Chronic voluntary ethanol intake hypersensitizes 5‐HT1A autoreceptors in C57BL/6J mice

Alcoholism is a complex disorder involving, among others, the serotoninergic (5‐HT) system, mainly regulated by 5‐HT_1A autoreceptors in the dorsal raphe nucleus. 5‐HT_1A autoreceptor desensitization induced by chronic 5‐HT reuptake inactivation has been associated with a decrease in ethanol intake in mice. We investigated here whether, conversely, chronic ethanol intake could induce 5‐HT_1A autoreceptor supersensitivity, thereby contributing to the maintenance of high ethanol consumption. C57BL/6J mice were subjected to a progressive ethanol intake procedure in a free‐choice paradigm (3–10% ethanol versus tap water; 21 days) and 5‐HT_1A autoreceptor functional state was assessed using different approaches. Acute administration of the 5‐HT_1A receptor agonist ipsapirone decreased the rate of tryptophan hydroxylation in striatum, and this effect was significantly larger (+75%) in mice that drank ethanol than in those drinking water. Furthermore, ethanol intake produced both an increased potency (+45%) of ipsapirone to inhibit the firing of 5‐HT neurons, and a raise (+35%) in 5‐HT_1A autoreceptor‐mediated stimulation of [³⁵S]GTP‐γ‐S binding in the dorsal raphe nucleus. These data showed that chronic voluntary ethanol intake in C57BL/6J mice induced 5‐HT_1A autoreceptor supersensitivity, at the origin of a 5‐HT neurotransmission deficit, which might be causally related to the addictive effects of ethanol intake.     

Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5‐HT) receptor subtype next to the 5‐HT_1A autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5‐HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5‐HT₇ receptors. Experiments were performed with the specific 5‐HT₇ antagonist SB 258741 and the putative 5‐HT₇ agonist AS19. In this study WAY 100.635 was used to block 5‐HT_1A receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5‐HT in the ventral hippocampus as well as 5‐HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co‐administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5‐HT in ventral hippocampus, hinting at opposed 5‐HT₇ receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell‐shaped dose–effect curve: moderately increasing 5‐HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5‐HT₇ receptors and moderate affinity for 5‐HT_1A receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5‐HT neuronal activity mediated by 5‐HT₇ receptors. It can be speculated, that the restoration of 5‐HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5‐HT_1A receptors alone, in fact results from a shift in balance between 5‐HT_1A and 5‐HT₇ receptor function.     

Acute toxicity of ammonia and nitrite to different ages of Pacific cod (Gadus macrocephalus) larvae

Abstract

This study was conducted to evaluate the acute toxicity of ammonia and nitrite to three developmental stages of Pacific cod (Gadus macrocephalus) larvae (11, 22, and 35 days after hatching, with mean total lengths of 4.63 ± 0.14, 5.83 ± 0.17, and 7.46 ± 0.23 mm, respectively). The results showed for the first time that the acute toxicity of ammonia and nitrite is closely related to the age of Pacific cod larvae, and the acute toxicity of ammonia or nitrite increased with increased Pacific cod larval growth. Lethal concentrations (LC₅₀) of un-ionized ammonia nitrogen (UIAN) for a 48-h exposure in 11-day post-hatch, 22-day post-hatch, and 35-day post-hatch Pacific cod larvae were 1.72, 0.69, and 0.32 mg L^?1, respectively. The 48-h LC₅₀ of nitrite nitrogen to Pacific cod larvae 11-day post-hatch, 22-day post-hatch, and 35-day post-hatch were 831.76, 269.15, and 223.87 mg L^?1, respectively. The present findings demonstrate that the acute toxicity of ammonia for Pacific cod larvae is much higher than that of nitrite.     

Larvae of the small white butterfly,Pieris rapae,express a novel serotonin receptor

The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT_1A, 5‐HT_1B, 5‐HT_2A, 5‐HT_2B, and 5‐HT₇ receptors. We isolated a cDNA (Pr5‐HT₈) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT₈ mediated increased [Ca²⁺]_i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]_i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT₈ was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT₈. WAY 10635, a 5‐HT_1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca²⁺]_i increases. We infer that Pr5‐HT₈ represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT₈ in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

     

Effects of in ovo feeding of creatine pyruvate on the hatchability,growth performance and energy status in embryos and broiler chickens

The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the hatchability, growth performance and energy status of embryos and broilers (Arbor Acres) were investigated. Five treatments were arranged as non-injected treatment (Control), 0.6 ml physiological saline (0.75%) injected treatment (Saline), and IOF treatments injected with 0.6 ml physiological saline (0.75%) containing 3, 6 or 12 mg CrPyr (CrPyr₃, CrPyr₆ or CrPyr₁₂) into the amnion per fertile egg on day 17.5 of incubation. After hatching, 80 male chicks from each treatment with similar weight close to the average BW of their pooled group were selected and randomly assigned into eight replicates of 10 chicks each. The results showed that the hatchability was not affected among groups, whereas the hatching weight of broilers in CrPyr₁₂ was significantly higher than the control and saline groups (P<0.05). At 21 day post-hatch, the BWs of broilers in CrPyr₆ and CrPyr₁₂ were increased relative to the control and saline groups (P<0.05). Chickens in CrPyr₆ and CrPyr₁₂ exhibited higher BW gain and feed intake than the control and saline groups during 8 to 21 days post-hatch and the entire experiment period (P<0.05). Compared with the control and saline groups, the total and relative weight of pectoral muscle of embryos or chickens were greater in CrPyr₆ and CrPyr₁₂ at 19^th day of incubation (19 E), hatch, 3 and 21 days post-hatch (P<0.05). The concentrations of glucose and glycogen in liver were increased in CrPyr₆ and CrPyr₁₂ at 19 E and hatch (P<0.05). Neither glycogen nor glucose concentration in pectoral muscle was altered among treatments (P>0.05). Irrespective of dosage, the concentrations of creatine and phosphocreatine, and activities of creatine kinase in embryos were enhanced in CrPyr treatments at 19 E when compared with the control and saline groups (P<0.05). The activities of glucose-6-phosphatase in liver in CrPyr₆ and CrPyr₁₂ treatments were higher than the control and saline groups at 19 E (P<0.05). In conclusion, these results indicated that IOF of CrPyr, especially at the level of 12 mg/egg, could improve energy status of embryos and hatchlings, which was useful for enhancing hatching weight, BW and pectoral muscle weight until the end of the experiments at 21 days post-hatch in broilers.     

Organic Trace Mineral Levels in the First 96-H Post-Hatch Impact Growth Performance and Intestinal Gene Expression in Broiler Chicks

Alterations in nutrient intake in the avian neonatal post-hatch period can impact development, performance, and metabolism in adulthood. Very little is known about how mineral levels during the post-hatch period affect or “program” gene expression patterns later in life. The objective of this study was to determine the effect of post-hatch (0 to 96 h) dietary mineral supplementation on performance, tissue mineral content, and intestinal gene expression profiles in 21-day-old broiler chicks. One-day-old chicks were randomly assigned to one of two treatment groups consisting of N (organic Zn, Cu, and Mn provided at 100 % of recommendations (National Research Council 1994)) and/or L (organic Zn, Cu, and Mn provided at 20 % of recommendations (National Research Council 1994)) diets fed in two intervals (days 1–4, days 5–21) as follows: (1) N–L or (2) L–L. Performance parameters did not differ between treatments except that body weight gain was greater (P?< 0.05) in L–L birds than N–L birds over the experimental period. Bone mineral content was similar for both treatments at day 21. Intestinal gene expression profiling was examined using the Affymetrix GeneChip Chicken genome array. Ingenuity pathway analysis revealed differences in gene expression profiles between N and L treatments at day 5. At day 21, profiles were unique between N–L and L–L, suggesting that the diet fed until day 4 had an impact on gene expression patterns at day 21 even when birds were fed the same diets day 5–day 21. In this study, we demonstrated that diets fed for the 96 h post-hatch had long-term effects on gene expression, providing unique information as to why post-hatch diets are so important for the long-term bird health and productivity.     

Effect of genetic and pharmacological blockade of GABA receptors on the 5‐HT2C receptor function during stress

Serotonin (5‐HT)_2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5‐HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5‐HT_2C receptor‐induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5‐HT turnover by a 5‐HT_2C receptor agonist (RO 60‐0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA‐A or GABA‐B receptors in mice. Neither the GABA‐B receptor antagonist phaclofen nor the specific genetic ablation of either GABA‐B1a or GABA‐B1b subunits altered the inhibitory effect of RO 60‐0175, although 5‐HT turnover was markedly decreased in GABA‐B1a knock‐out mice in both basal and stress conditions. In contrast, the 5‐HT_2C receptor‐mediated inhibition of 5‐HT turnover was reduced by the GABA‐A receptor antagonist bicuculline. However, a significant effect of 5‐HT_2C receptor activation persisted in mutant mice deficient in the α₃ subunit of GABA‐A receptors. It can be inferred that non‐α₃ subunit‐containing GABA‐A receptors, but not GABA‐B receptors, mediate the 5‐HT_2C‐induced inhibition of stress‐induced increase in hippocampal 5‐HT turnover in mice.

     

Transient expression of serotonin 5‐HT4 receptors in the mouse developing thalamocortical projections

The serotonin 5‐HT₄ receptor (5‐HT₄‐R) is an unusually complex G‐protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5‐HT₄‐Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT‐PCR. The developing thalamocortical projections transiently expressed 5‐HT₄‐Rs in the embryonic brain and the 5‐HT₄‐R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13–17, the forebrain mRNA levels of the 5‐HT_4(a)‐R and 5‐HT_4(b)‐R splice variants increased nine‐ and fivefold, respectively, whereas the levels of the 5‐HT_4(e)‐R and 5‐HT_4(f)‐R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5‐HT₄‐R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

Regulation of the 5‐HT3A Receptor‐Mediated Current by Alkyl 4‐Hydroxybenzoates Isolated from the Seeds of Nelumbo nucifera

Four known alkyl 4‐hydroxybenzoates, i.e., methyl 4‐hydroxybenzoate ( 1 ), ethyl 4‐hydroxybenzoate ( 2 ), propyl 4‐hydroxybenzoate ( 3 ), and butyl 4‐hydroxybenzoate ( 4 ), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D‐ and 2D‐NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5‐HT‐stimulated inward current (I_5‐HT) mediated by the human 5‐HT₃A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I_5‐HT, but 4 reduced it. These results indicate that 4 is an inhibitor of the 5‐HT₃A receptors expressed in Xenopus oocytes.     

Serotonin promotes feminization of the sexually dimorphic nucleus of the preoptic area,but not the calbindin cell group

Testosterone and its metabolites masculinize the brain during a critical perinatal window, including the relative volume of sexually dimorphic brain areas such as the sexually dimorphic nucleus of the preoptic area (SDN), which is larger in males than females. Serotonin (5HT) may mediate this hormone action, since 5HT given during the second week of life decreases (i.e., feminizes) SDN volume in males and testosterone‐treated females. Although previous work indicates that the 5HT_2A/2C receptor is sufficient to induce feminization, it is unclear whether other serotonin receptors are required and which subpopulation(s) of SDN cells are specifically organized by 5HT. Therefore, we injected male and female Sprague‐Dawley rat pups with saline, a nonselective 5HTR agonist, a 5HT_2A/2C agonist, or a 5HT_2A/2C antagonist over several timecourses in early life, and measured the Nissl‐SDN as well as a calbindin+ subdivision of the SDN, the CALB‐SDN. When examined on postnatal day 18 or early adulthood, the size of the Nissl‐SDN was feminized in males treated with any of the serotonergic drugs, eliminating the typical sex difference. In contrast, the sex difference in CALB‐SDN size was maintained regardless of serotoninergic drug treatment. This pattern suggests that although gonadal hormones shape the whole SDN, individual cellular phenotypes respond to different intermediary signals to become sexually dimorphic. Specifically, 5HT mediates sexual differentiation of non‐calbindin population(s) within the SDN. The results also caution against using measurement of the CALB‐SDN in isolation, as the absence of an effect on the CALB‐SDN does not preclude an effect on the overall nucleus. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1241–1253, 2016     

Kisspeptin1 modulates odorant‐evoked fear response via two serotonin receptor subtypes (5‐HT1A and 5‐HT2) in zebrafish

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT_1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT₁ and 5‐HT₂ receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT_1A and 5‐HT₂ receptors.

     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.     

Effects of in ovo feeding of l-arginine on breast muscle growth and protein deposition in post-hatch broilers

In ovo feeding (IOF) of l-arginine (Arg) can affect growth performance of broilers, but the response of IOF of Arg on breast muscle growth is unclear, and the mechanism involved in protein deposition remains unknown. Hense, this experiment was conducted to evaluate the effects of IOF of Arg on breast muscle growth and protein-deposited signalling in post-hatch broilers. A total of 720 fertile eggs were collected from 34-week-old Arbor Acres breeder hens and distributed to three treatments: (1) non-injected control group; (2) 7.5 g/l (w/v) NaCl diluent-injected control group; (3) 0.6 mg Arg/egg solution-injected group. At 17.5 days of incubation, fertile eggs were injected 0.6 ml solutions into the amnion of the injected groups. Upon hatching, 80 male chicks were randomly assigned to eight replicates of 10 birds each and fed ad libitum for 21 days. The results indicated that IOF of Arg increased relative breast muscle weight compared with those of control groups at hatch, 3-, 7- and 21-day post-hatch (P<0.05). In the Arg-injected group, the plasma total protein and albumen concentrations were higher at 7- and 21-day post-hatch than those of control groups (P<0.05). The alanine aminotransferase activity in Arg group was higher at hatch than that of control groups (P<0.05). The levels of triiodothyronine at four time points and thyroxine hormones at hatch, 7- and 21-day post-hatch in Arg group were higher than those of control groups (P<0.05). In addition, IOF of Arg increased the amino acid concentrations of breast muscle at hatch, 7- and 21-day post-hatch (P<0.05). In ovo feeding of Arg also enhanced mammalian target of rapamycin, ribosomal protein S6 kinase-1 and eIF4E-bindingprotein-1 messenger RNA expression levels at hatch compared with those of control groups (P<0.05). It was concluded that IOF of Arg treatment improved breast muscle growth, which might be associated with the enhancement of protein deposition.     

Effect of intrauterine infusion of recombinant bovine interferon αI1 on luteal phase duration and oxytocin-induced release of 13,14-dihydro-15-keto-prostaglandin F2α in postpartum beef cows

The objective of this study was to determine if oxytocin-induced release of prostaglandin F₂α (PGF_2α; measured by the stable metabolite, 13,14-dihydro-15-keto-prostaglandin F₂α (PGFM)) was inhibited following intrauterine infusion of bovine interferon-α_I1 (rboIFNα_I1) into postpartum cows anticipated to have short estrous cycles following first ovulation postpartum. Cows expected to have short estrous cycles were assigned to receive twice daily intrauterine infusions of either placebo (SCP; n = 11) or 2 mg rboIFNα_I1 (SCIFN; n = 14) on Days 1–16 following hCG injection (2500 IU; day 0) on Days 30 or 31 postpartum. On Day 5 following hCG, each cow was injected with 100 IU oxytocin (i.v.) to induce the release of uterine PGF_2α (as measured by PGFM). Other treatment groups consisted of cows expected to have normal estrous cycle lengths following pretreatment with a 9 day norgestomet implant on Days 21 or 22 postpartum followed by hCG injection to induce ovulation. Cows expected to have normal estrous cycle lengths received twice daily intrauterine infusions of either placebo from Days 1 to 16 of the cycle and 100 IU oxytocin (i.v.) on Day 5 (NCPE; n = 11) or twice daily infusions of placebo (NCPL; n = 7) or rboIFNα_I1 (NCIFN; n = 10), from Day 13 post-hCG injection until luteolysis. Oxytocin was injected (100 IU; i.v.) into cows in the NCPL and NCIFN groups on Day 16. The calculated areas under the curve (arbitrary PGFM units) were: 164 ± 18 units, 96 ± 16 units, 93 ± 18 units, 137 ± 27 units and 53 ± 20 units for SCP, SCIFN, NCPE, NCPL and NCIFN, respectively (SCIFN < SCP; NCIFN < NCPL; P < 0.015). Mean luteal phase length was calculated as the number of days from injection of hCG until progesterone declined to below 0.5 ng ml⁻¹ and was: 6.7 ± 1.0 days, 10.5 ± 0.9 days, 12.0 ± 1.0 days, 18.0 ± 1.3 days and 20.7 ± 1.1 days for SCP, SCIFN, NCPE, NCPL and NCIFN, respectively (SCP < SCIFN = NCPE < NCPL = NCIFN; P < 0.01). In summary, luteal phase lengths were increased and oxytocin-induced release of PGFM was reduced by rboIFNα_I1 infusion in cows anticipated to have short luteal phases.     

Evidence of thermolabile sex determination in pejerrey*

Temperature regimes of 17 ± 1°C and 21 ±1°C early in development of pejerrey Odontesthes bonariensis produced nearly all females, whereas at 25 ± 1°C variable, sometimes male-biased sex-ratios were obtained. The critical period of thermolabile sex determination seemed to occur between 25 and 50 days post-hatch (about 11 and 21 mm s.i.) at low temperatures (17–20°C) and between 0 and 25 days (about 7 and 15 mm) at high temperatures (22–25°C). The likelihood of expression of temperature-dependent sex determination in natural populations and the possible adaptive significance of environmental sex determination in pejerrey are discussed.     

Combined Treatment of Xenon and Hypothermia in Newborn Rats - Additive or Synergistic Effect?

BackgroundBreathing the inert gas Xenon (Xe) enhances hypothermic (HT) neuroprotection after hypoxia-ischemia (HI) in small and large newborn animal models. The underlying mechanism of the enhancement is not yet fully understood, but the combined effect of Xe and HT could either be synergistic (larger than the two effects added) or simply additive. A previously published study, using unilateral carotid ligation followed by hypoxia in seven day old (P7) rats, showed that the combination of mild HT (35°C) and low Xe concentration (20%), both not being neuroprotective alone, had a synergistic effect and was neuroprotective when both were started with a 4 h delay after a moderate HI insult. To examine whether another laboratory could confirm this finding, we repeated key aspects of the study.Design/MethodsAfter the HI-insult 120 pups were exposed to different post-insult treatments: three temperatures (normothermia (NT) NT_37°C, HT_35°C, HT_32°C) or Xe concentrations (0%, 20% or 50%) starting either immediately or with a 4 h delay. To assess the synergistic potency of Xe-HT, a second set (n = 101) of P7 pups were exposed to either HT_35°C+Xe_0%, NT+Xe_20% or a combination of HT_35°C+Xe_20% starting with a 4 h delay after the insult. Brain damage was analyzed using relative hemispheric (ligated side/unligated side) brain tissue area loss after seven day survival.ResultsImmediate HT_32°C (p = 0.042), but not HT_35°C significantly reduced brain injury compared to NT_37°C. As previously shown, adding immediate Xe_50% to HT_32°C increased protection. Neither 4 h-delayed Xe_20%, nor Xe_50% at 37°C significantly reduced brain injury (p>0.050). In addition, neither 4 h-delayed HT_35°C alone, nor HT_35°C+Xe_20% reduced brain injury. We found no synergistic effect of the combined treatments in this experimental model.ConclusionsCombining two treatments that individually were ineffective (delayed HT_35°C and delayed Xe_20%) did not exert neuroprotection when combined, and therefore did not show a synergistic treatment effect.     

Attenuation of 1-(m-Chlorophenyl)-Biguanide Induced Hippocampus-Dependent Memory Impairment by a Standardised Extract of Bacopa monniera (BESEB CDRI-08)

Bacopa monniera is a well-known medhya-rasayana (memory enhancing and rejuvenating) plant in Indian traditional medical system of Ayurveda. The effect of a standardized extract of Bacopa monniera (BESEB CDRI-08) on serotonergic receptors and its influence on other neurotransmitters during hippocampal-dependent learning was evaluated in the present study. Wistar rat pups received a single dose of BESEB CDRI-08 during postnatal days 15–29 showed higher latency during hippocampal-dependent learning accompanied with enhanced 5HT_3A receptor expression, serotonin and acetylcholine levels in hippocampus. Furthermore, 5HT_3A receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) impaired learning in the passive avoidance task followed by reduction of 5HT_3A receptor expression, 5HT and ACh levels. Administration of BESEB CDRI-08 along with mCPBG attenuated mCPBG induced behavioral, molecular and neurochemical alterations. Our results suggest that BESEB CDRI-08 possibly acts on serotonergic system, which in turn influences the cholinergic system through 5-HT₃ receptor to improve the hippocampal-dependent task.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.