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The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardio-vascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for [[beta].sub.1] subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3-4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor. cardiovascular | knockout mice | cGMP | platelet aggregation | smooth muscle relaxation

Proper functioning of the human circadian timing system is crucial to physical and mental health. Much of what we know about this system is based on experimental protocols that induce the desynchronization of behavioral and physiological rhythms within individual subjects, but the neural (or extraneural) substrates for such desynchronization are unknown. We have developed an animal model of human internal desynchrony in which rats are exposed to artificially short (22-h) light--dark cycles. Under these conditions, locomotor activity, sleep--wake, and slow-wave sleep (SWS) exhibit two rhythms within individual animals, one entrained to the 22-h light--dark cycle and the other free-running with a period >24 h ([tau]>24 h). Whereas core body temperature showed two rhythms as well, further analysis indicates this variable oscillates more according to the [tau]>24 h rhythm than to the 22-h rhythm, and that this oscillation is due to an activity-independent circadian regulation. Paradoxical sleep (PS), on the other hand, shows only one free-running rhythm. Our results show that, similarly to humans, (i) circadian rhythms can be internally dissociated in a controlled and predictable manner in the rat and (ii) the circadian rhythms of sleep--wake and SWS can be desynchronized from the rhythms of PS and core body temperature within individual animals. This model now allows for a deeper understanding of the human timekeeping mechanism, for testing potential therapies for circadian dysrhythmias, and for studying the biology of PS and SWS states in a neurologically intact model. suprachiasmatic

Noncoplanar polychlorinated biphenyls (PCBs) are widely dispersed in human environment and tissues. Here, an exemplar noncoplanar PCB was fed to rat dams during gestation and throughout three subsequent nursing weeks. Although the hearing sensitivity and brainstem auditory responses of pups were normal, exposure resulted in the abnormal development of the primary auditory cortex (A1). A1 was irregularly shaped and marked by internal nonresponsive zones, its topographic organization was grossly abnormal or reversed in about half of the exposed pups, the balance of neuronal inhibition to excitation for A1 neurons was disturbed, and the critical period plasticity that underlies normal postnatal auditory system development was significantly altered. These findings demonstrate that developmental exposure to this class of environmental contaminant alters cortical development. It is proposed that exposure to noncoplanar PCBs may contribute to common developmental disorders, especially in populations with heritable imbalances in neurotransmitter systems that regulate the ratio of inhibition and excitation in the brain. We conclude that the health implications associated with exposure to noncoplanar PCBs in human populations merit a more careful examination. autism | brain development | cortical maps | environment | sensory systems

Adult mice carrying a null mutation of the prostanoid receptor EP3R (EP3[R.sup.-/-] mice) exhibit increased frequency of feeding during the light cycle of the day and develop an obese phenotype under a normal fat diet fed ad libitum. EP3[R.sub.-/-] mice show increased motor activity, which is not sufficient to offset the increased feeding leading to increased body weight. Altered 'nocturnal' activity and feeding behavior is present from a very early age and does not seem to require age-dependent factors for the development of obesity. Obesity in EP3[R.sub.-/-] mice is characterized by elevated leptin and insulin levels and >20% higher body weight compared with WT littermates. Abdominal and subcutaneous fat and increased liver weight account for the weight increase in EP3[R.sub.-/-] mice. These observations expand the roles of prostaglandin [E.sub.2] signaling in metabolic regulation beyond the reported stimulation of leptin release from adipose tissue to involve actions mediated by EP3R in the regulation of sleep architecture and feeding behavior. The findings add to the growing literature on links between inflammatory signaling and obesity. body weight | insulin | leptin | prostanoid receptor | signaling

Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar ( adult stemcells | embryos | cloning | keratinocyte | nuclear transfer

Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-[kappa]B activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 [micro]M, and are adjustably shut off at biotin levels exceeding 10 [micro]M (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing. biological circuit | biopharmaceutical manufacturing | gene switch | synthetic biology | biotin

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal [Ca.sup.2+]/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK 'knockdown' mice in which CASK expression was suppressed by [approximately equal to] 70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/ Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a [Ca.sup.2+]/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, [Ca.sup.2+]-triggered presynaptic release, or postsynaptic receptor functions. CaM kinase | MAGUK | neurexin | neurotransmitter release | synapse

Fragile X syndrome is a common heritable form of mental retardation in humans. Recent neuroanatomical studies indicate an apparent immature appearance of neurons in fragile X syndrome patients and fragile X mental retardation protein (FMRP)-knockout mice, an animal model of this condition. In this work, we investigated possible alterations in synaptic plasticity in the neocortex of FMRP-knockout mice. Extracellular field potentials were recorded from the deep-layer visual neocortex. Long-term potentiation (LTP) was severely attenuated in brain slices from knockout mice relative to that observed in slices from wild-type mice. Considering that neocortical LTP can involve both NMDA receptor-dependent and -independent mechanisms, we attempted to distinguish the nature of LTP attenuated in the knockout condition. In slices from wild-type mice, LTP was partially attenuated by the NMDA receptor antagonist 3-[([+ or -])-2-carboxypiperazin-4-yl]-propyl-1-phosphate (CPP); however, the general metabotropic glutamate receptor (mGluR) antagonist [alpha]-methyl-4-carboxyphenylglycine (MCPG) strongly attenuated LTP, resulting in a response indistinguishable from that observed in slices from knockout mice. The selective mGluRs antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) attenuated LTP to a similar degree as did MCPG in wild-type slices, but MPEP did not alter the reduced potentiation in knockout slices. Our results suggest that LTP in layer V visual neocortex depends primarily on mGlu[R.sub.5] activation. Our data also indicate that mGlu[R.sub.5]mediated synaptic plasticity is absent in the neocortex of FMRP-knockout mice. Such an alteration may contribute to the cognitive and learning deficits exhibited in these mice as well as in fragile X syndrome. fragile X mental retardation protein | long-term potentiation | synaptic plasticity | visual cortex

There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with late-onset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/ SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations. vaccination | immunotherapy | antibody

Type I diabetes in both humans and nonobese diabetic (NOD) mice results from autoreactive T cell destruction of insulin-producing cells. Cure of type 1 diabetes may require both reversal of autoimmunity and regeneration of [beta] cells. Induction of chimerism via allogeneic hematopoietic cell transplantation has been shown to reestablish tolerance in both prediabetic and diabetic NOD mice. However, it is unclear whether this therapy augments [beta] cell regeneration. Furthermore, this procedure usually requires total body irradiation conditioning of recipients. The toxicity of total body irradiation conditioning and potential for graft-versus-host disease (GVHD) limit the application of allogeneic hematopoietic cell transplantation for treating type 1 diabetes. Here we report that injection of donor bone marrow and [CD4.sup.+] T cell-depleted spleen cells induced chimerism without causing GVHD in overtly diabetic NOD mice conditioned with anti-CD3/CD8 and that induction of chimerism in new-onset diabetic NOD mice led to elimination of insulitis, regeneration of host [beta] cells, and reversal of hyperglycemia. Therefore, this radiation-free GVHD preventive approach for induction of chimerism may represent a viable means for reversing type 1 diabetes. hematopoietic cell transplantation | type 1 diabetes | anti-CD3-conditioning | reversal of diabetes | reversal of autoimmunity

Mast cells are immune sentinels that participate in the defense against bacteria and parasites. Resident within the joint, mast cells become activated in human rheumatoid arthritis and are implicated in the pathogenesis of experimental murine synovitis. However, their arthritogenic role remains undefined. Using a model of autoantibody-induced arthritis, we show that mast cells contribute to the initiation of inflammation within the joint by elaboration of IL-1. Mast cells become activated to produce this cytokine via the IgG immune complex receptor Fc[gamma]/RIII. Interestingly, mast cells become dispensable for the perpetuation of arthritis after delivery of IL-1, highlighting the contribution of this lineage to arthritis induction. These findings illuminate a mechanism by which mast cells can participate in the pathogenesis of autoimmune inflammatory arthritis and provide insights of potential relevance to human rheumatoid arthritis. cytokine | mouse model | synovitis

Sleep is one of the most noticeable and widespread phenomena occurring in multicellular animals. Nevertheless, no consensus for a theory of its origins has emerged. In particular, no explicit, quantitative theory exists that elucidates or distinguishes between the myriad hypotheses proposed for sleep. Here, we develop a general, quantitative theory for mammalian sleep that relates many of its fundamental parameters to metabolic rate and body size. Several mechanisms suggested for the function of sleep can be placed in this framework, e.g., cellular repair of damage caused by metabolic processes as well as cortical reorganization to process sensory input. Our theory leads to predictions for sleep time, sleep cycle time, and rapid eye movement time as functions of body and brain mass, and it explains, for example, why mice sleep [approximately equal to] 14 hours per day relative to the 3.5 hours per day that elephants sleep. Data for 96 species of mammals, spanning six orders of magnitude in body size, are consistent with these predictions and provide strong evidence that time scales for sleep are set by the brain's, not the whole-body, metabolic rate. allometric scaling | brain | cellular repair | metabolic rate | sleep times

Notch-mediated induction of Nodal at the vertebrate node is a critical step in initiating left-right (LR) asymmetry. In mice and zebrafish we show that Baf60c, a subunit of the Swi/Snf-like BAF chromatin remodeling complex, is essential for establishment of LR asymmetry. Baf60c knockdown mouse embryos fail to activate Nodal at the node and also have abnormal node morphology with mixing of crown and pit cells. In cell culture, Baf60c is required for Notch-dependent transcriptional activation and functions to stabilize interactions between activated Notch and its DNA-binding partner, RBP-J. Brg1 is also required for these processes, suggesting that BAF complexes are key components of nuclear Notch signaling. We propose a critical role for Baf60c in Notch-dependent transcription and LR asymmetry. chromatin | Swi/Snf | node

Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the [alpha]-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin ([V.sub.actin]) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower [V.sub.actin] in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated. animal models | biophysics | genetics | single molecule | laser trap

Neurons of the cerebral neocortex in mammals, including humans, are generated during fetal life in the proliferative zones and then migrate to their final destinations by following an inside-to-outside sequence. The present study examined the effect of ultrasound waves (USW) on neuronal position within the embryonic cerebral cortex in mice. We used a single BrdU injection to label neurons generated at embryonic day 16 and destined for the superficial cortical layers. Our analysis of over 335 animals reveals that, when exposed to USW for a total of 30 min or longer during the period of their migration, a small but statistically significant number of neurons fail to acquire their proper position and remain scattered within inappropriate cortical layers and/or in the subjacent white matter. The magnitude of dispersion of labeled neurons was variable but systematically increased with duration of exposure to USW. These results call for a further investigation in larger and slower-developing brains of non-human primates and continued scrutiny of unnecessarily long prenatal ultrasound exposure. brain malformations | cerebral cortex | embryonic development

The perirhinal cortex and hippocampus have close anatomical links, and it might, therefore, be predicted that they have close, interlinked roles in memory. Lesion studies have, however, often failed to support this prediction, providing dissociations and double dissociations between the two regions on tests of object recognition and spatial memory. In a series of rat studies we have compared these two regions using the expression of the immediate early gene c-fos as a marker of neuronal activity. This gene imaging approach makes it possible to assess the relative involvement of different brain regions and avoids many of the limitations of the lesion approach. A very consistent pattern of results was found as the various hippocampal subfields but not the perirhinal cortex show increased c-fos activity following tests of spatial learning. In contrast, the perirhinal cortex but none of the hippocampal subfields show increased c-fos activity when presented with novel rather than familiar visual objects. When novel scenes are created by the spatial rearrangement of familiar objects it is the hippocampus and not the perirhinal cortex that shows c-fos changes. This double dissociation for gene expression accords with that found from lesion studies and highlights the different contributions of the perirhinal cortex and hippocampus to memory.

Investigation of the anatomical substructure of the medial temporal lobe has revealed a number of highly interconnected areas, which has led some to propose that the region operates as a unitary memory system. However, here we outline the results of a number of studies from our laboratories, which investigate the contributions of the rat's perirhinal cortex and postrhinal cortex to memory, concentrating particularly on their respective roles in memory for objects. By contrasting patterns of impairment and spared abilities on a number of related tasks, we suggest that perirhinal cortex and postrhinal cortex make distinctive contributions to learning and memory: for example, that postrhinal cortex is important in learning about within-scene position and context. We also provide evidence that despite the strong connectivity between these cortical regions and the hippocampus, the hippocampus, as evidenced by lesions of the fornix, has a distinct function of its own--combining information about objects, positions, and contexts.

Changes in the lungs due to smoking include inflammation, epithelial damage, and remodeling of the airways. Airway inflammation is likely to play a critical role in the genesis and progression of tobacco smoke-induced airway disease. Soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous chemical mediators that play an important role in inflammation. Epoxyeicosatrienoic acids (EETs) have demonstrated antiinflammatory properties, and hydrolysis of these epoxides by sEH is known to diminish this activity. To examine whether acute tobacco smokeinduced inflammation could be reduced by a sFH inhibitor, 12-(3-adamantane-1-yl-ureido)-dodecanoic acid n-butyl ester was given by daily s.c. injection to spontaneously hypertensive rats exposed to filtered air or tobacco smoke for a period of 3 days (6 h/day). Acute exposure to tobacco smoke significantly increased by 3.2-fold (P < 0.05) the number of cells recovered by bronchoalveolar lavage. The sEH inhibitor significantly decreased total bronchoalveolar lavage cell number by 37% in tobacco smoke-exposed rats with significant reductions noted in neutrophils, alveolar macrophages, and lymphocytes. A combination of sEH inhibitor and EETs was more significant in its ability to further reduce tobacco smoke-induced inflammation compared with the sEH inhibitor alone. The sEH inhibitor led to a shift in some plasma epoxides and diols that are consistent with the hypothetical action of these compounds. We conclude that an sEH inhibitor, in the presence or absence of EETs, can attenuate, in part, inflammation associated with acute exposure to tobacco smoke. epoxyeicosatrienoic acids | pulmonary | antiinflammatory

A number of recent conditioned taste aversion (CTA) experiments have demonstrated a super-latent inhibition (LI) effect--namely, a time-induced increase in the effects of stimulus preexposure when the interval between acquisition and test is spent in a context that is different from the other experimental contexts. Two CTA experiments with rats were conducted to examine the role of primacy in producing super LI. In Experiment 1, one of two flavours was preexposed, following which a second flavour was preexposed. After the second preexposure, animals were conditioned by pairing a compound of the two preexposed flavours with LiCl. The test stage was conducted l or 21 days alter conditioning, with the interval being spent in either the same or different contexts. In the test, animals were confronted with two bottles, each with one of the two preexposed flavours. Super-LI was obtained only for the first preexposed flavour in the 21-day delay group that spent the interval in a different context. Experiment 2 was designed to ensure that the effects in Experiment 1 represented LI, and to control for order of presentation of the flavours and time between preexposure and acquisition. The results replicated those of Experiment 1. The two experiments support the importance of primacy in the general super-LI experiment where CS alone preexposure precedes CS-US.

Byline: B. N. Akifiev (1), E. B. Dizhe (1), A. M. Efremov (2), D. A. Mogilenko (1,2), G. N. Oleinikova (1), I. A. Lapikov (1,2), O. Yu. Zhdanova (1), O. V. Kidgotko (1), S. V. Orlov (1,2), A. P. Perevozchikov (1,2) Keywords: C57Bl/6 mice; intravenous hydrodynamic injections; plasmid human apolipoprotein A-I gene expression vectors; gene therapy; significance of the exon--intron gene structure for long-term expression in vivo Abstract: Human apolipoprotein A-I gene (apoA-I) inserted into a plasmid expression vector was transferred in vivo into C57Bl/6 mice using hydrodynamic injections into the tail vein. Two types of plasmid expression vectors were used: (1) pCMVcapoAI which contained cDNA of apoA-I driven by the human cytomegalovirus (CMV) early gene promoter and (2) pAlg, which contained a genomic locus of intron-containing apoA-I driven by its own extended 5 -regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to the appearance of human apoA-I mRNA in the liver and human ApoA-I protein in the serum of injected mice. The dynamics of human ApoA-I content in the sera of mice injected with pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, the concentration of human ApoA-I in mouse serum was maximal one day after injection and decreased to zero within the next two weeks. In the case of pAlg, the content of human ApoA-I in serum was maximal (up to 20 ug/ml) on days 5--7 after injection and then gradually decreased for several months (six months after injection, for example, it decreased to 25% of the maximal value). Experiments on 'saved' pAlg plasmid isolated from the nuclei of hepatocytes 50 days after injection showed that the plasmid was retained for a long time in the form of an episome. A significant content of human ApoA-I in serum and its long-term persistence after injecting mice with pAlg may be accounted for by the properties of APOAI and/or the exon--intron structure of the apoA-I gene. Injecting mice with different variants of APOAI coupled with the luciferase gene did not lead to long-term expression of luciferase in the liver. It is concluded that the presence of introns in the apoA-I gene is required for its efficient and long-term expression after transfer to mice by means of hydrodynamic injections. Author Affiliation: (1) Institute of Experimental Medicine, Russian Academy of Medical Sciences, St. Petersburg, 197376, Russia (2) St. Petersburg State University, St. Petersburg, 199034, Russia Article History: Registration Date: 06/01/2005

Because [delta]-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5- (4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA ([10.sup.-9] M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v, also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v, instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats. AM251 | CB1 receptor | medial basal hypothalamus

We characterize the survival, migration, and differentiation of human neurospheres derived from CNS stem cells transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion. Transplanted neurospheres survived robustly in naive and ischemic brains 4 wk posttransplant. Survival was influenced by proximity of the graft to the stroke lesion and was negatively correlated with the number of IB4-positive inflammatory cells. Targeted migration of the human cells was seen in ischemic animals, with many human cells migrating long distances ([approximately equal to]1.2 mm) predominantly toward the lesion; in naive rats, cells migrated radially from the injection site in smaller number and over shorter distances (0.2 mm). The majority of migrating cells in ischemic rats had a neuronal phenotype. Migrating cells between the graft and the lesion expressed the neuroblast marker doublecortin, whereas human cells at the lesion border expressed the immature neuronal marker [beta]-tubulin, although a small percentage of cells at the lesion border also expressed glial fibrillary acid protein (GFAP). Thus, transplanted human CNS (hCNS)-derived neurospheres survived robustly in naive and ischemic brains, and the microenvironment influenced their migration and fate.

In young animals, monocular deprivation leads to an ocular dominance shift, whereas in adults after the critical period there is no such shift. Chondroitin sulphate proteoglycans (CSPGs) are components of the extracellular matrix (ECM) inhibitory for axonal sprouting. We tested whether the developmental maturation of the ECM is inhibitory for experience-dependent plasticity in the visual cortex. The organization of CSPGs into perineuronal nets coincided with the end of the critical period and was delayed by dark rearing. After CSPG degradation with chondroitinase-ABC in adult rats, monocular deprivation caused an ocular dominance shift toward the nondeprived eye. The mature ECM is thus inhibitory for experience-dependent plasticity, and degradation of CSPGs reactivates cortical plasticity., Cortical circuits are sensitive to experience during well-defined intervals of early postnatal development called critical periods (1). After the critical period, plasticity is reduced or absent. Monocular deprivation (MD) is [...]

Byline: I. I. Astapova (1), A. N. Smirnov (2), P. M. Rubtsov (1) Keywords: exon--intron structure of genes; gene duplication; 5'RACE; alternative splicing; repetitive elements of genome; ID elements; PCR Abstract: Estrogen sulfotransferases (STE) are members of a large superfamily of sulfotransferases. The expression of rat Ste genes is regulated in a tissue- and sex-specific manner. The cDNAs of two closely related rat estrogen sulfotransferases have been cloned earlier. By PCR performed on rat genomic DNA, we have amplified fragments of two estrogen sulfotransferase genes, Ste1 and Ste2, and established their exon--intron organization. By rapid amplification of the 5'-terminal cDNA sequences, the two variants of 5'-untranslated regions of both Ste1 and Ste2 mRNA were found. These variants are produced through transcription initiation from sites located in front of the alternative exons 1a and 1b and alternative splicing. Intron 1 of Ste1 gene contains the repeated element of ID family inserted after duplication of the ancestor gene. Author Affiliation: (1) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia (2) Biological Faculty, Moscow State University, Moscow, 119899, Russia Article History: Registration Date: 09/10/2004

Byline: L. E. Panin (1), F. V. Tuzikov (2), N. A. Tuzikova (2), O. I. Gimautdinova (1), L. M. Polyakov (1) Keywords: cortisol; tetrahydrocortisol; lipoproteins; apolipoprotein A-I; DNA; oligonucleotides; small-angle X-ray scattering Abstract: The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC--ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC).sub.n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (K .sub.a) was 1.66*10.sup.6 M.sup.--1. Substitution of THC with cortisol considerably decreases the K .sub.a. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair. Author Affiliation: (1) Institute of Biochemistry, Siberian Division, Russian Academy of Medical Sciences, Novosibirsk, 630117, Russia (2) Institute of Molecular Biology, State Research Center for Virology and Biotechnology VECTOR, Russian Ministry of Health, Kol'tsovo, Novosibirsk Region, 633159, Russia Article History: Registration Date: 09/10/2004

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by [B.sub.2] bradykinin and [M.sub.1] muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the I[P.sub.3] receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing I[P.sub.3] to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [[[Ca.sup.2+]].sub.i] rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [[[Ca.sup.2+]].sub.i] were blocked by depletion of [Ca.sup.2+] internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing [Ca.sup.2+] from I[P.sub.3]-sensitive [Ca.sup.2+] stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

In the present study, high levels of peptidylglycine [Alpha]-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive [Alpha]-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17[Beta]-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17[Beta]-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

Neu differentiation factor (NDF/neuregulin) is widely expressed in the central and peripheral nervous systems, where it functions as a mediator of the interactions between nerve cells and Schwann, glia, oligodendrocyte, and muscle cells, to control cellular proliferation, differentiation, and migration. NDF binds to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Here we demonstrate that NDF and its ErbB-4 receptor are highly reactive to changes in ambient neuronal activity in the rodent brain in a region-selective manner. Generation of epileptic seizures by using kainic acid, a potent glutamate analog, elevated levels of NDF transcripts in limbic cortical areas, hippocampus, and amygdala. Concomitantly, ErbB-4 mRNA was increased with a similar spatial distribution, but transcription of the other NDF receptor, ErbB-3, did not change. A more moderate stimulation, forced locomotion, was accompanied by an increase in NDF transcripts and protein in the hippocampus and in the motor cortex. Similar changes were found with ErbB-4, but not ErbB-3. Last, a pathway-specific tetanic stimulation of the perforant path, which produced long-term potentiation, was followed by induction of NDF expression in the ipsilateral dentate gyrus and CA3 area of the hippocampus. Taken together, these results indicate that NDF is regulated by physiological activity and may play a role in neural plasticity.

Rats learn a novel foraging pattern better with their right-side whiskers than with their left-side whiskers. They also learn better with the left cerebral hemisphere than with the right hemisphere. Rotating an already reamed maze relative to the external environment most strongly reduces right-whisker performance: starting an already reamed maze at a different location most strongly reduces left-whisker performance. These results suggest that the right-periphery-left-hemisphere system accesses a map-like representation of the foraging problem, whereas the left-periphery-right-hemisphere system accesses a rote path. Thus, as in humans, functional asymmetries in rats can be elicited by both peripheral and cortical manipulation, and each hemisphere makes qualitatively distinct contributions to a complex natural behavior., Cerebral and peripheral sensory-motor asymmetries in humans have been a central theoretical topic in cognitive neuroscience for more than a century. Today's theories of asymmetries converge on three main ideas [...]

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood-brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-[Beta]-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-[Beta]-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

In order to test experimentally a novel model for myogenic contraction of the arterioles, presented in Part I of this series, a sequence of in-vivo studies was performed on arterioles of rat cremaster muscle using a modified 'Box Method.' The tissue was enclosed in a sealed chamber in which the extravascular pressure could be changed as a means of stimulating the myogenic response. The microvascular pressures were measured using an improved cremaster preparation with intact distal feeder. The experiment consists of measurements of a static myogenic response and the response to a step extravascular pressure, as well as passive viscoelastic properties of the arteriole. These measurements served to determine the parameters involved in the theoretical model. The model prediction were then compared with in-vivo observations during a ramp and during oscillatory extravascular pressure changes in the same arterioles. The results indicate that the model is capable of quantitatively predicting time dependent in-vivo changes in response to transmural pressure. The measured model parameters suggest an increase in myogenic activity from proximal arcade arteriotes to more distal transverse arterioles in cremaster muscle of Wistar rats.

The N-methyl-D-aspartate (NMDA) receptor-mediated long-term potentiation (LTP) is not essential for normal spatial learning. Both the non-spatially pretrained and the control rats give efficient watermaze performance. The NMDA receptor antagonist 2R,4R,5S-2-amino-4,5-(1,2-cyclo hexyl)-7-phosphonoheptano acid inhibits the dentate gyrus LTP in rats but has no effect on the normal spatial learning in watermaze.

D(-)-2-amino-5-phosphonovaleric acid (AP5) inhibits learning and memory by antagonizing the N-methyl-D-aspartate (NMDA) receptor in rats. The inhibitory effect of this antagonist on the hippocampal long-term potentiation and spatial learning is eliminated by pretraining the rats in a different watermaze. Pretraining of the rats with a non-spatial learning does not improve the learning impairment caused by AP5. This indicates that the NMDA receptors may not be associated with the encoding of spatial representation of individual environments.

The kinetics of graft rejection and severity of intragraft inflammatory responses are analyzed. Semiquantitative competitive reverse transcriptase-PCR and the immunohistology in a rat model of chronic rejection of renal allografts are assessed. The rejection of kidney allografts in rats is the result of the up-regulation of local macrophages products such as, MCP-1, IL-6 and nitric oxide synthase. The level of TGF-beta is enhanced in allografts after 16 and 32 weeks and is related to fibrosis and tubular atrophy.

Byline: R. L. Bogorad (1), V. S. Smyslova (1), A. N. Smirnov (1), P. M. Rubtsov (2), O. V. Smirnova (1) Keywords: hepatocytes; prolactin receptors; isoforms; RT-PCR; obstructive cholestasis Author Affiliation: (1) Biological Faculty, Moscow State University, Moscow, 119899, Russia (2) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia Article History: Registration Date: 09/10/2004

Thyroid hormone (triiodothyronine or T3) is known to be involved in energy metabolism and growth, and it exerts its action through binding to specific transcription factors that regulate gene expression. [...]

Pyramidal cells in layer V of the neocortex form a major excitatory projection system in the brain. Their activity is modulated by a network of excitatory and inhibitory inputs from [...]

The conditioning of cocaine's subjective actions with environmental stimuli may be a critical factor in long-lasting relapse risk associated with cocaine addiction. To study the significance of learning factors in persistent addictive behavior as well as the neurobiological basis of this phenomenon, rats were trained to associate discriminative stimuli ([S.sup.D]) with the availability of i.v. cocaine vs. nonrewarding saline solution, and then placed on extinction conditions during which the i.v. solutions and [S.sup.D]s were withheld. The effects of reexposure to the [S.sup.D] on the recovery of responding at the previously cocaine-paired lever and on Fos protein expression then were determined in two groups. One group was tested immediately after extinction, whereas rats in the second group were confined to their home cages for an additional 4 months before testing. In both groups, the cocaine [S.sup.D], but not the non-reward [S.sup.D], elicited strong recovery of responding and increased Fos immunoreactivity in the basolateral amygdala and medial prefrontal cortex (areas Cg1/Cg3). The response reinstatement and Fos expression induced by the cocaine [S.sup.D] were both reversed by selective dopamine [D.sub.1] receptor antagonists. The undiminished efficacy of the cocaine [S.sup.D] to elicit drug-seeking behavior after 4 months of abstinence parallels the long-lasting nature of conditioned cue reactivity and cue-induced cocaine craving in humans, and confirms a significant role of learning factors in the long-lasting addictive potential of cocaine. Moreover, the results implicate [D.sub.1]-dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala as substrates for cocaine-seeking behavior elicited by cocaine-predictive environmental stimuli.

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in [Mg.sup.2+]-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or [Mg.sup.2+]-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Gene transfer technology shows promise for the study and treatment of seizure disorders in which altered GABA function is implicated. The present study examined the effects of blocking or enhancing [...]

The purpose of this study was to determine the effect of aging on skeletal inflammatory cytokine factors related to muscle regeneration after overload in young (5M). and aged (25M) male [...]

Long-Term Neuropathological and Neurochemical Effects of Nucleus Basalis Lesions in the Rat ALZHEIMER'S DISEASE (AD), THE most common cause of dementia in the elderly, is consistently associated with a loss [...]

Expression of Functional Cell-Cell Channels from Cloned Rat Liver Gap Junction Complementary DNA IT IS GENERALLY ASSUMED THAT GAPjunctions represent assemblies of cell-cell channels. This is based on several lines [...]

Transformation of Rat Fibroblasts by FSV Rapidly Increases Glucose Transporter Gene Transcription ONCOGENIC TRANSFORMATION BYviruses involves complex changes in the expression of numerous cellular genes (1). Critical to the understanding [...]

Decreased Hippocampal Inhibition and a Selective Loss of Interneurons in Experimental Epilepsy IN HUMAN EPILEPSY, AN EPILEPTIC'focus,' made up of an aggregate of abnormally discharging cells, develops as a result [...]

Delayed Transneuronal Death of Substantia Nigra Neurons Prevented by gamma-Aminobutyric Acid Agonist WITHIN THE CENTRAL NERVOUSsystem (CNS), neurons may die when deprived of their afferent input (1). This form of [...]

Suprachiasmatic Nucleus Vasopressin Messenger RNA: Circadian Variation in Normal and Brattleboro Rats CONCENTRATIONS OF THE NEURO-peptide vasopressin in the cerebrospinal fluid (CSF) vary in a circadian rhythm, with morning levels [...]

Trigeminal deafferentation, which removes somatosensation selectively from the mouth and face, severely disrupts food intake in rats, cats, and pigeons (1-6). The disruption is not attributable solely to a simple [...]