Long-term injury induced plasticity in {\it Aplysia\/} neurons: Behavioral, electrophysiological, and morphological studies

Nerve injury is known to produce a variety of electrophysiological and morphological neuronal alterations (reviewed by Titmus and Faber, 1990; Bulloch and Ridgeway, 1989; Walters, 1994). Determining if these alterations are adaptive and how they are activated and maintained could provide important insight into basic cellular mechanisms of injury-induced plasticity. Furthermore, characterization of injury-induced plasticity provides a useful assay system for the identification of possible induction signals underlying these neuronal changes. Understanding fundamental mechanisms and underlying induction signals of injury-induced neuronal plasticity could facilitate development of treatment strategies for neural injury and neuropathic pain in humans.^ This dissertation characterizes long-lasting, injury-induced neuronal alterations using the nervous system of Aplysia californica as a model. These changes are examined at the behavioral, electrophysiological, and morphological levels. Injury-induced changes in the electrophysiological properties of neurons were found that increased the signaling effectiveness of the injured neurons. This increase in signalling effectiveness could act to compensate for partial destruction of the injured neuron's peripheral processes. Recovery of a defensive behavioral response which serves to protect the animal from further injury was found within 2 weeks of injury. For the behavioral recovery to occur, new neural pathways must have been formed between the denervated area and the CNS. This was found to be mediated at least in part by new axonal growth which extended from the injured cell back along the original pathway (i.e. into the injured nerve). In addition, injury produced central axonal sprouting into different nerves that do not usually contain the injured neuron's axons. This could be important for (i) finding alternative pathways to the periphery when the original pathways are impassable and (ii) the formation of additional synaptic connections with post-synaptic targets which would further enhance the signalling effectiveness of the injured cell. ^

Intra-hippocampal administration of the VEGF receptor blocker PTK787/ZK222584 impairs long-term memory.

A number of studies have established a role for vascular endothelial growth factor (VEGF) in angiogenesis. Recent reports have shown that VEGF overexpression in the hippocampus improves learning and memory and is associated with enhanced neurogenesis. PTK787/ZK222584 (PTK/ZK) is a reported inhibitor of VEGFR signaling that is currently being tested for its effects on lung and colon cancer. However, the influence of this drug on cognition has not been examined. In the present study, we questioned if post-training administration of PTK/ZK influences hippocampus-dependent memory. When administered to rats immediately following massed training in the Morris water maze, PTK/ZK impaired spatial memory retention tested 48 h later. This impairment was evidenced by increased latency to the hidden platform and fewer platform crossings. However, this impairment was not associated with a change in neurogenesis during this time frame. PTK/ZK infusion did not reduce VEGFR or AKT phosphorylation, but increased the phosphorylation of ERK. These studies suggest that VEGFR inhibitors such as PTK/ZK may negatively influence cognition.

Signals underlying the induction and expression of long-term, injury-related plasticity in sensory neurons of Aplysia

An important goal in the study of long-term memory is to understand the signals that induce and maintain the underlying neural alterations. In Aplysia, long-term sensitization of defensive reflexes has been examined in depth as a simple model of memory. Extensive studies of sensory neurons (SNs) in Aplysia have led to a cellular and molecular model of long-term memory that has greatly influenced memory research. According to this model, induction of long-term memory in Aplysia depends upon serotonin (5-HT) release and subsequent activation of the cAMP-PKA pathway in SNs. The evidence supporting this model mainly came from studies of long-term synaptic facilitation (LTF) using dissociated (and therefore axotomized) cells growing in culture. However, studies in more intact preparations have produced complex and discrepant results. Because these SNs function as nociceptors, and display similar alterations (long-term hyperexcitability [LTH], LTF, and growth) in models of memory and nerve injury, this study examined the roles of 5-HT and the cAMP-PKA pathway in the induction and expression of long-term, injury-related LTH and LTF in Aplysia SNs. ^ The results presented here suggest that 5-HT is not a primary signal for inducing LTH (and perhaps LTF) in Aplysia SNs. Prolonged treatment with 5-HT failed to induce LTH of Aplysia SNs in either ganglia or dissociated-cell preparations. Treatment with a 5-HT antagonist, methiothepin, during noxious nerve stimulation failed to reduce 24 hr LTH. Furthermore, while 5-HT can induce LTF of SN synapses, this LTF appears to be an indirect effect of 5-HT on other cells. When neural activity was suppressed by elevating divalent cations or by using tetrodotoxin (TTX), 5-HT failed to induce LTF. Unlike LTF, LTH of the SNs could not be produced, even when 5-HT treatment occurred in normal artificial sea water (ASW), suggesting that LTH and LTF are likely to depend on different signals for induction. However, methiothepin reduced the later expression of LTH induced by nerve stimulation, suggesting that 5-HT contributes to the maintenance of LTH in Aplysia SNs.n of somata from the ganglion (which axotomizes SNs) or crushing peripheral n. ^ In summary, this study found that 5-HT and the cAMP-PKA pathway are not involved in the induction of long-term, injury-related LTH of Aplysia SNs, but persistent release of 5-HT and persistent PKA activity contribute to the maintenance of LTH induced by injury. (Abstract shortened by UMI.)^

Perspective transformation: Women in long-term recovery from alcohol abuse

Objective. The purpose of this study was to determine the meaning of personal transformation for twenty women in long term, stable recovery from alcohol abuse; to identify themes or patterns of this recovery, and; to determine the extent to which they experienced the phenomenon of perspective transformation. ^ Method. Volunteers were recruited by advertisement, word of mouth, and through a closed circuit web based broadcast. A descriptive, exploratory study, which analyzed perspective transformation from the standpoint of five action phases, was conducted. Data was collected using in-depth personal interviews and questionnaires. Subjects' responses were analyzed by qualitative methods. Triangulation was performed on the grouped data comparing the interviews to the data produced by the questionnaires. Quantitative analysis of questionnaire items explored behavioral changes experienced before and after alcoholism recovery. ^ Results. Five phases of recovery were identified. Phase I which involved recognition that alcohol was a problem and change might be possible took several years during which 3 major transitions occurred: (1) from often being alienated to having relationships with family and friends; (2) from daily upheavals to eventually a more peaceful existence, and; (3) from denial that alcohol was a problem to acceptance and willingness to change. Recovery was often seen in a spiritual context, which also required ongoing support. During Phase II there was an assessment of self, others, and the environment which revealed a pattern of intense unhappiness and negative feelings toward self and others with a disregard for cultural norms. Phase III revealed a period of desperation as life became unmanageable, but gradual willingness to accept support and guidance and a desire to improve self and help others. This led to improvement of existing role performance and the willingness to try out new roles. In Phase IV there was a pattern of personal growth which included: the establishment of boundaries, setting priorities, a willingness to place others' needs above their own, acceptance of responsibility, and learning to cope without alcohol, often with the use of tools learned in AA. During Phase V, many experienced knowledge of frailties but growing respect for self and others, with an improved ability to function in giving relationships. Implications for Prevention and Recovery: Early education concerning addiction and recovery may play a crucial role in prevention and early recovery, as it did for children of women in this study. Recovery requires persistent effort and organized support. ^

Design of long-term animal bioassay for low-dose extrapolation using a multistage model

Conventional designs of animal bioassays allocate the same number of animals into control and dose groups to explore the spontaneous and induced tumor incidence rates, respectively. The purpose of such bioassays are (a) to determine whether or not the substance exhibits carcinogenic properties, and (b) if so, to estimate the human response at relatively low doses. In this study, it has been found that the optimal allocation to the experimental groups which, in some sense, minimize the error of the estimated response for low dose extrapolation is associated with the dose level and tumor risk. The number of dose levels has been investigated at the affordable experimental cost. The pattern of the administered dose, 1 MTD, 1/2 MTD, 1/4 MTD,....., etc. plus control, gives the most reasonable arrangement for the low dose extrapolation purpose. The arrangement of five dose groups may make the highest dose trivial. A four-dose design can circumvent this problem and has also one degree of freedom for testing the goodness-of-fit of the response model.^ An example using the data on liver tumors induced in mice in a lifetime study of feeding dieldrin (Walker et al., 1973) is implemented with the methodology. The results are compared with conclusions drawn from other studies. ^

Long Term Follow-up of Morbidity and Quality of Life Associated with Isolated Gastroschisis

Gastroschisis is a birth defect in which an opening in the abdominal wall allows herniation of the viscera. Prenatal counseling regarding gastroschisis typically discusses that, although these infants often endure a difficult neonatal course, they experience few long-term complications. However, information regarding long-term outcomes is based on limited studies that lack specificity. Therefore, we aimed to study the long-term morbidity and quality of life in children born with gastroschisis in a large and diverse population drawn from the Texas Birth Defects Registry (TBDR). Study packets with informed consent, a questionnaire, and the Pediatric Quality of Life Inventory Generic Core Scale 4.0 (PedsQL 4.0) in English and Spanish were mailed to 1,112 parents of children born with isolated gastroschisis in Texas between 1999 and 2008 via the TBDR. Information was abstracted from the TBDR for 58 mothers of children with gastroschisis who returned study materials. Three hundred fifty five packets were returned to sender, giving a response rate of 7.7%. Children born with gastroschisis had quality of life scores that were not significantly different than expected (p = 0.981). However, factors such as having a learning disability (p = 0.001) and missing school due to gastrointestinal issues (p = 0.020) were found to significantly decrease quality of life. Overall, children with gastroschisis had a significantly increased risk for learning disabilities regardless of whether they were preterm (p = 0.021) or full term (p = 0.021). Additionally, there appeared to be an increased risk for auditory impairment in Caucasian children (p < 0.0005). Therefore, while overall long-term quality of life is not significantly altered for children born with gastroschisis, the previously unreported increased risk for learning disabilities and possible association with hearing impairment are important findings that should be conveyed to prospective parents.

In-utero exposure to chemotherapy: What are the long term effects?

There is not a large body of evidence on in-utero exposure to chemotherapy for pregnancy-associated cancers to help determine the long term effects on offspring. This study is a systematic review of long term follow-up to find evidence for adverse outcomes in exposed offspring. In order for studies to be eligible for this systematic review, they had to have a median follow up of at least 24 months with the resulting child. PubMed, Medline, and Scopus were the databases used, and we included all eligible articles, regardless of the date of publication. The search resulted in six articles meeting the eligibility requirements. The review of findings of these studies suggested that there is not enough evidence to make a determination of the risk of chemotherapy for the offspring. Exposed children in the sample of reviewed papers did have some medical conditions, but the rate and type did not differ from the non-exposed population. However, a limitation of this literature review is the very small sample size of publications on this important topic. This finding of few studies on this topic is an important result of this systematic review. More research and long term follow-up studies must be conducted to address this issue.^

The cGMP -PKG pathway is critical for inducing long-term sensitization of identified nociceptive sensory neurons

In various species, peripheral injury produces long-lasting sensitization of central and peripheral neurons representing the affected area. In Aplysia, memory-like traces (lasting days or weeks) of noxious peripheral stimulation include enhancement of central synaptic transmission and enhanced excitability of the central soma and peripheral branches of nociceptive sensory neurons. An important role for the cAMP-PKA-CREB pathway in consolidating long-term memory and inducing transcription-dependent synaptic potentiation has also been indicated by studies in rodents and Drosophila. ^ Much less attention has been paid to the cGMP-PKG pathway for transcription-dependent plasticity. Nevertheless, the cGMP-PKG pathway has been implicated in activity-dependent neural alterations lasting hours, and may trigger some forms of persistent pain. Recent evidence indicates PKG can regulate gene expression in the brain and several properties make it an attractive candidate for inducing long-term memories. ^ This dissertation reports that brief, noxious stimulation of a behaving, semi-intact preparation from mollusc, Aplysia californica, produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway and which lasts for at least 24 hours. Intracellular injection of cGMP is sufficient to induce LTH. Similarly, body wall injury induces LTH which can be blocked with specific inhibitors of the NO-cGMP-PKG pathway such as L-NMMA, ODQ, Rp-8-cGMPS, PKI-G and KT5823 by isolated perfusion of pleural ganglion sensory cells in or directly by intracellular injection. In contrast, specific inhibitors of the cAMP-PKA pathway (Rp-8-cAMPS, PKI-A and H-89) failed to block injury-induced LTH. Interestingly, co-injection of the cAMP-responsive element (CRE) blocked the induction of both cAMP and injury-induced LTH, but not cGMP-induced LTH. Furthermore, co-injection of cAMP and cGMP with the Ca2+ buffer BAPTA in reduced Ca2+ seawater blocked cAMP-, but not cGMP-induced LTH. These findings demonstrate that the NO-cGMP-PKG pathway and at least one other pathway (perhaps mediated by Ca2+), but not the cAMP-PKA pathway, are critical for inducing LTH during transient, noxious stimulation.^