Synaptic optical imaging platforms: Examining pharmacological modulation of neurotransmitter release at discrete synapses

Abstract

Chemical synapses are not only fundamental functional units of the brain but also anatomical and functional biomarkers of numerous brain disorders. Therefore, new experimental readouts of synaptic function are needed--with the spatial resolution of single synapses and the scale to image large ensembles of synapses in specific circuits--for the study of both acute and chronic effects of pharmacological agents on synaptic plasticity in living mammals. In this article we discuss the design and use of fluorescent false neurotransmitters (FFNs) as an important step in the development of versatile synaptic imaging platforms. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Keywords: Brain receptors; Fluorescent probes; In vivo imaging; Synapses; Synaptic transmission.

Fig. 1. Excitatory and dopaminergic inputs into the dorsal striatum (dSTR)

(A) Sagittal slice of the mouse brain showing glutamatergic inputs (yellow) into the dSTR (blue region) from layer 5 of the motor cortex (MCTX5, yellow region) and dopaminergic inputs (purple) into the dorsal striatum from the substantia nigra (SN, pink region). Glutamatergic axons from the cortex (shown) and thalamus (not shown) as well as dopaminergic axons form the SN (shown) spread widely and form complex arborizations in the dSTR (estimated 10⁵ synaptic boutons per SN neuron). Image of the background sagittal slice is adapted from the Allen Developing Mouse Brain Atlas. (B) Graphic showing the complex regulation of individual synapses on medium spiny neurons (MSNs) in the dSTR. Glutamate (GLU, yellow circles) modulates MSNs by binding to the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, the N-methyl-D-aspartic acid (NMDA) receptor and the metabotropic glutamate receptors (not shown). Dopamine (DA, purple circles) modulates MSNs by binding to D1 and D2 like receptors. The MSNs are further modulated by cholinergic synapses (via the muscarinic acetylcholine receptor M1) and GABAergic synapses (not shown) as well as a variety of neuropeptides. Both dopaminergic and glutamatergic neurons are regulated presynaptically by D2 receptors and nicotinic and muscarinic acetylcholine receptors. The glutamatergic inputs are further regulated by cannabinoid release from MSNs.

Fig. 2. Fluorescent False Neurotransmitters (FFNs) are optical tools that enable the visualization of synaptic vesicle content release at individual presynaptic terminals

FFN102 is a fluorescent dopamine transporter (DAT) substrate, which selectively labels presynaptic dopamine terminals in the dorsal striatum of the mouse brain. It is accumulated into synaptic vesicles via the vesicular monoamine transporter 2 (VMAT2). Its fluorescence is attenuated (blue circles) inside the acidic synaptic vesicles and unquenched (blue stars) upon stimulation when it is released to the extracellular physiological pH. FFN102 therefore allows for optical measurement of released vesicle content.

Fig. 3. Unexpected effect of methylphenidate on the release of dopamine and FFN102 in mouse brain

(A) Structure of methylphenidate. (B) Acute striatal slices loaded with FFN102 were electrically stimulated at a frequency of 10 Hz starting at time point t = 0. Stimulation continues until the final time point. Error bars represent S.E. The change in fluorescence signal obtained by 2-photon microscopy (field of view of 37.5 × 37.5 μM including ~150 presynaptic boutons) upon stimulation was 19.6 ± 1.4% in control slices (mean ± S.E., n = 5) and 8.8 ± 1.2% in slices with 30 μM methylphenidate (MPH, mean ± S.E., n = 5). Methylphenidate inhibited the evoked increase in fluorescence (p < 0.0001, two-way ANOVA; p < 0.0001 for methylphenidate versus control). Dopamine release (measured by constant potential amperometry) was inhibited by the same extent (not shown).