Single synaptic observation of cholinergic neurotransmission on living neurons: concentration and dynamics

Abstract

Acetylcholine, the first neurotransmitter identified more than a century ago, plays critical roles in human activities and health; however, its synaptic concentration dynamics have remained unknown. Here, we demonstrate the in situ simultaneous measurements of synaptic cholinergic transmitter concentration and release dynamics. We used nanoscale electroanalytical methods: nanoITIES electrode of 15 nm in radius and nano-resolved scanning electrochemical microscopy (SECM). Time-resolved in situ measurements unveiled information on synaptic acetylcholine concentration and release dynamics of living Aplysia neurons. The measuring technique enabled the quantitative sensing of acetylcholine with negligible interference of other ionic and redox-active species. We measured cholinergic transmitter concentrations very close to the synapse, with values as high as 2.4 mM. We observed diverse synaptic transmitter concentration dynamics consisting of singlet, doublet and multiplet events with a signal to noise ratio of 6 to 130. The unprecedented details about synaptic neurotransmission unveiled are instrumental for understanding brain communication and diseases in a way distinctive from extra-synaptic studies.

Chemical sensing with electrodes offers chemical identity, quantification, and spatiotemporal information about biological processes in vivo. These advantages make electroanalytical chemistry one of the most widely used tools in the detection of signaling molecules and redox neurotransmitters^{1–, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}. Acetylcholine, the first neurotransmitter identified in 1914 ^{20, 21} plays a key role in learning, memory and human health; defects in its release have been associated with aging and neurodegeneration. Elucidating its release concentration dynamics at the source of its release, the synaptic cleft, is instrumental in understanding neurodegenerative diseases. However, this has been challenging due to the nanometer size of the cleft, with a typical width of 300 nm and a gap of < 100 nm (Fig. 1)^{22–, 23, 24, 25, 26, 27} and technical limitation in available nanoprobes for the simultaneous detection of its concentration and release dynamics since acetylcholine is a redox inactive molecule; although carbon nanofiber electrodes have been reported, their detection mainly targets dopamine and norepinephrine, where significant progress has been made^{13, 28, 29}.

Figure 1.

Study of cholinergic neurotransmission at single synaptic cleft with nanoelectrode and scanning electrochemical microscope. (A) Illustration of synaptic cleft dimensions^{22–, 23, 24, 25, 26, 27}. (B) Cultured living Aplysia pedal ganglion neurons used for the experiment, where the axon from cell 1 (pink) formed a synaptic connection with the body of cell 2. Scale bar: 200 μm. (C) A nanoITIES pipet electrode was positioned around the synaptic cleft to measure the concentration and release dynamics of acetylcholine (ACh⁺) simultaneously using amperometry; the positioning of the nanoelectrode was achieved using the scanning electrochemical microscope (Fig. E) with a spatial resolution of 5 nm. The zoom shows the nanoITIES formed at the tip of the nanoITIES pipet electrode, and ionic transmitter (ACh⁺) transfers across the interface, generating a current and thus getting detected. (D) Cyclic voltammogram corresponding to ACh detection, where the detection potential follows Nernstian equation, and a steady state transfer potential, E_ACh= - 0.48 V vs E_{1/2, TBA}, selective for cholinergic neurotransmitter detection was used in amperometry to study its synaptic concentration dynamics (results shown in Figs. 2 and 3). (E) A Scanning Electrochemical Microscope (SECM) and a lab-built side view optical microscope were used for the positioning of the nanoelectrode around synapses with nm spatial resolution. The lab-built side view optical microscope provided rough positioning before the fine positioning of 5 nm spatial resolution with SECM. After SECM positioning (Supporting Information, figs. S9, S10), the optical microscopic view of the nanoelectrode and the synapse are shown in Fig. F, where it can be seen that it is very hard to locate the synapse by visual observation alone. The combined use of the side view optical microscope and nano-positioning platform, SECM, is critical. (F) A stimulating pipet was used to provide high concentration K⁺ stimulation. Reflection was used for the rough positioning of the nanoelectrode and stimulating pipet in the x, y and z axes by optical microscope, which was followed by the nanometer positioning of the nanoelectrode around the synapse achieved using nano-resolution SECM with details described in the supporting information. Scale bar: 150 μm. (G) High resolution scanning electron microscope (SEM) picture of the nanopipet tip with radius (a) to be around 15 nm.

Here we measured in situ simultaneously synaptic acetylcholine concentration and release dynamics with a nanoelectrode of ~15 nm in radius (Fig. 1G). The neuronal model used in the present study is Aplysia californica, previously used by Kandel et al. to understand the synapse-specific long-term facilitation^{30, 31}; studied pedal ganglion neurons are cholinergic^{32, 33, 34} and they were cultured following the well-established protocols ^{35, 36}, with details shown in the Supporting Information. We employed nanoresolved scanning electrochemical microscopy (SECM)^{37, 38, 39, 40, 41, 42} (Fig. 1E) to position the nanoelectrode near the synaptic cleft, formed between the axon of one neuron (pink) and the neurite of another neuron (blue) (Figs. 1B, 1F). We detected acetylcholine, based on the charge transfer across a nano interface between two immiscible electrolyte solutions (ITIES), at the nanoITIES pipet electrode ^{6, 10, 35, 43, 44}. Fig. 1D shows the cyclic voltammogram of acetylcholine detection on the nanoITIES pipet electrode, with calibration curve shown in Fig. S1. We measured the current-time trace at the steady state detection potential, selective for cholinergic transmitter detection (E_ACh) against other substances that have been identified or suggested to be released from the Aplysia neurons and their vesicles, including serotonin, gamma-aminobutyric acid, dopamine, glutamate etc., as well as the pH change accompanying exocytosis and high concentration K⁺ in the stimulating solution, (Control experiments on selectivity shown in Figs. S2-S5, discussion detailed in the supplementary text of the supporting information), to learn about its synaptic release dynamics and concentration profiles.

The results of synaptic cholinergic neurotransmission are shown in Fig. 2 (More results are shown in Figs. S6 and S7). We measured the synaptic release from Aplysia neurons in response to high concentration K⁺ stimulation and recorded intense release peaks, which are raw data without data processing. Control experiments (Fig. S6) confirm that the measured release appear to be from a single synapse near where the nanoelectrode was located, using a lab-built side view optical microscope and nano-resolution SECM with procedures described under methods section. The detection has high sensitivity as evidenced by a signal to noise ratio from 6 to 130. Direct measurements around the cleft avoid the dilution of transmitter due to its diffusion into the extracellular medium^{45, 46, 47}, easing the required performance specifications of the small-volume electrochemical measurement. We measured the acetylcholine concentration around the synapse to be as high as 2.4 mM (Figs. 2–3). This measured mM regime of near-synaptic acetylcholine concentration is the same order of magnitude as the number estimated from multiple neuromuscular junctions using stimulated Raman scattering in a recent study⁴⁸, and is consistent with the number estimated based on hypothesis and theoretical simulation^{49, 50}.

Figure 2.

Single synaptic cholinergic neurotransmission measured in situ. (A-C) Current-time trace (amperometry) representing synaptic transmitter concentration and release dynamics simultaneously, where diverse cholinergic concentration dynamics were observed consisted of singlet (Fig. A), doublet (Fig. B), and multiplet (Fig. C). Single current (concentration) maxima occur during singlet release (50% occurrence frequency out of 16 events total); a second current maximum occurs before the first current peak decreases to the base value for the doublet events (~30% occurrence frequency); multiple concentration peaks (more than two) were observed for multiplet with lower occurrence frequency (~20%) (Fig. S7D). (D) Proposed mechanism on variation in synaptic transmitter release dynamics. Neurotransmitter is released into the synaptic cleft from a single vesicle (Left). Neurotransmitter is released into the synaptic cleft from two vesicles, V1 and V2, simultaneously (Middle) or multiple vesicles simultaneously, which are going through either different stages of exocytosis as shown here, or similar stages of exocytosis (Right). (E) An alternative mechanism is possible for explaining doublets and multiplets based on the phenomenon of partial release^54–67. A vesicle goes through partial release twice, generating a doublet (Middle); the two individual peaks (Peak 1 and Peak 2) correspond to each partial release event.

Figure 3.

Simultaneous determination of the synaptic cholinergic transmitter concentration dynamics and the number of transmitter molecules (N) during discrete synaptic release events (A). N_A is Avogadro’s number, Q is the charge based on integration of amperometric current peak, z is the charge of the transmitter molecule (equals 1 for acetylcholine), and F is Faraday’s constant. Synaptic transmitter concentration profile was obtained from amperometric peak based on current expression at the nanoelectrode, $c=\frac{i}{4xzFDa}$ (Supporting Information). (B) Number of neurotransmitter molecules and number of moles released during singlet (N = 8), doublet (N = 4) and multiplet events (N = 4) measured from the single synapse shown in Fig. 1B in response to 6 repetitive chemical stimulations. Release events were constantly observed during each of six chemical stimulations. A variation in synaptic cholinergic transmitter concentration was observed, which is likely due to variation in synaptic vesicles sizes as observed by TEM⁵².

The synaptic concentration of neurotransmitter dynamically changes, governed by the combination of multiple processes including its release from presynaptic vesicles, its reuptake by membrane proteins, its breakdown by the enzymatic reactions, and its diffusion out of the cleft into the extracellular space. The neurotransmitter concentration profiles (Figs. 2A-C, figs. S7A-C) represent this dynamic process. We measured synaptic transmitter concentration dynamic profiles to be composed of singlet, doublet and multiplet (Fig. 2D), with 50% occurrence frequency for singlet and a lower occurrence frequency for doublet and multiplet (Fig. S7D). Our observed occurring frequency of the diverse dynamics for synaptic acetylcholine release is consistent with that of synaptic norepinephrine release measured with carbon nanofiber electrode²⁹. More examples of singlet, doublet and multiplet are shown in Fig. S7. For singlet type peaks, the concentration corresponding to neurotransmitter release increases to reach the maxima and then decreases to the base value. In contrast, for doublet and multiplet events, the current did not decrease to the base value after the first peak, and instead it increased to generate the second or even more peaks.

We did quantitative analysis to understand the variation in synaptic transmitter release dynamics. Single vesicular dopamine release studied via carbon electrodes demonstrated half amperometric peak width of hundreds ms, increasing with vesicle sizes⁵¹. For acetylcholine, the singlet events have half amperometric peak widths of hundreds ms, with multiple values (Fig. S8). This suggests that the singlets be the synaptic single vesicular events, which was further supported by the analysis described in the next paragraph. The variation in half-peak widths are likely due to a distribution of vesicle sizes of Aplysia⁵². Half amperometric peak widths for the doublets totaled two half peak widths of singlets, and that for the multiplets totaled the peak width of multiple singlets (Fig. S8); this applies to all the doublets and multiplets that we observed.

The average number of acetylcholine molecules was 1.0 × 10⁶ for singlet events, and 2.0 × 10⁶ for doublet events (Table. 3B). These quantities are consistent with the amount of acetylcholine needed to produce an end plate potential when acetylcholine was perfused to the neuron muscular junction electrophoretically⁵³. Doublets have twice the molecules of the singlets on average (Table 3B). This observation, along with amperometric peak widths discussed above, suggests that doublet and multiplet peaks represent simultaneous release from two or more vesicles (Fig. 2D). Besides, the lower range of the total number of the molecules for doublets and multiplets are similar orders of magnitude to that of the singlets (Table 3B); this suggests the partial release occurrence (Fig. 2E), as proposed in recent studies^{54–, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67} during the first peak or the first couple peaks for some of the doublets and multiplets, respectively.

In summary, we have successfully measured cholinergic transmitter release concentration dynamics at the single living Aplysia synapse. The work presented here is the first study of the intra-synaptic electrochemical detection of non-redox active transmitter. Our observed diverse release dynamics (singlet, doublet and multiplet) and its occurrence frequency for synaptic acetylcholine release concurred well with that of synaptic norepinephrine release reported in a recent study²⁹. Quantitative analysis of half amperometric peak width and of the number of molecules released suggests doublet and multiplet be observation of multiple vesicular events; partial release was suggested as well. Measuring the intra-synaptic dynamics of neurotransmitter release is a critical step in our ability to understand transmission and its deficiencies that are explicated in aging and neurodegenerative diseases. The new information on the diversity in cholinergic transmitter dynamics and synaptic concentration uncovered will be very valuable for fundamental and biomedical sciences, contributing to our understanding of brain communication and various diseases from a distinctive perspective. Future work includes studying the synaptic release heterogeneity from different kinds of synapses and neuronal types.