The heterozygous mutation B6;C3-Opa1(Q285STOP), which models autosomal dominant optic atrophy, leads to a 50% reduction in Opa1 transcript and protein in the mouse retina and neural tissues and is associated with visual dysfunction and structural changes in the murine retina and optic nerve. In this article we use this model to quantify and evaluate the dendritic morphology of retinal ganglion cells. Retinal ganglion cells in Opa1(+)(/)(-) mutant mice (n = 16) and accompanying age- and sex-matched controls (n = 11) (age ranges of <10, 10-15 and >20 months) were labelled DiOlistically with carbocyanine dyes to quantify changes in dendritic tree architecture as a function of age. We observed localized dendritic reduction to sublamina b of the inner plexiform layer without retinal ganglion cell loss, showing dendritic pruning of on- but not off-centre retinal ganglion cells, and this effect was exacerbated with age. The mean dendritic field area was reduced in on-centre retinal ganglion cells of 10- to 15-month-old mice (-24.24%; C(V) = 0.68; P < 0.05) and >20-month-old mice (-43.22%; C(V) = 0.75; P < 0.05) compared with age-matched wild-type controls. Similar changes were seen in average total dendritic length in on-centre retinal ganglion cells of 10- to 15-month-old mice (-31.66%; C(V) = 0.67; P < 0.05) and >20-month-old mice (-49.55%; C(V) = 0.63; P < 0.05). Sholl analysis showed a marked difference in the dendritic arborization of on-centre retinal ganglion cells in the 10- to 15-month-old group (area under the curve -21.67%; P > 0.05) and of the >20-month-old group (area under the curve -42.12%; P < 0.05) compared with the control group. There was no detectable change in dendritic morphology in <10-month-old Opa1(+/)(-) mutant mice compared with wild-type (P > 0.05). No significant changes (P > 0.05) were seen in off-centre retinal ganglion cells. Finally, there was also no significant change (P > 0.05) in the retinal ganglion cell count across all age groups. In conclusion, we show dendritic pruning in on-centre retinal ganglion cells of the Opa1(+/)(-) mouse model of autosomal dominant optic atrophy from as early as 10 months of age. These results highlight the importance of normal mitochondrial fusion balance, as influenced by the OPA1 protein in maintaining the dendritic morphology of retinal ganglion cells. Dendritic pruning precedes the onset of clinical visual loss and structural changes in the optic nerve in the absence of significant cell loss.

Dominant optic atrophy (DOA) is the most common inherited optic neuropathy affecting 1 in every 12,000 people. It presents with bilateral visual loss, central visual fields defects, colour vision disturbance and optic disc pallor. OPA1 has been identified as the responsible gene and its locus mapped to chromosome 3q28-q29. Mutations in this gene are responsible for the clinical phenotype in over 70% of patients with DOA. Histopathological studies in tissues from patients reveal loss of retinal ganglion cells but the paucity of viable human tissue has raised the importance of an animal model to study the pathophysiology of the disease. In the last decade considerable work has gone into the generation of animal, most notably mouse, models of Opa1 DOA. Two murine models of DOA have been published, designated B6;C3-Opa1(Q285STOP) and B6;C3-Opa1(329-355del) and they provide valuable insights with respect to neurological and visual phenotyping, mitochondrial dysfunction, optic nerve and axonal changes, retinal ganglion cell depletion and dendritic atrophy. Here we summarise the current state of knowledge of the mechanisms of disease based on data from these models of Opa1 DOA.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.Cell Death and Differentiation advance online publication, 6 August 2010; doi:10.1038/cdd.2010.95.

Satellite cells are quiescent cells located under the basal lamina of skeletal muscle fibres that contribute to muscle growth, maintenance, repair and regeneration. Mouse satellite cells have been shown to be muscle stem cells, able to regenerate muscle fibres and self-renew. As human skeletal muscle is also able to regenerate following injury, we assume that the human satellite cell is, like its murine equivalent, a muscle stem cell. In this review, we compare human and mouse satellite cells and highlight their similarities and differences. We discuss gaps in our knowledge of human, compared to mouse, satellite cells and suggest ways in which we may advance studies on human satellite cells, particularly by finding new markers and attempting to re-create the human satellite cell niche in vitro.

Na(+)-NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H(+)-pumping complex I. It has generally been assumed that the sodium pump of Na(+)-NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na(+)-NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN(C), while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN(B) to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that Na(+)-NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes.

Skeletal muscle is maintained and repaired by resident stem cells called muscle satellite cells, but there is a gradual failure of this process during the progressive skeletal muscle weakness and wasting that characterises muscular dystrophies. The pathogenic mutation causes muscle wasting, but in conditions including Duchenne muscular dystrophy, the mutant gene is not expressed in satellite cells, and so muscle maintenance/repair is not directly affected. The chronic muscle wasting however, produces an increasingly hostile micro-environment in dystrophic muscle. This probably combines with excessive satellite cell use, to eventually culminate in an indirect failure of satellite cell-mediated myofibre repair. By contrast, in disorders such as Emery-Dreifuss muscular dystrophy, the pathogenic mutation not only instigates muscle wasting, but could also directly compromise satellite cell function, leading to less effective muscle homeostasis. This may again combine with excessive use and a hostile environment to further compromise satellite cell performance. Whichever the mechanism, the ultimate consequence of perturbed satellite cell activity is a chronic failure of myofibre maintenance in dystrophic muscle. Here, we review whether the pathogenic mutation can directly contribute to satellite cell dysfunction in a number of muscular dystrophies.

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients and may also confound the analysis of dystrophin restoration in treated patients, if, as previously observed in the mdx mouse, revertant fibres increase with age. In 62% of the diagnostic biopsies reports of 65 DMD patients studied, traces or revertants were recorded with no correlation between traces or revertants, the patients' performance, or corticosteroids response. In nine of these patients, there was no increase in traces or revertants in biopsies taken a mean of 8.23 years (5.8-10.4 years) after the original diagnostic biopsy. This information should help in the design and execution of clinical trials focused on dystrophin restoration strategies.

In the photocycle of bacteriorhodopsin at pH 7, proton release from the proton releasing group (PRG) to the extracellular medium occurs during formation of the M intermediate. This proton release is inhibited at acidic pH, below the pKa of the PRG, ~6 in M, and instead occurs later in the cycle as the initial state is restored from the O intermediate. Here, structural changes related to deprotonation of the PRG have been investigated by time-resolved FTIR spectroscopy at 25 degrees C. The vibrational features at 2100-1790 cm-1, 1730-1685 cm-1, 1661 cm-1, and 1130-1045 cm-1 have greater negative intensity in the pure M-minus-BR spectrum and even in the M-minus-BR spectrum, that is present earlier together with the L-minus-BR spectrum, at pH 7, than in the corresponding M-minus-BR spectra at pH 5 or pH 4. The D212N mutation abolishes the decreases in the intensities of the broad feature between 1730 and 1685 cm-1 and the band at 1661 cm-1. The 1730-1685 cm-1 feature may arise from transition dipole coupling of the backbone carbonyl groups of Glu204, Phe208, Asp212 and Lys216 interacting with Tyr57 and C15-H of the chromophore. The 1661 cm-1 band, which is insensitive to D2O substitution, may arise by interaction of the backbone carbonyl of Asp212 with C15-H. The 2100-1790 cm-1 feature with a trough at 1885 cm-1 could be due to a water cluster. Depletion of these bands upon deprotonation of the PRG is attributable to disruption of a coordinated structure, held in place by interactions of Asp212. Deprotonation of the PRG is accompanied also by disruption of the interaction of the water molecule near Arg82. The liberated Asp212 may stabilize the protonated state of Asp85, and thus confer uni-directionality to the transport.

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at-risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA-deficient breast cancers.

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.

Stem cell therapy holds promise for treating muscle diseases. Although satellite cells regenerate skeletal muscle, they only have a local effect after intra-muscular transplantation. Alternative cell types, more easily obtainable and systemically-deliverable, were therefore sought. Human synovial stem cells (hSSCs) have been reported to regenerate muscle fibres and reconstitute the satellite cell pool. We therefore determined if these cells are able to regenerate skeletal muscle after intra-muscular injection into cryodamaged muscles of Rag2-/gamma chain-/C5-mice. We found that hSSCs possess only limited capacity to undergo myogenic differentiation in vitro or to contribute to muscle regeneration in vivo. However, this is enhanced by over-expression of human MyoD1. Interestingly, hSSCs express extracellular matrix components laminin alpha2 and collagen VI within grafted muscles. Therefore, despite their limited capacity to regenerate skeletal muscle, hSSCs could play a role in treating muscular dystrophies secondary to defects in extracellular matrix proteins.

Abstract Aims The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and western blotting is often used in these cases, but is not always possible if the sample is scarce. We therefore sought to develop a method to quantify relative levels of sarcolemma-associated proteins using digitally captured images of immunolabelled sections of skeletal muscle. Methods To validate our relative quantification method, we labelled dystrophin and other sarcolemmal proteins in transverse sections of muscle biopsies taken from Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) patients, a manifesting carrier of DMD and normal controls. Results Using this method to quantify relative sarcolemmal protein abundance, we were able to accurately distinguish between the different patients on the basis of the relative amount of dystrophin present. Conclusions This comparative method adds value to techniques that are already part of the diagnostic process and can be used with minimal variation of the standardised protocols, without using extra amounts of valuable biopsy samples. Comparative quantification of sarcolemmal proteins on immunostained muscle sections will be of use to establish both the abundance and localization of the protein. Moreover, it can be applied to assess the efficacy of experimental therapies where only partial restoration or up-regulation of the protein, may occur.

Purpose. The quantification of early retinal ganglion cell damage in ocular hypertension and glaucoma. Methods. Thirty subjects under treatment for open angle glaucoma, 23 subjects with ocular hypertension and a control group of 28 normal subjects were investigated using monocular pattern electroretinograms (ERG), L&amp; M cone ERGs and S-cone ERGs. Glaucoma diagnosis was based on masked assessment of digital stereoscopic optic nerve head images by 3 glaucoma specialists. The optic nerve head and retinal nerve fibre layer was assessed by scanning laser ophthalmoscopy and optical coherence tomography. Results. All types of ERG had reduced mean amplitudes in ocular hypertension and open angle glaucoma groups compared to the control group. In the ocular hypertension group the N95 and the L&amp;M-pathway PhNR were significantly attenuated being reduced by 17% and 18% compared to the control group respectively (30% and 22% for the open angle glaucoma group respectively). For the open angle glaucoma subjects, the PERG P50-N95 was found to be the most sensitive electrophysiological test and the cup:disc area with the scanning laser ophthalmoscopy the most sensitive imaging parameter. Modest but not statistically significant correlations were found between the imaging and electrophysiological parameters. Conclusion. With disc appearance used for the classification of open angle glaucoma and ocular hypertension, significant electrophysiological losses are found in both conditions. The modest correlation between the structural and electrophysiological measures suggests that these assess different aspects of the pathological process; electrophysiology can be used to quantify retinal ganglion cell dysfunction that occurs prior to cell death.

Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles.

Glaucoma is characterised by the preferential death of retinal ganglion cells (RGCs). However, mammalian models indicate that neurons pass through a period in which they manifest signs of neuronal damage, but have yet to fully commit to death. Mounting evidence suggests that one of the clearest indications of this process is the reduction in RGC dendritic arborisation, resulting in functional compromise. The extent to which this may be reversible is unclear, since the molecular events that precede changes in dendritic structure have received little attention. Furthermore, there are likely to be many factors involved in this process potentially acting in different individual cells at different times. Recent work in Drosophila shows that dendritic reorganisation/remodelling involves local activation and tight regulation of caspase activity. Here, we propose a model in which the balance between caspases and inhibitors of apoptosis (IAPs) contributes towards the regulation of dendritic remodelling. Thus, RGC dendrite reorganisation and cell death represent opposite ends of a spectrum of events regulated by apoptosis signalling pathways. We summarise relevant events in apoptosis, focusing on caspases and IAPs. We also discuss mechanisms of dendrite development, structure and reorganisation and the implications for early diagnosis and treatment of glaucoma and neurodegenerative disease.

During the past two decades clinical and research efforts have led to increasingly sophisticated and effective methods and instruments designed to detect exaggeration or fabrication of neuropsychological dysfunction, as well as somatic and psychological symptom complaints. A vast literature based on relevant research has emerged and substantial portions of professional meetings attended by clinical neuropsychologists have addressed topics related to malingering (Sweet, King, Malina, Bergman, & Simmons, 2002). Yet, despite these extensive activities, understanding the need for methods of detecting problematic effort and response bias and addressing the presence or absence of malingering has proven challenging for practitioners. A consensus conference, comprised of national and international experts in clinical neuropsychology, was held at the 2008 Annual Meeting of the American Academy of Clinical Neuropsychology (AACN) for the purposes of refinement of critical issues in this area. This consensus statement documents the current state of knowledge and recommendations of expert clinical neuropsychologists and is intended to assist clinicians and researchers with regard to the neuropsychological assessment of effort, response bias, and malingering.

BACKGROUND: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. METHODS: We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. FINDINGS: Two patients received 0.09 mg AVI-4658 in 900 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44-79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26.4%), and the mean intensity was 17% (range 11-21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0.002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. INTERPRETATION: Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. FUNDING: UK Department of Health.

Prior chemical and physical analysis of lunar soil suggests a composition of dust particles that may contribute to the development of acute and chronic respiratory disorders. In this study, fine Al(2)O(3) (0.7 mum) and fine SiO(2) (mean 1.6 mum) were used to assess the cellular uptake and cellular toxicity of lunar dust particle analogs. Respiratory cells, murine alveolar macrophages (RAW 264.7) and human type II epithelial (A549), were cultured as the in vitro model system. The phagocytic activity of both cell types using ultrafine (0.1 mum) and fine (0.5 mum) fluorescent polystyrene beads was determined. Following a 6-h exposure, RAW 264.7 cells had extended pseudopods with beads localized in the cytoplasmic region of cells. After 24 h, the macrophage cells were rounded and clumped and lacked pseudopods, which suggest impairment of phagocytosis. A549 cells did not contain beads, and after 24 h, the majority of the beads appeared to primarily coat the surface of the cells. Next, we investigated the cellular response to fine SiO(2) and Al(2)O(3) (up to 5 mg/ml). RAW 264.7 cells exposed to 1.0 mg/ml of fine SiO(2) for 6 h demonstrated pseudopods, cellular damage, apoptosis, and necrosis. A549 cells showed slight toxicity when exposed to fine SiO(2) for the same time and dose. A549 cells had particles clustered on the surface of the cells. Only a higher dose (5.0 mg/ml) of fine SiO(2) resulted in a significant cytotoxicity to A549 cells. Most importantly, both cell types showed minimal cytotoxicity following exposure to fine Al(2)O(3). Overall, this study suggests differential cellular toxicity associated with exposure to fine mineral dust particles.

AIM: To determine if retinal ganglion cell (RGC) loss influences the loss of surrounding RGCs to generate clustered patterns of cell death in human glaucoma. We hypothesise that retinal ganglion cell loss accelerates the loss of surrounding cells to generate, at a local, cellular scale, clustered patterns of retinal of RGC death; the absence of these interactions would result in a diffuse pattern of RGC loss. METHOD: Six glaucomatous retinas and six age-matched control retinas were prepared as wholemounts and stained by 4',6-diamidino-2-phenylindole solution (3mug/mL). An area corresponds to central 14 degree of the visual field was imaged. The nearest neighbour distribution was determined for cells in both normal and glaucomatous RGCL. RESULTS: We observed clustered RGC loss in human glaucoma on a background of diffuse loss. The mean nearest neighbour distance (NND) of the glaucomatous retinas was significantly higher compared with controls (p<0.001). The distribution of NND in glaucomatous retinas was skewed to the higher values with a higher positive kurtosis relative to controls. The quantitative analysis of the pattern of cell loss is supported by the visual inspection of the patterns of cell loss. DISCUSSION: Nearest neighbour analysis is consistent with the presence of two patterns of cell loss in the RGCL in glaucoma. While the diffuse of cell loss can account for an overall reduction in the RGC population and additional non random pattern is consistent with the hypothesis that RGC loss has a local influence on the viability of surrounding cells.

In developmental and reproductive toxicity studies, drinking water is a common means of delivering the test agent. Reduced consumption of toxicant-containing water raises questions about indirect effects of reduced maternal fluid consumption resulting from unpalatability, versus direct effects of the test compound. Issues to consider include: objective assessment of dehydration and thirst, the relative contributions of innate and learned behaviors to drinking behavior and flavor preference, and the objective assessment of physiologic stress. Not only do lab animals under ad lib conditions consume more water than the minimum required to maintain fluid balance, animals faced with water restriction have substantial physiologic capacity for protection of metabolic processes. Measures of blood biochemistry can provide quantifiable, objective indications of fluid balance, but changes in these parameters could result from other causes such as effects of a test toxicant. Consummatory behaviors in response to perceived need are highly influenced by learning. Hence, the drinking behavior, water intake, and flavor acceptance/preference of animals used in toxicology experiments could be subject to learning experiences with the test compound. Physiological symptoms of stress produced by water deprivation may be distinguishable from the symptoms associated with other generalized stressors, such as food deprivation, but doing so may be beyond the scope of most developmental or reproductive toxicity studies. Use of concurrent controls, paired to test groups for water consumption, could help distinguish between the direct effects of a test toxicant as opposed to effects of reduced water consumption alone.