Harvey A. Swadlow - US grants

A novel method is being developed for study of sub-threshold synaptic events that occur within individual neurons in the awake mammalian brain. Unlike traditional methods which require impalement and resultant damage to a nerve cell by an intracellular microelectrode, the present method uses an electrode which is extracellular. Far less cellular damage results, therefore, and sub-threshold synaptic events can be studied for very long periods of time. The method entails passing extracellular current pulses through a recording microelectrode and measuring the threshold of the neuron under study to this direct electrical stimulation. This research explores the application of this new method to two problem areas in cortical neurophysiology: (1) the analysis of sub- threshold receptive fields and components of receptive fields in sensory neocortex, and (2) the analysis of sub-threshold synaptic inputs to neurons studied with chronically implanted electrodes over very long periods of time. This research will contribute to a better understanding of the cortical neuronal substrates of sensory perception and the dynamic mechanisms by which the properties of individual neurons are modified by experience over long periods of time.

The undisputed role of the cerebral cortex in perception, action and higher cognitive processes has led to an intense effort to understand the functional properties of this tissue. A primary goal of this effort has been to understand the mechanisms whereby individual cortical neurons obtain their unique repertoire of response properties and how these properties contribute to overall cortical function. The proposed research is aimed at understanding the elaboration of response properties seen in identified neurons in the intact, thalamo-cortical somatosensory system. Two sets of experiments will be conducted, all in fully awake rabbits. (1) The first set is aimed at understanding the transformations performed upon inputs to the cortex by the intracortical circuitry and how these transformations lead to parallel and distinct efferent outflows. The activity of thalamo-cortical projection neurons, corticocortical and corticifugal efferent neurons and putative interneurons will be simultaneously recorded and subjected to cross-correlational analysis. Previous work has resulted in a model of S-1 cortical circuitry that has generated explicit predictions of the excitatory and inhibitory synaptic interactions to be expected among these identified elements. These experiments will test this model and result in its further elaboration. (2) The second set of experiments examines the nature of a large population of neurons throughout sensory cortex that have no demonstrable (supra-threshold) receptive fields. It has recently been shown, however, that most such neurons do have sub-threshold receptive fields. This proposal will use a new technique for analyzing sub-threshold synaptic events to identify which classes of cortical neurons demonstrate sub- threshold receptive fields and to explore the spatial and temporal characteristics of such fields. In addition, the pharmacological conditions under which sub-threshold receptive fields become supra- threshold will be determined. The data will offer a unique view of the physiology of individual cortical neurons studied under natural conditions, and will provide insight into interactive networks of cortical neurons and their varied functional properties.

The cerebral neocortex plays a crucial role in perception, action and higher cognitive processes and this has led to an intense effort to understand the functional properties of
this tissue. Major goals of this effort have been to elucidate the mechanisms whereby individual cortical neurons obtain their diverse response properties and how ensembles
of cortical neurons interact to produce global perceptual and behavioral capabilities. The proposed research, conducted in the rabbit, is aimed at understanding the
mechanisms governing cortical function in the awake, intact, thalamocortical somatosensory system.
The work is aimed at understanding the transformations performed upon thalamocortical inputs to the cortex by the intracortical circuitry, the role of feed-forward inhibitory interneurons
in producing these transformations, and how these transformations lead to parallel and distinct efferent outflows. Using novel multi-electrode methodology, the activity of thalamocortical projection neurons, cortical output (projection neurons), and putative inhibitory interneurons will be simultaneously recorded, and interactions among these identified elements will be analyzed using methods of cross-correlation and microstimulation. Paired intracortical recordings (intracellular/extracellular) will also yield information about interactions between interneurons and their neighboring intracortical targets. Special emphasis will be placed on understanding: (a) the rules governing the specificity of functional connections among the populations of thalamocortical and cortical neurons under study, and (b) the mechanisms underlying feed-forward intracortical
inhibition.
The data obtained from these experiments will offer a unique view of the physiology of individual cortical neurons studied under natural conditions, and will provide insight into interactive networks of cortical neurons and their varied functional properties. Such information is
essential if we are to understand how neocortical networks generates perception, action, and higher cognitive processes.

neurophysiology; neural information processing; cerebral cortex; somesthetic sensory cortex; thalamus; neocortex; neuroanatomy; electrical potential; interneurons; cell type; single cell analysis; neurons; synapses; electrophysiology; laboratory rabbit; microelectrodes;

This proposal is aimed at understanding the mechanisms by which thalamocortical sensory information gains access to neocortical networks and is processed during different behavioral states. The mammalian dorsal thalamus serves as a gateway through which all sensory information, other than olfaction, must pass before gaining access to sensory neocortex. The individual neurons of thalamic nuclei operate in two distinct modes: "relay" mode and "burst" mode. Relay mode occurs primarily in the alert state, and synaptic transmission through the thalamus is highly effective. In contrast, burst mode has been primarily associated with inattention, drowsiness, and sleep, and synaptic transmission through the thalamus is depressed. Considerable effort has gone into understanding the mechanisms underlying these thalamic modes and their role in perception, and thalamic bursts have been linked to mechanisms of attention. However, a crucial link in our understanding of the perceptual consequences of thalamic bursting has remained unexplored: we do not know the fate of bursting thalamic impulses at the thalamocortical synapse. One could expect either a decrease in synaptic efficacy, as occurs at the retino-geniculate synapse during burst mode, or an increase in the efficacy with which bursting thalamic impulses activate neocortical circuits. Our preliminary work has shown a potent enhancement in the synaptic impact of thalamocortical bursts onto a class of cortical inhibitory neuron within somatosensory "barrel" cortex. The proposed experiments will extend these results by (a) examining the effect of bursting thalamocortical impulses on excitatory cortical populations studied during different behavioral/EEG states, and (b) examining the intracortical spread of excitation that results from thalamic bursts. All experiments will be performed in awake subjects using novel extracellular and intracellular multi-electrode methods. The activity of thalamocortical projection neurons and cortical neurons of different classes within S1 barrels will be simultaneously studied, and interactions among these identified neurons will be analyzed using methods of cross-correlation, spike-triggered averaging of post-synaptic potentials, and current source-density analysis. We hypothesize that bursting thalamic impulses generate an enhanced thalamocortical and intracortical spread of excitation. These data will offer a unique view of thalamocortical networks studied under natural conditions in the awake state.

DESCRIPTION: (Applicant's Abstract) This proposal is aimed at understanding the mechanisms by which visual information gains access to visual cortical networks and is processed during different operational modes of the lateral geniculate nucleus. The individual neurons of the lateral geniculate nucleus operate in two distinct modes: "relay" mode and "burst" mode. Relay mode occurs primarily in the alert state, and synaptic transmission through this nucleus is highly effective. In contrast, burst mode has been primarily associated with inattention, drowsiness, and sleep, and synaptic transmission through the thalamus is depressed. Considerable effort has gone into understanding the mechanisms underlying these thalamic modes and their role in visual perception, and thalamic bursts have been linked to mechanisms of attention. However, a crucial link in our understanding of the perceptual consequences of thalamic bursting has remained unexplored: we do not know the fate of bursting thalamic impulses at the geniculocortical synapse. Our preliminary work in the somatosensory system has shown a potent enhancement in the synaptic impact of thalamocortical bursts onto a class of cortical inhibitory neuron. This result, if applicable to the geniculocortical visual system, would impact current thinking on the role of geniculocortical bursts in visual processing. The proposed experiments will extend these results to the visual thalamocortical system and will (1) examine the synaptic impact of both visually elicited and "spontaneous" bursting geniculocortical impulses on excitatory and inhibitory visual cortical populations of layer 4, and (2) examine the intracortical spread of excitation that results from both spontaneous and visually elicited geniculocortical bursts. All experiments will be performed in awake subjects using novel extracellular and intracellular multi-electrode methods. The activity of geniculocortical projection neurons and visual cortical neurons of different classes within visual cortex will be simultaneously studied, and interactions among these identified neurons will be analyzed using methods of cross-correlation and spike-triggered averaging of post-synaptic potentials. We hypothesize that bursting geniculocortical impulses generate an enhanced activation and spread of excitation within the visual cortical network. These data will offer a unique view of visual processing by geniculocortical networks studied under natural conditions in the awake state.

Award Abstract
The sensory neocortex of the brain is essential for sensation and perception, and this has led to an intense effort to understand the functional properties of this tissue. Pyramidal cells comprise about 80% of all neurons in the cortex, and a special type of pyramidal cell in layer-5 that shows intrinsic bursts of activity is one of the most anatomically and physiologically distinctive of pyramidal sub-populations. These neurons have especially thick apical dendrites and they project descending axons that branch to innervate multiple subcortical areas of the brain. The superior colliculus is one of the subcortical structures innervated by these bursting pyramidal neurons and is responsive to visual stimulation. Such "corticotectal" pyramidal neurons are known to strongly influence the response properties of neurons in the superior colliculus, but the mechanism of this influence is still unknown. It is not known, for example, whether receptive field properties are conveyed to superior colliculus neurons directly by corticotectal input, or whether the corticotectal influence modulates other input to the neurons of the superior colliculus. This proposal focuses on the means by which corticotectal neurons influence their targets in the superior colliculus in awake rabbits, and how EEG state (alert vs. drowsy state) may affect corticotectal communication. First, Dr. Swadlow will gain a comprehensive, global view of the synaptic impact of single corticotectal neurons on the superior colliculus, and the dynamics of this impact. He will do this by electrophysiological examination of monosynaptic currents generated within the superior colliculus by the impulses (bursts and single spikes) of single corticotectal neurons. Next, Dr. Swadlow will gain a local view of the synaptic impact of single corticotectal neurons on members of specific neural sub-populations within the superior colliculus, and the dynamics of this impact. To do this, he will obtain simultaneous extracellular electrophysiological recordings from corticotectal neurons and from specific sub-populations within the superior colliculus, and apply cross-correlation methods to infer synaptic connectivity. In addition, Dr. Swadlow will record intracellularly from corticotectal neurons and examine the sub-threshold sensory events related to bursting behavior. The proposed studies will provide important insights into both the manner in which sensory cortical information is processed in an awake, perceiving subject and how cortical neurons communicate with and influence neurons in distant regions of the brain. Such information is crucial if we are to understand the mechanisms of perception in awake, perceiving mammals. The proposed studies will also further the training of future neurobiologists through involvement of undergraduate students in the PI's research.

DESCRIPTION (provided by applicant): The proposed work aims to understand early cortical mechanisms of vision in alert and in non-alert subjects. Thanks to the development of behavioral methods for the control of eye position, great advances have been made in understanding central mechanisms of visual perception of alert, attentive subjects. However, there is little understanding of cortical processes that come into play when alertness wanes. The awake, non-alert state is not equivalent to anesthesia, or to sleep states. When non-alert, we are capable of perception, but our perceptual capacities differ. It is commonly believed that "accidents happen" when we are not alert, but the extent to which early thalamic or visual cortical mechanisms may be responsible for this (as opposed to higher cognitive processes) is an open question. This proposal relies on a unique model system that is very well-suited to address this question: the awake, non-behaving rabbit, an animal who's "inner mental life" transparently shifts between alert and non-alert states, and who's stable eyes and diffident nature make it an ideal subject for these experiments. The proposed research will examine how changes in the brain state of awake subjects, and the dramatic changes in response gain that are associated with them, influence the multiple, sequential stages of information processing that occur within the visual thalamocortical and intracortical network. We will examine visual response properties, examine mechanisms, and develop a biologically realistic model of how excitatory and inhibitory neurons of the input layer of the cortex (layer 4) respond to state-changes, and will examine how state-modified visual information is conveyed by output pathways of visual cortex to subsequent processing stations. This work will lead to a better understanding of cortical mechanisms of visual processing in a dynamic, awake brain. From a health perspective, these studies will have an important impact on our understanding of how alertness/vigilance deficits can impact visual perception and performance, and will provide the basis for future clinical studies of human mental health and behavioral disorders. PUBLIC HEALTH RELEVANCE The current work will have an important impact on our understanding of how alertness/vigilance deficits can impact visual perception and performance. A disruption in response gain within thalamocortical systems has been associated with mental diseases that involve changes in sensory processing and the level of vigilance. This proposal will reveal the mechanisms that modulate response gain within the visual thalamocortical system and, by doing so, it will provide the basis for future clinical studies of human mental health and behavioral disorders.

DESCRIPTION (provided by applicant): The hallmark of neuronal systems lies in the vast, modifiable functional interconnections among the elements. Understanding brain function requires a detailed knowledge of how such functional connectivity is achieved, but the tools for analysis of synaptic communication in the waking brain are very limited. In vitro, cell-to-cell connectivity has been studied using paired intracellular recordings or minimal electrical stimulation of thalamic or other afferents. These methods have yielded a wealth of information about cell-to-cell interactions, but in vitro slices eliminate many of the synaptic and modulatory inputs that operate in intact brains. Moreover, these methods are difficult to apply in vivo, where the primary tool for examining synaptic interactions has been extracellular cross-correlation. The above in vitro tools, as well as in vivo extracellular cross-correlation, generally yield an analysis of the synaptic impact of a single presynaptic neuron on a single (or on a very few) postsynaptic target cells. However, some experimental questions require a view of the overall, global synaptic impact generated by a single presynaptic neuron on its targeted cortical domain. We have been developing a method that yields such a view, referred to as spike-triggered current source-density analysis. This method allows an analysis of the synaptic impact generated by a single presynaptic neuron on a neuronal population contained within a restricted cortical cylinder of ~200 microns diameter. This method has proved very useful in the study of systems in which the presynaptic neuron generates a relatively powerful and focal impact on the targeted domain. The goal of the proposed work is to gain a better understanding of both the strengths and limitations of this method, and to extend its use to weaker and less focal neuronal connections, which are more prevalent in the brain. By doing so, the proposed work will provide the scientific community with a new and powerful tool for the study of functional connectivity in the intact, awake brain. The current work will have an important impact on our ability to monitor functional neuronal connectivity in intact, awake brains. A disruption in neural communication has been associated with a wide range of mental diseases. This proposal involves development of new methods to study communication between neurons and will provide the basis for future studies of human mental health and behavioral disorders.

This proposal aims to understand the changes in visual processing that occur when subjects shift between alert and non-alert waking states. Thanks to the development of behavioral methods for the control of eye position, great advances have been made in understanding central mechanisms of visual perception of alert, attentive subjects. However, there is little understanding of cortical processes that come into play when alertness wanes. The awake, non-alert state is not equivalent to anesthesia, or to sleep states. When non-alert, we are capable of perception, but our perceptual capacities differ. It is commonly believed that accidents happen when we are not alert, but the extent to which early thalamic or visual cortical mechanisms may be responsible for this (as opposed to higher cognitive processes) is an open question. This proposal relies on a unique model system that is very well-suited to address this question: the awake rabbit, an animal who's inner mental life transparently shifts between alert and non- alert states, and who's stable eyes and diffident nature make it an ideal subject for these experiments. The proposed research will examine how changes in the brain state of awake subjects influence the multiple, sequential stages of information processing that occur within the visual thalamocortical and intracortical network. The experiments will measure state- dependent changes in the visual response properties of excitatory and inhibitory neurons at the input and output layers of the cortex and will investigate the underlying mechanisms leading to these changes, at the subthreshold and spiking level. This work will lead to a better understanding of cortical mechanisms of visual processing in a dynamic, awake brain. From a health perspective, these studies will have an important impact on our understanding of how alertness/vigilance deficits can impact visual perception and performance, and will provide the basis for future clinical studies of human mental health and behavioral disorders.

DESCRIPTION (provided by applicant): A major goal of the proposed work is to better understand how thalamocortical inputs to rabbit visual cortical (V1) neurons contribute to the synthesis of the diverse receptive fields properties seen in the input layers of V1. In both layers 4 and 6, these include (a) simple cells with highly oriented and directional receptive fields composed of separate parallel ON and/or OFF sub-zones, and (b) putative fast spike inhibitory interneurons (SINs) with overlapping ON-OFF, receptive fields that lack significant orientation/direction selectivity. While considerable work has been done on these questions for simple cells in layer 4 of feline V1, little is known about receptive field synthesis in V1 of rabbts and rodents, and the nature of the thalamocortical contribution to this synthesis. V1 of rabbits and rodents is similar to that of carnivores and primates in having many simple cells that are highly selective for the orientation of a visual stimulus. However, unlike carnivores and primates, rabbits and rodents lack orientation columns. Moreover, their visual thalamus (LGN) contains neurons that provide directional and orientation information directly to V1. Whereas in cats and primates, orientation and directional selectivity are thought to be synthesized within V1, in rabbits and rodents these properties could potentially be inherited from the thalamus. The proposed experiments will address this question directly, in both layers 4 and 6. We will record from different cell types in the LGN of awake rabbits, some of which have directional and or orientation selective receptive fields. We will make simultaneous recordings from simple cells and SINs in the input layers (4 and 6), the simple cells being highly tuned for orientation/direction, the SINs being poorly tuned. We will use cross-correlation methods to see which LGN cells make functional contacts with which cortical cells. Our preliminary results suggest that layer 4 simple cells do not inherit their orientation/directional preference from LGN directional selective neurons. Therefore, the orientation preference of layer 4 simple cells may be largely created from the receptive field arrangement of ON and OFF LGN afferents, as in the cat. Alternatively, it may be inherited from LGN inputs with concentric receptive fields that show an orientation bias. By measuring the receptive field properties of the LGN inputs to a L4 cortical simple cell, our experiments will be able to distinguish between these two possible mechanisms.

This proposal aims to understand the changes in visual processing that occur when subjects shift between alert and non-alert waking states. While great advances have been made in understanding central mechanisms of visual perception of alert, attentive subjects, there is little understanding of cortical processes that come into play when alertness wanes. The awake, non-alert state is not equivalent to anesthesia, or to sleep states. When non-alert, we are capable of perception, but our perceptual capacities differ. It is commonly believed that accidents happen when we are not alert, but the extent to which early thalamic or visual cortical mechanisms may be responsible for this (as opposed to higher cognitive processes) is an open question. This proposal relies on a unique model system that is very well-suited to address this question: the awake rabbit, an animal who's inner mental life transparently and frequently shifts between alert and nonalert EEG-defined states, and who's stable eyes and diffident nature make it an ideal subject for these experiments. The proposed research will examine how changes in the brain state of awake subjects influence the multiple, sequential stages of information processing that occur within the visual thalamocortical, intracortical, and cortical output networks. The experiments will compare state dependent changes in the visual response properties of excitatory and inhibitory neurons at the input layer and within several output systems of the cortex and will investigate the underlying mechanisms leading to these changes, at the subthreshold and spiking level. This work will lead to a better understanding of cortical mechanisms of visual processing in a dynamic, awake brain. From a health perspective, these studies will have an important impact on our understanding of how alertness/vigilance deficits can impact visual perception and performance, and will provide the basis for future clinical studies of human mental health and behavioral disorders.

PROJECT SUMMARY The lateral geniculate nucleus (LGN) of rabbits and rodents contains a population of neurons that show strong directional selectivity (DS) to visual stimulation. Although these LGN DS neurons are known to project to the primary visual cortex (V1), their synaptic targets, and their role in the synthesis of V1 receptive fields are unknown. Whereas in cats and primates, LGN neurons display little or no orientation and directional selectivity and these properties are thought to be largely synthesized within V1, in rabbits and rodents these properties could potentially be inherited, in part, from the LGN DS neurons. The proposed experiments will address this question, aiming to understand how LGN DS neurons contribute to the synthesis of the diverse receptive field seen in V1 neurons. This will be accomplished using two sets of complementary methods, in fully awake rabbits. First (Aim 1), we will determine which layers of V1 receive a strong input from LGN DS neurons. We will do this using single-axon spike-triggered current source-density analysis, a method that provides a view of the laminar profile of the presynaptic (axonal) and monosynaptic local field potentials and currents generated by the spikes of single thalamocortical neurons within the topographically aligned region of recipient cortex. Next (Aim 2), we will record the spike trains of both LGN DS neurons, and retinotopically aligned cortical neurons of different receptive field classes. We will determine which cortical neurons within the synaptic recipient zone of the LGN DS neuron receives synaptic input (using methods of extracellular cross-correlation), and how the directional preferences of the LGN DS neurons relate to the preferences of their synaptic targets. Direction and orientation selectivity are among the most salient response properties of visual cortical neurons and this project is aimed at understanding how these properties emerge.