Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy

Following 10 daily pairings of multiple conditioned stimuli with injection of cocaine (15 mg/kg), the presentation of the stimuli alone elicited behaviors in rats similar to those induced by cocaine. The behaviors included increased duration or frequency of rearing, sniffing, head bobbing, and horizontal locomotor activity (crossing). The level of the conditioned response for several of these behaviors approximated that induced by the drug itself. The conditioned drug effect showed decay over 15 days but little extinction during 4 daily trials. Brain concentrations of the dopamine metabolites, homovanillic acid and dihydroxyphenylacetic acid, were similar in the conditioned and pseudoconditioned control groups in both the caudate and mesolimbic areas. The behavioral results demonstrate that, in a classical conditioning paradigm, previously neutral stimuli can elicit behaviors similar to those induced by cocaine and that certain conditioned responses show time related decline. This agrees with the reported conditioning of amphetamine's behavioral effects but differs in terms of the action on brain dopamine turnover.     

Studies of sulfate utilization by algae: 10. Nutritional and enzymatic characterization of chlorella mutants impaired for sulfate utilization

Seven mutants of Chlorella pyrenoidosa (Emerson strain 3) impaired for sulfate utilization have been isolated after treatment of the wild-type organism with nitrosoguanidine by replica plating on media containing thiosulfate and l-methionine. These mutants fall into three classes based on their ability to grow on sulfate, accumulate compounds labeled from sulfate-³⁵S, and reduce adenosine 3′-phosphate 5′-phosphosulfate-³⁵S (PAPS-³⁵S) to thiosulfate-³⁵S. Mutant Sat₂⁻ cannot grow on sulfate, but it accumulates thiosulfate-³⁵S and homocysteic acid-³⁵S from sulfate-³⁵S in vivo. In addition, extracts of mutant Sat₂⁻ reduce PAPS-³⁵S to thiosulfate-³⁵S, indicating the possession of enzyme fractions S and A, both of which are required for thiosulfate formation. Mutants Sat₁⁻, Sat₃⁻, Sat₄⁻, Sat₅⁻, and Sat₆⁻ cannot grow on sulfate, and their extracts lack the ability to reduce PAPS-³⁵S to thiosulfate-³⁵S. Mutant Sat₇⁻R₁, a probable revertant, can grow on sulfate but still lacks the ability to reduce PAPS-³⁵S to thiosulfate-³⁵S in vitro. Complementation experiments in vitro show that the block in formation of acid-volatile radioactivity in every case is due to the absence of activity associated with fraction S. All mutants can grow on thiosulfate and all possess the activating enzymes which convert sulfate to PAPS. Through a comparison of nutritional and enzymatic characteristics, the first outlines of a branched and complicated pathway for sulfate reduction in Chlorella are beginning to emerge.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.     

A comparison of light-dependent RNA metabolism in wild-type Euglena with that of mutants impaired for chloroplast development

Summary The possibility that ³²PO ₄ ^3- (³²P_i) labeling of both chloroplast and non-chloroplast RNAs during light-induced chloroplast development in Euglena is due, in part, to the break-down of existing RNAs and their resynthesis into labeled RNAs has been examined by comparing the RNA content of dark-grown, non-dividing cells after completion of light-induced chloroplast development with that of identical cells maintained in darkness for the same period of time. The involvement of the photo-conversion of protochlorophyll to chlorophyll and other photoreceptor systems in the labeling of RNA during chloroplast development has been considered by comparing the labeling pattern obtained with wild-type cells with the patterns obtained with mutants of Euglena which either lack detectable amounts of protochlorophyll and chlorophyll or form only rudimentary chloroplasts upon light induction.No significant difference in RNA content between dark-grown, non-dividing cells containing fully developed chloroplasts and the same cells maintained in darkness for the development period can be detected. This observation is interpreted to mean that in non-dividing cells precursors for chloroplast-associated RNAs are derived from pools and pre-existing RNAs, including non-chloroplast RNAs, and that the matebolic entrapment of ³²P_i involves a light-dependent turnover and DNA-directed RNA synthesis in wild-type cells.The RNA profiles on sucrose gradients of mutants of Euglena show no remarkable deviation from the profile established for wild-type cells. The labeling patterns obtained after 24 hours of incubation in light and in darkness differ from that obtained for wild-type cells in that all mutants show less of a light-minus-dark difference than wild-type and that mutants lacking plastid-associated DNA and detectable amounts of chlorophyll incorporate considerably more ³²P_i into RNA in darkness than wild-type. One such mutant shows no significant difference in its light-dark labeling pattern.These observations indicate that cells possessing normal proplastids capable of forming functional chloroplasts regulate metabolism of RNA in darkness in a different manner than with either rudimentary chloroplasts or containing no detectable plastids structures. The possible involvement of more than one photoreceptor system in metabolic control is discussed.Supported by a grant from the National Institutes of Health, GM 14595     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

Derivation and characterization of an efficiently myocarditic reovirus variant.

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.