Gerald H. Clayton

Ontogeny of cation–Cl− cotransporter expression in rat neocortex

Neuronal precursors and immature cortical neurons actively accumulate Cl- and as a consequence depolarize in response to GABAA receptor activation. With maturity, intracellular Cl- decreases resulting in a shift towards GABAA inhibition. These observations suggest that changes in expression of cation-Cl- cotransporters may have a significant role in the ontogeny of neuronal Cl- homeostasis. Using ribonuclease protection analysis and in situ hybridization we examined the developmental expression of all presently known members of the cation-Cl- cotransporter gene family in rat brain. Of the inwardly directed cotransporters, NKCC-1, NKCC-2, and NCC-1, only NKCC-1 was detected at significant levels in brain. NKCC-1 was expressed in neurons, appearing first in cortical plate but not in ventricular or subventricular zone. Expression levels peaked by the third postnatal week and were maintained into adulthood. The outwardly directed cotransporters, KCC-1 and KCC-2, demonstrated significantly different levels and time courses of expression. KCC-1 was expressed prenatally at very low levels which increased little over the course of development. In contrast, KCC-2 expression appeared perinatally and increased dramatically after the first week of postnatal life. Differential changes in expression of this gene family occurred during periods of critical shifts in chloride homeostasis and GABA response suggestive of a role in these processes. Furthermore the absence of expression of known inwardly directed cotransporters in Cl- accumulating neuroepithelia and lack of evidence for glial expression suggests that as yet unidentified members of this gene family may be involved in chloride homeostasis in immature neuronal precursors and neuroglia.

Corticosteroid treatment and functional improvement in Duchenne muscular dystrophy: long-term effect.

Balaban B, Matthews DJ, Clayton GH, Carry T: Corticosteroid treatment and functional improvement in Duchenne muscular dystrophy: Long-term effect. Am J Phys Med Rehabil 2005;84:843–850. Objective:To determine and compare the long-term effects of prednisone and deflazacort on the functional status of children with Duchenne muscular dystrophy. Design:A total of 49 boys with Duchenne muscular dystrophy, between the age of 12 and 15 yrs, who were observed over a 7-yr period were reviewed retrospectively. Eighteen had been treated with prednisone, 12 with deflazacort, and 19 had no drug treatment. All boys treated with steroids received medication for >2 yrs before losing their ambulation. Lower and upper limb motor functions, pulmonary function, prevalence of surgery for scoliosis, and side effects were compared. Results:Boys in the steroid groups were significantly more functional and performed better on all tests than boys not treated (P < 0.05). There was no significant difference between the deflazacort- and prednisone-treated groups (P > 0.05). The number of boys having scoliosis surgery in treated groups was significantly less than nontreated boys (P < 0.05). The control group’s pulmonary capacity was decreasing and significantly less than both prednisone- and deflazacort-treated boys. Both deflazacort and prednisone had beneficial effect on pulmonary function and scoliosis. Cataracts, hypertension, behavioral changes, excessive weight gain, and vertebral fracture were noted as serious side effects. Conclusions:Prednisone and deflazacort have a significant beneficial effect on slowing the disease progress. Their usage in Duchenne muscular dystrophy may prolong ambulation and upper limb function with similar potency. Both steroids also improve pulmonary function, in addition to delaying the need for spinal interventions, with similar therapeutic profiles.

Programmed Cell Death in Developing Grafts of Fetal Substantia Nigra

Intracerebral transplants of ventral mesencephalic (VM) tissue have been well characterized. VM grafts contain numerous tyrosine hydroxylase immunoreactive neurons which send axons into the host brain. Transplanted neurons in VM grafts develop normally in that they contain tyrosine hydroxylase and GAP43. An overlooked aspect of graft development is cell death. It has been suggested that cell death in VM grafts was mostly necrotic. However, recent work in this laboratory suggested that developing grafts contain numerous apoptotic cells. In the present paper morphological, histochemical, and molecular correlates of apoptosis were used to assay cell death during VM graft development. At early times (5-15 days) after grafting VM grafts contained numerous apoptotic cells. In older grafts (21 and 28 days) few apoptotic cells were observed. In situ end labeling of fragmented DNA with biotinylated dUTP showed that early grafts contained numerous positive cells. The expression of RP8, a molecular correlate of apoptotic cell death, occurred in early grafts, but was not detectable in older grafts. These results indicate that apoptosis is a normal part of VM graft development. As in naturally developing neural systems, cell death in grafts may function to eliminate cells that fail to connect to appropriate targets.

The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study

GAP43 is a developmentally regulated phosphoprotein which is almost exclusively found in neurons. Numerous correlative studies have shown that GAP43 is expressed at high levels during neurite extension, axonal elongation and synaptogenesis. In this study we used in situ hybridization to examine GAP43 expression during late embryonic and early postnatal development. The highest relative levels of GAP43 at all stages were present in the neocortex. Levels in this and other regions peaked between postnatal days 5 and 10. These results indicate that high levels of GAP43 mRNA correlate most highly with the latter stages of axon outgrowth and with the early stages of synapse formation.

Pediatric Spinal Cord Tumors and Masses

Abstract Background/Objective: Spinal cord tumors are a relatively rare diagnosis, accounting for 1% to 10% of all pediatric central nervous system tumors. Understanding the etiology and clinical outcomes of these tumors is therefore very important. This study presents detailed information regarding clinical presentation, histological findings, outcomes, functional assessment, and management of a series of patients with this diagnosis. Method: Retrospective, descriptive study. Subjects: Thirty-five children with a final diagnosis of spinal cord tumor or mass, excluding dysraphism. Results: Neurodevelopmental tumors (dermoid tumors, epidermoid tumors, and teratomas) were the most common tumor type (31 %), followed by astrocytomas (29%) and neuroblastomas (14%). Other types included schwannomas, meningiomas, giant cell tumors, extradural cystic masses, leukemic-related masses, and masses related to neurofibromatosis. Mean age at diagnosis was 6.6 years (SD = 5.5 y) and did not vary significantly by tumor type except for children with neuroblastoma (mean = 0.4 y, SD = 0.5 y). More boys (57%) were identified in the series than girls (43%); however, there was no association between tumor type and sex. Presenting complaints of pain were noted in 57% and were localized to the back, neck, or extremities. Extremity weakness was reported as an initial presenting symptom in 46%. Three children had scoliosis as a presenting issue and 14 had gait abnormalities. Regardless of treatment modality, mobility was retained in 83% of children with or without gait aids. Neurogenic bowel and/or bladder were present in 23% of the population. Conclusions: This study corroborates other studies indicating that intramedullary tumors are the predominant form of pediatric spinal cord tumor. This population, however, presented with an unusually large number of developmental tumors, contrary to several published studies. The disparity may be the result of this institution acting as a regional referral center, thus increasing the number of this type of patient. The population is too small to make any other conjecture. The predominance of astrocytomas and neuroblastomas among those patients with poor outcomes and the prevalence of developmental tumors suggest the need for broader investigation. Although, in general, spinal cord tumors are relatively rare, this preliminary study supports the need to further evaluate associations between tumor type, presenting symptoms, treatment, and functional outcome in children with spinal cord tumors.

DEVELOPMENTAL EXPRESSION OF CLC-2 IN THE RAT NERVOUS SYSTEM

Abstract Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammons Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of ClC-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

Expression of mRNA for the elav-like neural-specific RNA binding protein, HuD, during nervous system development

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry HuD expression was shown to be expressed at high levels at the earliest time point observed (E15), but declined significantly during the first postnatal week to levels which were maintained into adulthood. In the adult, HuD expression became restricted primarily to large pyramidal-like neurons. Exceptions of note were many smaller neurons within a variety of thalamic nuclei. Expression of HuD was observed to be coincident with terminal differentiation of all neuronal structures evaluated regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS.

Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System

ABSTRACT Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.

Specific outgrowth from neurons of ventral mesencephalic grafts to the catecholamine-depleted striatum of adult hosts

Grafts of fetal ventral mesencephalon including substantia nigra have been used to correct some motor deficits produced by unilateral destruction of the dopaminergic nigrostriatal pathway in rats. Histochemical studies have shown that dopaminergic neurons within the graft send processes from the graft to the host neuropil, wherein they form synapses. The results of numerous immunocytochemical studies indicate, however, that a large proportion of neurons in grafts are not catecholaminergic. Whether or not the nondopaminergic neurons in grafts project to the host brain is unknown. The purpose of the present study was to combine immunocytochemistry and retrograde tracing with fluorogold to identify the cell types which project from grafts to the host striatum. Tissue from the ventral mesencephalon of E15 fetuses was placed into the 6-hydroxydopamine denervated striatum of graft recipients. Six weeks to 6 months following transplantation, fluorogold was pressure injected under stereotaxic control immediately adjacent to the ventral mesencephalic grafts; after 4 days CNS tissue was prepared for light microscopic immunocytochemistry. Ventral mesencephalic grafts contained cell bodies immunoreactive for enkephalin, GAD, substance P, and serotonin in addition to those immunoreactive for tyrosine hydroxylase. Some cells of each immunochemically defined type were retrogradely labeled by the fluorogold injection into the host brain. Nevertheless, more catecholaminergic and serotonergic cells projected from grafts to the fluorogold injection site than did other cell types. Since many of the nonmonoaminergic neurons in grafts are probably projection neurons, our results suggest that the extent of neurite outgrowth from grafted cells is influenced by the surrounding target tissue.

Sports and Disability

Participation in recreational and competitive sports at an early age has long been touted as a positive influence on growth and development, and for fostering lifelong healthy lifestyles. The benefits of an active lifestyle include not only fitness, but the promotion of a sense of inclusion and improved self‐esteem. These benefits are well documented in all populations, and their importance has been summarized in the recent Healthy People 2010 guidelines. The American Academy of Pediatrics has recently produced a summary statement on the benefits of activity for disabled children. They note that children with disabilities tend to have an overall lower level of fitness and an increased level of obesity. For this population, developing a lifelong desire to be active can be a simple means for limiting illness and much of the morbidity associated with sedentary lifestyles often associated with disability. For disabled youth, participation in disabled sports programs available nationally and internationally can be an effective means to promote such precepts. The goal of this focused review is to improve the learners knowledge of the positive impact that active lifestyles can have on overall health in the disabled youth population and, as a result, modify their practice by incorporating recreational and competitive sport activities as part of improving overall patient care.