Neurosteroids modulation of mouse neocortical network: a multielectrode based study

Neurosteroids (NSs) such as allopregnanolone (ALLO) and tetrahydrodeoxy-corticosterone (THDOC ) are well known modulators of GABAA receptor function. While a large amount of data are available at the single cell level, their “global modulation” of network firing activity was not investigated in detail. NSs levels vary in the diverse brain regions and may change under different physiological and pathological conditions [1-3]. The NSs synthesizing enzymes, 5α-reductase type I and 3α-hydroxysteroid dehydrogenase have also different expression profiles, i.e. in the neocortex they co-localize in glutamatergic but not in GABAergic neurons [4]. Therefore these endogenous substances in excitatory neurons that co-express GABAA receptors and NSs synthetic enzymes can act in an autocrine manner [5] while in inhibitory cells their modulation is different. The goal of our study was to investigate the effects of physiological NSs concentrations on the activity of networks formed by acutely dissociated mouse neocortical neurons. By using the multi-electrode arrays (MEA) technique the spontaneous reverberating activity of excitatory and inhibitory neurons was simultaneously recorded and analyzed in control and after perfusion with NSs. Their effects were analyzed, by using statistical methods [6], on physiological variables such as excitability (spikes-per neuron) and number of neurons engaged in bursts. THDOC, at physiological concentrations, selectively decreased inhibitory interneuron activity, whereas at concentrations higher than 100 nM it inhibited both excitatory and inhibitory clusters. The analysis of the network activity after long time wash-out (8 hrs) highlight that the NS effect on inhibitory clusters persisted for hours producing a sort of long-term depression (LTD) in their excitability. To further clarify this point we applied THDOC twice (after a 2h wash-out). The first application induced, as expected, a persistent depression of inhibitory neurons but after the second administration of THDOC no further LTD was observed. This experiment suggests that the first application of the NS produces some stable modifications, a sort of “memory” in the network that could be relevant also in vivo. THDOC and ALLO, at low concentrations, are allosteric modulators of GABAergic neurotransmission but in the μM concentration range they also act as GABAA receptor agonists [7]. In agreement with these properties, our analysis of the global excitability of the network showed a sharp increase in NSs inhibitory effects at concentrations between 100 and 1000 nM, probably due to a direct agonistic activity at GABAA receptors. Many single-cell studies of the effects of ALLO and THDOC have been performed, but no differences were reported between their effects. We show here that THDOC and ALLO, although consistently producing network inhibition at high concentrations, at low concentrations (10-100 nM) have different effects on excitatory and inhibitory clusters; moreover, the network recovered more easily from ALLO than from THDOC effect. Previous studies suggested that tonic GABAergic currents are highly sensitive to NSs [8]. To investigate whether this happens also in our experimental mode we applied Gabazine (GBZ), a GABAA receptor antagonist, at concentrations that only block the phasic GABAergic current. GBZ 100 nM increased the activity of inhibitory neurons, leaving almost unaffected the excitatory neuron excitability suggesting that interneurons are controlling each other mainly through a phasic inhibition. In these conditions the excitability of the network was reduced but also the sensitivity to THDOC was unexpectedly decreased by approximately one order of magnitude compared to control. A detailed analysis [6, 9] of the burst properties showed that in the presence of NSs, the neuronal activity changed and became heterogeneous because of the appearance of different states with occupancy probabilities strongly dependent on the drug concentration. In particular, we observed the random appearance of novel up-states, characterized by excitability features and engaged neurons different from those observed in control. While the analysis of the “global network effect” of NSs provide information about the average changes in excitability of inhibitory and excitatory clusters, the “states analysis “ highlighted changes in the network connectivity: in the presence of neuromodulators different connectivity modes appear. What is the physiological significance of this effect? Specific firing patterns recorded in selected neuronal populations encode information during physiological or pathological conditions [10] and changes in the connectivity induced by endogenous compounds are able to modify the response of the network. Taken together, our results provide a new description of the mode of action of NSs and give some new insight in understanding the complexity of the network response to these endogenous modulators.