosine A1 and A2A receptors are expressed in striatal projection neurons (SPNs). A1 receptors ... more Adenosine A1 and A2A receptors are expressed in striatal projection neurons (SPNs). A1 receptors are located in direct (dSPN) and indirect SPNs (iSNP). A2A receptors are only present in iSPNs. Dopamine D2 receptors are also expressed in iSPNs and interactions between D2 and A2A receptors have received attention. iSPNs activity increases during parkinsonism (PD) and A2A receptors may be responsible by enhancing Ca2+ currents (iCa2+). Therefore, A2A receptors blockade is a therapeutic approach. We asked whether A2A receptors need the interaction with D2 receptors (D2R) to exert their actions. By using isolated and identified iSPNs to avoid indirect influences, we show that D2R action habilitates A2A receptors (A2AR) modulation. iCa2+ through voltage gated Ca2+ channels (CaV) was used as a signal to observe this interaction. Voltage-clamp recordings in acutely dissociated iSPNs, current-clamp recordings in slices and calcium imaging in transgenic A2A-Cre mice, showed that D2R reduction...
Amantadine and clozapine have proved to reduce abnormal involuntary movements (AIMs) in preclinic... more Amantadine and clozapine have proved to reduce abnormal involuntary movements (AIMs) in preclinical and clinical studies of L-DOPA-Induced Dyskinesias (LID). Even though both drugs decrease AIMs, they may have different action mechanisms by using different receptors and signaling profiles. Here we asked whether there are differences in how they modulate neuronal activity of multiple striatal neurons within the striatal microcircuit at histological level during the dose-peak of L-DOPA in ex-vivo brain slices obtained from dyskinetic mice. To answer this question, we used calcium imaging to record the activity of dozens of neurons of the dorsolateral striatum before and after drugs administration in vitro. We also developed an analysis framework to extract encoding insights from calcium imaging data by quantifying neuronal activity, identifying neuronal ensembles by linking neurons that coactivate using hierarchical cluster analysis and extracting network parameters using Graph Theory. The results show that while both drugs reduce LIDs scores behaviorally in a similar way, they have several different and specific actions on modulating the dyskinetic striatal microcircuit. The extracted features were highly accurate in separating amantadine and clozapine effects by means of principal components analysis (PCA) and support vector machine (SVM) algorithms. These results predict possible synergistic actions of amantadine and clozapine on the dyskinetic striatal microcircuit establishing a framework for a bioassay to test novel antidyskinetic drugs or treatments in vitro.
In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked lon... more In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked long‐lasting plateau potentials in neostriatal neurons. Plateau potentials were often followed by spontaneous voltage transitions between two preferred membrane potentials. While the induction of plateau potentials was disrupted by non‐NMDA and NMDA glutamate receptor antagonists, the maintenance of spontaneous voltage transitions was only blocked by NMDA receptor and L‐type Ca2+ channel antagonists. The frequency and duration of depolarized events, resembling up‐states described in vivo, were increased by NMDA and L‐type Ca2+ channel agonists as well as by GABAA receptor and K+ channel antagonists. NMDA created a region of negative slope conductance and a positive slope crossing indicative of membrane bistability in the current‐voltage relationship. NMDA‐induced bistability was partially blocked by L‐type Ca2+ channel antagonists. Although evoked by synaptic stimulation, plateau potentials...
A pipeline is proposed here to describe different features to study brain microcircuits on a hist... more 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 includesex-vivoNMDA-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 conne...
This work investigated if diverse properties could be ascribed to evoked inhibitory postsynaptic ... more This work investigated if diverse properties could be ascribed to evoked inhibitory postsynaptic currents (IPSCs) recorded on rat neostriatal neurons when field stimulation was delivered at two different locations: the globus pallidus (GP) and the neostriatum (NS). Previous work stated that stimulation in the GP could antidromically excite projection axons from medium spiny neurons. This maneuver would predominantly activate the inhibitory synapses that interconnect spiny cells. In contrast, intrastriatal stimulation would preferentially activate inhibitory synapses provided by interneurons. This study shows that, in fact, intensity-amplitude experiments are able to reveal different properties for IPSCs evoked from these two locations (GP and NS). In addition, while all IPSCs evoked from the GP were always sensitive to omega-conotoxin GVIA (Ca(V2.2)2.2 or N-channel blocker), one-half of the inhibition evoked from the NS exhibited little sensitivity to omega-conotoxin GVIA. Characteristically, all omega-conotoxin GVIA-insensitive IPSCs exhibited strong paired pulse depression, whereas omega-conotoxin GVIA-sensitive IPSCs evoked from either the GP or the NS could exhibit short-time depression or facilitation. omega-Agatoxin TK (Ca(V2.1)2.1+ or P/Q-channel blocker) blocked IPSCs evoked from both locations. Therefore 1) distinct inhibitory inputs onto projection neostriatal cells can be differentially stimulated with field electrodes; 2) N-type Ca2+ channels are not equally expressed in inhibitory terminals activated in the NS; and 3) synapses that interconnect spiny neurons use both N- and P/Q-type Ca2+ channels.
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