Abstract
The balance between glutamate- and GABA-mediated neurotransmission in the brain is fundamental in the nervous system, but it is regulated by the “tonic” release of a variety of endogenous factors. One such important group of molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in vivo physiologically relevant concentrations, the effects of NSs and BDZs as GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 μM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after wash-out, the excitability of inhibitory neurons continued to be depressed, leading to a network long-term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex vivo networks show a sensitivity to NSs and BDZs comparable to that expressed in vivo, but also provide a new global in vitro description that can help in understanding their activity in more complex systems.
Highlights
Neurosteroids (NSs) such as tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) are synthesized endogenously within the brain and potently modulate GABAergic synaptic transmission (Puia et al, 1990, 2003; Belelli and Lambert, 2005); their global function in regulating network firing activity is still largely unclear
It is not clear whether these cells are exposed to a stable extracellular concentration of NSs (Puia et al, 2003) that guarantees a tonic level of network inhibition, or the neuronal network is finely tuned by local stimulation of NS synthesis
The heterogeneity analysis indicates that all the state 2 bursts were characterized by a much shorter duration, compared with state 1. This suggests that the neurons’ ability to maintain the temporal connectivity may be impaired during the up-state. These results suggest that ALLO, by enhancing the GABAA currents on both excitatory and inhibitory neurons, www.frontiersin.org produced similar effects on state 1 mode and completely different effects on the connectivity of state 2
Summary
Neurosteroids (NSs) such as tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) are synthesized endogenously within the brain and potently modulate GABAergic synaptic transmission (Puia et al, 1990, 2003; Belelli and Lambert, 2005); their global function in regulating network firing activity is still largely unclear. The ALLO and THDOC synthesizing enzymes, respectively, 5α-reductase (5α-R) type I and 3α-hydroxysteroid dehydrogenase (3α-HSD), co-localize in glutamatergic but not in GABAergic neurons (Agis-Balboa et al, 2006). Excitatory neurons that co-express GABAARs and NS synthetic enzymes can be regulated in an autocrine manner (Gunn et al, 2011). It is not clear whether these cells are exposed to a stable extracellular concentration of NSs (Puia et al, 2003) that guarantees a tonic level of network inhibition, or the neuronal network is finely tuned by local stimulation of NS synthesis
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