UC San Diego

Excitatory synapses possess a vast array of proteins, including glutamate receptors such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which mediate fast synaptic transmission. There are four different AMPA receptor subunits: GluA1, GluA2, GluA3, and GluA4). AMPA receptors are generally found in the adult rodent brain in two heteromeric forms: GluA1/GluA2 receptors and GluA3/GluA2 receptors. Trafficking of GluA1/GluA2 receptors is activity-dependent, while GluA3/GluA2 heteromers are constitutively cycling and replacing synaptic GluA1/GluA2 and GluA3/GluA2 receptors. Given the importance of the GluA1 subunit in plasticity, on which the absence of GluA3 has little effect, GluA3 has not been well-studied. As it turns out, GluA3 is required for the expression of the synaptic dysfunction caused by amyloid-β (Aβ), a small peptide thought to be responsible for the pathogenesis of Alzheimer’s disease. In the absence of GluA3, Aβ does not cause synaptic depression, memory deficits, or block long-term potentiation (LTP). These results will be described in Chapter 2.

Studies have demonstrated that Aβ causes synaptic dysfunction by activating signaling pathways involved in long-term depression (LTD). Previous studies from our lab established successfully engineered associative fear memories by pairing optogenetic activation of the auditory cortex (AuC) and medial geniculate nucleus (MGN) with footshock in cued fear conditioning. Replacing the traditional conditioned stimulus (CS) with an optogenetically-delivered input (ODI) allowed us to utilize LTP and LTD protocols to activate and inactivate memories. To examine if Aβ in Alzheimer’s disease behaves as LTD in vivo, Aβ and a blue-shifted form of channelrhodopsin (ChR) known as oChIEF were co-expressed in the AuC and MGN. A class of Alzheimer’s disease therapeutics known as γ-secretase modulators (GSMs) were used to control Aβ levels, and a behaviorial paradigm was established to determine whether Aβ could remove inactivate memories like LTD, and later be recovered with LTP in the presence of GSMs. This study will be described in Chapter 3.

Lastly, given the importance of LTP in learning and memory, a new method for identifying recently potentiated synapses was investigated. One existing strategy for assessing learning-induced plasticity involves the use of two-photon laser scanning microscopy (TPLSM) in vivo, which can provide high visual and temporal resolution on synapses undergoing potentiation. However, a caveat to this technique is that TPLSM techniques are labor intensive and thus low throughput. Another strategy for brain regions involved in a learning task is examining the localization of immediate- early gene (IEG) transcripts and proteins. Despite this method being relatively high throughput, the correlation between synaptic strength and IEG expression is poorly understood. Thus, we established a new technique, which we call SYNaptic Proximity Ligation Assay. SYNPLA applies the antibody-based methodology of PLA to the diversity of transsynaptic proteins to label synapses. In Chapter 4, SYNPLA’s labeling of all synapses or recently potentiated synapses will be described.