Forward locomotion of Drosophila melanogaster larvae is composed of rhythmic waves of contractions that are thought to be produced by segmentally organized central pattern generators. We present a systematic description of spike activity patterns during locomotive contraction waves in semi-intact wild-type and mutant larval preparations. We have shown previously that T␤h nM18 mutants, with altered levels of octopamine and tyramine, have a locomotion deficit. By recording en passant from the segmental nerves, we investigated the coordination of the neuronal activity driving contraction waves of the abdominal body-wall muscles. Rhythmic bursts of activity that occurred concurrently with locomotive waves were frequently observed in wild-type larvae but were rarely seen in T␤h nM18 mutants. These centrally generated patterned activities were eliminated in the distal stumps of both wild-type and T␤h nM18 larvae after severing the segmental nerve from the CNS. Patterned activities persisted in the proximal stumps deprived of sensory feedback from the periphery. Simultaneous recordings demonstrated a delay in the bursting activity between different segments, with greater delay for segments that were farther apart. In contrast, bilateral recordings within a single segment revealed a well synchronized activity pattern in nerves innervating each hemisegment in both wild-type and T␤h nM18 larvae. Significantly, rhythmic patterns of bursts and waves could be evoked in T␤h nM18 mutants by head or tail stimulation despite their highly irregular spontaneous activities. These observations suggest a role of the biogenic amines in the initiation and modulation of motor pattern generation. The technique presented here can be readily extended to examine the locomotion motor program of other mutants.

Biogenic amines are believed to play important roles in producing behaviors. Although some biogenic amines have been extensively studied in both vertebrates and invertebrates, little is known about the effects of trace amines like tyramine and octopamine. We investigated how trace amines affect behaviors using quantitative morphometric methods on Drosophila Tbetah(nM18) and iav(N) mutants that have altered levels of tyramine and octopamine. Locomotion of wild-type and mutant third instar larvae was analyzed using Dynamic Image Analysis System (DIAS) software. We found that Tbetah(nM18) mutants, with elevated tyramine levels and reduced octopamine levels, had a severe locomotion phenotype. Mutant larvae spent much more time in pausing episodes than wild-type larvae and displayed a reduction in speed and linear translocation. The locomotion phenotype was partially rescued by feeding Tbetah(nM18) larvae octopamine, an effect that could be nullified with simultaneous feeding of tyramine. Feeding Tbetah(nM18) larvae yohimbine, an agent that inhibits the activity of Drosophila tyramine receptors, also improved some locomotion parameters. Feeding both octopamine and yohimbine further improved rescue efficiency. Simultaneously reducing the octopamine and tyramine levels as in iav(N) larvae, in contrast, led to a less severe behavioral phenotype than that of Tbetah(nM18) mutants. Feeding iav(N) larvae either tyramine or octopamine exerted only a minor improvement in locomotion. These results suggest that tyramine and octopamine have opposite effects on Drosophila larval locomotion regulation and that a balance between the two is important in producing normal behavior.

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Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.

Glutamine synthetase (GS) is expressed in the neural retina only in Muller glia cells and is inducible with cortisol. A chicken genomic clone that contains at least part of the coding region for the GS enzyme was used to investigate developmental changes in the level of GS mRNA in embryonic chicken retina. A major GS transcript (-3 kilobases) detected by the probe begins to accumulate sharply on day 15 of embryonic development. When cortisol is prematurely supplied to early embryonic retina, it induces precocious accumulation of GS mRNA and of the GS enzyme. At later ages, these effects of cortisol are significantly greater, which suggests that competence to In embryonic and mature retina, GS is restricted to Muller glia cells (1). In chicken embryo retina (the most thoroughly studied system), GS level is very low until day 15-16 of development. It then begins to increase sharply in response to systemic elevation of adrenal corticosteroid hormones, and it increases in 6 days >100-fold. However, GS can be induced also precociously, already in 8-day retina, by prematurely adding a corticosteroid inducer such as cortisol to embryos or to organ cultures of isolated retina tissue (2, 3). Previous indirect evidence suggested that cortisol induced GS in the retina by eliciting GS mRNA accumulation (4, 5). Here, we used a chicken GS clone to investigate changes in the level of GS mRNA in the retina during normal development and when GS is prematurely induced.CA-II is present at high levels already in early embryonic retina (by day 5 of development) when it is found in all retina cells (6). As differentiation progresses, neurons stop expressing CA-II; by day 13, this enzyme is found only in Muller glia cells. During retina maturation, CA-II accumulates to a new high level that persists in the adult (6). It is important to note that cell proliferation in the neural retina of chicken embryo begins to decline after day 8 and ceases by day 12 of development (3). Using a CA-II cDNA clone, we investigated the accumulation and localization of CA-II mRNA during retina development in relation to changes in CA-II enzyme level. MATERIALS AND METHODSNeural Retina. Neural retina tissue was isolated under sterile conditions from eyes of chicken embryos (White Leghorn) at different stages of development and was stored at -70°C or directly used for RNA preparation.RNA Preparation and Analysis. Retina tissue was disrupted by Dounce homogenization in an ice-cold solution of 0.15 M NaCl/0.01 M Tris HCl, pH 7.5/2 mM MgCl2/0.75% Nonidet P-40. Nuclei were removed by centrifugation (8 min at 1500 x g). The supernatant was adjusted to 2.5 mM EDTA/0.5% NaDodSO4 and cytoplasmic RNA was isolated by phenol/ chloroform extraction and ethanol precipitation. Poly(A)+ RNA was selected by using an oligo(dT)-cellulose column (Collaborative Research, Waltham, MA, type III). Total RNA was prepared by the guanidinium isothiocyanate/CsCl method (7). Total RNA (10 ,tg) or poly(A)+ RNA (5 ,ug) was denatured by heating at 60°C in 2.2 M formaldehyde/50...

Accumulation of c-src mRNA gradually increased during early development of the neural retina in chicken embryos and reached a peak by days 11 to 13 of embryonic life. Thereafter, its amount declined to a low level which persisted also in adult retina. The early increase in c-src mRNA correlated inversely with the decrease in the amount of H3.2 replication histone mRNA and with the decline in the rate of cell growth. The accumulation profile of c-src mRNA corresponded to that of pp6Oc-' protein, suggesting that the latter is regulated at the level of transcription.

Objective Neuropathic pain is common and often difficult to treat because it generally does not respond well to the currently available pain medications or nerve blocks. Recent studies in both humans and animals have suggested that exercise may induce a transient analgesia and reduce acute pain in normal healthy individuals. We examined whether swim therapy could alleviate neuropathic pain in rats. Design Rats were trained to swim over a two week period in warm water. After the rats were trained, neuropathic pain was induced by constricting the right sciatic nerve and regular swimming was resumed. The sensitivity of each hind paw was monitored using the Hargreaves test and von Frey test to evaluate the withdrawal response thresholds to heat and touch. Results The paw ipsilateral to the nerve ligation expressed pain-like behaviors including thermal hyperalgesia and mechanical allodynia. Regular swim therapy sessions significantly reduced the mechanical allodynia and thermal hyperalgesia. Swim therapy had little effect on the withdrawal thresholds for the contralateral paw. In addition, swim therapy alone did not alter the thermal or mechanical thresholds of normal rats. Conclusions The results suggest that regular exercise, including swim therapy, may be an effective treatment for neuropathic pain caused by nerve injuries. This study, showing that swim therapy reduces neuropathic pain behavior in rats, provides a scientific rationale for clinicians to test the efficacy of exercise in the management of neuropathic pain. It may prove to be a safe and cost-effective therapy in a variety of neuropathic pain states.

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