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J Physiol Volume 581, Number 3, 890-892, June 15, 2007 DOI: 10.1113/jphysiol.2007.133538
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CLASSICAL PERSPECTIVES

Ricardo Miledi and the foundations of synaptic and extra-synaptic neurotransmitter receptor physiology

 Fabrizio Eusebi1

1 Dipartimento di Fisiologia Umana & Farmacologia, Centro di Eccellenza BEMM, Universita' di Roma ‘Sapienza’, P.le A. Moro 5, I00185 Roma; Istituto di Medicina e Scienza dello Sport CONI, via dei Campi Sportivi 46, 00197 Roma; and Neuromed, Via Atinese 18, I86077 Venafro, Italy Email: fabrizio.eusebi{at}uniroma1.it

Considered together, the two papers by Miledi (1960a,b) represent a milestone in synapse physiology: they provided completely new findings and striking concepts concerning chemosensitivity at, and around, the neuromuscular junction. These papers also suggested a variety of new avenues for investigation (some of which are still unexplored), and they also predicted new lines of research, such as into neurotrophism and neuroregeneration, which represent major topics in modern neuroscience.

Ricardo Miledi, after carefully exploring the chemo-sensitivity to acetylcholine (ACh) at rat diaphragm neuromuscular junctions, first introduced the concept of extrasynaptic receptors: these were located symmetrically, hundreds of micrometres from the end-plate (functionally identified as a region of about 30 µm diameter), where focal miniature end-plate potentials (mEPPs) were recorded (Fig. 1; i.e. Fig. 3 of Miledi, 1960b). From the original findings, Miledi (1960b) raised two general questions important for a better understanding of synaptic transmission. One was whether the extrasynaptic receptors are activated by the amount of ACh released normally by nerve impulses. He concluded that this was a rather likely event, also because it explained the ‘slow potential wave’ that was reported previously by Eccles et al. (1942) at endplates of eserinized muscle while they were doing research to help Allied countries during Word War II against chemical warfare agents. Noteworthy, Miledi gave the first evidence of the so-called spillover of the neurotransmitter, an event that occurs abundantly at central synapses. The second question addressed by Miledi was whether the extent of the extrajunctional region is fixed or variable. Considered together with previous findings, which showed that the motor nerve exerts a long-term restricting effect on the chemo-sensitivity of the muscle membrane (Miledi, 1959), findings reported in Miledi (1960b) led him to conclude that the extrajunctional region ‘may decrease or increase, depending on the intensity of action of the controlling neural factor’. This insight paved the way for future investigations on the neurotrophic role of motor axons on muscle (see for instance Witzemann et al. 1991), and introduced the concept of neuromodulation of synaptic activity by endogenous agents (Miledi, 1960a).


Figure 1
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  		Figure 1.  Simultaneous intracellular recording of potentials at two places in the same fibre
One microelectrode (bottom traces) was near the neuromuscular junction; the other (top traces) was 225 µm away from the first. A, B and C illustrate potentials produced by a 0.37 s pulse of ACh applied at –263 µm, +75 µm and +325 µm, respectively, from the electrode placed near the junction (see diagram, where the positions of the ACh pipette are represented by black triangles). Voltage and time calibration for all records as in A. The intensity of ACh pulses was not monitored in this case but the ACh sensitivity at B was greater than at either A or C. (Fig. 3 in Miledi, 1960b.)

 
His insights into the ACh sensitivity at neuromuscular junctions also allowed Miledi to open the door to dramatic advances in our understanding of mechanisms underlying denervation and events coupled to this. Starting from the concept that nerve cells usually have a number of fibres impinging on them (Wyckoff & Young, 1956), Miledi examined frog skeletal muscle fibres, which are innervated at more than one point (Katz & Kuffler, 1941), to study the effects of partial, and complete, denervation. First of all, it was reported that the whole length of the muscle (ca 30 mm) became sensitive to ACh some 10 weeks after complete denervation (Miledi, 1960a), compared to only 1 mm ACh in normal, undenervated muscle fibres. This phenomenon, called denervation supersensitivity, had been known previously (Kuffler, 1943; Cannon & Rosenbueth, 1949; Miledi, 1959), and Miledi provided a map of the distribution of the ACh sensitivity in a muscle fibre 66 days after complete denervation (Fig. 2; i.e. Fig. 1 in Miledi, 1960a). Surprisingly, the ACh sensitivity of the area restricted to, and closely surrounding, the neuromuscular junction in the normal muscle fibres is comparable to that observed at the denervated end-plate. This area is identified by the presence of low-frequency mEPPs that occur at denervated end-plates. When he began the work on denervated muscle, Miledi (1959) discovered that several days after nerve section the mEPPs disappeared but, very surprisingly, a few days later the spontaneous mEPP activity reappeared at the majority – perhaps all – of the endplates. This finding was subsequently reported in detail (Katz & Miledi, 1959; Birks et al. 1960). These mEPPs are due to the release of ACh quanta from the Schwann cells, which after motor nerve degeneration, move to occupy the now-vacated synaptic position. This phenomenon occurs also in mammalian (rat) muscles, but here the Schwann cell mEPPs are seen in only a few fibres partly because the cells contact the muscle fibres only transiently and then move away (Miledi & Slater, 1968). It is still not known why the frog Schwann cells remain in contact with the denervated muscle for several months whilst the mammalian cells withdraw. It was suggested that this difference may partly account for the differences in denervation atrophy seen between frogs and rats. Finally, it was noted (Miledi, 1960b) that many small minis (min-mEPPs) occur merging with the baseline electric noise. It is possible that the ACh synthesis is slow and the release process is accelerated so the vesicular packages are released before they are full. This point would still be interesting to investigate. The amplitude distribution of Schwann cell mEPPs is highly skewed to the left (Miledi, 1960a), which may be due to a very low content of neurotransmitter in the Schwann cell vesicles, in contrast to the standard nerve mEPPs This point is directly related to another open question: whether the high ACh sensitivity, maintained after denervation at the junctional end-plate sites, is consequent to Schwan cell ‘trophic’ quantal activity. A big effort to demonstrate the trophic role of Schwann cells is documented in recent literature (e.g. Peng et al. 2003). Another open question is whether the presence of high ACh receptor density at the site of the old end-plate confers any advantage for innervation.


Figure 2
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  		Figure 2.  Distribution of acetylcholine sensitivity in a muscle fibre (resting potential 90 mV) 66 days after complete denervation ({circ}) and at a normal end-plate (resting potential 94 mV) in the control muscle (bullet)
Abscissae, distance along fibre; ordinates, sensitivity to iontophoretic pulses of ACh, log scale. Inset: upper traces, examples of ACh potentials of denervated fibre at specified distances; lower traces, records of current flowing through acetylcholine pipette. Pulse duration is 3.7 ms in A–E, and 37 ms in F–H; time base calibration in G applies to records A–G; voltage and current calibration for all records in H. Monitor calibration: vertical bar in H = 2.2 x 10–7 A. Muscle supersensitivity, 100. The muscle supersensitivity is expressed as the ratio, control : denervated, of minimal ACh concentrations which evoke muscle twitches. (Fig. 1 in Miledi, 1960a.)

 
An important issue raised from findings reported in Miledi (1960a) is the different rates of desensitization at sites showing different ACh sensitivities in denervated muscle fibre. Specifically, Miledi reported that: (i) the responses to repeated pulses of ACh diminished progressively in amplitude; (ii) the decline in the amplitude of consecutive responses is greater and faster the smaller the initial local sensitivity; (iii) the reduction of ACh response is accompanied by a slower decay; (iv) the rate of recovery is independent of the local sensitivity. While points (i) and (iv) suggest a classical desensitization, points (ii) and (iii) can be tentatively explained by a subunit-dependent desensitization. In fact the switch from adult to embryonic type AChR confers slower desensitization and smaller conductance (Naranjo & Brehm, 1993), and an explanation of this desensitization pattern is that fast desensitizing adult-type and slow desensitizing embryonic-type receptor-channels might coexist at neuromuscular junctions (Henderson et al. 1987; Shepherd & Brehm, 1997). This important point focuses on ACh receptor subunit composition and on glial neurotrophisms that still remain to be fully investigated.

Miledi's analysis was extended to partially denervated muscle fibres by placing one microelectrode at a denervated endplate and another at an innervated end-plate of the same fibre. In this way it was found that supersensitivity developed at and beyond the denervated end-plate while the ACh sensitivity at the innervated end-plate was normal. Moreover, the pattern of desensitization in the denervated region was the same as that after total denervation. Strikingly Fig. 6 in Miledi (1960a) shows membrane potential noise is more easily detected in denervated muscle (perhaps due to longer channel duration), which Katz & Miledi (1970) later described. Finally, after considering all of the results, Miledi came to the notable conclusions that (i) after denervation, it is not the neuromuscular inactivity, but the removal of the neural influence which causes supersensitivity, and (ii) the sensitivity is simply an index of the density of the receptor units. It is noteworthy that the concepts of receptor density and neurotrophism were rather new in those days, and the work showed nicely that axon degeneration causes the Schwann cells to acquire neuronal characteristics and the muscle to acquire extra ACh receptors, making the Schwann cell, muscle and axon a complex system where multiple, as yet unidentified, signals are exchanged.

In conclusion, both of the highly innovative papers by Miledi (1960a,b) not only introduced original concepts and opened new roads that many scientists have followed in subsequent decades, but, satisfyingly after half a century, they still suggest some fine experiments to do.

References

Birks R, Katz B & Miledi R (1960). Physiological and structural changes at the amphibian myoneural junction, in the course of nerve degeneration. J Physiol 150, 145–168.[Free Full Text]

Cannon WB & Rosenbueth A (1949). The Supersensitivity of Denervated Structures. Macmillan Co., New York.

Eccles JC, Katz B & Kuffler SW (1942). Effects of eserine on neuromuscular transmission. J Neurophysiol 5, 211–230.[Free Full Text]

Henderson LP, Lechleiter JD & Brehm P (1987). Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle. J Gen Physiol 89, 999–1014.[Abstract/Free Full Text]

Katz B & Kuffler SW (1941). Multiple motor innervation of the frog's sartorius muscle. J Neurophysiol 4, 209–223.[Free Full Text]

Katz B & Miledi R (1970). Membrane noise produced by acetylcholine. Nature 226, 962–963.[CrossRef][Medline]

Katz B & Miledi R (1959). Spontaneous subthreshold activity at denervated amphibian end-plates. J Physiol 146, 44–45P.

Kuffler SW (1943). Specific excitability of the end-plate region in normal and denervated muscle. J Neurophysiol 6, 99–110.[Free Full Text]

Miledi R (1959). Acetylcholine sensitivity of partially denervated frog muscle fibres. J Physiol 147, 45–46P.

Miledi R (1960a). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. J Physiol 151, 1–23.[Free Full Text]

Miledi R (1960b). Junctional and extrajunctional acetylcholine receptors in skeletal muscle fibres. J Physiol 151, 24–30.[Free Full Text]

Miledi R & Slater CR (1968). Electrophysiology and electron-microscopy of rat neuromuscular junctions after nerve degeneration. Proc R Soc Lond B Biol Sci 169, 289–306.[Medline]

Naranjo D & Brehm P (1993). Modal shifts in acetylcholine receptor channel gating confer subunit-dependent desensitization. Science 260, 1811–1814.[Abstract/Free Full Text]

Peng HB, Yang J-F, Dai Z, Lee CW, Hung HW, Feng ZH & Ko C-P (2003). Differential effects of neurotrophins and Schwann cell-derived signals on neuronal survival/growth and synaptogenesis. J Neurosci 23, 5050–5060.[Abstract/Free Full Text]

Shepherd D & Brehm P (1997). Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle. J Neurophysiol 78, 2966–2974.[Abstract/Free Full Text]

Witzemann V, Brenner HR & Sakmann B (1991). Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses. J Cell Biol 114, 125–141.[Abstract/Free Full Text]

Wyckoff RWG & Young JZ (1956). The motoneuron surface. Proc R Soc Lond B Biol Sci 144, 440–450.[Medline]





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