Synaptic Transmission and Neurotransmitter Release Mechanisms

Synaptic Transmission

Overview of Neurotransmitter Release

• A chemical synapse converts an electrical signal to a chemical signal and then back to electrical.

Part 1: Mechanisms of Release

• How are electrical events in the presynaptic terminal coupled to secretion of the neurotransmitter?

• Experiments were conducted using the squid giant axon bundle and stellate ganglion, focusing on the squid giant synapse.

• Katz and Miledi (1967) investigated the relationship between the presynaptic action potential and neurotransmitter (NT) release.

  • TTX (tetrodotoxin) was used to block Na+ channels gradually.

  • A minimum of 40mV depolarization in the presynaptic terminal is necessary for a postsynaptic response.

  • Normal fluxes of Na^+ and K^+ ions during the presynaptic action potential are not necessary for transmitter release; depolarization is sufficient to trigger release.

Calcium's Role in Neurotransmitter Release

• External Ca^{2+} is crucial for release (del Castillo and Katz).

• In the squid axon, each action potential produces a small influx of Ca^{2+} through voltage-dependent Ca^{2+} channels (Hodgkin and Baker).

• The remaining presynaptic current after application of TTX and TEA is carried by Ca^{2+} ions (Katz and Miledi).

• Rodolfo Llinás conclusively proved Ca^{++} is necessary and sufficient to cause release.

  • Voltage-clamp the presynaptic terminal of the squid giant synapse in the presence of TTX and TEA.

  • Depolarization leads to Ca^{2+} influx and NT release.

  • Further depolarization (to Ca^{2+} equilibrium potential, E_{Ca^{++}}) results in no calcium influx and no NT release.

  • Simulating an action potential presynaptically allowed measurement of the time course of the Ca^{++} current.

• Further evidence that presynaptic Ca^{++} is sufficient to induce release comes from studies with “caged” Ca^{++} (Zucker, 1993; Schneggenburger et al., 2002).

  • Glutamate Uncaging at the Calyx of Held was also used.

• The Ca^{++}-Dependence of Release is Highly Non-Linear (Dodge & Rahaminoff, 1967; Schneggenburger & Neher, 2000).

  • The exponent is approximately 4.

• Voltage-gated Ca^{++} channels are located directly opposite ACh receptors in the synaptic cleft.

  • Fluorescent conotoxin and bungarotoxin are used to visualize this.

• Fatt and Katz in 1951 showed that using a low concentration of curare to partially block synaptic transmission at the frog neuromuscular junction allows for resolution of end plate potentials (EPPs), which precede muscle action potentials.

Part 2: Quantal Hypothesis of Neurotransmitter Release

• Fatt and Katz, 1952, made recordings in TTX that reveal spontaneous miniature EPPs (mEPPs) occur at the end plate (0.5-1mV).

• The quantal hypothesis poses the question: Are the minis the building blocks of the EPP?

• Quantal content (m) is the mean EPP size (V{avg}) divided by the mEPP, or quantal size (q): m = \frac{V{avg}}{q}.

• Fatt and Katz proposed the Quantal Hypothesis: Single quantal events observed spontaneously represent the building blocks for the synaptic potentials evoked by stimulation.

• Under conditions of low Ca^{++} and high Mg^{++} concentration, the magnitude of EPP fluctuates in a stepwise manner.

• José del Castillo and Bernard Katz proposed a statistical model to describe the relationship between quanta and evoked release:

  • Does quantal release fit a Poisson distribution?

  • Evoked EPP amplitudes increase in a stepwise manner, indicating the size of the response increases in discrete “chunks” or quanta.

Analyzing Evoked EPSP Amplitudes

• Amplitude histograms of mEPPs are made to analyze quantal size.

• Observations include spontaneous events, control conditions, and effects of drugs A & B, including failures.

Part 3: Vesicle Hypothesis of Neurotransmitter Release

• EM micrographs had shown “omega figures” in the presynaptic terminal, hypothesized to be vesicles in the act of fusing.

• Heuser and Reese (1979) used “slam freezing” and “freeze fracturing” techniques to observe vesicles in the act of fusing.

  • Procedure: Stimulate nerve, Drop (~3 msec), Freeze (4°K).

  • Frog NMJ

  • Use 4-AP to enhance release

• Freeze-fracture EM reveals ‘pits’ representing vesicles fusing. Vesicle openings match calculated # of quanta released!

Presynaptic Homeostasis

• Presynaptic Homeostasis at the Drosophila NMJ influences synaptic strength.

• Key factors (presynaptic and postsynaptic):

  • Number of release sites/vesicles (N).

  • Action potential waveform.

  • Ca^{2+} channel #/ -function / -type.

  • Ca^{2+} buffering.

  • Vesicle – Ca^{2+} channel distance.

  • Ca^{2+} sensitivity.

  • Release site/active zone/vesicle #.

  • Release competence.

  • Neurotransmitter molecules/vesicle.

  • Neurotransmitter receptor #/-type.

  • Release probability (p)

  • Quantal size (q)

• EPSP(C) = N \cdot p \cdot q

• In synapses with high Pr, one needs to use a binomial model where: m=np.

  • m = quantal content.

  • n = quanta available for release (number of active zones).

  • p = average release probability.

• Presynaptic Homeostatic Synaptic Plasticity in fly NMJ (Grae Davis UCSF, Martin Müller U. Zurich).

  • Delvendahl and Müller, Curr Op. in Neuro. 2019

  • +Philanthotoxin (AMPA blocker)

Considerations in the CNS

• When looking at minis, mini frequency reflects n, while mini size (q) reflects the amount of postsynaptic receptors.

• In CNS synapses, quantal content is ~1, and the amount of NT released in a vesicle saturates the postsynaptic terminal.

• Most CNS synapses have 1 active zone, but 1 axon can make several synapses onto one cell. n=3

Review

• Synaptic transmission is triggered by Ca^{++} flowing into the presynaptic terminal.

• Neurotransmitter is released in multimolecular packets called quanta.

• Each quanta results in a mini EPP, and the evoked response is composed of multiple quanta.

• Each vesicle represents a single quanta.

• Synaptic strength is a combination of pre and postsynaptic factors, each of which can be modulated to cause synaptic plasticity.

Here are some key terms related to synaptic transmission:

• Neurotransmitter release

• Presynaptic terminal

• Postsynaptic response

• Voltage-dependent Ca^{2+} channels

• Quantal hypothesis

• Miniature EPPs (mEPPs)

• Quantal content

• Vesicle hypothesis

• Freeze fracturing

• Presynaptic homeostasis

• Release probability (p)

• Quantal size (q)