Translational neuronal ensembles: Neuronal microcircuits in psychology, physiology, pharmacology and pathology.

Multi-recording techniques show evidence that neurons coordinate their firing forming ensembles and that brain networks are made by connections between ensembles. While "canonical" microcircuits are composed of interconnected principal neurons and interneurons, it is not clear how they participate in recorded neuronal ensembles: "groups of neurons that show spatiotemporal co-activation". Understanding synapses and their plasticity has become complex, making hard to consider all details to fill the gap between cellular-synaptic and circuit levels. Therefore, two assumptions became necessary: First, whatever the nature of the synapses these may be simplified by "functional connections". Second, whatever the mechanisms to achieve synaptic potentiation or depression, the resultant synaptic weights are relatively stable. Both assumptions have experimental basis cited in this review, and tools to analyze neuronal populations are being developed based on them. Microcircuitry processing followed with multi-recording techniques show temporal sequences of neuronal ensembles resembling computational routines. These sequences can be aligned with the steps of behavioral tasks and behavior can be modified upon their manipulation, supporting the hypothesis that they are memory traces. In vitro, recordings show that these temporal sequences can be contained in isolated tissue of histological scale. Sequences found in control conditions differ from those recorded in pathological tissue obtained from animal disease models and those recorded after the actions of clinically useful drugs to treat disease states, setting the basis for new bioassays to test drugs with potential clinical use. These findings make the neuronal ensembles theoretical framework a dynamic neuroscience paradigm.

Synaptic determinants of cholinergic interneurons hyperactivity during parkinsonism.

Parkinson's disease is a neurodegenerative ailment generated by the loss of dopamine in the basal ganglia, mainly in the striatum. The disease courses with increased striatal levels of acetylcholine, disrupting the balance among these modulatory transmitters. These modifications disturb the excitatory and inhibitory balance in the striatal circuitry, as reflected in the activity of projection striatal neurons. In addition, changes in the firing pattern of striatal tonically active interneurons during the disease, including cholinergic interneurons (CINs), are being searched. Dopamine-depleted striatal circuits exhibit pathological hyperactivity as compared to controls. One aim of this study was to show how striatal CINs contribute to this hyperactivity. A second aim was to show the contribution of extrinsic synaptic inputs to striatal CINs hyperactivity. Electrophysiological and calcium imaging recordings in Cre-mice allowed us to evaluate the activity of dozens of identified CINs with single-cell resolution in ex vivo brain slices. CINs show hyperactivity with bursts and silences in the dopamine-depleted striatum. We confirmed that the intrinsic differences between the activity of control and dopamine-depleted CINs are one source of their hyperactivity. We also show that a great part of this hyperactivity and firing pattern change is a product of extrinsic synaptic inputs, targeting CINs. Both glutamatergic and GABAergic inputs are essential to sustain hyperactivity. In addition, cholinergic transmission through nicotinic receptors also participates, suggesting that the joint activity of CINs drives the phenomenon; since striatal CINs express nicotinic receptors, not expressed in striatal projection neurons. Therefore, CINs hyperactivity is the result of changes in intrinsic properties and excitatory and inhibitory inputs, in addition to the modification of local circuitry due to cholinergic nicotinic transmission. We conclude that CINs are the main drivers of the pathological hyperactivity present in the striatum that is depleted of dopamine, and this is, in part, a result of extrinsic synaptic inputs. These results show that CINs may be a main therapeutic target to treat Parkinson's disease by intervening in their synaptic inputs.

Activation of parvalbumin-expressing neurons reconfigures neuronal ensembles in murine striatal microcircuits.

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.

Comparison of Actions between L-DOPA and Different Dopamine Agonists in Striatal DA-Depleted Microcircuits In Vitro: Pre-Clinical Insights.

Parkinson's disease (PD) is a neurodegenerative illness presenting motor and non-motor symptoms due to the loss of dopaminergic terminals in basal ganglia, most importantly, the striatum. L-DOPA relieves many motor signs. Unfortunately, in the long term, L-DOPA use causes motor disabilities by itself and does not act in comorbid conditions such as depression. These deficiencies have led to search for drugs such as dopamine (DA) receptor agonists (DA-agonists) that allow the reduction of L-DOPA dose. Previously, we have identified the attributes of non-stimulated (resting) and cortical stimulated (active) striatal microcircuits following the activity of dozens of neurons simultaneously using calcium imaging in brain slices. We also have characterized the changes that take place in DA-depleted microcircuits in vitro. In control conditions, there is low spontaneous activity. After cortical stimulation (CtxS) sequences and alternation of neuronal ensembles activity occur, including reverberations. In contrast, DA-deprived circuits exhibit high spontaneous activity at rest, and a highly recurrent ensemble curtails alternation. Interestingly, CtxS briefly relieves these Parkinsonian signs in DA-depleted tissue. Here we compare the actions of some DA-agonists used in PD therapeutics on the pathological dynamics of DA-depleted microcircuits at rest and with CtxS; taking L-DOPA as reference. D2-class agonists better reduce the excessive spontaneous activity of DA-depleted microcircuits. All DA-agonists tend to maintain ensemble alternation seen in control circuits after CtxS. However, quantitative analyses suggest differences in their actions: in general, DA-agonists only approximate L-DOPA actions. Nonetheless no treatment, including L-DOPA, completely restores microcircuit dynamics to control conditions.

Cortical stimulation relieves parkinsonian pathological activity in vitro.

Previously, we have shown that chemical excitatory drives such as N-methyl-d-aspartate (NMDA) are capable of activating the striatal microcircuit exhibiting neuronal ensembles that alternate their activity producing temporal sequences. One aim of this work was to demonstrate whether similar activity could be evoked by delivering cortical stimulation. Dynamic calcium imaging allowed us to follow the activity of dozens of neurons with single-cell resolution in mus musculus brain slices. A train of electrical stimuli in the cortex evoked network activity similar to the one induced by bath application of NMDA. Previously, we have also shown that the dopamine-depleted striatal microcircuit increases its spontaneous activity generating dominant recurrent ensembles that interrupt the temporal sequences found in control microcircuits. This activity correlates with parkinsonian pathological activity. Several cortical stimulation protocols such as transcranial magnetic stimulation reduce motor signs of Parkinsonism. Here, we show that cortical stimulation in vitro temporarily eliminates the pathological activity from the dopamine-depleted striatal microcircuit by turning off some neurons that sustain this activity and recruiting new ones that allow transitions between network states, similar to the control circuit. When cortical stimulation is given in the presence of L-DOPA, parkinsonian activity is eliminated during the whole recording period. The present experimental evidence suggests that cortical stimulation such as that generated by transcranial magnetic stimulation, or otherwise, may allow reduce L-DOPA dosage.

Differences in synaptic integration between direct and indirect striatal projection neurons: Role of CaV 3 channels.

Different corticostriatal suprathreshold responses in direct and indirect striatal projection neurons (SPNs) of rodents have been reported. Responses consist in prolonged synaptic potentials of polysynaptic and intrinsic origin, in which voltage-gated Ca2 ⁺ currents play a role. Recording simultaneous Ca2 ⁺ imaging and voltage responses at the soma, while activating the corticostriatal pathway, we show that encoding of synaptic responses into trains of action potentials (APs) is different in SPNs: firing of APs in D1-SPNs increase gradually, in parallel with Ca2 ⁺ entry, as a function of stimulus intensity. In contrast, D2-SPNs attain a maximum number of evoked spikes at low stimulus intensities, Ca2 ⁺ entry is limited, and both remain the same in spite of increasing stimulus strength. Stimulus needs to reach certain intensity, to have propagated Ca2 ⁺ potentials to the soma plus a sudden step in Ca2 ⁺ entry, without changing the number of fired APs, phenomena never seen in D1-SPNs. Constant firing in spite of changing stimulus, suggested the involvement of underlying inactivating potentials. We found that Caᵥ3 currents contribute to Ca2+ entry in both classes of SPNs, but have a more notable effect in D2-SPNs, where a low-threshold spike was disclosed. Blockade of CaV 3 channels retarded the steep rise in firing in D2-SPNs. Inhibition block increased the number of spikes fired by D2-SPNs, without changing firing in D1-SPNs. These differences in synaptic integration enable a biophysical dissimilarity: dendritic inhibition appears to be more relevant for D2-SPNs. This may imply distinctions in the set of interneurons affecting each SPN class.

Calcium currents in striatal fast-spiking interneurons: dopaminergic modulation of CaV1 channels.

BACKGROUND: Striatal fast-spiking interneurons (FSI) are a subset of GABAergic cells that express calcium-binding protein parvalbumin (PV). They provide feed-forward inhibition to striatal projection neurons (SPNs), receive cortical, thalamic and dopaminergic inputs and are coupled together by electrical and chemical synapses, being important components of the striatal circuitry. It is known that dopamine (DA) depolarizes FSI via D1-class DA receptors, but no studies about the ionic mechanism of this action have been reported. Here we ask about the ion channels that are the effectors of DA actions. This work studies their Ca2+ currents. RESULTS: Whole-cell recordings in acutely dissociated and identified FSI from PV-Cre transgenic mice were used to show that FSI express an array of voltage gated Ca2+ channel classes: CaV1, CaV2.1, CaV2.2, CaV2.3 and CaV3. However, CaV1 Ca2+ channel carries most of the whole-cell Ca2+ current in FSI. Activation of D1-like class of DA receptors by the D1-receptor selective agonist SKF-81297 (SKF) enhances whole-cell Ca2+ currents through CaV1 channels modulation. A previous block of CaV1 channels with nicardipine occludes the action of the DA-agonist, suggesting that no other Ca2+ channel is modulated by D1-receptor activation. Bath application of SKF in brain slices increases the firing rate and activity of FSI as measured with both whole-cell and Ca2+ imaging recordings. These actions are reduced by nicardipine. CONCLUSIONS: The present work discloses one final effector of DA modulation in FSI. We conclude that the facilitatory action of DA in FSI is in part due to CaV1 Ca2+ channels positive modulation.

Pathophysiological signatures of functional connectomics in parkinsonian and dyskinetic striatal microcircuits.

A challenge in neuroscience is to integrate the cellular and system levels. For instance, we still do not know how a few dozen neurons organize their activity and relations in a microcircuit or module of histological scale. By using network theory and Ca(2+) imaging with single-neuron resolution we studied the way in which striatal microcircuits of dozens of cells orchestrate their activity. In addition, control and diseased striatal tissues were compared in rats. In the control tissue, functional connectomics revealed small-world, scale-free and hierarchical network properties. These properties were lost during pathological conditions in ways that could be quantitatively analyzed. Decorticated striatal circuits disclosed that corticostriatal interactions depend on privileged connections with a set of highly connected neurons or "hubs". In the 6-OHDA model of Parkinson's disease there was a decrease in hubs number; but the ones that remained were linked to dominant network states. l-DOPA induced dyskinesia provoked a loss in the hierarchical structure of the circuit. All these conditions conferred distinct temporal sequences to circuit activity. Temporal sequences appeared as particular signatures of disease process thus bringing the possibility of a future quantitative pathophysiology at a histological scale.

KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons.

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.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

Modulation of direct pathway striatal projection neurons by muscarinic M4-type receptors.

Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D(1 or 2)-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca(2+)-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca(2+)-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed.

Global actions of nicotine on the striatal microcircuit.

THE QUESTION TO SOLVE IN THE PRESENT WORK IS: what is the predominant action induced by the activation of cholinergic-nicotinic receptors (nAChrs) in the striatal network given that nAChrs are expressed by several elements of the circuit: cortical terminals, dopamine terminals, and various striatal GABAergic interneurons. To answer this question some type of multicellular recording has to be used without losing single cell resolution. Here, we used calcium imaging and nicotine. It is known that in the presence of low micromolar N-Methyl-D-aspartate (NMDA), the striatal microcircuit exhibits neuronal activity consisting in the spontaneous synchronization of different neuron pools that interchange their activity following determined sequences. The striatal circuit also exhibits profuse spontaneous activity in pathological states (without NMDA) such as dopamine depletion. However, in this case, most pathological activity is mostly generated by the same neuron pool. Here, we show that both types of activity are inhibited during the application of nicotine. Nicotine actions were blocked by mecamylamine, a non-specific antagonist of nAChrs. Interestingly, inhibitory actions of nicotine were also blocked by the GABAA-receptor antagonist bicuculline, in which case, the actions of nicotine on the circuit became excitatory and facilitated neuronal synchronization. We conclude that the predominant action of nicotine in the striatal microcircuit is indirect, via the activation of networks of inhibitory interneurons. This action inhibits striatal pathological activity in early Parkinsonian animals almost as potently as L-DOPA.

Direct evaluation of L-DOPA actions on neuronal activity of Parkinsonian tissue in vitro.

Physiological and biochemical experiments in vivo and in vitro have explored striatal receptor signaling and neuronal excitability to posit pathophysiological models of Parkinson's disease. However, when therapeutic approaches, such as dopamine agonists, need to be evaluated, behavioral tests using animal models of Parkinson's disease are employed. To our knowledge, recordings of population neuronal activity in vitro to assess anti-Parkinsonian drugs and the correlation of circuit dynamics with disease state have only recently been attempted. We have shown that Parkinsonian pathological activity of neuronal striatal circuits can be characterized in in vitro cerebral tissue. Here, we show that calcium imaging techniques, capable of recording dozens of neurons simultaneously with single-cell resolution, can be extended to assess the action of therapeutic drugs. We used L-DOPA as a prototypical anti-Parkinsonian drug to show the efficiency of this proposed bioassay. In a rodent model of early Parkinson's disease, Parkinsonian neuronal activity can be returned to control levels by the bath addition of L-DOPA in a reversible way. This result raises the possibility to use calcium imaging techniques to measure, quantitatively, the actions of anti-Parkinsonian drugs over time and to obtain correlations with disease evolution and behavior.

Early endocrine and molecular changes in metabolic syndrome models.

The twenty-first century arrived in the middle of a global epidemic of metabolic syndrome (MS) and type 2 diabetes mellitus (DM2). It is generally accepted that an excess of nutrients linked to a low physical activity triggers the problem. However, the molecular features that interact to develop the MS are not clear. In an effort to understand and control them, they have been extensively studied, but this goal has not been achieved yet. Nonhuman animal models have been used to explore diet and genetic factors in which experimental conditions are controlled. For example, only one factor in the diet, such as fats or carbohydrates can be modified to better understand a single change that would be impossible in humans. Most of the studies have been done in rodents. However, it is difficult to directly compare them, because experiments are different in more than one variable; genetic strains, amount, and the type of fat used in the diet and sex. Thus, the only possible criteria of comparison are the relevance of the observed changes. We review different animal models and add some original observations on short-term changes in metabolism and beta cells in our own model of adult Wistar rats that are not especially prone to get fat or develop DM2, treated with 20% sucrose in drinking water. One early change observed in pancreatic beta cells is the increase in GLUT2 expression that is located to the membrane of the cells. This change could partially explain the presence of insulin hypersecretion and hyperinsulinemia in these rats. Understanding early changes that lead to MS and in time to pancreatic islet exhaustion is an important biomedical problem that may contribute to learn how to prevent or even reverse MS, before developing DM2.