Molecular determinants of hippocampal
fast excitatory and inhibitory synaptic transmission
Neurons in our brain communicate through electrochemical synapses. These synapses get their functionality from the proteins that are expressed. This thesis focused on increasing our understanding of the most abundant fast inhibitory and excitatory synapses: the GABA synapse containing GABAAreceptors and the glutamate synapses containing AMPA receptors.
Functional and pharmacological properties of GABAARs depend on their subunit composition, and this is essential for setting neuronal network activity at specific frequencies. GABAAARs are built up from a combination of five subunits selected from: α1-6, β1-3, γ1-3 and δ. In Chapter 2, we addressed the question of which GABAAR subunit is most important in setting fast network oscillation frequencies in the hippocampus. In particular, the role of two GABAergic subunits were investigated: α1 and α2. We used different genetic and pharmacological strategies to manipulate the functionality of GABAARs containing either the α1 or the α2 subunits. This thesis work revealed that the α2 subunit is located in the synapses between fast spiking interneurons and pyramidal neurons of the CA3 are of the hippocampus. The α2 subunits were most important in setting the fast network oscillation frequency. In contrast, the α1 subunit had a minor role in setting rhythmic activity in the hippocampus.
Neuronal networks need excitation and inhibition in a proper balance. Therefore, we also studied the role of the two newly discovered AMPAR auxiliary proteins Shisa7 and Shisa9 in excitatory synaptic plasticity and network activity in Chapters 3 and 4.
The following research questions were addressed:
1. Do interactions of the C-terminal of the Shisa9 protein affect AMPAR functioning and synaptic properties? Do such interactions affect the fast network oscillations?
2. Is Shisa7 an AMPAR auxiliary subunit? Does Shisa7 affect AMPAR functioning? Does Shisa 7 affect the synaptic properties? Does Shisa7 affect glutamatergic plasticity? Does Shisa7 have a role in memory?
To address these questions we used a multidisciplinary approach combining molecular interventions with electrophysiological techniques of patch-clamp and multi-electrode arrays for single cell and field potential recordings, respectively, on in vitro mice hippocampal brain preparations. In addition, we used mouse behavior tests such as fear conditioning in order to test if AMPAR auxiliary proteins have a role in hippocampal memory formation.
This thesis work reveals that excitatory synaptic transmission and plasticity is tuned by both members of the Shisa family. In chapter 3 we found that C-terminal interactions between Shisa9 and other proteins are important for AMPAR function. Interfering with these interactions resulted in shorter AMPAR currents and reduced short-term plasticity. Furthermore, fast network oscillations in the hippocampus were affected when Shisa9 could no longer interact with intracellular proteins. In chapter 4 we found that Shisa7 is indeed a bonafide auxiliary subunit of the AMPAR. The absence of the Shisa7 protein resulted in shorter AMPAR currents and it also reduced the degree of long term synaptic plasticity. Finally, the observed synaptic changes correlated with reduced memories of fearful events.
The presented studies gave us a better understanding of the role of the AMPAR auxiliary proteins Shisa9 and Shisa7 as well as the differential role of the GABAergic subunits α1 and α2, which provides grounds to better understand how the brain balances excitation and inhibition. Disruption of the excitatory-inhibitory balance can lead to dramatic malfunctioning of the neuronal network activity, exemplified by diseases such as epilepsy, anxiety, cognitive deficits, schizophrenia, depression, and substance abuse.