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Pre-Print - Ricardo Miledi

The calcium hypothesis of neurotransmission” is a central dogma in the field of neurophysiology. In these papers Ricardo Miledi and his frequent collaborator Bernard Katz systematically walk through the possibilities for ionic control of neurotransmitter release and ...

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Ricardo Miledi

Contributor: Kristen E. Frenzel

Topics: neurophysiology, synaptic release, calcium, Mexican neuroscientist

Teaching Resources:

A study of synaptic transmission in the absence of nerve impulses.

Katz B, Miledi R., J Physiol. 1967 Sep;192(2):407-36.

Transmitter release induced by injection of calcium ions into nerve terminals.

Miledi R. Proc R Soc Lond B Biol Sci. 1973 Jul 3;183(1073):421-5.

 

Description:

The calcium hypothesis of neurotransmission” is a central dogma in the field of neurophysiology. In these papers Ricardo Miledi and his frequent collaborator Bernard Katz systematically walk through the possibilities for ionic control of neurotransmitter release and definitively demonstrate Ca2+ as the critical ionic player. These two papers address the necessity and sufficiency of Ca2+ for neurotransmitter release, as measured by postsynaptic potentials in the squid giant synapse. The one-two punch of these papers nails the calcium hypothesis to the door and fuels this line of inquiry for years to come. 
 

The purpose of the September 1967 paper was to describe in detail experiments that reveal the nature of the presynaptic depolarization needed to generate a postsynaptic response, but in the absence of the all-or-nothing event of an action potential. The data in this paper had been previously published in several papers throughout 1965 and 1966 but this paper synthesizes the data, providing a complete set of detailed experiments that reveal the ionic nature of neurotransmission.  These experiments utilized tetrodotoxin and tetraethylammonium to effectively block the voltage-gated channels critical for action potential generation, and elegantly reveal the necessity of Ca2+ influx for generating a postsynaptic potential.
 

In the first set of experiments, Katz and Miledi used tetrodotoxin to block voltage-gated sodium channels and simultaneously inject current to depolarize the presynaptic membrane. Specifically, the data from Figure 4 and the statement on page 415 suggest that the cause of neurotransmitter release is not the influx of sodium ions that occurs during an action potential but another event stimulated by presynaptic depolarization. The data from Figures 10 and 11 address the hypothesis that the efflux of potassium through voltage-gated channels is the necessary event, yet the data effectively demonstrate that blocking both influx of sodium (with tetrodotoxin) and efflux of potassium (with tetrethylammonium) does not diminish the postsynaptic response after presynaptic depolarization. These data definitively ruled out the need for sodium and potassium as ions necessary for neurotransmitter release and were foundational in the hypothesis put forth as “the calcium hypothesis”, simply stated as depolarization --- (insert arrows here) calcium influx --- (insert arrows here) quantal neurotransmitter release.


In their analysis of the data shown in Figure 10, Katz and Miledi realized that the recordings of the “on” and “off” responses produced during the membrane depolarization and repolarization, respectively, provided information regarding the source of synaptic delay (i.e., the delay in postsynaptic response following presynaptic release of neurotransmitter). Before the publication of this paper, it was largely assumed that synaptic delay is due to the time it takes for neurotransmitter to diffuse across the synaptic cleft.  The lack of delay in the “off” response though (seen in panels 5-8 of Figure 10), compared to the delay seen in the “on” response, indicated that the synaptic delay is due to the time it takes to open the voltage-gated calcium channels. 


The introduction to the 1973 paper concisely synthesizes the overwhelming evidence to date for the necessity for Ca2+ in neurotransmission. The direct test for the sufficiency of calcium in neurotransmission is injection of calcium into the presynaptic terminal in the absence of an action potential and measuring a postsynaptic response. However, this experiment is technically not possible at the frog neuromuscular junction because the presynaptic terminal is too small to accommodate the injection and recording electrodes. In the giant synapse of squid, Miledi needed to overcome a technical issue that the response to the release of a few quanta at the giant synapse (i.e., the miniature excitatory postsynaptic potential) was generally too small to detect. Overcoming this technical hurdle by using very small squid allowed Miledi to proceed with the experiments that tested the sufficiency of calcium in neurotransmission. Direct injection of Ca2+ ions into the presynaptic terminal in the presence and the absence of Ca2+ in the extracellular solution (Figures 2 and 3) provide concrete support for the calcium hypothesis laid out almost a decade previous. Data shown in Figure 3 also reinforce the findings reported in their 1967 paper regarding synaptic delay, as there was very little delay in postsynaptic response following the presynaptic injection of Ca2+ .


Value: 

Neurophysiology textbooks sometimes utilize the exact data from these two papers or modified versions of these figures and students might find it informative to read the associated text to understand the context of the experiments and the logical arguments built by the authors in support of the calcium hypothesis. As well, the pros and cons of the two primary synaptic model systems, the frog neuromuscular junction and the squid giant synapse, are elegantly described in the 1967 Katz and Miledi article. The techniques described are straightforward neurophysiology techniques that are accessible for mid-level undergraduate students to understand, and reinforce the concepts of membrane potential and equilibrium potential. As well, the concepts of necessity and sufficiency for a biological process are easily understood based on experiments in these two papers.  

 

Biographical information:

Ricardo Miledi was 1 of 7 children, born in Mexico City in 1927. When taking the broad view of the research career of Ricardo Miledi, it is clear that a) science has no borders b) networking opened doors and c) persistence is key to success. He attended medical school in Mexico but decided that his interests in how and why the body worked in normal conditions and in disease states would be more interesting than directly treating the patient's symptoms. He parlayed this interest into a research fellowship (as a way to satisfy the social service component of his medical training) in the lab of Arturo Rosenblueth. Working with Arturo Rosenblueth in 1954, Dr. Miledi had a serendipitous meeting with two renowned scientists, Albert Grass and Stephen Kuffler from the Marine Biological Laboratory in Woods Hole, MA. At this point in his life, he was a married 27-year old man who was working in a lab as a research fellow and his research career had not yet started. He jumped at the opportunity to spend the summer of 1955 at the Marine Biological Laboratory at Wood's Hole, Massachusetts. There, he began his study of synapses in the common squid and began to see the importance of calcium in synaptic transmission, particularly when he would forget to add calcium to his solutions. Miledi found his way to Canberra, Australia to work with John Eccles, and through this relationship met his future collaborator and Nobel Laureate Bernard Katz, who offered him a position in the Department of Biophysics at University College London (UCL) and worked on acetylcholine receptors at the frog neuromuscular junction, laying important groundwork for subsequent experimentation in understanding postsynaptic neurotransmitter receptor biology. In the early 1960s, he circled back around to his interest in Ca2+ and synaptic release and revisited the squid giant synapses as the model system for investigation of the fundamental nature of Ca2+ in synaptic vesicle release. His work with squid while at UCL was not without its challenges. Obtaining live squid suitable for research in England was not possible, despite trips to the marine center in Plymouth to collect squid. Again, a serendipitous discussion with a colleague in London turned him towards centering his research in squid at an aquarium in Naples, Italy. His fruitful work in Naples provided the data for the seminal work in the mid 1960s and into the 1970s to test “the calcium hypothesis”. 

Miledi's work evolved away from a study of calcium-mediated neurotransmitter release to structural and functional analysis of proteins involved in synaptic communication. He pioneered the use of frog oocytes for heterologous expression of neurotransmitter receptors and channels, extensively publishing detailed analyses of these proteins and their role in normal and disease states until his death at age 90 in 2017.

 
Audience:

These papers would be appropriate for mid-level to advanced undergraduate courses including introductory neuroscience, neurobiology or neurophysiology course. 

 

Teaching Resources:

Much of the detail about Miledi and his life comes from the outstanding review by Jade-Ming Jeng (2002) who had the opportunity to interview Miledi for a Timeline article in Nature Reviews Neuroscience. These papers can be used to highlight the trajectory of a Mexican undergraduate STEM student to a renowned scientist, and his research story highlights the global nature of scientific inquiry. 

 

As a teaching resource, these two papers can be used to walk through the ionic nature of neurotransmitter release and to push students to check their biases with regard to data. Students tend to come into an introductory neuroscience or neurobiology course with the basic idea that action potentials trigger neurotransmitter release, and that synaptic delay is due to diffusion of neurotransmitter. However, these data require the students to check their biases about the need for influx of Na+/efflux of K+ per se in neurotransmitter release. The experimental setups in both papers can be used to introduce, or solidify, understanding about electrophysiological techniques that are still used today, and to reinforce skills in interpreting data figures and drawing conclusions. After working through specific figures in the 1967 paper, students can then be challenged to consider what experiments would test the sufficiency of calcium in neurotransmitter release, and many of them come up with the exact experiments performed by Miledi in 1973. Regarding factors contributing to synaptic delay, they can be shown data from experiments in which the synapse was cooled (e.g., Figure 3 of Katz and Miledi, 1965), which exaggerates the delay, and they can consider the source of the delay (this question can be used before reading the 1967 paper, or be used as a follow-up or an exam question).  Recognition that they (the undergraduate students) can also design an experiment similar to the one published by Miledi in the 1973 paper helps them identify as scientists.

 

These studies did not involve the use of voltage clamp, but subsequent experiments by Rodolfo Llinas, in which the presynaptic membrane was clamped at the desired potentials, substantiated the findings of Katz and Miledi.  The work of Katz and Miledi therefore is a good example that observational skills, more so than technological advances, are key to doing good science.  It is also worth pointing out that the work by Katz and Miledi predates the evidence for the fluid mosaic model of the lipid bilayer, as well as our modern-day models of ion channel structure.  Discussions of this work can lead naturally to the question of “what is the calcium sensor for release?” and discussion of T- and V-SNAREs.

References:

Jeng, JM; Nature Reviews Neuroscience Vol.3: 71–76 (2002).

Katz B, Miledi R., J.Physiol. 181: 656-670 (1965)  

Katz B, Miledi R., J Physiol. 192(2):407-36  (1967) 

Miledi R. Proc R Soc Lond B Biol Sci. Jul 3;183 (1073):421-5 (1973)

https://senate.universityofcalifornia.edu/in-memoriam/files/ricardo-miledi.html (accessed July 24, 2019)

https://en.wikipedia.org/wiki/Ricardo_Miledi (accessed July 24, 2019)

There is a nice review of this work in a 1982 review by Llinas, accessed through researchgate: https://www.researchgate.net/publication/16905432_Calcium_in_Synaptic_Transmission

One can find an obituary on nature.com written just after Miledi's passing.

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