A major question in neurobiology is: How do neurons, or small groups of them, become modified to encode memory of previous experience, or to provide more effective transmission? Part of the answer lies in studying individual synapses, which are the points of communication between nerve cells. We have found that synapses show several different adaptive modifications. In some neurons, a brief episode of activity leads to recruitment of previously non-transmitting synapses, thereby strengthening the effect of a nerve cell on its target. With prolonged activity, neurons gradually become more able to sustain synaptic transmission. These adaptive effects are mediated by second- messenger systems, and the long-lasting responses require neuronal protein synthesis and structural modification. The work is carried out on single identifiable neurons, particularly in crustaceans and in the fruit fly, Drosophila, which is used for studies of the effects on synaptic transmission of genetic mutations known to affect learning and memory. Additional studies are being carried out on regulation of calcium in different types of nerve cells, on presynaptic inhibition, and on effects of neurohormones on synaptic transmission.
 

Brief Outline of Past and Present Research Themes

Dr. Harold Atwood's research program has addressed the physiology of muscular contraction, motor control, synaptic transmission, and synaptic modification in the nervous systems and neuromuscular systems of both arthropods (insects and crustaceans) and mammals. By far the largest part of his work has been on neuromuscular systems of crustaceans, which have been frequently studied by physiologists because their muscle cells and motor neurons attain a relatively large size and are relatively few in number (and thus readily identifiable in the intact animal or in isolated tissues). Beginning in the early 1960's, Dr. Atwood and his students and co- workers discovered several new features of crustacean neuromuscular systems which helped to explain behavioural mechanisms. They also showed that the nerve cells of these animals undergo several activity-dependent long-term changes which have adaptive value for the animal, and appear to occur also in other animal species. The findings have general significance for the performance and experience-dependent modification of nervous and neuromuscular systems.

Investigating muscular contraction in crustaceans, Dr. Atwood discovered in 1963 that crustacean muscles contain fast-acting and slow-acting muscle fibers (muscle cells) which are selectively recruited for fast and slow movements respectively. Subsequent studies with co-workers showed that the ultrastructure and biochemistry of the different muscle fibers are differentiated to accommodate the physiological demands they experience. In addition, studies on single innervated crab muscle cells showed that contraction is regulated by electrical events of the muscle cellþs membrane and not by direct chemical actions of the neurotransmitter. Significantly, the properties of synaptic transmission from motor neuron to muscle are matched with those of the muscle fibers: fast-acting muscle fibers are typically innervated by neurons which evoke large electrical responses in the muscle fiber, leading to large contractions, while slow-acting muscle fibers are typically supplied by neurons which evoke smaller electrical responses resulting in slow, graded contractions. Furthermore, specialized endings of a single motor neuron often produce different responses among the muscle fibers they innervate. Thus, a single neuron can selectively and progressively recruit the muscle fibers it innervates: at low frequencies of activity, only a few fibers contract, while at higher frequencies, as electrical activity increases, many more muscle fibers contract. In 1965 and 1967, Dr. Atwood showed that these features could be explained in large measure by the differences in release of excitatory neurotransmitter from the individual nerve endings on their target muscle fibers: some nerve endings release more neurotransmitter, and thus cause a larger electrical response, than others. The same type of recruitment mechanism has since been observed by others in the nervous systems of different animals, ranging from leeches to mammals, and probably constitutes one of the many basic operational mechanisms of nervous systems in general.

Investigations combining ultrastructural and physiological observations of synapses (the locations of transmission between one nerve cell and another, or between a nerve cell and a muscle cell) led to several general principles of organization and operation. It was found in 1967 that inhibitory nerve cells, which innervate crustacean muscles in parallel with the excitatory motor neurons, form well-defined synapses on the endings of the latter. These synapses account for the phenomenon of presynaptic inhibition (decrease in the release of excitatory neurotransmitter upon activation of the inhibitory nerve cell) which had first been described in crustacean muscles by Josef Dudel and Stephen Kuffler (1961). Further work by Dr. Atwood and his students led to a general model of this synaptic action, derived from the observed morphology and electrical properties of the nerve terminals, and to the observation that presynaptic inhibition apparently leads to fewer excitatory synapses effectively releasing neurotransmitter. Thus, an important principle of short-term response modification is variation in the number of a neuron's contributing synapses. In presynaptic inhibition, synapses are temporarily 'decommissioned' to weaken the response; but in other cases, they are added to strengthen it.

Recruitment of additional synapses to strengthen a response, or strengthening of the responses of active synapses, were found to contribute to long- and short-term facilitation in crustaceans. Short-term facilitation is enhancement of synaptic transmission resulting from previous nerve impulses in a pathway, usually due to increased release of neurotransmitter, as originally described by Katz and Miledi in vertebrates, and by Dudel and Kuffler in crustaceans. Starting in the early 1970's, Dr. Atwood and co- workers produced three-dimensional reconstructions of nerve terminals from electron micrographs which allowed them to count all the individual synapses contributing to a local synaptic response. They also found structural differences between 'strong' and 'weak' synapses, a finding which has been confirmed and amplified by C.K. Govind and others. In combination with statistical analyses of neurotransmission, begun by Atwood and Parnas in 1968 following the earlier work of Dudel and Kuffler, these studies led to the conclusion that individual synapses differ in effectiveness. Many synapses appear to be 'silent' (do not release neurotransmitter) when a nerve cell is active at low frequencies.

A longer-lasting recruitment of synapses occurs during 'long-term facilitation' (described by Sherman and Atwood for crustaceans in 1971); this is a very persistent enhancement of neurotransmission induced by a period of high-frequency activity. It has much in common with some forms of 'long-term potentiation' now being intensively investigated in the mammalian nervous system as a possible mechanism contributing to learning and memory. Dr. Atwood and co-workers found that the long-term effect in crustaceans was enhanced by accumulation of sodium and calcium ions in the presynaptic nerve terminal; this increases the release of neurotransmitter. They later showed that long-lasting recruitment of additional synapses takes place in the presynaptic nerve endings to maintain the response at a higher level. They also found that, as in the facilitatory responses analyzed by Eric Kandel and co- workers in molluscan nervous systems, the long-term effect in crustaceans depended upon the activation, in the presynaptic nerve ending, of the cyclic adenosine monophosphate (cyclic AMP) intracellular second-messenger system, which enhances neurotransmission. However, they also found in 1975 and 1988 that, unlike several other analyzed cases, induction of this system depended primarily upon electrical activity in the nerve ending, and could occur without translocation of calcium ions into the nerve cell. Voltage-dependent activation of the cyclic AMP system has now been described by Daniel Storm and co-workers in other cell types, and may be of more general occurrence than is presently known.

An entirely different type of long-term modification in crustacean nerve cells was discovered in the early 1980's by Atwood and Lnenicka. When they altered the ongoing activity of a selected nerve cell in an intact animal, they found that a semi-permanent change in its synaptic transmission and morphology occurred. This modification was found (by Nguyen and Atwood) to depend upon protein synthesis in the affected nerve cell, and to involve several different intracellular mechanisms. The significance of the effect for nerve cells in general is that in many cases their properties are strongly influenced and continuously altered by ongoing activity ('experience'). This was found to be the case in a visual pathway of an insect nervous system by Bloom and Atwood in 1980.

Observations of this type in crustaceans and other animals raise the question of the relative importance of genetic limitations and 'experience' in shaping the capabilities of the nervous system. The fruit fly, Drosophila, offers an opportunity to investigate this question; it has been extensively studied from the genetic standpoint, and its neuromuscular systems have much in common with those of crustaceans. Dr. Atwood began physiological and ultrastructural studies of synaptic transmission with Chun-Fang Wu, a leading authority on Drosophila neurophysiology, in 1991. He developed new physiological procedures (including a new physiological solution, since the one commonly in use for the previous two decades was found to rapidly damages the exposed cells). Once the basic physiology of selected synapses was worked out, work on physiological consequences of genetic alteration of synapses was begun. Significantly, genetically induced structural perturbations can be physiologically compensated to maintain an appropriate level of synaptic transmission, indicating synaptic adaptation and an optimal 'set point' for development of neurotransmission in a specific neural pathway. The nervous system can overcome certain genetic liabilities. Synaptic molecules discovered in Drosophila were shown to have close counterparts in crustaceans; thus, the way was opened for functional assays of gene products initially found in Drosophila, using crustacean neurons which provide an experimentally more tractable system for physiological investigations.

Altogether, the work on crustacean and insect nerve cells has established several basic operational features, and provided insight into activity-dependent changes which are particularly well expressed in crustacean neurons, but are known to be of widespread occurrence in other animals. The combination of ultrastructural and physiological analysis of specific synapses developed by Dr. Atwood and co-workers has provided unique insights into factors determining the strength of responses in the nervous system, and how these responses can be modified.