A Method for Detecting Plasticity in the Brain
Post by Leigh Christopher
What's the science?
The brain is plastic, meaning that the strength between synapses (connections between neurons) is altered after learning something new or creating a memory. Long term potentiation is the biological process of the strengthening of synaptic connections. This process is mediated by the insertion of AMPA receptors containing a GluA1 subunit into the synapse, which is followed by a replacement of these receptors with GluA1 lacking AMPA receptors. Therefore, the presence of GluA1 acts as a signal of recent learning-induced plasticity in the brain. Current methods used to detect synaptic plasticity are either slow or lack resolution. This week in PNAS, Dore and colleagues present a new method called SYNPLA (synaptic proximity ligation assay) for detecting the insertion of GluA1 containing AMPA receptors, in order to identify recent synaptic plasticity.
How did they do it?
SYNPLA uses proximity ligation assay (PLA), a method that detects two proteins that are close together. This method relies on the use of antibodies to flag the proteins of interest. A second set of antibodies, each paired to oligonucleotides (short segments of DNA) are then used to detect the first set of antibodies. Lastly, a second complementary pair of oligonucleotides are added, and if they are close to one another, they will ligate and form a circle. This sequence can then by amplified (1000 times) to form a ball of DNA that is probed and observed with light microscopy as points where co-localized proteins exist (referred to here as PLA puncta). First, the authors expressed antibody detectable NRXN (a presynaptic protein), and antibody detectable NLGN (a postsynaptic protein) in neurons, and performed PLA in order to demonstrate that they were able to label synapse formation during development in cultured neurons. The authors then tested whether they could detect postsynaptic AMPA receptors containing GluA1 in cultured neurons and cultured hippocampal slices following chemically induced LTP (i.e. plasticity that is thought to occur during learning). Next, they went on to assess whether SYNPLA could detect learning-induced plasticity in rats in vivo. They injected either the auditory cortex or thalamus with a virus expressing antibody detectable NRXN (presynaptic protein) to detect the co-localization of this protein with postsynaptic AMPA containing GluA1. Rats underwent a defense conditioning paradigm where they heard an auditory tone, followed by a foot shock – this paradigm is known to induce fear learning and synaptic plasticity in the amygdala (fear center of the brain). They also performed SYNPLA on tissue sections of the amygdala as well as the lateral habenula which is known to process aversive stimuli.
What did they find?
SNYPLA was able to successfully detect and label synapse formation during development with a high specificity and signal-to-noise ratio. The authors were also able to detect the insertion of postsynaptic GluA1 containing AMPA receptors (a sign of potentiation) in neuron cultures and cultured hippocampal slices following chemically induced LTP, as demonstrated by a large increase in PLA puncta. Following the defense conditioning paradigm, the authors found that rats who underwent paired conditioning (paired tone and foot shock) showed increased levels of PLA puncta in the amygdala compared to control rats or rats who underwent unpaired conditioning, demonstrating that SNYPLA was able to detect synaptic plasticity in the amygdala in vivo following learning. They also observed increased PLA puncta in the lateral habenula (a region of the brain thought to be active during punishment or disappointment) for rats who underwent both the paired and unpaired conditioning paradigm compared to control rats, suggesting that plasticity occurs in this region whenever an aversive shock is administered (and not just for learning a fear response).
What's the impact?
This is the first study to present a fast, high-resolution method for detecting learning-induced synaptic plasticity. Understanding which specific synapses have been modified by learning or memory is difficult. SYNPLA can quickly identify synaptic plasticity at specific synapses in defined pathways in the brain and can be used at the whole-brain level as a screening tool to detect recent learning and memory.
Dore et al. SYNPLA, a method to identify synapses displaying plasticity after learning. PNAS (2020). Access the original scientific publication here.