- Email: [email protected]
The view from inside
Brain Research Bulletin, Vol. 50, Nos. 5/6, pp. 305–306, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(99)00160-4
The view from inside Per Andersen* Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway [Received 6 May 1999; Accepted 8 May 1999] shortly afterwards there was a series of notable breakthroughs. After Nastuk and Hodgkin recorded intracellularly from muscle cells in 1950 [14], Fatt and Katz [8] succeeded in recording the first intracellular synaptic potential in the form of end plate potentials from frog muscles. The next year, Brock, Coombs and Eccles [4] gave the first demonstration of excitatory postsynaptic potentials (EPSP) from spinal cord motoneurones in cats. One year later, Eccles’ group [3] reported the hyperpolarizing inhibitory postsynaptic potentials (IPSP) as nearly a mirror image of the EPSP, only slightly delayed in onset. The properties of the EPSP suddenly gave a comprehensive explanation of Sherrington’s term “central excitatory state (c.e.s.)” in which the activation of spinal cord elements were followed by a period with lowered excitability for a second impulse arriving from a homonymous muscle nerve. EPSP components from individual muscle spindle afferents summed linearly, explaining the effect of increasing strength or speed of muscle shortening on the reflex size. Afferent signals from muscle spindles in homonymous muscles also summed, while a nerve volley through Ia afferents from an antagonistic muscle caused inhibition. By careful analysis of the onset times of EPSPs and IPSPs, Eccles and colleagues [3,7] showed that there was a delay between the two, sufficient for the intercalating action of an inhibitory interneurone. Ever since this fundamental discovery, this concept has deeply influenced our thoughts about circuit organization in the nervous system. For a long time, Eccles’ tenet of an intercalated interneurone was believed to be a general phenomenon in all brain circuits until Eccles’ pupil, Ito, showed that Purkinje cells inhibited vestibular cells directly and another of Eccles’ collaborators, Wilson, showed that certain of the vestibulo-spinal effects on neck motoneurones also were direct. Intracellular recording allowed an assessment of the relative functional strength of various converging influences. Eccles and Lundberg and colleagues demonstrated the intricate network of synaptic effects from various sense organs onto the different hind limb motoneurones and thereby controlled the muscle activity in an integrated fashion, a prerequisite for an understanding of postural and walking reflexes. Eccles’ group, while still in Dunedin, succeeded not only in postulating an inhibitory neuron in the antagonistic reflex pathway to motoneurones, but to find such interneurones. Shortly afterwards, Eccles, Fatt and Koketsu [7] showed that the motor axon collaterals of motoneurones synapses on another type of interneurone, the Renshaw cell, and even identified the transmitter involved in acetylcholine.
A dramatic change in our understanding of the inner machinery of the nervous system occurred when intracellular recording methods were introduced around the middle of this century. Quite suddenly, we were told how nerve cells could sum the effect from several sources, how inhibition worked to control the net effect of afferent barrage of impulses and, in fact, how the synaptic effect itself was the result of multi-component packages of transmitters. Before the advent of intracellular recording, our view of the nervous system as it was reflected by influential textbooks was dominated by accounts of reflexes, effects of electrical stimulation and of lesions. From Sherrington’s [18] and Pavlov’s [15] pioneering studies, the multitude of reflexes aptly illustrated the great power and variability of the relatively simple neuronal networks. However, recordings of the overall effect could usually not unravel the underlying mechanisms. Electrical stimulation was remarkably influential, serving as an instrument for localization of various major nervous subsystems and their functions. However, scientists soon realized that the stimulation electrode forced the surrounding neurones into an artificial discharge, both with regard to pattern and intensity, often not seen under physiological conditions. Responses to electrical stimulation, therefore, could sometimes appear as a caricature of the functions they were aimed at disclosing. Clinical and experimental brain lesions have historically led to major discoveries in the neurosciences. By the observed deficits the removal of an area can tell us about the normal effect of this region. Again, the approach had limitations, partly because of interruption of a previous imbalance between two competing influences and partly because of compensatory mechanisms which often camouflage much of the original damage. On this background, the ability to record from individual nerve and muscle cells by an impaling electrode gave unprecedented insights into basic mechanisms. Recording from single nervous elements had already showed its power. Adrian and his colleagues [1] succeeded in recording from single nerve fibres of muscle and from peripheral nerves, and the introduction of metal microelectrodes by Renshaw, Forbes and Morison [17] in 1940 was soon to give a wealth of information, not least by the pioneering analysis of the visual system by Hubel and Wiesel [12]. It was, however, the introduction of glass micropipettes as recording electrodes which made the greatest change in recording opportunities. The start was made by Gerard and his colleagues [9] and a successful introduction of glass pipettes in the giant nerve fibres of the squid by Hodgkin and Huxley [10,11] heralded the revolution to come. Unfortunately, World War II postponed much of the effort, but
* Address for correspondence: Prof. Per Andersen, Institute for Basic Medical Sciences, University of Oslo, Pb 1104 Blindern, 0317 Oslo, Norway. Fax: ⫹47-22 85 12 49; E-mail: [email protected]
305
306 Intracellular recording form spinal motoneurones also allowed a detailed characterization of biophysical mechanisms involved in EPSPs and IPSPs. For the latter, a string of ionic candidates responsible for the inhibitory current was identified, with chloride influx and potassium efflux as the two most important choices [5]. Later, both mechanisms have been found to be present. Shortly after the demonstration of the intracellular endplate potential, Del Castillo and Katz [6] made the seminal discovery of spontaneous miniature signals with the same form and polarity as the 50 times larger end plate potential. Their analysis of the miniature endplate potentials, or minips by aficionados, showed the quantal nature of this process in that a nerve impulse releases about 200 such minips nearly simultaneously to make a full endplate potential. Since then, this revelation has made the framework for analysis of all other synapses, and has proven extraordinarily successful. Only recently has it been found that many cortical synapses usually releases transmitter quanta with very low probability such that no, or only one, quantum of transmitter is released per impulse. In fact, at a subset of cortical synapses, there is no release at all, the so-called silent synapses. The importance of intracellular recording techniques in muscle and the spinal cord quickly spread to studies of other parts of the nervous system. Pioneering studies were made by Phillips [16] in the motor cortex. Following the initial studies of Albe-Fessard and Buser [2] in the hippocampus, Kandel, Spencer, and Brinley [13] succeeded in recording both excitatory and inhibitory postsynaptic potentials from hippocampal pyramidal cells and their relation to epileptiform activity after tetanic activation. Today, the introduction of patch clamp recording has further importantly increased our ability to analyse synaptic integration in nerve cells. Coupled to several types of imaging methods and the use of various marker molecules, there is today an enormous increase in the opportunities for intracellular recording. As always, the combination of new methods with well designed problems of biological importance has been responsible for the recent revolutionary progress in understanding nervous function. In this context, the introduction of intracellular recording techniques represents one of the true highlights of twentieth century neuroscience.
ANDERSEN REFERENCES 1. Adrian, E. D.; Bronk, D. W. The discharge of impulses in motor nerve fibres. Part II. The frequency of discharge in reflex and voluntary contractions. J. Physiol. (Lond.) 67:119 –151; 1929. 2. Albe-Fessard, D.; Buser, P. E´tude de re´ponses neuroniques a` l’aide de microe´lectrodes intrasomatiques. Rev. Neurol. 87:455; 1952. 3. Bradley, K.; Easton, D. M.; Eccles, J. C. An investigation of primary or direct inhibition. J. Physiol. (Lond.) 122:474 – 488; 1953. 4. Brock, L. G.; Coombs, J. S.; Eccles, J. C. The recording of potentials from motoneurones with an intracellular electrode. J. Physiol. (Lond.) 117:431– 460; 1952. 5. Coombs, J. S.; Eccles, J. C.; Fatt, P. The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol. (Lond.) 130: 326 –373; 1955. 6. Del Castillo, J.; Katz, B. Quantal components of the end-plate potential. J. Physiol. (Lond.) 124:560 –573; 1954. 7. Eccles, J. C.; Fatt, P.; Koketsu, K. Colinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (Lond.) 126:524 –562; 1954. 8. Fatt, P.; Katz, B. An analysis of the end-plate potential recorded with an intra-cellular electrode. J. Physiol. (Lond.) 115:320 –370; 1951. 9. Gerard, R. W.; Marshall, W. H.; Saul, L. J. Electrical activity of the cat’s brain. Arch. Psychiat. Neurol. (Chicago) 36:675–738; 1936. 10. Hodgkin, A. L.; Huxley, A. F. Action potentials recorded from inside a nerve fibre. Nature 144:710 –711; 1939. 11. Hodgkin, A. L.; Huxley, A. F. Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. (Lond.) 116:449 – 472; 1952. 12. Hubel, D. H.; Wiesel, T. N. Receptive fields, binocular interaction and functional architecture in the cat’s visual cortex. J. Physiol. (Lond.) 160:106 –154; 1962. 13. Kandel, E. R.; Spencer, W. A.; Brinley, F. J. Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization. J. Neurophysiol. 24:225–242; 1961. 14. Nastuk, W. L.; Hodgkin, A. L. The electrical activity of single muscle fibres. J. Cell. Comp. Physiol. 35:39 –74; 1950. 15. Pavlov, I. P. The scientific investigation of the physical faculties or processes in the higher animals. Science 24:613– 619; 1906. 16. Phillips, C. G. Intracellular records from Betz cells in the cat. Quart. J. Exp. Physiol. 41:58 – 69; 1956. 17. Renshaw, B.; Forbes, A.; Morison, B. R. Activity of isocortex and hippocampus: Electrical studies with micro-electrodes. J. Neurophysiol. 3:74 –105; 1940. 18. Sherrington, C. S. The Integrative action of the nervous system. New Haven and London: Yale University Press; 1906.
PII S0361-9230(99)00160-4
The view from inside Per Andersen* Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway [Received 6 May 1999; Accepted 8 May 1999] shortly afterwards there was a series of notable breakthroughs. After Nastuk and Hodgkin recorded intracellularly from muscle cells in 1950 [14], Fatt and Katz [8] succeeded in recording the first intracellular synaptic potential in the form of end plate potentials from frog muscles. The next year, Brock, Coombs and Eccles [4] gave the first demonstration of excitatory postsynaptic potentials (EPSP) from spinal cord motoneurones in cats. One year later, Eccles’ group [3] reported the hyperpolarizing inhibitory postsynaptic potentials (IPSP) as nearly a mirror image of the EPSP, only slightly delayed in onset. The properties of the EPSP suddenly gave a comprehensive explanation of Sherrington’s term “central excitatory state (c.e.s.)” in which the activation of spinal cord elements were followed by a period with lowered excitability for a second impulse arriving from a homonymous muscle nerve. EPSP components from individual muscle spindle afferents summed linearly, explaining the effect of increasing strength or speed of muscle shortening on the reflex size. Afferent signals from muscle spindles in homonymous muscles also summed, while a nerve volley through Ia afferents from an antagonistic muscle caused inhibition. By careful analysis of the onset times of EPSPs and IPSPs, Eccles and colleagues [3,7] showed that there was a delay between the two, sufficient for the intercalating action of an inhibitory interneurone. Ever since this fundamental discovery, this concept has deeply influenced our thoughts about circuit organization in the nervous system. For a long time, Eccles’ tenet of an intercalated interneurone was believed to be a general phenomenon in all brain circuits until Eccles’ pupil, Ito, showed that Purkinje cells inhibited vestibular cells directly and another of Eccles’ collaborators, Wilson, showed that certain of the vestibulo-spinal effects on neck motoneurones also were direct. Intracellular recording allowed an assessment of the relative functional strength of various converging influences. Eccles and Lundberg and colleagues demonstrated the intricate network of synaptic effects from various sense organs onto the different hind limb motoneurones and thereby controlled the muscle activity in an integrated fashion, a prerequisite for an understanding of postural and walking reflexes. Eccles’ group, while still in Dunedin, succeeded not only in postulating an inhibitory neuron in the antagonistic reflex pathway to motoneurones, but to find such interneurones. Shortly afterwards, Eccles, Fatt and Koketsu [7] showed that the motor axon collaterals of motoneurones synapses on another type of interneurone, the Renshaw cell, and even identified the transmitter involved in acetylcholine.
A dramatic change in our understanding of the inner machinery of the nervous system occurred when intracellular recording methods were introduced around the middle of this century. Quite suddenly, we were told how nerve cells could sum the effect from several sources, how inhibition worked to control the net effect of afferent barrage of impulses and, in fact, how the synaptic effect itself was the result of multi-component packages of transmitters. Before the advent of intracellular recording, our view of the nervous system as it was reflected by influential textbooks was dominated by accounts of reflexes, effects of electrical stimulation and of lesions. From Sherrington’s [18] and Pavlov’s [15] pioneering studies, the multitude of reflexes aptly illustrated the great power and variability of the relatively simple neuronal networks. However, recordings of the overall effect could usually not unravel the underlying mechanisms. Electrical stimulation was remarkably influential, serving as an instrument for localization of various major nervous subsystems and their functions. However, scientists soon realized that the stimulation electrode forced the surrounding neurones into an artificial discharge, both with regard to pattern and intensity, often not seen under physiological conditions. Responses to electrical stimulation, therefore, could sometimes appear as a caricature of the functions they were aimed at disclosing. Clinical and experimental brain lesions have historically led to major discoveries in the neurosciences. By the observed deficits the removal of an area can tell us about the normal effect of this region. Again, the approach had limitations, partly because of interruption of a previous imbalance between two competing influences and partly because of compensatory mechanisms which often camouflage much of the original damage. On this background, the ability to record from individual nerve and muscle cells by an impaling electrode gave unprecedented insights into basic mechanisms. Recording from single nervous elements had already showed its power. Adrian and his colleagues [1] succeeded in recording from single nerve fibres of muscle and from peripheral nerves, and the introduction of metal microelectrodes by Renshaw, Forbes and Morison [17] in 1940 was soon to give a wealth of information, not least by the pioneering analysis of the visual system by Hubel and Wiesel [12]. It was, however, the introduction of glass micropipettes as recording electrodes which made the greatest change in recording opportunities. The start was made by Gerard and his colleagues [9] and a successful introduction of glass pipettes in the giant nerve fibres of the squid by Hodgkin and Huxley [10,11] heralded the revolution to come. Unfortunately, World War II postponed much of the effort, but
* Address for correspondence: Prof. Per Andersen, Institute for Basic Medical Sciences, University of Oslo, Pb 1104 Blindern, 0317 Oslo, Norway. Fax: ⫹47-22 85 12 49; E-mail: [email protected]
305
306 Intracellular recording form spinal motoneurones also allowed a detailed characterization of biophysical mechanisms involved in EPSPs and IPSPs. For the latter, a string of ionic candidates responsible for the inhibitory current was identified, with chloride influx and potassium efflux as the two most important choices [5]. Later, both mechanisms have been found to be present. Shortly after the demonstration of the intracellular endplate potential, Del Castillo and Katz [6] made the seminal discovery of spontaneous miniature signals with the same form and polarity as the 50 times larger end plate potential. Their analysis of the miniature endplate potentials, or minips by aficionados, showed the quantal nature of this process in that a nerve impulse releases about 200 such minips nearly simultaneously to make a full endplate potential. Since then, this revelation has made the framework for analysis of all other synapses, and has proven extraordinarily successful. Only recently has it been found that many cortical synapses usually releases transmitter quanta with very low probability such that no, or only one, quantum of transmitter is released per impulse. In fact, at a subset of cortical synapses, there is no release at all, the so-called silent synapses. The importance of intracellular recording techniques in muscle and the spinal cord quickly spread to studies of other parts of the nervous system. Pioneering studies were made by Phillips [16] in the motor cortex. Following the initial studies of Albe-Fessard and Buser [2] in the hippocampus, Kandel, Spencer, and Brinley [13] succeeded in recording both excitatory and inhibitory postsynaptic potentials from hippocampal pyramidal cells and their relation to epileptiform activity after tetanic activation. Today, the introduction of patch clamp recording has further importantly increased our ability to analyse synaptic integration in nerve cells. Coupled to several types of imaging methods and the use of various marker molecules, there is today an enormous increase in the opportunities for intracellular recording. As always, the combination of new methods with well designed problems of biological importance has been responsible for the recent revolutionary progress in understanding nervous function. In this context, the introduction of intracellular recording techniques represents one of the true highlights of twentieth century neuroscience.
ANDERSEN REFERENCES 1. Adrian, E. D.; Bronk, D. W. The discharge of impulses in motor nerve fibres. Part II. The frequency of discharge in reflex and voluntary contractions. J. Physiol. (Lond.) 67:119 –151; 1929. 2. Albe-Fessard, D.; Buser, P. E´tude de re´ponses neuroniques a` l’aide de microe´lectrodes intrasomatiques. Rev. Neurol. 87:455; 1952. 3. Bradley, K.; Easton, D. M.; Eccles, J. C. An investigation of primary or direct inhibition. J. Physiol. (Lond.) 122:474 – 488; 1953. 4. Brock, L. G.; Coombs, J. S.; Eccles, J. C. The recording of potentials from motoneurones with an intracellular electrode. J. Physiol. (Lond.) 117:431– 460; 1952. 5. Coombs, J. S.; Eccles, J. C.; Fatt, P. The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol. (Lond.) 130: 326 –373; 1955. 6. Del Castillo, J.; Katz, B. Quantal components of the end-plate potential. J. Physiol. (Lond.) 124:560 –573; 1954. 7. Eccles, J. C.; Fatt, P.; Koketsu, K. Colinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (Lond.) 126:524 –562; 1954. 8. Fatt, P.; Katz, B. An analysis of the end-plate potential recorded with an intra-cellular electrode. J. Physiol. (Lond.) 115:320 –370; 1951. 9. Gerard, R. W.; Marshall, W. H.; Saul, L. J. Electrical activity of the cat’s brain. Arch. Psychiat. Neurol. (Chicago) 36:675–738; 1936. 10. Hodgkin, A. L.; Huxley, A. F. Action potentials recorded from inside a nerve fibre. Nature 144:710 –711; 1939. 11. Hodgkin, A. L.; Huxley, A. F. Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. (Lond.) 116:449 – 472; 1952. 12. Hubel, D. H.; Wiesel, T. N. Receptive fields, binocular interaction and functional architecture in the cat’s visual cortex. J. Physiol. (Lond.) 160:106 –154; 1962. 13. Kandel, E. R.; Spencer, W. A.; Brinley, F. J. Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization. J. Neurophysiol. 24:225–242; 1961. 14. Nastuk, W. L.; Hodgkin, A. L. The electrical activity of single muscle fibres. J. Cell. Comp. Physiol. 35:39 –74; 1950. 15. Pavlov, I. P. The scientific investigation of the physical faculties or processes in the higher animals. Science 24:613– 619; 1906. 16. Phillips, C. G. Intracellular records from Betz cells in the cat. Quart. J. Exp. Physiol. 41:58 – 69; 1956. 17. Renshaw, B.; Forbes, A.; Morison, B. R. Activity of isocortex and hippocampus: Electrical studies with micro-electrodes. J. Neurophysiol. 3:74 –105; 1940. 18. Sherrington, C. S. The Integrative action of the nervous system. New Haven and London: Yale University Press; 1906.