Fiona Inglis, PhD - US grants

Nerve cells, or neurons, receive inputs from other neurons predominantly within their dendrites, highly branched structures emanating from the cell body. In many neurons, the patterns of dendrites are refined during development, through a process that is dependent on the activity of the input neurons. This process has been hypothesized to eliminate parts of the dendrites that receive erroneous inputs, thus "hardwiring" the neurons for maximal efficiency.

This project employs cultures containing spinal cord motor neurons to investigate how these neurons use electrical activity to accomplish refinement of their dendrites. Real-time imaging will be performed to examine how rates of growth, elongation and elimination of dendritic segments are affected by the neurotransmitter glutamate binding to specific glutamate receptors on the cell surface. Fluorescent-tagged glutamate receptors will be introduced into neurons, to determine whether these are co-localized at areas of dynamic growth or retraction of dendrites. In addition, level of gene expression for each receptor will be measured using a quantitative PCR method, to determine how the availability of each receptor is controlled during the development of motor neurons. Finally, levels of glutamate receptor expression will be experimentally reduced, to investigate whether dendrite growth is compromised. These studies are important because they will provide information regarding how developing neurons attain patterns of connectivity by altering their ability to respond to neurotransmitters.

The broader impact of this proposal includes significant enhancement of graduate and undergraduate scholarship and research at Tulane University. The integration of current research strategies into graduate and undergraduate training and classroom discussions will promote neuroscience as a subject for study throughout the higher education system.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human immunodeficiency virus (HIV) infection is a global health burden, affecting an estimated 40 million people worldwide. Over half those infected suffer from neurological symptoms, ranging from mild cognitive deficits to severe dementia and motor disorders;however the cellular mechanisms underlying neurological alterations following infection with HIV remain unclear. This project makes use of tissue samples collected from rhesus macaque monkeys inoculated with the simian immuno-deficiency virus (SIV), as a model for HIV. The aim of this study is to establish a time-course for changes in neuronal morphology and connectivity following SIV infection in distinct brain regions that are likely to contribute to neurological deficits observed in patients with HIV. Our preliminary studies have focused on the hippocampus, a region of brain associated with spatial cognitive functions, and in which there is evidence for altered neuronal morphology in post-mortem tissue from patients infected with HIV. Our preliminary data indicate that following inoculation with SIV, a complex but robust pattern of changes occurs in the dendrite complexity of pyramidal neurons within the hippocampal CA1 subfield, the terminal zone of the tri-synaptic pathway. Following SIV infection, we observed an increase in the total amount of arbor within basal dendrites, and a corresponding increase in the inter-branch segment length, suggesting that dendritic segments are extending beyond their normal terminal fields within these neurons. In contrast, we did not observe an increase in total arbor length in apical dendrites, but instead found an increase in the number of branch segments, and a corresponding reduction in inter-branch length, suggesting that apical dendrite arbor undergoes re-arrangement in a manner consistent with the addition of new branch-points. These results are significant, because alterations in dendrite complexity and length are key indicators of changes in firing patterns of neurons, and of network properties within the brain. Our findings suggest that network firing is altered in hippocampal neurons in a manner that may depend on distinct afferent innervation of apical vs. basal dendrites. These results are in contrast to findings in post-mortem brain that display reduced dendritic arbor in hippocampal neurons, and may suggest that significant rearrangement of neuronal architecture precedes degenerative changes in neurons. We have now extended our analyses of hippocampal neurons from infected and control animals to include other hippocampal subfields, the CA3 and dentate gyrus, since these three regions support distinct aspects of cognitive function that may be differentially affected by SIV infection. We have also begun examining neurons within the dorsal thalamus, a region critical for attentional functions and sensory gating. We are continuing to collect samples for RNA analyses, and will use these, and archival specimens collected at the TNPRC in order to determine whether alterations in dendritic morphology occur in parallel with changes in key signaling molecules, such as glutamate receptors and their associated signaling molecules.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Human immunodeficiency virus (HIV) infection is a global health burden, affecting an estimated 40 million people worldwide. Over half those infected suffer from neurological symptoms, ranging from mild cognitive deficits to severe dementia and motor disorders;however the cellular mechanisms underlying neurological alterations following infection with HIV remain unclear. This project makes use of tissue samples collected from rhesus macaque monkeys inoculated with simian immunodeficiency virus (SIV) as a non-human primate model for HIV. The aim of this study is to examine neuronal dendrite morphology as a predictor of neuronal connectivity, and to establish a time-course for changes in morphology as related to the progression of SIV. Our studies employ Golgi staining, a classic but unparalleled staining technique for neurons which enables measurements of key features of architecture known to be associated with alterations in connectivity and excitability. We have now collected tissue from several brain regions that are likely to contribute to neurological deficits observed in patients with HIV. These include the prefrontal cortex (associated with working memory), the thalamus (associated with attentional functions) and the hippocampus (associated with spatial memory and mnemonic functions). While we intend to characterize alterations of dendrite morphology within each region in full, our analyses to date have concentrated on the hippocampus: this region has been the focus of extensive studies of learning and memory, and its anatomy and physiology are well defined. Our analyses have concentrated on three hippocampal regions, namely the dentate gyrus, CA3 and CA1;together, these regions represent the three principal regions of the tri-synaptic pathway within the hippocampus, and the site of long-term potentiation, a form of synaptic plasticity thought to be the basis for hippocampal learning. Extensive evidence exists to show that disruption of hippocampal function leads to cognitive impairment in animals and humans: in animal models, disruption of cellular events supporting LTP, and structural alterations in hippocampal dendrites, are associated with inability to perform a variety of cognitive tasks. Studies of patients with HIV consistently demonstrate alterations in functional markers within the hippocampus. Thus, understanding the temporal pattern of neurological changes within the hippocampus following SIV infection represents an important opportunity to design strategies to limit or even reverse cognitive disturbances associated with HIV. To date, we have compared indices of dendrite morphology in three treatment groups: controls, animals infected with SHIV, and animals infected with SIV. For these studies, tissues were collected from animals following chronic infection (on average 1 year after inoculation);however future studies aim to examine whether altered dendrite morphology is a feature of acute infection. Our current studies demonstrate that inoculation with SIV or SHIV results in alterations in hippocampal dendrite morphology that would not be predicted from results of existing post-mortem studies performed at disease endpoints within human patients. Within the granule cells of the dentate gyrus -- the first neurons within the trisynaptic pathway -- we observed increases in dendritic segment number and length following inoculation with SIV or SHIV, compared to controls. The increases in dendritic complexity in response to SHIV inoculation were highly significant, whereas alterations in arbor following SIV are more modest, and failed to reach statistical significance within our current sample size. Preliminary studies of morphology of CA3 pyramidal neurons (representing the second synapse within the trisynaptic pathway) are somewhat hampered by the greater variability in dendrite morphology within this region;however, our preliminary data suggest that SIV and SHIV inoculation results in increased amounts of dendritic arbor within basal dendrites, but reduces the extent of arbor within apical dendrites. While greater sample sizes will be required to verify this pattern of alterations, these results are consistent with increased connectivity within the tri-synaptic path, but reduced connectivity from extra-hippocampal regions, such as thalamus and entorhinal cortex, that are known to synapse directly with CA3 apical dendrites. This pattern is essentially repeated within the CA1 region, the terminus of the tri-synaptic pathway: here, robust increases (approximately 50%) in dendrite length were observed within basal dendrites. Thus, the patterns of alterations in dendritic complexity that we observe are consistent with discrete, anatomically defined changes in activity within specific hippocampal circuits, and further suggest that drug therapies targeted at restoring the balance of hippocampal activity and connectivity may be useful in reversing cognitive deficits. The importance of these results is demonstrated by the lack of congruence between our results and those of human post-mortem studies, in which by necessity measurements of dendrite morphology are made very late in the progression of the disease. In contrast, to our findings, postmortem studies within the hippocampus have demonstrated loss of hippocampal dendritic arbor. Thus, strategies aimed solely at regrowth of dendritic arbor may be ineffectual in preventing or reversing abnormalities in dendritic connectivity, particularly during early stages of the disease. Our on-going studies aim to increase sample sizes in order to provide confirmation of these patterns of dendrite morphology, to examine acute vs. chronic effects on dendritic arbor, and to extend our studies to other regions of the brain likely to support cognitive functions. We have also begun measurements of dendritic arbor within the prefrontal cortex, an area in which loss of function is associated with reduced inhibitory behaviors, a possible contributing factor to increased risk-taking that occurs in many patients with HIV.