No abstract

Neuronal ensembles, coactive groups of neurons found in spontaneous and evoked cortical activity, are causally related to memories and perception, but it still unknown how stable or flexible they are over time. We used two-photon multiplane calcium imaging to track over weeks the activity of the same pyramidal neurons in layer 2/3 of the visual cortex from awake mice and recorded their spontaneous and visually evoked responses. Less than half of the neurons were commonly active across any two imaging sessions. These 'common neurons' formed stable ensembles lasting weeks, but some ensembles were also transient and appeared only in one single session. Stable ensembles preserved ~68 % of their neurons up to 46 days, our longest imaged period, and these 'core' cells had stronger functional connectivity. Our results demonstrate that neuronal ensembles can last for weeks and could, in principle, serve as a substrate for long-lasting representation of perceptual states or memories.

Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals of both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we find that, after optogenetic or electrical stimulation, coactivated neurons increase their correlated activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Optogenetic and electrical stimulation also induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed unexpected strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reductions in spike threshold. A pharmacological agent that blocks changes in membrane resistance can revert this effect. These significant increases in excitability may partly generate the observed biphasic synaptic plasticity. We propose that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an 'iceberg' model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.

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.

The firing of striatal projection neurons (SPNs) exhibits afterhyperpolarizing potentials (AHPs) that determine discharge frequency. They are in part generated by Ca2+-activated K+-currents involving BK and SK components. It has previously been shown that suprathreshold corticostriatal responses are more prolonged and evoke more action potentials in direct pathway SPNs (dSPNs) than in indirect pathway SPNs (iSPNs). In contrast, iSPNs generate dendritic autoregenerative responses. Using whole cell recordings in brain slices, we asked whether the participation of Ca2+-activated K+-currents plays a role in these responses. Secondly, we asked if these currents may explain some differences in synaptic integration between dSPNs and iSPNs. Neurons obtained from BAC D1 and D2 GFP mice were recorded. We used charybdotoxin and apamin to block BK and SK channels, respectively. Both antagonists increased the depolarization and delayed the repolarization of suprathreshold corticostriatal responses in both neuron classes. We also used NS 1619 and NS 309 (CyPPA), to enhance BK and SK channels, respectively. Current enhancers hyperpolarized and accelerated the repolarization of corticostriatal responses in both neuron classes. Nevertheless, these drugs made evident that the contribution of Ca2+-activated K+-currents was different in dSPNs as compared to iSPNs: in dSPNs their activation was slower as though calcium took a diffusion delay to activate them. In contrast, their activation was fast and then sustained in iSPNs as though calcium flux activates them at the moment of entry. The blockade of Ca2+-activated K+-currents made iSPNs to look as dSPNs. Conversely, their enhancement made dSPNs to look as iSPNs. It is concluded that Ca2+-activated K+-currents are a main intrinsic determinant causing the differences in synaptic integration between corticostriatal polysynaptic responses between dSPNs and iSPNs.