Rats

We previously reported a solid-phase extraction (SPE) method for determination of the neurotoxin domoic acid (DA) in both seawater and phytoplankton by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose of sample desalting without DA pre-concentration. In the present study, we optimized the SPE procedure with seawater and phytoplankton samples directly acidified with aqueous formic acid without addition of organic solvents, which allowed sample desalting and also 20-fold pre-concentration of DA in seawater and phytoplankton samples. In order to reduce MS contamination, a diverter valve was installed between LC and MS to send the LC eluant to waste, except for the 6-min elution window bracketing the DA retention time, which was sent to the MS. Reduction of the MS turbo gas temperature also helped to maintain the long-term stability of MS signal. Recoveries exceeded 90% for the DA-negative seawater and the DA-positive cultured phytoplankton samples spiked with DA. The SPE method for DA extraction and sample clean-up in seawater was extended to mammalian fluids and tissues with modification in order to accommodate the fluid samples with limited available volumes and the tissue extracts in aqueous methanol. Recoveries of DA from DA-exposed laboratory mammalian samples (amniotic fluid, cerebrospinal fluid, plasma, placenta, and brain) were above 85%. Recoveries of DA from samples (urine, feces, intestinal contents, and gastric contents) collected from field stranded marine mammals showed large variations and were affected by the sample status. The optimized SPE-LC-MS method allows determination of DA at trace levels (low pg mL −1 ) in seawater with/without the presence of phytoplankton. The application of SPE clean-up to mammalian fluids and tissue extracts greatly reduced the LC column degradation and MS contamination, which allowed routine screening of marine mammalian samples for confirmation of DA exposure and determination of fluid and tissue DA concentrations in experimental laboratory animals.

The purpose of this paper is to present the experimental device and the work in progress performed in search for objective organic correlation of damage to hearing, examining possible acoustic otofunctional effects on the cochlear epithelium of the rat due to exposure to microwaves (900 MHz). Two experiments using male Sprague-Dawley rats were carried out with a far-field exposure in a cubic chamber. No statistically significant evidence was obtained at both specific absorption rate (SAR) values. The exposure system and the diagnostic apparatus are extremely useful to investigate a potential effect on the auditory system: however, with the parameters applied in these experiments, no evidence was observed.

Non-invasive vaccine delivery is a top priority for public health agencies because conventional immunization practices are unsafe and associated with numerous limitations. Recently, the skin has emerged as a potential alternative route for non-invasive delivery of vaccine. Topical immunization (TI), introduction of antigen through topical application onto the intact skin, has many practical merits compared to injectable routes of administration. One of the possibilities for increasing the penetration of bioactives through the skin is the use of vesicular systems. Specially designed lipid vesicles are attracting intense attention and can be used for non-invasive antigen delivery. In the present study, elastic vesicle transfersomes, non-ionic surfactant vesicles (niosomes) and liposomes were used to study their relative potential in non-invasive delivery of tetanus toxoid (TT). Transfersomes, niosomes and liposomes were prepared and characterized for shape, size and entrapment efficiency. These vesicles were extruded through polycarbonate filter (50-nm pore size) to assess the elasticity of the vesicles. The immune stimulating activity of transfersomes, niosomes and liposomes were studied by measuring the serum anti-TT IgG titre following topical immunization. The immune response elicited by topical immunization was compared with that elicited by same dose of alum-adsorbed tetanus toxoid (AATT) given intramuscularly. The results indicate that optimal formulations of transfersomes, niosomes and liposomes could entrap 72.7 ± 3.4, 42.5 ± 2.4 and 41.3 ± 2.2% of antigen and their elasticity values were 124.4 ± 4.2, 29.3 ± 2.4 and 21.7 ± 1.9, respectively. In vivo study revealed that topically given TT containing transfersomes, after secondary immunization, could elicit immune response (anti-TT-IgG) that was equivalent to one that produced following intramuscularly alum-adsorbed TT-based immunization. In comparison to transfersomes, niosomes and liposomes elicited weaker immune response. Thus transfersomes hold promise for effective non-invasive topical delivery of antigen(s).

Endometriosis is a painful disorder defined by extrauteral endometrial growths whose contribution to pain symptoms is poorly understood. Endometriosis is created in rats by autotransplanting on abdominal arteries pieces of either uterus (ENDO), which form cysts, or fat (shamENDO), which do not form cysts. ENDO, but not shamENDO induces vaginal hyperalgesia. We tested the hypothesis that the cysts are necessary to maintain vaginal hyperalgesia by assessing the effect of surgically removing them. Complete cyst removal eliminated ENDO-induced vaginal hyperalgesia up to four months post-operatively. Sham-cyst-removal in ENDO rats, in which cysts were not removed, or partial cyst-removal increased the ENDO-induced hyperalgesia. The decreases and increases both took three to six weeks to develop. Changes in ENDO-induced hyperalgesia did not occur in a control group of ENDO rats who had no surgery after ENDO. In a double-surgery control group, neither shamENDO surgery nor a subsequent sham surgery that mimicked "removal" of non-existent cysts influenced vaginal nociception. In a no-surgery control group, vaginal nociception remained stable for > six months. The increases in ENDO-induced hyperalgesia produced by the sham-cyst-removal surgery were smaller in proestrus than in other estrous stages. During the other stages (but not during proestrus), sympathetic innervation of the cysts increased. These results suggest that maintenance of ENDO-induced vaginal hyperalgesia requires continued presence of at least some ectopic endometrial tissue, and that surgical treatment that fails to remove ectopic endometrial tissue can exacerbate the hyperalgesia, possibly due in part to an increase in the cysts' sympathetic innervation.

Intracellular recordings were obtained from pyramidal neurons in layer 5 of rat somatosensory and visual cortical slices maintained in vitro. When directly depolarized, one subclass of pyramidal neurons had the capacity to generate intrinsic burst discharges and another generated regular trains of single spikes. Burst responses were triggered in an all-or-none manner from depolarizing afterpotentials in most bursting neurons. Regular spiking cells responded to electrical stimulation of ascending afferents with a typical EPSP-IPSP sequence, whereas IPSPs were hard to detect in bursting cells. Orthodromic activation of the latter evoked a prominent voltage-dependent depolarization that could trigger a burst response. Intracellularly labelled bursting and regular spiking cells were located in layer 5b, but had distinctly different morphologies. Bursting neurons had a large pyramidal soma, a gradually emerging apical dendrite, and an extensive apical and basal dendritic tree. Their axonal collateral arborization was predominantly limited to layers 5/6. In contrast, regular spiking cells had a more rounded soma with abruptly emerging apical dendrite, a smaller dendritic arborization, and 2 to 8 ascending axonal collaterals that arborized widely in the supragranular layers. Both bursting and regular spiking cells had main axons that entered the subcortical white matter.

Little is known about why clinical depression feels so bad, perhaps because optimal neural circuit-based animal models of depression do not yet exist. Our goal here was to develop a strategy of inducing and measuring depressive-like states in the rat using neural circuits as both the independent and major dependent variables. We hypothesized that repeated electrical stimulation of the brain (ESB) within the dorsal periaqueductal gray (dPAG) aversion circuits would lead to a long-lasting suppression of 50 kHz ultrasonic vocalizations (USVs), a validated measure of positive social affect. Fifteen consecutive daily 10 min sessions of intermittent PAG-ESB reduced systematically evoked 50 kHz USVs for up to 29 days following termination of ESB treatment, along with altering traditional measures of negative affect, including behavioral agitation, sucrose intake, and decreased exploratory behavior. These findings suggest a new affective circuit-based preclinical model of depression.

The longitudinal growth of long bones is attributed to epiphyseal growth. However, the effects of low-level laser therapy (LLLT) in such structures has still not been studied extensively in the literature. Therefore, the aim of this study was to evaluate the use of LLLT, 670 nm, at three different doses on the epiphyseal growth of the right tibia of rats. Twenty-one Wistar rats, aged four weeks, were subjected to the application of LLLT, with dosage according to the group (G4: were submitted to the application of 4 J/cm 2 ; G8: were submitted to the application of 8 J/cm 2 ; G16: were submitted to the application of 16 J/cm 2 ). After completion of protocol they were kept until they were 14 weeks of age and then submitted to a radiological examination (evaluation of limb length) and euthanised. The histological analysis of the growth plates (total thickness and hypertrophic and proliferative zones) was then performed. Comparisons were made with the untreated left tibia. No differences were observed in any of the reviews (radiological and histological), when comparing the right sides (treated) to the left (untreated). It was concluded that the treatment with LLLT within the parameters used caused changes neither in areas of the epiphyseal cartilage nor in the final length of limbs.

Situations in which rewards are unexpectedly obtained or withheld represent opportunities for new learning. Often, this learning includes identifying cues that predict reward availability. Unexpected rewards strongly activate midbrain dopamine neurons. This phasic signal is proposed to support learning about antecedent cues by signaling discrepancies between actual and expected outcomes, termed a reward prediction error. However, it is unknown whether dopamine neuron prediction error signaling and cue-reward learning are causally linked. To test this hypothesis, we manipulated dopamine neuron activity in rats in two behavioral procedures, associative blocking and extinction, that illustrate the essential function of prediction errors in learning. We observed that optogenetic activation of dopamine neurons concurrent with reward delivery, mimicking a prediction error, was sufficient to cause long-lasting increases in cue-elicited reward-seeking behavior. Our findings establish a causal role for temporally precise dopamine neuron signaling in cue-reward learning, bridging a critical gap between experimental evidence and influential theoretical frameworks. npg

Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis. Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot. Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressi...

Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30mg

Cerebellar afferent Raphe interpositus Biotinylated dextran amine Single axon reconstruction a b s t r a c t Integration of cortical Purkinje cell inputs and brain stem inputs is essential in generating cerebellar outputs to the cerebellar nuclei (CN). Currently, collaterals of climbing and mossy fiber axons, noradrenergic, serotoninergic and cholinergic axons, and collaterals of rubrospinal axons are known to innervate the CN from the brain stem. We investigated whether other afferents to the CN from the medulla exist in the rat. Retrograde labeling revealed the presence of neurons that project to the CN but not to the cerebellar cortex in the median reticular formation in the rostrodorsal medulla (tentatively named 'caudal raphe interpositus area', CRI). Anterograde tracer injection into the CRI labeled abundant axonal terminals in the CN, mainly in the ventral parvocellular part of the posterior interposed and lateral nucleus. Axonal reconstruction showed that a single CRI axon projected to the CN with 170-1086 varicosities, more broadly and densely than collaterals of a mossy or climbing fiber axon. CRI axons had no or a few collaterals that projected to the granular and Purkinje cell layers of the cerebellar cortex with some small terminals, indicating that these axons are different from mossy fiber axons. CRI axons also had http://dx.Abbreviations: 4V, forth ventricle; 5-HT, 5-hydroxytriptamine; V, trigeminal nucleus; VI, abducens nucleus; VII, facial nucleus; AIN, anterior interposed nucleus; BDA, biotinylated dextran amine; C, caudal; CN, cerebellar nuclei; CRI, caudal raphe interpositus area; D, dorsal; DAB, 3,3 0 -diaminobenzidine; DLH, dorsolateral hump of the anterior interposed nucleus; DLP, dorsolateral protuberance; DMC, dorsomedial crest of the anterior interposed nucleus; gVII, genu of the facial nerve; GL, granular layer; GRN, gigantocellular reticular nucleus; HRP, horseradish peroxidase; icp, inferior cerebellar peduncle; IO, inferior olive; IN, interposed nucleus; L, lateral; L.II-VI, lobules II-VI; LN, lateral nucleus; LN-parvo, ventral parvocellular subdivision of the lateral nucleus; Lt, left; LVN, lateral vestibular nucleus; M, medial; ML, molecular layer; MN, medial nucleus; MN-CM, caudomedial subdivision of the medial nucleus; MN-M, middle subdivision of the medial nucleus; mlf, medial longitudinal fasciculus; MVN, medial vestibular nucleus; nVII, tract of the facial nerve; NRTP, nucleus reticularis tegmenti pontis; PC, Purkinje cell; PCG, pontine central gray; PCL, Purkinje cell layer; PHA-L, Phaseolus vulgaris leucoagglutinin; PIN, posterior interposed nucleus; PIN-parvo, ventral parvocellular subdivision of the posterior interposed nucleus; PrH, prepositus hypoglossi (I. Sugihara).

Cannabinoids have been reported to provide neuroprotection in acute and chronic neurodegeneration. In this study, we examined whether they are also effective against the toxicity caused by 6-hydroxydopamine, both in vivo and in vitro, which may be relevant to Parkinson's disease (PD). First, we evaluated whether the administration of cannabinoids in vivo reduces the neurodegeneration produced by a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. As expected, 2 weeks after the application of this toxin, a significant depletion of dopamine contents and a reduction of tyrosine hydroxylase activity in the lesioned striatum were noted, and were accompanied by a reduction in tyrosine hydroxylase-mRNA levels in the substantia nigra. None of these events occurred in the contralateral structures. Daily administration of D 9 -tetrahydrocannabinol (D 9 -THC) during these 2 weeks produced a significant waning in the magnitude of these reductions, whereas it failed to affect dopaminergic parameters in the contralateral structures. This effect of D 9 -THC appeared to be irreversible since interruption of the daily administration of this cannabinoid after the 2-week period did not lead to the re-initiation of the 6-hydroxydopamine-induced neurodegeneration. In addition, the fact that the same neuroprotective effect was also produced by cannabidiol (CBD), another plant-derived cannabinoid with negligible affinity for cannabinoid CB 1 receptors, suggests that the antioxidant properties of both compounds, which are cannabinoid receptorindependent, might be involved in these in vivo effects, although an alternative might be that the neuroprotection exerted by both compounds might be due to their anti-inflammatory potential. As a second objective, we examined whether cannabinoids also provide neuroprotection against the in vitro toxicity of 6-hydroxydopamine. We found that the non-selective cannabinoid agonist HU-210 increased cell survival in cultures of mouse cerebellar granule cells exposed to this toxin. However, this effect was significantly lesser when the cannabi-noid was directly added to neuronal cultures than when these cultures were exposed to conditioned medium obtained from mixed glial cell cultures treated with HU-210, suggesting that the cannabinoid exerted its major protective effect by regulating glial influence to neurons. In summary, our results support the view of a potential neuroprotective action of cannabinoids against the in vivo and in vitro toxicity of 6hydroxydopamine, which might be relevant for PD. Our data indicated that these neuroprotective effects might be due, among others, to the antioxidant properties of certain plant-derived cannabinoids, or exerted through the capability of cannabinoid agonists to modulate glial function, or produced by a combination of both mechanisms. D

Our aim was to study histopathological changes in lung tissue at the light microscopic and ultrastructural level during recovery from immunosuppression and Pneumocystis carinii pneumonia. Male Wistar rats were immunosuppressed by per oral dexamethasone for 12 weeks to induce P. carinii pneumonia, after which dexamethasone was stopped. Recovery was monitored 1, 2 and 4 weeks after cessation of the immunosuppression. In immunosuppressed animals, CD4+ and CD8+ lymphocytes were both decreased in situ. CD8+ lymphocytes increased above control level at week one. Like CD8+ cells, the ED1+ macrophages increased rapidly in situ. This was accompanied by a progressively increasing migration (more transient for lymphocytes) of macrophages into bronchoalveolar fluid, associated with morphological signs of activation and phagocytosis and proliferation of type II pneumocytes. Bronchoalveolar lavage (BAL) fluid tumour necrosis factor-alpha (TNF-alpha) increased from subnormal levels to a 4 week pea...

Addictive diseases, including addiction to heroin, prescription opioids, or cocaine, pose massive personal and public health costs. Addictions are chronic relapsing diseases of the brain caused by drug-induced direct effects and persisting neuroadaptations at the epigenetic, mRNA, neuropeptide, neurotransmitter, or protein levels. These neuroadaptations, which can be specific to drug type, and their resultant behaviors are modified by various internal and external environmental factors, including stress responsivity, addict mindset, and social setting. Specific gene variants, including variants encoding pharmacological target proteins or genes mediating neuroadaptations, also modify vulnerability at particular stages of addiction. Greater understanding of these interacting factors through laboratory-based and translational studies have the potential to optimize early interventions for the therapy of chronic addictive diseases and to reduce the burden of relapse. Here, we review the molecular neurobiology and genetics of opiate addiction, including heroin and prescription opioids, and cocaine addiction.

Several colubrid snakes produce venomous oral secretions. In this work, the venom collected from Venezuelan opisthoglyphous (rear-fanged) Philodryas olfersii snake was studied. Different proteins were present in its venom and they were characterized by 20% SDS-PAGE protein electrophoresis. The secretion exhibited proteolytic (gelatinase) activity, which was partially purified on a chromatography ionic exchange mono Q2 column. Additionally, the haemorrhagic activity of Philodryas olfersii venom on chicken embryos, mouse skin and peritoneum was demonstrated. Neurotoxic symptoms were demonstrated in mice inoculated with Philodryas olfersii venom. In conclusion, Philodryas olfersii venom showed proteolytic, haemorrhagic, and neurotoxic activities, thus increasing the interest in the high toxic action of Philodryas venom. Key-words: Colubridae. Haemorrhage. Neurotoxic. Philodryas olfersii. Proteolytic activity. Venom.

Physical activities for a few days can increase brain-derived neurotrophic factor (BDNF) mRNA in rat hippocampus. To investigate the influence of maternal exercise during pregnancy on rat pup hippocampal BDNF mRNA, we studied its expression by a semi-quantitative RT-PCR method after young pregnant rats were exercised on a motor driven treadmill. Pups of exercised mothers had significantly increased hippocampal BDNF

Rats were trained to discriminate amphetamine, 1 mg/kg given intraperitoneally, from saline injection in a two-lever operant drug discrimination task. Pseudoephedrine (a sympathomimetic drug with central and peripheral actions) at doses of 10 mg/kg failed to substitute for amphetamine, at 20 mg/kg partial substitution occurred, while at a 40 mg/kg full substitution was seen. The specificity of the amphetamine cue at the training dose used (1 mg/kg) was shown by the finding that a peripherally acting sympathomimetic drug phenylephrine at doses from 0.2 to 0.8 mg/kg failed to substitute for amphetamine. The potential for abuse of pseudoephedrine administered at high doses is discussed.

Recent experimental evidence indicates that progesterone (PROG) protects against various models of brain injury, including ischemic stroke. Most human studies of pharmacologic treatments for acute cerebral stroke have failed despite initial success in animal models. To simulate better the typical human stroke without reperfusion, the present study was conducted to examine the efficacy of PROG on infarct volume and functional outcome in a permanent model of stroke, using direct cauterization of the middle cerebral artery (MCA). Twenty-four male adult Sprague-Dawley rats underwent pMCAO by electro-coagulation and sham operation. After induction of permanent MCA occlusion (pMCAO), the rats received an initial intraperitoneal injection of PROG (8 mg/kg) or vehicle at 1h post-occlusion followed by subcutaneous injections at 6, 24 and 48 h. Functional deficits were tested on the rotarod and grip strength meter at 24, 48 and 72 h after pMCAO. The rats were killed 72 h after surgery and isolated brain was sectioned into coronal slices and stained with 2, 3, 5triphenyltetrazolium chloride (TTC). PROG-treated rats showed a substantial reduction (54.05%) in the volume of the infarct (% contralateral hemisphere) compared to vehicle controls. In addition there was a significant improvement in ability to remain on an accelerating rotarod and increased grip strength observed in the pMCAO rats treated with PROG compared to vehicle. Taken together, these data indicate that PROG is beneficial in one of the best-characterized models of stroke, and may warrant further testing in future clinical trials for human stroke.

We tested the hypothesis that the phosphatidylinositol-3 kinase (PI3K/Akt) pathway mediates some of the neuroprotective effects of progesterone (PROG) after ischemic stroke. We examined whether PROG acting through the PI3K/Akt pathway could affect the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Rats underwent permanent focal cerebral ischemia (pMCAO) by electro-coagulation and received intraperitoneal injections of PROG (8mg/kg) or vehicle at 1h post-occlusion and subcutaneous injections at 6, 24, and 48h. PAkt/Akt levels, apoptosis and apoptosis-related proteins (pBAD, BAD, caspase-3, and cleaved caspase-3) were analyzed by TUNEL assays, Western blotting and immunohistochemistry at 24h post-pMCAO. VEGF and BDNF were analyzed at 24, 72h and 14 days post-pMCAO with Western blots. Following pMCAO, PROG treatment significantly (p<0.05) reduced ischemic lesion size and edema. Treatment with PROG significantly (p<0.05) decreased VEGF at 24 and 72h but increased VEGF expression 14d after injury. The treatment also increased BDNF, and attenuated apoptosis by increasing Akt phosphorylation compared to vehicle-alone. The selective PI3K inhibitor Wortmannin compromised PROG-induced neuroprotective effects and reduced the elevation of pAkt levels in the ischemic penumbra. Our findings lead us to suggest that the PI3K/ Akt pathway can play a role in mediating the neuroprotective effects of PROG after stroke by altering the expression of trophic factors in the brain.

We compare the effects of psychoactive drugs such as morphine and amphetamine on the synaptic organization of neurons in the orbital frontal (OFC) and medial frontal (mPFC) regions in the rat. Both regions are altered chronically by exposure to intermittent doses of either drug but the effects are area-dependent. For example, whereas morphine produces increased spine density in OFC but decreased spine density in mPFC. The differential response of the OFC and mPFC to drugs is paralleled by an areal-dependent effect of gonadal hormones on these regions as well: males have greater dendritic arborization in the mPFC whereas females have a greater arborization in the OFC. We also compared the effects of neonatal injury to the OFC and mPFC on cognitive, motor, and social behaviors as well as on the anatomical organization of the remaining brain. Again, there were differential effects of the treatments to the OFC and mPFC. Neonatal OFC lesions allowed virtually complete functional recovery of cognitive and motor behaviors, which was correlated with mild abnormalities in cerebral development compared to the more severe deficits and morphological sequelea following mPFC lesions at the same ages. One exception was the effect of OFC on social behavior, which was severe regardless of whether the injury was in infancy or adulthood. It is proposed that both drug-induced and developmental abnormalities in the integrity of OFC neurons may lead to deficits in social behavior or other behavioral pathologies, possibly including depression.

The subcellular localization of five isoforms of facilitated-diffusion glucose transporters (GLUTs), from GLUT1 to GLUT5, in rat pancreatic islets was studied by immunohistochemistry using rabbit polyclonal antisera against mouse or rat GLUT peptides. Animals were perfusion-fixed with phosphate-buffered 4% paraformaldehyde and the pancreases were removed. Some specimens were embedded in paraffin, serially sectioned, and immunostained for glucagon, insulin, somatostatin, and the GLUTs for light microscopic observation. Others were prepared for immunoelectron microscopy by the post-embedding method. By these methods, GLUT2 immunostaining was observed on the lateral membranes of pancreatic beta-cells, whereas GLUT3 immunoreaction was predominantly localized in the cytoplasm of beta-cells and was not found in alpha-cells. In contrast, GLUT5 immunostaining was preferentially localized in the cytoplasm of alpha-cells compared to that of beta-cells. However, GLUT1 and GLUT4 were either barely or not at all detectable in any cells. These results suggest that rat islets take up glucose by at least three different processes and that blood glucose levels could be modulated differentially by: a high Km glucose transporter, GLUT2, in beta-cells; by a low Km glucose transporter, GLUT3, in beta-cells; and by a low Km glucose transporter, GLUT5, in alpha-cells.

Atrazine, a chlorotriazine herbicide, is used to control annual grasses and broadleaf weeds. In this review, we summarize our laboratory's work evaluating the neuroendocrine toxicity of atrazine (and related chlorotriazines) from an historic perspective. We provide the rationale for our work as we have endeavored to determine: 1) the underlying reproductive changes leading to the development of mammary gland tumors in the atrazine-exposed female rat; 2) the cascade of physiological events that are responsible for these changes (i.e., the mode of action for mammary tumors); 3) the potential cellular mechanisms involving adverse effects of atrazine; and 4) the range of reproductive alterations associated with this pesticide.

The ovaries' innervation arrives via the superior ovarian nerve, which originates from the celiac ganglion. Using True Blue as an antidromic marker, the present study analyzed the changes in the anatomical relation between each ovary and the prevertebral celiac-superior mesenteric ganglia during the estrous cycle. The number of labeled neurons increased from the day of diestrus 1 to the day of proestrus. The largest number of labeled cells was observed when tracer was injected into the left ovary on proestrus. The number of labeled cells was significantly higher when the tracer was injected into the left ovary on proestrus than when it was done in the right one. When tracer was injected into the left ovary, the average labeled area of cells increased significantly from diestrus 1 to proestrus, and declined at estrus. In contrast, when True Blue was injected into the right ovary, the average labeled area was similar in diestrus 1 and diestrus 2, and the values increased in proestrus and estrus. The results indicate an apparent asymmetry in the activity of neural connections between ovaries and the prevertebral celiac-superior mesenteric ganglia, and that the number of active neurons of these connections varies during the estrous cycle.

Senna (Tinnevelly senna fruits), a known laxative derived from plants, was administered by gavage to Sprague-Dawley (Crl:CD (SD) BR) rats once daily at dose levels of 0, 25, 100 and 300 mg/kg/day for up to 104 consecutive weeks. Based upon clinical signs related to the laxation effect of senna, the highest dose (300 mg/kg/day) was considered to be a maximum tolerated dose. Sixty animals per sex were assigned to the control and dose groups. Assessments included clinical chemistry, hematology, full histology (control and high-dose groups; in addition, low and mid dose: intestinal tract, adrenals, liver, kidneys, brain and gross lesions) and toxicokinetics. The primary treatment-related clinical observation was mucoid feces seen at 300 mg/kg/day. When compared to controls, animals administered 300 mg/kg/day had slightly reduced body weights, increased water consumption and notable changes in electrolytes in serum (increases in potassium and chloride) and urine (decreases in sodium, pot...

Lysosomes of embryonic rat fibroblasts cultivated in vitro normally contain heavy metals, as shown with a modified sulfide-silver method (SSM). Cultures which received lead added to the cultivation medium showed an enhanced SSM-positivity. However, since the SSM demonstrates several different heavy metals the sulfides of which are weakly soluble in water, it was not possible to distinguish between naturally occurring heavy metals-largely iron-and added lead (supposed to have been taken up by the cells). By treating the cells with a 0.2 M TCA solution after an initial exposure to HS-it was possible to dissolve FeS without affecting the PbS to any noticeable extent. When subsequently the development process of the SSM was applied to the cells, no metals could be demonstrated in the control cells whereas those exposed to lead showed presence of granules, most of which were identical with lysosomes as visualized with a Gomori type reaction. A lysosomal uptake of lead could thus be demonstrated with the modified SSM when combined with a simple dissolving process.

The active ingredient of marijuana is(−)-Δ 9 -tetrahydrocannabinol(Δ 9 -THC).Δ 9 -THC and other natural and synthetic cannabinoids such as CP-55, 940 inhibit spontaneous activity and produce catalepsy in animals in a receptor-mediated fashion. Tolerance develops to the motor ...

Release of glutamate from synaptic terminals in the central nervous system elicits fast excitatory postsynaptic potentials by activating AMPA and NMDA receptors (AMPARs and NMDARs). Both these receptors are embedded within a dense protein matrix forming a distinct specialization known as the postsynaptic density (PSD; Sheng, 2001). PSD-95, a member of the MAGUK (membrane-associated guanylate kinase) superfamily of proteins, is one of the most abundant proteins in the PSD (Hunt et al. 1996). This protein is preferentially, although not exclusively, targeted to dendritic spines where it is thought to interact directly or indirectly with both NMDARs and AMPARs

Hyperbaric oxygenation is effective in augmenting the delivery of oxygen to tissue, but also causes oxidative stress. As part of our focus on improving peripheral nerve salvage from ischemic fiber degeneration, we evaluated whether hyperbaric oxygenation rescues peripheral nerve, rendered ischemic by microembolization, from ischemic fiber degeneration. The supplying arteries of rat sciatic nerve were embolized with microspheres of 14 p m diameter at moderate (2 x lo6) and high (5.6 x lo6) doses. Rats were randomized to receive hyperbaric oxygenation treatment (2.5 atm 100% oxygen for 2 hours/day for 7 days beginning within 30 minutes of ischemia), or room air. End points for the embolized limb were (1) behavioral scores (0-11 in increasing levels of limb function), (2) nerve action potential of sciatic-tibial nerve, (3) nerve blood flow, and (4) histological grade as percentage of fibers undergoing ischemic fiber degeneration (0 = <5%; 1 = 5-25%; 2 = 26-50%; 3 = 51-75960; 4 = >76%). Nerve blood flow and nerve action potential were uniformly absent and more than !N% of fibers had degenerated in both control and treatment groups receiving high doses. Control and treatment groups receiving moderate doses were well matched by level of ischemia (8.5 2 0.3 {N = 18) vs 7.7 k 0.4 m1/100 gm/min [N = 181, p > 0.05) but were significantly different by behavior score (5.6 k 0.7 vs 9.2-+ 0.5 {N = 191, p < 0.001), nerve action potential (1.4 +-1.0 vs 3.9 f 0.5 {N = 6) , p < 0.05), and histology (2.4 f 0.4 {N = 57 vs 0.8 f 0.5 {N = 41, p < 0.05). On single teased fiber evaluation, the predominant abnormality was E (zonal degeneration). We conclude that hyperbaric oxygenation will effectively rescue fibers from ischemic fiber degeneration, providing the ischemia is not extreme.

Two experiments were conducted with growing male rats to determine the effects of 120 ppm of dietary sarsaponin (S) on nitrogen (N) metabolism when urea or protein are added to the diet. Growth, feed efficiency, N digestibility and balance, urinary N and ammonia-N (NH3-N), and cecal urease and NH3-N were measured. Growth and feed utilization were unaffected by dietary S. Adding urea or protein to the diet increased apparent N digestibility and increased urinary-N excretion. Urea did not affect N balance, whereas growth, feed utilization and N balance were maximized with 22% compared with either 16 or 28% dietary protein. Urinary NH3-N excretion was decreased by S when urea was added to the diet but was not affected when fed with increasing dietary protein. Cecal urease was decreased by S when urea was added or when the protein level was increased in the diet; effects on cecal NH3-N varied between the two experiments. Plasma urea-N was decreased by S. It is concluded that S has minor...

The aim of this study was to investigate the influence of the long-term treatment of rats with letrozole on the testis morphology. The pharmacologically induced estrogen deficiency caused statistically significant decreases of both intratesticular and serum levels of estradiol, and morphological changes in the seminiferous epithelium and in the interstitial tissue of the testes. Six months of treatment resulted in the sloughing of premature germ cells of the seminiferous epithelium into the tubular lumen and in intraepithelial vacuolization. Multinucleated giant cells composed of premature germ cells, conglomerates of various cell nuclei and cell debris as well as irregularities and infoldings of the tubular basement membrane were also seen. Moreover, deep invaginations of the lamina propria with myoid cells were observed. Cells in the interstitial tissue showed changes similar to that observed in aging processes. The cytoplasm of LH-R-positive Leydig cells was loaded with lipofuscin granules. The number of lipofuscin-loaded cells was significantly increased in the interstitial tissue of testis in letrozole-treated rats. The results indicate the direct influence of estrogens on seminiferous tubules and the interstitial tissue morphology.

Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [ 1 ,4-'4C]succinate, the reaction velocity being judged through the generation of I4CO2 in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP-malate dehydrogenase. In the presence of 1.0 mM succinate, the reaction velocity averaged 5.53 f 0.44pmol min-' pg-' islet protein. The K,,, for succinate was close to 0.4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD-isocitrate dehydrogenase or the 2-ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca2+. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O2 uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate-limiting step of the Krebs cycle in models of B-cell dysfunction. KEY woRos-Pancreatic islets; succinate dehydrogenase.

To understand the interactions between iron and zinc during absorption in iron- and zinc-deficient rats, and their consequences on intestinal oxidant-antioxidant balance. Twenty-four weanling Wistar-Kyoto rats fed an iron- and zinc-deficient diet (&lt; 6.5 mg Fe and 4.0 mg Zn/kg diet) for 4 wk were randomly divided into three groups (n = 8, each) and orally gavaged with 4 mg iron, 3.3 mg zinc, or 4 mg iron + 3.3 mg zinc for 2 wk. At the last day of repletion, 3 h before the animals were sacrificed, they received either 37 mBq of (55)Fe or (65)Zn, to study their localization in the intestine, using microautoradiography. Hemoglobin, iron and zinc content in plasma and liver were measured as indicators of iron and zinc status. Duodenal sections were used for immunochemical staining of ferritin and metallothionein. Duodenal homogenates (mitochondrial and cytosolic fractions), were used to assess aconitase activity, oxidative stress, functional integrity and the response of antioxidant e...

A sonicated liposome suspension of y-aminobutyric acid (GABA) and phosphatidylserine (liposomeentrapped GABA), intraperitoneally administered in rats, inhibited EEG epileptic activity induced by penicillin, whereas GABA did not. A significant increase (20.4%) in brain radioactivity accumulation occurred at 5 min after i.p. administration of ('4C]GABA associated with phosphatidylserine in comparison with the administration of [ 14C]GABA; such an increase persisted after 20 min. However, the accumulation of radioactivity into brain synaptosomes demonstrated a 24.1% increase at 5 min and subsequently showed a 43.3% increase at 20 min after injection of liposome-entrapped GABA. The above findings suggest that phosphatidylserine stimulates exogenous GABA uptake into brain GABAergic nerve terminals. Q 1988 Academic Press, Inc.

Ultrasonic vocalization (USV) was found useful for differentiating fear and anxiety in rats. These affective states were established through a Pavlovian conditioning procedure. Danger stimulus, preceding unavoidable tail shock, elicited acute fear. Intertrial situational cues evoked anxiety. A safety signal (SS) indicating the omission of shock inhibited fear. Sustained 22-kHz USV characterized anxiety and was present between trials. A signal of danger resulted in immediate inhibition of vocalization, while a SS reversed this effect. These results are discussed in the context of three theories: Pavlovian, Bollesian and Konorskian. The anxiolytic drugs diazepam and buspirone (1 and 5 mg/kg) suppressed vocalization in the intertrial and SS periods. The reaction to the signal of danger remained complete inhibition of USV. Anxiogenic pentyletetrazole (1 and 5 mg/kg) enhanced intertrial vocalization, but did not affect its reoccurrence during the SS. Anxiogenic FG7142 (5 mg/kg) did not affect intertrial vocalization, but blocked its reappearance on the SS. It is suggested that the behavioral target of both anxiogenic drugs is different */pentyletetrazole supposedly exerts its anxiogenic effect by increasing situational anxiety, whereas FG7142 suppresses inhibition of fear.

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli 3-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid 3-galactosidase proteins. Conditions for transfection were varied to optimize the expression of 13-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli fgalactosidase and distinct from the major lysosomal "acid" fl-galactosidase. The fused preproinsulin-fl-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric P-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-,B-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed. Gene fusions utilizing the Escherichia coli lacZ structural gene provide an especially suitable method for studying gene expression and regulation (1). Its product, f3-galactosidase, can be as

The pharmacodynamics and pharmacokinetics of bupivacaine in solution and in liposome preparations following subcutaneous administration were studied in rats. Multilamellar vesicles entrapping bupivacaine solution were prepared. The local anaesthetic effect was estimated by the tail-flick test in Wistar rats treated with 1 mg bupivacaine in 0.2-ml. preparations. Plasma concentrations of bupivacaine were determined by high-performance liquid chromatography. The results showed that both bupivacaine solution and bupivacaine liposomes revealed local anaesthetic effects in the initial tail-flick test (15 min after injection). With bupivacaine liposomes, the duration of action was 5-fold (447 ± 28.9 vs 87 ± 6.7 min), the maximum possible effect was 2-fold (100 ± 0 vs 47.6 ± 13%), and the peak plasma concentration (Cmax) was less than one-fifth (0.12 ± 0.04 vs 0.65 ± 0.04 μg mL−1) that with bupivacaine solution. The sensory block effect of bupivacaine solution completely resolved at 90 min,...

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/␥-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/␥-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/␥-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/␥-filamin might play a role in Z-disc assembly.

Laboratory housing conditions have significant physiological and psychological effects on rodents, raising both scientific and humane concerns. Published studies of rats, mice and other rodents were reviewed to document behavioural and psychological problems attributable to predominant laboratory housing conditions. Studies indicate that rats and mice value opportunities to take cover, build nests, explore, gain social contact, and exercise some control over their social milieu, and that the inability to satisfy these needs is physically and psychologically detrimental, leading to impaired brain development and behavioural anomalies (e.g. stereotypies). To the extent that space is a means to gain access to such resources, spatial confinement likely exacerbates these deficits. Adding environmental 'enrichments' to small cages reduces but does not eliminate these problems, and I argue that substantial changes in housing and husbandry conditions would be needed to further reduce them.

creted 23.9% after the oral dose. Thus, based on urinary excretion of total radioactivity in dogs, it appears that only 5040% of furosemide is absorbed from an oral solution. The plasma and tissue levels and the pharmacokinetic parameters of furosemide in dogs and monkeys following 5-mg/kg oral and intravenous doses will be described in a subsequent paper. These data in dogs confirm the lack of complete bioavailability of furmmide from an oral solution dosage form. Less than 1% of radioactivity in the plasma of dog and monkey is attributable to metabolites.