Constitutive activity of 5-HT2C receptors is present after incomplete spinal cord injury but is not modified after chronic SSRI or baclofen treatment.

After spinal cord injury (SCI), reflexes become hyperexcitable, leading to debilitating muscle spasms and compromised motor function. Previous work has described adaptations in spinal systems that might underlie this hyperexcitability, including an increase in constitutively active 5-HT2C receptors in spinal motoneurons. That work, however, examined adaptations following complete transection SCI, whereas SCI in humans is usually anatomically and functionally incomplete. We therefore evaluated whether constitutive activity of 5-HT2C receptors contributes to reflex hyperexcitability in an incomplete compression model of SCI and to spasms in vitro and in vivo. Our results confirm that 5-HT2C receptor constitutive activity contributes to reflex excitability after incomplete SCI. We also evaluated whether constitutive activity could be altered by manipulation of neural activity levels after SCI, testing the hypothesis that it reflects homeostatic processes acting to maintain spinal excitability. We decreased neural activity after SCI by administering baclofen and increased activity by administering the selective serotonin reuptake inhibitor (SSRI) fluoxetine. We found that drug administration produced minimal alterations in in vivo locomotor function or reflex excitability. Similarly, we found that neither baclofen nor fluoxetine altered the contribution of constitutively active 5-HT2C receptors to reflexes after SCI, although the contribution of 5-HT2C receptors to reflex activity was altered after SSRIs. These results confirm the importance of constitutive activity in 5-HT2C receptors to spinal hyperexcitability following SCI in the clinically relevant case of incomplete SCI but suggest that this activity is not driven by homeostatic processes that act to maintain overall levels of spinal excitability.NEW & NOTEWORTHY After spinal cord injury (SCI), most people will develop muscle spasms below their level of injury that can severely impact function. In this work, we examine the adaptations that occur within the spinal cord after SCI that contribute to these motor dysfunctions. We also evaluate one hypothesis about how these adaptations develop, which will potentially lead to intervention strategies to improve functional outcomes in persons with SCI.

Modified sham feeding of sweet solutions in women with and without bulimia nervosa.

Although it is possible that binge eating in humans is due to increased responsiveness of orosensory excitatory controls of eating, there is no direct evidence for this because food ingested during a test meal stimulates both orosensory excitatory and postingestive inhibitory controls. To overcome this problem, we adapted the modified sham feeding technique (MSF) to measure the orosensory excitatory control of intake of a series of sweetened solutions. Previously published data showed the feasibility of a "sip-and-spit" procedure in nine healthy control women using solutions flavored with cherry Kool Aid and sweetened with sucrose (0-20%). The current study extended this technique to measure the intake of artificially sweetened solutions in women with bulimia nervosa (BN) and in women with no history of eating disorders. Ten healthy women and 11 women with BN were randomly presented with cherry Kool Aid solutions sweetened with five concentrations of aspartame (0, 0.01, 0.03, 0.08 and 0.28%) in a closed opaque container fitted with a straw. They were instructed to sip as much as they wanted of the solution during 1-minute trials and to spit the fluid out into another opaque container. Across all subjects, presence of sweetener increased intake (p<0.001). Women with BN sipped 40.5-53.1% more of all solutions than controls (p=0.03 for total intake across all solutions). Self-report ratings of liking, wanting and sweetness of solutions did not differ between groups. These results support the feasibility of a MSF procedure using artificially sweetened solutions, and the hypothesis that the orosensory stimulation of MSF provokes larger intake in women with BN than controls.

Intake, sweetness and liking during modified sham feeding of sucrose solutions.

Although sweet solids and liquids are palatable to humans and ingested frequently when readily available, the quantitative relationship between sweet taste and intake has not been reported in humans. To investigate the quantitative relationship between sweet taste and intake directly, we adapted the modified sham feeding technique, previously used in humans for the study of the orosensory control of autonomic, neuroendocrine, and metabolic mechanisms, to measure the intake of solutions both unsweetened and sweetened with four concentrations of sucrose. By limiting the sucrose stimuli to the mouth, the modified sham feeding technique measures the orosensory stimulation of intake by sucrose in the absence of inhibitory postingestive stimulation. Nine healthy women were randomly presented with two series of five solutions of cherry Kool Aid unsweetened or sweetened with one of four concentrations of sucrose (2.5%, 5%, 10%, or 20%) in a closed opaque container fitted with a straw. They were instructed to sip as much as they wanted of the liquid during 2-min trials and to spit the fluid out into another opaque container. At the end of each trial, they used Visual Analogue Scales to rate the perceived intensities of sweetness and liking of the liquid that they had just sipped and spit. Intake, liking and perceived sweetness were significantly affected by sucrose concentration (p values

Increased vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGFbeta) in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization.

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and B10.A mice by immunization with S-antigen (S-Ag) to study the potential roles of vascular endothelial growth factor (VEGF) and the beta1 and beta2 isoforms of transforming growth factor (TGFbeta1 and TGFbeta2) during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFbeta1 and TGFbeta2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier (BRB) failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFbeta1 and TGFbeta2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFbeta1 or TGFbeta2. In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFbeta1 or TGFbeta2. Since TGFbeta can inhibit endothelial cell proliferation, it is likely that an increase in TGFbeta may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors.

Blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains.

Intraocular coronavirus inoculation results in a biphasic retinal disease in susceptible mice (BALB/c) characterized by an acute inflammatory response, followed by retinal degeneration associated with autoimmune reactivity. Resistant mice (CD-1), when similarly inoculated, only develop the early phase of the disease. Blood-retinal barrier (BRB) breakdown occurs in the early phase in both strains, coincident with the onset of inflammation. As the inflammation subsides, the extent of retinal vascular leakage is decreased, indicating that BRB breakdown in experimental coronavirus retinopathy (ECOR) is primarily due to inflammation rather than to retinal cell destruction. Vascular endothelial growth factor (VEGF) is upregulated only in susceptible mice during the secondary (retinal degeneration) phase.

Heteroduplex resolution using T7 endonuclease I in microbial community analyses.

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection.

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.

Development of an animal model of fluoxetine akathisia.

1. Akathisia describes the pattern of intense inner restlessness often associated with neuroleptic and antidepressant treatment. 2. The authors postulated that drug-induced akathisia would be characterized by more position changes and less time spent immobile, in the absence of significant increase in ambulation. In contrast, a psychomotor stimulant would produce both activation and ambulation. 3. Procedures and instruments were developed to test this hypothesis. Adult rats were habituated for 72 hours to the testing environment, and their precise pattern of movements was tracked and recorded (10 reading per second; resolution 0.04 mm) by an infrared motion analysis system. Activity was recorded for a 90 min period after a single injection of sub-stereotypic doses of d-amphetamine (0, 0.3, 1.0 mg/kg) or racemic fluoxetine (0, 3.0, 10.0, 20.0, or 30.0 mg/kg, s.c.). 4. Amphetamine produced both activation and ambulation. Activation was indicated by a decrease in time spent immobile, and an increase in the temporal scaling exponent, which reflects the degree the animal is "acting' in its environment, and the number of position changes. Enhanced locomotion was inferred from marked increases in both the total distance traversed and the ratio of forward movements-to-reversals and a decrease in the spatial scaling exponent, indicative of a less complex and more linear movement pattern. 5. Fluoxetine caused animals to spend more time active, but exerted little effect on locomotion. Activation was indicated by a decrease in time spent immobile and an increase in the temporal scaling exponent and number of position changes. Fluoxetine failed to significantly effect either the ratio of forward movements-to-reversals or the spatial scaling exponent. 6. These findings provide an operational definition and methodology that can be used to differentiate between psychostimulant effects and akathisic effects. This approach may have utility for screening drugs for akathisic potential, for exploring underlying mechanisms, and for developing novel treatments.

Chronic benign pain.

Chronic benign pain (CBP) can be defined as a type of unpleasant sensory experience that arises from inflammation, visceral stress or damage, or other such pathophysiologic process(es), and that is not associated with a metastatic process. A patient's complaint of pain should be taken seriously by the practitioner, both in terms of the discomfort evoked and the likelihood that the potential cause of the pain requires diagnostic evaluation. This article reviews the diagnosis and treatment of the following common conditions associated with CBP syndromes: fibromyalgia, lower back pain syndrome, sickle-cell disease, reflex sympathetic dystrophy syndrome, and peripheral neuropathies.

Development of an affordable hi-resolution activity monitor system for laboratory animals.

We describe a hardware and software system for recording and analyzing the spatial and temporal pattern of locomotor activity of laboratory animals. The system offers maximal spatial resolution 500-fold greater than existing light beam monitors. An infrared motion analysis systems (MacReflex, Qualysis) simultaneously tracks the location of up to 20 subjects (identified by reflective markers) to within 0.04 mm at a rate of up to 50 Hz. Macintosh software provides measures of distance traveled, amount of area traversed, number of position changes (microevents), average time between movements, number of left and right turns, number of forward movements and reversals, as well as temporal and spatial scaling exponents. This system was validated by comparing these parameters to direct observer scoring of video tapes and other commercially available activity monitors. Our findings show that applying reflective markers to the subjects does not significantly alter activity levels. The effect of pharmacological manipulation with d-amphetamine is provided to show the value of the different activity parameters. The main advantages of this system are very high spatial resolution, capacity to monitoring up to 20 animals simultaneously at reasonable cost, and lack of sensitivity of the system to ambient lighting. The main limitation is the need to apply reflective markers.

Bladder freeze ulceration and sodium saccharin feeding in the rat: examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes.

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.

Molecular and cultural assessment of chytrid and Spizellomyces populations in grassland soils.

We developed a molecular method for the detection and quantification of members of the genus Spizellomyces in the environment and used this technique, together with traditional cultural techniques, to measure the effects of cultivation and nitrogen availability on Spizellomyces populations in grassland soils. Primer sets specific for Spizellomyces acuminatus and S. kniepii were developed by sequencing internal transcribed spacer 2 (ITS2) of the gene encoding ribosomal RNA for 9 isolates within the genus Spizellomyces, 5 representatives of different genera within the order Spizellomycetales and one member of the order Chytridiales. These primers were used with fungal-specific primers in a nested PCR approach to generate a specific molecular signal for S. acuminatus and S. kneipii in a soil from which S. acuminatus had previously been recovered. Using MPN-PCR (a quantitative molecular technique) and traditional cultural techniques, we found that chytridiomycetous fungi, including members of the genus Spizellomyces, are abundant in the grassland ecosystems studied. No significant differences in occurrence were observed between native and disturbed control soils but it appeared in 2 separate MPN assays and one MPN-PCR assay that chytrid populations increased in response to disturbance. No significant differences in chytrid or Spizellomyces populations were observed with variations in nitrogen availability. The primer sets and protocols developed in this study worked well to complement traditional cultural data to better assess Spizellomyces populations in the environment. These molecular approaches should provide a foundation for further work with these interesting and oft neglected fungi.

Modified sham feeding of sweet solutions in women with anorexia nervosa.

Anorexia Nervosa (AN) is a disorder of self-starvation characterized by decreased meal size and food intake. While it is possible that reduced food intake in AN reflects an excess of inhibitory factors, e.g., cognitive inhibition related to fear of weight gain or abnormal postingestive negative feedback, it is also possible that decreased intake reflects diminished orosensory stimulation of food intake. This has been difficult to test directly because the amount of food ingested during a test meal by patients with AN reflects an integration of orosensory excitatory, and cognitive, learned, and postingestive inhibitory controls of eating. To begin to dissociate these controls, we adapted the modified sham feeding technique (MSF) to measure the intake of a series of sweetened solutions in the absence of postingestive stimulation. Subjects with AN (n=24) and normal controls (NC, n=10) were randomly presented with cherry Kool Aid solutions sweetened with five concentrations of aspartame (0, 0.01, 0.03, 0.08 and 0.28%) in a closed opaque container fitted with a straw. They were instructed to sip as much as they wanted of the solution during 15 1-minute trials and to spit the fluid out into another opaque container. Subjects with AN sipped less unsweetened solution than NC (p<0.05). Because this difference appeared to account completely for the smaller intakes of sweetened solutions by AN, responsiveness of intake to sweet taste per se was not different in AN and NC. Since MSF eliminated postingestive and presumably cognitive inhibitory controls, and the orosensory response to sweet taste was not different in AN than NC, we conclude that decreased intake by AN subjects under these conditions reflects the increased inhibition characteristic of this disorder that is presumably learned, with a possible contribution of decreased potency of orosensory stimulation by the sipped solutions.

Starvation effects on Escherichia coli and aquatic bacterial responses to nutrient addition and secondary warming stresses.

A gram-negative polar flagellated rod, isolated from a Colorado mountain stream and considered to be an Aeromonas sp., a mixed aquatic microbial population, and a culture of Escherichia coli were starved to determine their responses to the short-term presence of nutrients and mild warming stress (49.5 C for 2 min) in relation to starvation time. At the beginning of the starvation period, the Aeromonas isolate was extremely sensitive to the secondary warming stress. This response was markedly diminished after a 3-week starvation period. The mixed aquatic microbial population showed a similar trend in becoming less sensitive to stress with increased starvation. E coli under similar conditions, became more sensitive to the secondary stress after exposure to glucose. Respiration measurements after glucose additions also indicated that E. coli responded in a different manner to starvation stress and glucose presence than the Aeromonas isolate. The increased sensitivity of E. coli to secondary stress and short-term nutrient availability after starvation may contribute to the exclusion of this organism from aquatic environments.

Eating disorders.

Eating disorders rank among the most debilitating psychiatric disturbances that affect young women. Knowledge has increased in recent years about the two major eating disorders, anorexia nervosa (AN) and bulimia nervosa (BN); however, much remains unknown. This review article will provide an overview of the epidemiology, proposed risk factors and clinical features of AN, and BN, as well as current recommendations for evaluation and treatment of these disorders.

Comparative single-strand conformation polymorphism (SSCP) and microscopy-based analysis of nitrogen cultivation interactive effects on the fungal community of a semiarid steppe soil.

The effects of nitrogen accretion on fungal diversity and community structure in early-seral (cultivated) and native (uncultivated) shortgrass steppe soils were evaluated using single-strand conformation polymorphism (SSCP) and microscopy in a comparative experiment. Selected haplotypes generated from fungal 18S gene fragments were also sequenced for species identification. Microscopy-based analyses showed significantly shorter fungal hyphal lengths in the early-seral control plots in comparison with the native control plots (P<0.0003), independent of nitrogen addition. Although diversity indices did not show significant differences between the plots, SSCP analyses indicated that fungal community structure differed in the native and early-seral control sites. In nitrogen-amended sites, gene sequences from dominant haplotypes indicated a shift to a more common nitrogen-impacted fungal community. While nitrogen amendments appear to be more important than cultivation in influencing these soil fungal communities, hyphal lengths were only decreased due to cultivation. The use of microscopic and molecular techniques, as carried out in this study, provided integrative information concerning fungal community responses to wide spread stresses being imposed globally on terrestrial ecosystems, that is not provided by the individual techniques.

Filamentous fungi: the indeterminate lifestyle and microbial ecology.

The filamentous fungi have dynamic and variable hyphal structures within which cytoplasm can be moved, synthesized, and degraded, in response to changes in environmental conditions, resource availability, and resource distribution. Their study has gone through several phases. In the first phase, direct observation was emphasized without undue concern for interior structures or in the presence of cytoplasm. By the mid-1970s, single biochemical proxies (ergosterol, marker fatty acids, chitin derivatives, etc.) were being used increasingly. The use of these surrogate single measurements continues, in spite of their inability to provide information on the physical structure of the filamentous fungi. Molecular approaches also are being used, primarily through the use of bulk nucleic acid extraction and cloning. Because the sources of the nucleic acids used in such studies usually are not known, taxonomic and phylogenetic information derived by this approach cannot be linked to specific fungal structures. Recently, a greater emphasis has been placed on assessing physical aspects of indeterminate fungal growth, involving the assessment of cytoplasm-filled and evacuated (empty) hyphae. Both of these parameters are important for describing filamentous fungal growth and function. The use of phase contrast microscopy and varied general stains, as well as fluorogenic substrates with observation by epifluorescence microscopy, has made it possible to provide estimates of cytoplasm-filled hyphal lengths. Using this approach, it has been possible to evaluate the responses of the indeterminate fungal community to changes in environmental conditions, including soil management. It is now possible to obtain molecular information from individual bacteria and fungal structures (hyphae, spores, fruiting bodies) recovered from environments, making it possible to link individual fungal structures with their taxonomic and phylogenetic information. In addition, this information can be considered in the context of the indeterminate filamentous fungal lifestyle, involving the dynamics of resource allocation to hyphal structural development and synthesis of cytoplasm. Use of this approach should make it possible to gain a greater appreciation of the indeterminate filamentous fungal lifestyle, particularly in the context of microbial ecology.

Structural effects on Arthrobacter methylene hydroxylase activity.

Arthrobacter 4-44-2 (ATCC 25581), capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4-alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 16 carbons in length and was especially strong with methylcyclohexane among cyclic compounds tested. Induction was not observed with several related alcohols, 1-unsaturated compounds, or methoxy and ethoxy compounds tested. After induction, n-alkanes 14 and 16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with even-carbon alkanes of shorter chain length. Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products. Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting that a methyl group plays a role in orientation of the substrate for the methylene hydroxylation but that the methyl function was not as critical after completion of the hydroxylation step regardless of structural configuration. Acetate strongly repressed induction of n-hexadecane methylene hydroxylase activity. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems with methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated.