My interest today is in optimising optogenetic terminal stimulation. I have previously talked about optimising your opto stim frequency, and I had a little dig at a collaborator who stimulates at 30 Hz.
Optogenetic stimulation fidelity
To summarise my previous post regarding optogenetic stimulation fidelity: if you optogenetically stimulate a neurone too fast (and the definition of “too fast” depends on a number of factors including the next type, opsin, duration and intensity of light flash etc.), they cannot keep up, and instead of firing action potentials they become chronically depolarised without firing.
Essentially, the neurones need time to come recover their membrane potential back below a certain threshold (typically around -50 mV) before they can produce another action potential, thus they effectively become silenced when you want to be activating them.
However, it has since occurred to me that my collaborator only ever stimulates the projection sites of his ChR2-expressing neurones. The neurones he is interested in are found in the hindbrain, and as such he can’t reliably stimulate the soma.
So I had a thought: does stimulating neurone terminals work differently from soma because you don’t need to send the signal down an axon, and as such allow his high frequency stim to work? In particular, my collaborator maintains that high frequency stim results in more release of neuropeptides (eg. AgRP/NPY), rather than fast amino acid transmitters (eg. GABA). This has, in principle, been known for a long time1, but it doesn’t mean a) it’s true for all neurones everywhere b) it’s possible to stimulate neurones that fast in vivo using optogenetics.
In this post, I’ll be exploring the second point in more detail, by looking at the fundamental biology of a neuronal synapse, what causes release of neurotransmitter and how we can successfully control that with optogenetics.
Biology of a synapse
A quick biology lesson: we all know that neurones have action potentials, which is a transient spike in electrical activity across the membrane, and this is how they send information down an axon. However, it isn’t the voltage change that causes neurotransmitter release at the nerve terminal. At least, not directly. Instead, the increase in membrane potential causes an influx of calcium, and it’s the increase in calcium that causes vesicle fusion and release of the neurotransmitter (Figure 1)2.
So, my question regarding optogenetic stimulation of nerve terminals is this: if you overstimulate a nerve terminal into a chronically depolarised (silent) state, do you still drive release of neurotransmitter? This might sound paradoxical, but it could theoretically happen if the calcium release occurs at a slightly depolarised membrane potential (maybe -30 mV, which is easily obtainable by opto-overstimulation). In that case, action potentials would not be necessary, as we are already at the terminal end of the axon and don’t care about sending electrical signals, only about triggering vesicular fusion by increasing calcium.
To answer my question, we need to look at the channels that cause the increase in calcium upon membrane depolarisation, and in particular at what membrane potential the calcium release is triggered. If calcium release occurs at a low (-30 mV or below) membrane potential, then we could happily see neurotransmitter release from chronic depolarisation. However, if it occurs at a more depolarised level, it is extremely unlikely that the neurone terminal would reach that membrane potential from chronic overstimulation.
The channels we’re interested in are called, surprise surprise, voltage-activated calcium channels. This actually comprises a large family of channels, with multiple groups. I won’t go in to depth here, because there is so much literature concerning these channels. However, of the ten mammalian variants, there are three that are important in neurones for synaptic release (Table 1)3.
To quote the review by Dolphin3:
“For most synapses, CaV2.1 (P/Q)- and CaV2.2 (N)-type channels are involved in varying proportions in synaptic transmission, depending on the synapse in question and the developmental stage… At some synapses, CaV2.3 channels, activated by smaller depolarizations, play an important role, rarely as the main channel involved in vesicular release”
So there you have it, straight from the … Dolphin’s mouth. The two channels that are most important for vesicular fusion and the release of neurotransmitters are activated at very high membrane potentials (-5.7 mV and -13 mV), which are far too high to be activated by non-firing chronic depolarisation of the membrane.
I now feel confident in saying that high frequency optogenetic stimulation (eg. 30 Hz) of a nerve terminal, which is likely to induce chronic depolarisation rather than action potentials, is not likely to cause the release of neurotransmitter from the presynaptic terminal. I would therefore urge my fellow researchers to refrain from such high frequency optogenetic stimulation.
1. Dutton and Dyball J Physiol (Lond) 290, 433-440 (1979) Phasic firing enhances vasopressin release from the rat neurohypophysis
2. Südhof Cold Spring Harb Perspect Biol 4(1), a011353 (2012) Calcium control of neurotransmitter release
3. Dolphin Function 2(1), zqaa027 (2021) Functions of presynaptic voltage-gated calcium channels