Acute ethanol exposure leads to long-term effects on memory, behavior, and transcriptional regulation in the zebrafish brain.

Alcohol consumption is associated with alterations in memory and learning processes in humans and animals. In this context, research models such as the zebrafish (Danio rerio) arise as key organisms in behavioral and molecular studies that attempt to clarify alterations in the Central Nervous System (CNS), like those related to alcohol use. Accordingly, we used the zebrafish as a model to evaluate the effects of ethanol on the learning and memory process, as well as its relationship with behavior and transcriptional regulation of lrfn2, lrrk2, grin1a, and bdnf genes in the brain. To this end, for the memory and learning evaluation, we conducted the Novel Object Recognition test (NOR); for behavior, the Novel Tank test; and for gene transcription, qPCR, after 2 h, 24 h, and 8 days of ethanol exposure. As a result, we noticed in the NOR that after 8 days of ethanol exposure, the control group spent more time exploring the novel object than when compared to 2 h post-exposure, indicating that naturally zebrafish remember familiar objects. In animals in the Treatment group, however, no object recognition behavior was observed, suggesting that alcohol affected the learning and memory processes of the animals and stimulated an anxiolytic effect in them. Regarding transcriptional regulation, 24 h after alcohol exposure, we found hyper-regulation of bdnf and, after 8 days, a hypo-regulation of lrfn2 and lrrk2. To conclude, we demonstrated that ethanol exposure may have influenced learning ability and memory formation in zebrafish, as well as behavior and regulation of gene transcription. These data are relevant for further understanding the application of zebrafish in research associated with ethanol consumption and behavior.

Central action of CART induces neuronal activation in the paraventricular and dorsomedial hypothalamus of diet-induced obese and lean mice.

The cocaine- and amphetamine-regulated transcript (CART) is a peptide commonly studied in the feeding behavior, but it exerts an important role in the autonomic and cardiovascular control as well. It is known that exogenous administration of CART in the central nervous system can elicit increase in blood pressure of both conscious and anesthetized rodents, but little is known whether these central effects might differ between lean and obese animals. We have recently shown that diet-induced obese mice that developed hypertension presented an upregulation of CART levels in the dorsomedial nucleus of hypothalamus, while obese normotensive ones had not. Herein we investigate whether the central action of CART could activate differently the hypothalamic nuclei of diet-induced obese mice compared to the lean counterparts by using Fos protein expression, C57BL/6 mice were randomly assigned to two cohorts, one fed with a high-fat diet for 8 weeks (obese), and the other fed with regular rodent chow (lean). Both groups received an ICV injection of CART at the dose of 400µM, 1 mM or vehicle. Subsequently, the brains were processed for Fos protein immunohistochemical in order to identify hypothalamic neuronal activation. Significantly greater numbers of Fos-positive neurons were observed in the PVN and DMH of obese mice that received CART 1 mM, when compared to the lean control. These results indicate that the central action of CART induces neuronal activation in the hypothalamic nuclei of obese and lean mice, and this could be relevant to the different autonomic and cardiovascular adjustments that an organism exposed to different diet and metabolic condition.

Effects of the recombinant crustacean hyperglycemic hormones rCHH-B1 and rCHH-B2 on the osmo-ionic regulation of the shrimp Litopenaeus vannamei exposed to acute salinity stress.

The Pacific white shrimp Litopenaeus vannamei is a euryhaline organism that copes with salinity fluctuations in the environment; therefore, its osmotic and ionic regulation abilities are vital. Osmoregulation may be controlled by the crustacean hyperglycemic hormone (CHH), a neuropeptide mainly expressed in the eyestalks. In L. vannamei, CHH-B1 and CHH-B2 are CHH isoforms isolated from the eyestalks whose expression is influenced by environmental salinity. It has been suggested that they are involved in the response to salinity stress. To clarify this, we investigated the effect of the recombinant peptides, rCHH-B1 and rCHH-B2, on the osmo-ionic regulation of shrimp acutely exposed to different salinity conditions (8, 26 and 45‰). Both rCHHs promoted differential effects on the osmoregulatory capacity (OC) and the ionoregulatory capacity (IC) for hemolymph Na+ and Cl- during iso-osmotic (26‰) and hyper-osmotic (45‰) transfers. These changes were linked to the changes observed in Na+/K+ ATPase and carbonic anhydrase gene expression in gills, especially under high salinity conditions, suggesting that the hormones may regulate the expression of these genes. Glucose and protein levels measured during acute salinity transfer suggest their roles as sources of metabolic energy for osmotic regulation or as organic osmolytes. These results taken together suggest that both the CHH-B1 and CHH-B2 peptides are important regulators of the physiological response of L. vannamei to acute salinity fluctuations.

Activated microglia impairs neuroglial interaction by opening Cx43 hemichannels in hippocampal astrocytes.

Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin-based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS-induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL-1ß and TNF-α. Consequences of the hemichannel-mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation.

Semaphorin 3F and neuropilin-2 control the migration of human T-cell precursors.

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.

Immunization with a neural-derived peptide protects the spinal cord from apoptosis after traumatic injury.

Apoptosis is one of the most destructive mechanisms that develop after spinal cord (SC) injury. Immunization with neural-derived peptides (INDPs) such as A91 has shown to reduce the deleterious proinflammatory response and the amount of harmful compounds produced after SC injury. With the notion that the aforementioned elements are apoptotic inducers, we hypothesized that INDPs would reduce apoptosis after SC injury. In order to test this assumption, adult rats were subjected to SC contusion and immunized either with A91 or phosphate buffered saline (PBS; control group). Seven days after injury, animals were euthanized to evaluate the number of apoptotic cells at the injury site. Apoptosis was evaluated using DAPI and TUNEL techniques; caspase-3 activity was also evaluated. To further elucidate the mechanisms through which A91 exerts this antiapoptotic effects we quantified tumor necrosis factor-alpha (TNF-α). To also demonstrate that the decrease in apoptotic cells correlated with a functional improvement, locomotor recovery was evaluated. Immunization with A91 significantly reduced the number of apoptotic cells and decreased caspase-3 activity and TNF-α concentration. Immunization with A91 also improved the functional recovery of injured rats. The present study shows the beneficial effect of INDPs on preventing apoptosis and provides more evidence on the neuroprotective mechanisms exerted by this strategy.

Development of protective autoimmunity by immunization with a neural-derived peptide is ineffective in severe spinal cord injury.

Protective autoimmunity (PA) is a physiological response to central nervous system trauma that has demonstrated to promote neuroprotection after spinal cord injury (SCI). To reach its beneficial effect, PA should be boosted by immunizing with neural constituents or neural-derived peptides such as A91. Immunizing with A91 has shown to promote neuroprotection after SCI and its use has proven to be feasible in a clinical setting. The broad applications of neural-derived peptides make it important to determine the main features of this anti-A91 response. For this purpose, adult Sprague-Dawley rats were subjected to a spinal cord contusion (SCC; moderate or severe) or a spinal cord transection (SCT; complete or incomplete). Immediately after injury, animals were immunized with PBS or A91. Motor recovery, T cell-specific response against A91 and the levels of IL-4, IFN-γ and brain-derived neurotrophic factor (BDNF) released by A91-specific T (T(A91)) cells were evaluated. Rats with moderate SCC, presented a better motor recovery after A91 immunization. Animals with moderate SCC or incomplete SCT showed significant T cell proliferation against A91 that was characterized chiefly by the predominant production of IL-4 and the release of BDNF. In contrast, immunization with A91 did not promote a better motor recovery in animals with severe SCC or complete SCT. In fact, T cell proliferation against A91 was diminished in these animals. The present results suggest that the effective development of PA and, consequently, the beneficial effects of immunizing with A91 significantly depend on the severity of SCI. This could mainly be attributed to the lack of T(A91) cells which predominantly showed to have a Th2 phenotype capable of producing BDNF, further promoting neuroprotection.

Metallothionein 3 attenuated the apoptosis of neurons in the CA1 region of the hippocampus in the senescence-accelerated mouse/PRONE8 (SAMP8).

OBJECTIVE: Metallothionein 3 (MT-3) has been shown to protect against apoptotic neuronal death in the brains of patients with Alzheimer's disease. Zinc is a potent inhibitor of caspase-3 and its deficiency was found to promote apoptosis. Here, we measured the zinc and copper content in the brains of senescence-accelerated mouse/PRONE8 (SAMP8) and sought to investigate the effect of MT-3 on the apoptosis of neurons in the hippocampal CA1 region of these mice. METHOD: The zinc and copper content in the brain samples of SAMP8 and normal control SAMR1 mice were determined using an atomic absorption spectrophotometer. The mice were administered intraperitoneally for four weeks with MT-3 or MT1 and thereafter apoptosis was measured using the TUNEL method and the expression of anti-apoptotic protein Bcl-2 and proapoptotic protein Bax was examined by immunohistochemistry. RESULTS: Compared with that in SMAR1 mice, the content of zinc in the brains of SAMP8 mice was significantly reduced (P<0.05). Moreover, significant levels of apoptosis of neurons were observed in the hippocampus of SAMP8 mice, which, compared with those in SMAR1 mice, also showed significantly lower levels of Bcl-2 and higher levels of Bax (P<0.05). MT-3 increased zinc concentration in the hippocampus of SAMP8 mice and also significantly decreased apoptosis in these neurons dose-dependently and increased the levels of Bcl-2 and decreased the levels of Bax. CONCLUSION: MT-3 could attenuate apoptotic neuron death in the hippocampus of SAMP8, suggesting that the protein may lessen the development of neurodegeneration.

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels.

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-ß peptide (Aß) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3 h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aß-treated microglia (CM-Aß). The effects of CM-Aß were mimicked by TNF-α and IL-1ß and were abrogated by neutralizing TNF-α with soluble receptor and IL-1ß with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aß made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aß induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.

Metallothionein 3 attenuated the apoptosis of neurons in the CA1 region of the hippocampus in the senescence-accelerated mouse/PRONE8 (SAMP8) / Metalotioneina 3 atenuou a apoptose dos neurônios da região CA1 do hipocampo em camundongos PRONE8 com envelhecimento acelerado (SAMP8)

OBJECTIVE: Metallothionein 3 (MT-3) has been shown to protect against apoptotic neuronal death in the brains of patients with Alzheimer's disease. Zinc is a potent inhibitor of caspase-3 and its deficiency was found to promote apoptosis. Here, we measured the zinc and copper content in the brains of senescence-accelerated mouse/PRONE8 (SAMP8) and sought to investigate the effect of MT-3 on the apoptosis of neurons in the hippocampal CA1 region of these mice. METHOD: The zinc and copper content in the brain samples of SAMP8 and normal control SAMR1 mice were determined using an atomic absorption spectrophotometer. The mice were administered intraperitoneally for four weeks with MT-3 or MT1 and thereafter apoptosis was measured using the TUNEL method and the expression of anti-apoptotic protein Bcl-2 and proapoptotic protein Bax was examined by immunohistochemistry. RESULTS: Compared with that in SMAR1 mice, the content of zinc in the brains of SAMP8 mice was significantly reduced (P<0.05). Moreover, significant levels of apoptosis of neurons were observed in the hippocampus of SAMP8 mice, which, compared with those in SMAR1 mice, also showed significantly lower levels of Bcl-2 and higher levels of Bax (P<0.05). MT-3 increased zinc concentration in the hippocampus of SAMP8 mice and also significantly decreased apoptosis in these neurons dose-dependently and increased the levels of Bcl-2 and decreased the levels of Bax. CONCLUSION: MT-3 could attenuate apoptotic neuron death in the hippocampus of SAMP8, suggesting that the protein may lessen the development of neurodegeneration.

OBJETIVO: Metalotioneína 3 (MT-3) tem mostrado proteção contra a apoptose neuronal em cérebros de pacientes com doença de Alzheimer. Zinco é um potente inibidor da caspase-3, e sua deficiência pode promover a apoptose. No presente trabalho, foram dosados os níveis de zinco e cobre nos cérebros de camundongos PRONE8 com envelhecimento acelerado (SAMP8), visando investigar o efeito da MT-3 na apoptse dos neurônios da região hipocampal CA1 destes camundongos. MÉTODO: Os níveis de zinco e cobre em amostras cerebrais de camundongos SAMP8 e de controles normais SAMR1 foram determinados por absorção atômica em espectrofotometria. Foram administradas MT-3 ou MT-1 intraperitoneais durante quatro semanas, sendo em seguida avaliada a apoptose pelo método TUNEL , enquanto a expressão da proteína anti-apoptótica Bcl-2 e a proteína pró-apoptótica Bax foram avaliadas por imunohistoquímica. RESULTADOS: Em comparação aos camundongos SMAR1, o nível de zinco nas amostras cerebrais dos camundongos SAMP8 estava significativamente diminuído (P<0.05). Além disto, níveis significativos de apoptose foram observados no hipocampo dos camundongos SAMP8, o que, em comparação com os níveis em camundongos SMAR1, também mostrava níveis significativamente mais baixos de Bcl-2 e níveis mais altos de Bax (P<0.05). MT-3 aumentou a concentração de zinco no hipocampo dos camundongos SAMP8, além de diminuir significativamente a apoptose destes neurônios, de uma forma dose-dependente, ao mesmo tempo que aumentou níveis de Bcl-2 e diminuiu níveis de Bax. CONCLUSÃO: MT-3 pode atenuar a morte neuronal apoptótica no hipocampo de SAMP8, o que sugere que esta proteína possa diminuir a neurodegeneração.

The anorexigenic peptide cocaine-and-amphetamine-regulated transcript modulates rem-sleep in rats.

It is known that the sleep-waking cycle is modulated by several molecules that may also regulate food intake, among them several neuropeptides. The cocaine-and-amphetamine-regulated transcript has been studied in relation to food ingestion, but it seems to have several other functions that may include sleep regulation. In this context, we studied the effect of the intracerebroventricular administration of the cocaine-and-amphetamine-regulated transcript (0.15, 0.3, 0.6, 0.9nmol) on the sleep-waking cycle (12-h recordings), as well as its effect on food intake in rats. Additionally, we analyzed the neuronal activity as measured by c-Fos expression induced by the cocaine-and-amphetamine-regulated transcript in neurons of nuclei involved in the regulation of sleep and feeding behavior. Our main finding is that 0.3nmol of the cocaine-and-amphetamine-regulated transcript increases rapid-eye-movement sleep. In addition, our results further support that this neuropeptide triggers satiety; c-Fos expression suggested that the cocaine-and-amphetamine-regulated transcript activates specific hypothalamic nuclei without affecting other brain structures known to be involved in sleep regulation. These data further support the notion that a few neuropeptides are involved in the regulation of both the sleep-waking and the hunger-satiety cycles.

Complexin/synaptotagmin interplay controls acrosomal exocytosis.

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca(2+) and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca(2+). We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca(2+) and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.

An integrated approach to the characterization of cell movement.

BACKGROUND: Most phenomena in developmental biology involve or depend upon cell migration. This article describes a comprehensive framework for the characterization and analysis of trajectories defined by cell movement. The following two perspectives are considered: (a) the behavior of each individual cell and (b) interactions between neighboring pairs of cells. METHODS: The measurements considered for individual trajectories include the velocity magnitude and orientation, maximum spatial dispersion, displacement effectiveness, and displacement entropies. Interactions between two trajectories are characterized by comparing the respective velocities. RESULTS: The potential of the overall framework is illustrated using data of moving cells in different biological environments. The work shows that it is possible to use the new algorithm presented here to characterize cell motility. CONCLUSIONS: The features of the algorithm were successful in determining the motility changes under different experimental conditions.

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.

PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.

BDNF and NT-4 differentially modulate neurite outgrowth in developing retinal ganglion cells.

We show here that neurite outgrowth of ganglion cells (RGCs) was selectively enhanced following treatment with BDNF or NT-4 in short-term cultures of dissociated cells derived from the neuroretina of postnatal rats. NT-4 was more effective than BDNF. The effect of NT-3 was variable, whereas NGF and CNTF had no effects upon neurite elongation. The neuritogenic responses of RGCs to both BDNF and NT-4 were prevented by competition with soluble TrkB receptor, and abolished by K252a, a selective inhibitor of the tyrosine kinase activity of Trks. These results indicate that the differentiating effects of BDNF and NT-4 are mediated by TrkB receptors, naturally expressed by RGCs. Developing RGCs treated with these TrkB ligands displayed distinct, albeit partially overlapping, patterns of neurite morphology. BDNF supported predominantly polarized outgrowth, whereas NT-4 induced the appearance of intensely branched symmetrical arbors. The lack of RGCs showing combined morphologies (e.g., highly arborized unipolar cells) suggests distinct mechanisms underlying either elongation or branching, and implicates distinct responses of RGC subsets. We conclude that neurite growth in vitro is extensively promoted by neurotrophins in developing RGCs. Moreover, highly homologous neurotrophins such as BDNF and NT-4, presumably activating via TrkB receptors, selectively control the differentiation of distinct ganglion cell neuritic morphologies.

[Brain-derived growth factor: current aspects]. / Factor de crecimiento derivado del cerebro: aspectos de actualidad.

INTRODUCTION: The neurotrophins are a family of proteins which are closely related structurally and functionally and include nerve growth factor (NGF), brain derived nerve factor (BDNF) and neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and more recently neurotrophin-6 (NT-6). BDNF is one of the most important endogenous proteins for control of survival, growth and differentiation of certain neurone populations both in the peripheral and central nervous systems. DEVELOPMENT: The ARNmt of the BDNF is found in nearly all regions of the brain. The highest levels are those of the hippocampus and cerebral cortex. The large number of effects attributed to BDNF are probably due to the union of this protein to its specific receptor on the cell surface, which leads to the formation of a complex which enables transmission of the signal caused by activation of the specific neurone pathway. Since discovery, BDNF has been detected and/or measured by different methods from biological assay to the application of molecular biology techniques. These methods have permitted analysis of the biochemical characteristics of this factor and its behaviour in different tissues. CONCLUSIONS: In this paper we review the most relevant aspects of distribution, biological actions of BDNF on different neurone populations, its clinical usefulness in neurological disorders, routes of administration and side effects.

Ciliary neurotrophic factor stimulates in vivo myotube formation in mice.

It is known that ciliary neurotrophic factor (CNTF) administration reduces the atrophy observed with denervation, suggesting its role as a trophic factor for muscle cells. At the present, we studied the effects of 'in vivo' CNTF administration on the regenerative capacity of skeletal muscle fibres. Adult mice had their extensor digitorium longus muscle subjected to a denervation-devascularization lesion. CNTF (0.5 ng/microl) was administered using osmotic pumps implanted subcutaneously in unrestrained mice. CNTF was delivered into the muscle's region at a rate of 1 microl/h from 1 to 8 days after denervation. The results show that CNTF increased the number of regenerating myofibres by day 4. From day 7 on, the values seen on control and CNTF-treated groups were not significantly different. Our results show that 'in vivo' CNTF administration accelerates myotube differentiation.

Neurotrophin-3 enhances neurite outgrowth in cultured hippocampal pyramidal neurons.

Low density dissociated cultures of embryonic rat hippocampal cells were used to study the effects of neurotrophin-3 (NT-3) on neuronal morphogenesis. The results obtained indicate that NT-3 enhances neurite outgrowth and branching; this is a dose-dependent effect, detected in approximately 50% of the neurons, and prevented by K-252a, an inhibitor of the trk family of receptor protein kinases. NT-3 also accelerates the development of neuronal polarity, a phenomenon preceded by a dramatic accumulation of bundles of looped microtubules within axonal growth cones; these microtubule bundles contain tyrosinated, detyrosinated, and acetylated alpha-tubulin. Taken collectively, our data suggest that even though the basic shape of hippocampal neurons may be endogenously determined, critical aspects of their morphological development may be modulated by trophic factors such as NT-3. In addition, our observations suggest that at least some of the neuritogenic effects of NT-3 involve a stimulation of microtubule assembly and/or transport.

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina.

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.

Effects of crustacean hyperglycemic hormones from Carcinus maenas and Orconectes limosus on blood and muscle glucose and glycogen concentration of Chasmagnathus granulata.

The effects of purified crustacean hyperglycemic hormones (CHH) from Carcinus maenas or Orconectes limosus, and of eyestalk extract of Chasmagnathus granulata on the blood and muscle glucose and glycogen concentration of Chasmagnathus granulata were investigated. Different groups of animals (at least 7 animals per group) were injected with CHH from either C. maenas or O. limosus CHH dissolved in saline (16 pmol/animal) or crude eyestalk extract of C. granulata (1 eyestalk equivalent/animal). All injections had a volume of 10 microliters. Blood and muscle glucose and glycogen concentrations were determined immediately before the injections and after 30, 60 and 120 min. CHH administration from both species, as well as eyestalk extract, resulted in marked hyperglycemia. However, their effects were different. CHH from C. maenas also caused a decrease in the glycogen concentration of blood (from 89.8 +/- 4.3 to 76.6 +/- 3.1 mg/100 ml) and muscle (from 7.9 +/- 0.8 to 4.0 +/- 0.7 mg/g) and glucose concentration of muscle (from 2.4 +/- 0.3 to 1.2 +/- 0.2 mg/g). CHH from O. limosus caused an increase of glycogen concentration of muscle (from 4.9 +/- 1.1 to 9.0 +/- 1.1 mg/g). The injection of eyestalk extract resulted also in a decrease of hemolymph glycogen (from 157.7 +/- 20.6 to 30.2 +/- 7.7 mg/100 ml). Therefore, C. granulata may have different receptors for CHH in its different tissues, and/or in the same tissue, which act through different metabolic pathways to achieve the same final result, i.e., hyperglycemia.