A Combination Staining Method for Fibers and Cell Bodies of the Urodele Central Nervous System

A simple method for staining nerve cells and fibers of the salamander central nervous system is described. The procedure employs Carnoy\u27s fixation followed by Protargol impregnation and Nissl staining. This technique permits the simultaneous observation of intracellular neurofibrils, neuronal processes and basophilic components of the neuron. In addition, it eliminates the need to stain alternate sections with separate procedures to view the various components of the urodele central nervous system

Developing Allantois Is a Primary Site of 2\u27-Deoxycoformycin Toxicity

In this study, an assessment of normal mouse allantoic development and its sensitivity to 2\u27-(R)-deoxycoformycin (dCF; Pentostatin) exposure were examined. Both dissecting microscopy and scanning electron microscopy were used to describe the normal growth and morphogenesis of the mouse allantois over gestational days 7-10 as a preliminary step in evaluating potential abnormal allantoic ontogeny and its effect on umbilical cord and placental development. Two abnormal allantoic/umbilical cord phenotypes were observed subsequent to injecting pregnant mice with 5 mg dCF/kg, i.p., on gestational day 7 (GD 7) and evaluating litters on GD 10, 11,and 12. Abnormal phenotypes included: (1) an allantois which extended approximately halfway across the exocoelom but failed to establish a functional contact with the chorion; and (2) a phenotype characterized by reduced expansion of the allantois across the chorionic surface, a very thin umbilical cord, and aberrant vascularization throughout the structure. Both abnormal phenotypes exhibited either an agenesis or hypogenesis of the umbilical cord and chorioallantoic plate, respectively. Neither abnormal phenotype, however, exhibited errors in the directionality of allantoic growth toward the chorion nor in the formation of aberrant contacts between allantois and adjacent yolk sac or amnionic mesenchyme. Statistical interpretation of the experimental data strongly suggested that abnormalities in allantoic/umbilical cord development were directly associated with embryolethality as evidenced by a decline in the frequency of abnormal allantoic/umbilical cord phenotypes over GD 10-12 (73, 36, and 4%; respectively) and a concomitant increase in the frequency of implantation site resorptions over the same time period (7, 47, and 78%). These results strongly suggest that the developing allantois is very sensitive to the effects of dCF exposure, and that interference with its development leads to embryolethality by GD 12

Occurrence of Embryotoxicity in Mouse Embryos Following in Utero Exposure to 2′‐deoxycoformycin (Pentostatin)

Previous investigations had shown that i.p. injection of 2′‐deoxycoformycin (dCF; pentostatin; 5 mg/kg) on either E7 or E8 into pregnant mice results in a 61–81% resorption rate at E17. The incidence of visible gross malformations among the surviving conceptuses was exceptionally low (3%) at the time of necropsy on E17 and was unrelated to dCF dose (Knudsen et al., Teratology, 40:5–626, \u2789; Teratology, 45:91–103, \u2792). These findings demonstrated the embryotoxicity of dCF but provided no clues as to the site(s) of dCF action. To define the lesion site(s), we have now examined embryos at 72 h(E10), 96 h (E11), and 120 h (E12) following administration of a highly embryotoxic dose of 5 mg dCF/kg to dams on E7. Deoxycoformycin caused multiple abnormalities and growth retardation, and the temporal sequence between maximal abnormal embryo incidence and resorption frequency was established. The quantitative data show that the maximal occurrence of abnormal embryos on E10 (71%) was followed by a maximal resorption rate on E12 (78%). There was a strong correlation (r = −0.82; P \u3c 0.05) between the rapid decline of percent abnormal embryos over E10–E12 and the simultaneous increase in resorption rate, with linear regression analysis showing nearly equal but opposite slopes (−31.2% vs. +35.8% per gestational day, respectively). This suggests that one or more of the abnormalities seen at E10 is associated with the death and resorption of the embryo at E12. The dCF treatment perturbed a wide spectrum of developmental events, including neural tube closure, craniofacial and limb development, turning of the embryo, and growth retardation. None of the individual abnormalities, however, can quantitatively account for the high percentage of dead and resorbed embryos. Therefore, the specific cause of dCF‐induced embryolethality is not clear. There is evidence both for direct dCF toxicity at specific embryonic sites as well as for a generalized retardation in the rate of development

Adenosine Levels in the Postimplantation Mouse Uterus: Quantitation by Hplc‐fluorometric Detection and Spatiotemporal Regulation by 5′‐nucleotidase and Adenosine Deaminase

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryodecidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5′‐nucleotidase (5′‐NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno‐derivatized and quantitated by high‐performance liquid chromatography‐fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5′‐NT, an enzyme which catalyzes the irreversible dephos‐phorylation of 5′‐AMP to adenosine. 5′‐NT expression was shown by Northern blot analysis to peak in the embryo‐decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5′‐NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5′‐NT appeared on giant trophoblast (days 7–13) and the metrial gland (days 11–13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S‐adenosylhomocysteine hydrolase) were not detected in the embryo‐decidual unit suggesting that the net flux of utero‐placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6–11), secondary giant cells (days 7–13), and spongiotrophoblasts (days 8–13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5′‐NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities. Purine nucleoside phosphorylase and xanthine oxidase, which are two catabolic enzymes acting subsequent to 5′‐NT and ADA in the sequential degradation of AMP to xanthine, remained low and constant in the tissues examined suggesting that the catabolic pathway is geared toward regulation of adenosine levels. These results suggest the establishment of an adenosine gradient across the developing antimesometrium. It is proposed that the source of adenosine is AMP released during uterine cell death, and that adenosine, in turn, serves as a regulatory signal to coordinate early postimplantation morphogenetic events with the progression of cell death at the uterine‐embryo interface

Movement Disorders and Neurochemical Changes in Zebrafish Larvae After Bath Exposure to Fluoxetine (PROZAC)

This study examines the effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (PROZAC), on the ontogeny of spontaneous swimming activity (SSA) in developing zebrafish. The development of zebrafish motor behavior consists of four sequential locomotor patterns that develop over 1-5 days post fertilization (dpf), with the final pattern, SSA, established at 4-5 dpf. In stage specific experiments, larvae were exposed to 4.6 μM fluoxetine for 24 h periods beginning at 24 h post fertilization (hpf) and extending through 5 dpf. From 1-3 dpf, there was no effect on SSA or earlier stages of motor development, i.e., spontaneous coiling, evoked coiling and burst swimming. Fluoxetine exposure at 3 dpf for 24 h resulted in a transient decrease in SSA through 7 dpf with a complete recovery by 8 dpf. Larvae exposed to 4.6 μM fluoxetine for 24 h on 4 or 5 dpf showed a significant decrease in SSA by day 6 with no recovery through 14 dpf. Although SSA was significantly affected 24 h after fluoxetine exposure, there was little or no effect on pectoral fin movement. These results demonstrate both a stage specific and a long term effect of 4.6 μM fluoxetine exposure in 4 and 5 dpf larvae. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the relative levels of a serotonin transporter protein (SERT) transcript and the serotonin 1A (5-HT1A) receptor transcript in developing embryos/larvae over 1-6 dpf. Both transcripts were present at 24 hpf with the relative concentration of SERT transcript showing no change over the developmental time range. The relative concentration of the 5-HT1A receptor transcript, however, showed a two-tiered pattern of concentration. RT-PCR was also used to detect potential changes in the SERT and 5-HT1A receptor transcripts in 6 dpf larvae after a 24 h exposure to 4.6 μM fluoxetine on 5 dpf. Three separate regions of the CNS were individually analyzed, two defined brain regions and spinal cord. The two brain regions showed no effect on transcript levels subsequent to fluoxetine exposure, however, the spinal cord showed a significant decrease in both transcripts. These results suggest a correlation between decreased concentration of SERT and 5-HT1A receptor transcripts in spinal cord and decreased SSA subsequent to fluoxetine exposure