Neurobiology

Eric R. Kandel, M.D., Director
Craig Bailey, Ph.D., Research Scientist V
Claude Ghez, M.D., Research Scientist VI
Robert Hawkins, Ph.D., Research Scientist V
John Koester, Ph.D., Research Scientist VI
Irving Kupfermann, Ph.D., Research Scientist VI
John Martin, Ph.D., Research Scientist III,
Samuel Schacher, Ph.D., Research Scientist V
James H. Schwartz, M.D., Ph.D., Research Scientist VII

The Center for Neurobiology and Behavior consists of thirteen independent basic research laboratories, including two laboratories of the Howard Hughes Medical Institute. The overall research goal of the Center is to provide an analysis of neural development, behavior, learning, and diseases of the nervous system in terms of their underlying cellular and molecular mechanisms. The subjects used in these studies range from simple invertebrates to humans.

A cross-species approach to understanding the mechanisms of memory and learning has been applied, using two preparations: (1) the defensive gill and siphon withdrawal reflex of the sea hare, Aplysia californica, which undergoes habituation, sensitization, and classical conditioning; and (2) the mammalian hippocampus, which exhibits a pronounced type of long-lasting synaptic plasticity, long term potentiation (LTP), which is thought to underlie long term memory. These two complementary approaches have led to a number of fundamental observations concerning the mechanisms that underlie learning and memory.

James Schwartz has continued his studies of the biochemical mechanisms underlying synaptic plasticity. Neurobiologists believe that memory, learning, and other changes in brain function that occur as a result of experience are produced by alterations in the strengths of specific synapses in the central nervous system (synaptic plasticity). Depending on the type of experience or training, release of neurotransmitter at these synapses is enhanced or diminished for periods of minutes to hours (or permanently in the instance of long-term memory). Dr. Schwartz and his colleagues have been characterizing the second messenger cascades that mediate simple forms of learning, using biochemical, pharmacological, immunocytochemical, electrophysiological and recombinant DNA techniques. Most interesting are secondary effector enzymes, usually protein kinases primarily the cAMP-dependent protein kinase and protein kinase C. A physiologically important feature of these kinases is the property of persistence. Once stimulated by the second messenger, these enzymes become progressively autonomous or independent of subsequent second-messenger stimulation. In this way, the kinase activity persists for longer and longer periods of time. The molecular mechanism by which the cAMP-dependent protein kinase becomes persistently active involves selective proteolysis through the ATP-ubiquitin-proteasome pathway of protein degradation. He is examining this important cellular process in nervous tissue.

Craig Bailey has begun an analysis of the interaction between homosynaptic Hebbian (activity-dependent) facilitation and heterosynaptic (modulatory input-dependent) facilitation in Aplysia. He has examined how these two forms of facilitation interact at the level of an individual synaptic connection by using a culture preparation consisting of a single sensory neuron with a bifurcated axon that forms independent synaptic contacts with each of two spatially separated motor neurons. He finds that the homosynaptic facilitation produced by a train of action potentials is cell-wide, i.e., it is evident at all of the output terminals of the sensory neuron. By contrast, the heterosynaptic facilitation mediated by the modulatory transmitter serotonin can operate at the level of a single synapse. Homosynaptic activation gives rise to only a transient facilitation lasting a few hours, even when repeated in a spaced manner. The heterosynaptic facilitation produced by a single pulse of 5-HT, applied to one terminal of the sensory neuron, also lasts only minutes. But when one or more homosynaptic trains of spike activity are paired with even a single pulse of 5-HT applied to one of the two branches of the sensory neuron, the combined actions lead to a selective enhancement in synaptic strength only at the 5-HT treated branch that now lasts more than a day, and thus amplifies, by more than 20-fold, the duration of the individually produced homo- and heterosynaptic facilitation. This form of synapse-specific facilitation has unusual long-term properties. It does not require protein synthesis, nor is it accompanied by synaptic growth.

In related studies Samuel Schacher has found that stimulus-dependent release of different neuromodulators can influence a) local cytoskeletal organization at existing synapses, b) expression of mRNAs coding for specific isoforms in the cell body of specific neurons, and c) transport of mRNA towards synaptic sites where its local synthesis might influence synaptic function. These results suggest that cell-specific changes in the processing of mRNA in both the cell body and terminals of connected neurons and the local changes in proteins critical for growth and synaptic function may contribute to long-term plasticity at specific synapses.

Robert Hawkins and collaborators have continued to study cellular mechanisms of learning and memory in Aplysia and hippocampus. In Aplysia, they have used a simplified preparation for studying the siphon-withdrawal reflex to provide the first direct evidence that plasticity of monosynaptic postsynaptic potentials contribute to classical conditioning of the reflex. In addition, they have tested the contributions to that plasticity by two associative cellular mechanisms: activity-dependent presynaptic facilitation and Hebbian potentiation. Their results indicate that both mechanisms contribute and that they are not independent, but rather interact through retrograde signaling.

In studies of the hippocampus, the Hawkins lab has continued to study mechanisms of long-term potentiation. Recent studies have provided support for postsynaptic mechanisms, including changes in the number of clusters or puncta that label for the GluR1 subunit of the AMPA-type glutamate synaptic receptors. They have investigated whether similar changes occur presynaptically. They found that long-lasting potentiation in cultured hippocampal neurons is accompanied not only by an increase in the number of GluR1-immunoreactive puncta, as expected, but also by a rapid and long-lasting increase in the number of puncta that label for the presynaptic protein synaptophysin, and the number of sites where synaptophysin and GluR1 are co-localized. They obtained similar results using real time imaging of living hippocampal neurons expressing a synaptophysin-GFP fusion protein. Based on these initial results, Dr. Hawkins received a NARSAD Independent Investigator Award to continue these studies.

During the past year the Siegelbaum laboratory helped identify a new second messenger pathway, p38 MAP kinase, implicated in long-term depression in the mammalian hippocampus. Long-term depression (LTD) is a long-lasting decrease in the strength of synaptic transmission as a result of certain patterns of relatively weak electrical stimulation. LTD may down-regulate synaptic function in opposition to long-term potentiation (LTP), a strengthening of synaptic function that is thought to underlie learning and memory. Previous studies implicated the p42/p44 MAP kinase pathway in LTP. Their study shows that opposing forms of synaptic regulation recruit distinct branches of the MAP kinase pathway.

Eric Kandel and colleagues have found that the threshold for hippocampal-dependent synaptic plasticity and memory storage is determined by the balance between protein phosphorylation and dephosphorylation. The balance involves the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance they have examined the effects of inhibiting calcineurin on synaptic plasticity and memory storage. Using the doxycyline-dependent rtTA system to induce the expression of a calcineurin inhibitor in a reversible manner in the mouse brain, they find that the transient reduction of calcineurin activity results in facilitation of LTP both in vitro and in vivo. This facilitation persists over several days in the awake animal, and is accompanied by enhanced learning and strengthening of short- and long-term memory on several hippocampal-dependent spatial and non-spatial tasks. The LTP and memory improvements are reversed fully by withdrawal of doxycyline and thereby suppressing expression of the transgene. These results, together with their previous discovery that overexpression of calcineurin impairs PKA-dependent components of LTP and memory, demonstrate that endogenous calcineurin can modulate synaptic plasticity and memory in both directions depending on its level of activity.

Vincent Ferrera studies the neural basis of visual perception and visuomotor integration in alert non-human primates. He and his colleagues have recently completed a completed a study on the predictive responses of frontal eye field neurons to invisible moving targets. They found that neurons involved in spatial working memory also are also involved in planning responses to moving targets. In a second project on detection thresholds for spiral glass patterns, they provided evidence that the human visual system is specialized for detecting patterns with concentric or radial organization. On a technical front, they have also completed the development of a multi-channel neuronal recording and stimulation device which allows them to record data from several neurons simultaneously. This device will enable them to go beyond single neuron firing patterns to analyze network activity. They've also developed a system for pressure-injecting pharmacological agents that will be used to test ideas about orientation discrimination in primary visual cortex.

Ning Qian has conducted computational studies and psychophysical experiments that help to explain our ability to achieve depth perception from disparity between the two images of an object that are projected onto the two retinas. During the past year he and his colleagues completed three projects: (1) In modeling studies they revealed how in area V1 of visual cortex the neuronal responses to time-varying stimuli encode horizontal image disparity. (2) In a related study they developed a computational model that demonstrates how the motion repulsion illusion occurs as a result of binocular rivalry. (3) They created a new physiological theory that explains how the visual cortex calculates depth perception from vertical disparity.

Claude Ghez and colleagues are engaged in psychophysical and neuroimaging studies of neural control of reaching and motor learning in normal humans and in patients with Parkinsons and Huntingtons diseases or stroke. Their major focus is on the learning of spatial and dynamic sensorimotor transformations for reaching and on sequencing of movements. They find that reaching movements are planned in a hand-centered vectorial coordinate system based on independently learned scaling factors and reference frames and on internal models of inertial and other dynamic properties of the limb. Spatial models are learned from visual errors while dynamic models are learned using proprioception. Sequence learning involves an initial cognitive phase and a later dynamic phase in which the motor representation of the sequence is optimized. In related studies using O¹⁵ positron emission tomography (PET) they find that while the recalibration of visuomotor scaling involves only subcortical structures, the learning of novel reference frames involves frontal and parietal regions of cortex. Sequence learning additionally recruits prefrontal and premotor regions. Finally in studies of patients with Parkinsons Disease they find impairments in the acquisition and retrieval of spatial motor sequences associated both with increased activation of the same neural networks and of new contralateral ones.

René Hen has continued to use molecular genetic approaches to develop animal models of anxiety and depression. Serotonin is a neuromodulator that has been implicated in normal mood control as well as in a number of mood disorders such as anxiety, depression and aggressiveness. To model such disorders he has introduced mutations in various components of the serotonergic system. In particular, he and his colleagues have generated knockout mice that lack either the 5-HT1A or the 5-HT1B receptor. These mutant mice display a panoply of contrasting phenotypes. While the 5-HT1B knockout mice are more aggressive and less anxious than wild-type mice, the 5-HT1A knockout mice appear to be less aggressive and more anxious than the wild-types. They are currently using these two mouse models to identify the neural circuits that are responsible for their opposite emotional states. Specifically, they have explored the respective contributions of the presynaptic autoreceptors and the postsynaptic heteroreceptors by re-expressing the 5-HT1A and 5-HT1B receptors selectively in the raphe nuclei or in postsynaptic structures such as the hippocampus and amygdala. They have shown that expression of postsynaptic 5-HT1A receptors can rescue the anxious phenotype of the 5-HT1A knockout mice. Their preliminary results suggest also that these postsynaptic 5-HT1A receptors are required to mediate the antidepressant effect of selective serotonin reuptake inhibitors (SSRI) such as fluoxetine. They have also used cDNA microarrays, which can monitor the expression of thousands of different genes, to identify some of the molecular adaptations that may be responsible for the antidepressant effects of SSRIs.

Irving Kupfermann and colleagues have continued our study of the neural mechanisms underlying motivational states, using as a model system, feeding behavior in Aplysia. Feeding in Aplysia is a highly complex motor act which they have shown is controlled by higher order command cells. They previously found that it can be elicited by directly firing a single identified interneuron termed a CBI (cerebral-buccal interneuron). More recently they have identified additional feeding "command" neurons in the cerebral ganglion. One such neuron, instead of evoking the whole complex of feeding behavior, evokes the activity of only a single other neuron, a cell that projects to only one of the 30 muscles that execute, biting or swallowing. Thus this complex behavior is controlled by an overlapping hierarchy of higher order interneurons. In addition to their studies of feeding behavior they have examined the control of intestinal motility, which is coordinated with feeding and which can regulate meal size by altering the contents of the gut. Aplysia has been shown to contain a neuropeptide called neuropeptide Y (NPY). It is similar to vertebrate NPY, which when injected into the hypothalamus of vertebrates strongly elicits feeding behavior. In Aplysia, NPY strongly stimulates intestinal motility, with no apparent effect on feeding. NPY has been shown to be present in neurons in the brain of the animal, and therefore this peptide provides a means by which central neural processes can affect the gastrointestinal system.

John Koester studies the way in which the cellular biophysical and network properties of Aplysia neurons interact in the generation of behavior. A key element in this type of study is the necessity of determining the normal physiological activity pattern of a neuron. Toward this goal he has recently implemented a system that allows one to use the different axonal conduction velocities of the hundreds of individual axons in a nerve to filter out the activity of a particular identified neuron from baseline noise caused by the other axons in the nerve. This then allows one to determine how a specific neuron fires during normal behaviors in the intact, freely behaving animal. Using this technique he has been able to determine which subset of the patterns of neural activity that are recorded in vitro actually occur in vivo. Current studies focus on examining the mechanisms that underlie these firing patterns.

During the past year, John Martin has continued to examine development of the cortical motor systems. These systems are essential for the production of skilled limb movements and for learning new movements when changes in task conditions arise. The cortical motor systems develop during late preterm and early postnatal life in many animals, as well as humans, and are vulnerable to damage produced by birth trauma. For the first time Martin and colleagues have been able to show that terminal and preterminal corticospinal axon branches increase in complexity during a protracted early postnatal period. They have found that early in postnatal development, terminations from neighboring corticospinal neurons overlapped extensively. Later in development, corticospinal terminations were minimally overlapping, forming a mosaic-like pattern. This result is surprising because it shows a greater degree of topographic specificity than previously suspected for this system. They also found that corticospinal terminations in maturity had a high density of presynaptic sites, identified by confocal microscopy in which the axon is identified with a tracer and presynaptic sites, by the presence of synaptic vesicle protein labeling. This suggests that individual CS terminations can have significant effects on changing the excitability of individual spinal neurons. During the past year they also have completed an analysis of the development of the motor representation in primary motor cortex. This study revealed that formation of the motor map is delayed with respect to morphological development of the corticospinal terminations. They found that development of the somatotopic representation in motor cortex also followed a protracted time course. they are pursuing the mechanisms underlying these representational changes in the developing motor cortex.

During the past year Dr. Martin and colleagues have also begun to apply their experience in the developing corticospinal system to the study of mechanisms of recovery following spinal cord injury. One of the most devastating effects of upper spinal cord injury is interruption of descending motor control signals. They have received a two year research grant from the New York State Spinal Cord Injury Research Board (NYS SCIRB) to investigate how the extraordinary regenerative capacity of peripheral axons, together with the capability of the central nervous system to learn to adapt dynamically to peripheral changes, can be exploited for promoting recovery of function after chronic SCI. They plan to re-route spinal nerves to novel targets, with the aim of establishing functional connections between nerves that originate from the intact spinal cord rostral to SCI and targets isolated from the brain and the rest of the spinal cord by injury. This is the first year the NYS SCIRB has awarded research grants, and they are fortunate to be among the first group of neuroscientists to receive such an award.

Central cholinergic systems have long been implicated in various cognitive and psychiatric diseases. Lorna Role's laboratory studies the generation, maintenance and plasticity of cholinergic and cholinoceptive synapses with particular interest in the role of nicotinic receptors in synaptic modulation. Recent work focuses on examination of a novel class of signaling molecules, the neuregulins, which appear to be involved in synaptic maintenance during development and aging. Their studies employ molecular and biochemical examination of neuregulin signaling mechanisms as well as patch clamp and slice physiology of central neuronal systems. Neuregulin signaling appears to influence both pre and postsynaptic partners, as well as non-neural cells, by distinct downstream signaling cascades. Their studies test the possibility that such mechanisms are involved in sustaining synaptic connections and, hence, in promoting neural maintenance vs. degeneration. In studies related to nicotine per se, they are examining the effects of prenatal and postnatal exposure to the drug on the development and maintenance of cholinoceptic synapses in the limbic system. Recent work extends these analyses to test for interactions of nicotine exposure with various environmental factors thought to influence addiction and relapse, including changes in hormone status or imposed stress. Based on these accomplishments, Dr. Role recently received a NARSAD Grable Distinguished Investigator Award.