Designing a norepinephrine optical tracer for imaging individual noradrenergic synapses and their activity in vivo

Abstract

Norepinephrine is a monoamine neurotransmitter with a wide repertoire of physiological roles in the peripheral and central nervous systems. There are, however, no experimental means to study functional properties of individual noradrenergic synapses in the brain. Development of new approaches for imaging synaptic neurotransmission is of fundamental importance to study specific synaptic changes that occur during learning, behavior, and pathological processes. Here, we introduce fluorescent false neurotransmitter 270 (FFN270), a fluorescent tracer of norepinephrine. As a fluorescent substrate of the norepinephrine and vesicular monoamine transporters, FFN270 labels noradrenergic neurons and their synaptic vesicles, and enables imaging synaptic vesicle content release from specific axonal sites in living rodents. Combining FFN270 imaging and optogenetic stimulation, we find heterogeneous release properties of noradrenergic synapses in the somatosensory cortex, including low and high releasing populations. Through systemic amphetamine administration, we observe rapid release of cortical noradrenergic vesicular content, providing insight into the drug’s effect.

Electrochemistry at the Synapse

“…With FFNs that mimic dopamine, heterogeneity in release was observed that was controlled in part by D2 receptors. Recently, FFNs were developed that can measure norepinephrine distinct from dopamine (107). Methods to measure actual neurotransmitters are also being developed.…”

Nonlinear relationship between multimodal adrenergic responses and local dendritic activity in primary sensory cortices

“…Recent studies used two-photon Ca 2+ imaging to directly measure activity of NE axons in the cortex and to correlate their activity with pupil diameter and locomotion (Reimer et al 2016;Breton-Provencher and Sur 2018). Others have developed optical imaging methods to measure the binding of NE to various fluorescent indicators (Muller et al 2014;Dunn et al 2018;Feng et al 2019). However, little is known about the sensory cues that induce phasic NE-release in sensory cortex and the consequences of this release on synaptic integration in dendrites and spines, where the adrenergic receptors 85 are found (Herkenham 1987;Nicholas et al 1993;Aoki et al 1998;Wang et al 2007).…”

Evoked transients of pH-sensitive fluorescent false neurotransmitter reveal dopamine hot spots in the globus pallidus

“…A variety of FFNs now provide means to observe release during synaptic vesicle fusion from specific sites on axons, including striatal dopamine axons in vitro (Gubernator et al, 2009; Rodriguez et al, 2013; Pereira et al, 2016) and norepinephrine axons in vitro and in vivo (Dunn et al, 2018). Here, we introduce a new use for pH sensitive FFNs, which is to resolve release from dopamine axons in a region of very sparse innervation by ‘FFN flashes’, that is short duration calcium-dependent release transients during exocytosis as the fluorescence is unquenched upon exocytosis from the acidic vesicle lumen to the neutral extracellular space.…”

“…Dopamine is known to disinhibit GPe cells (Cooper and Stanford, 2001; Shin et al, 2003), and could modulate the balance between the arky- and protopallidal pathways. The recently introduced FFN270, which is a substrate for the norepinephrine transporter (Dunn et al, 2018), is also pH sensitive and could provide means to measure properties of the sparse and widely distributed network of norepinephrine axons in the nervous system.…”