Steffany A. L. Bennett

The estrous cycle affects cocaine self-administration on a progressive ratio schedule in rats

Although it has been demonstrated that many of the behavioral responses to psychomotor stimulants are gender dependent and hormonally sensitive, few studies have examined the possibility that the estrous cycle interacts with drug reinforcement in laboratory animals. The present experiment assessed the effect of the estrous cycle on two aspects of cocaine self-administration behavior: the breaking point on a progressive ratio (PR) schedule and the rate of cocaine intake on a fixed ratio one (FR1) schedule. On the PR schedule, the first lever response produced a drug infusion. Subsequent response requirements escalated with each injection until the behavior extinguished. Breaking points were defined as the final ratio completed. On a FR1 schedule, the estrous cycle had no effect on the rate of drug intake. On a PR schedule, female rats reached higher breaking points during estrus than during other stages of the estrous cycle. Furthermore, female rats displayed higher breaking points than male rats. It appears that the estrous cycle influences an animals motivation to self-administer cocaine.

Apoptosis-Inducing Factor Is a Key Factor in Neuronal Cell Death Propagated by BAX-Dependent and BAX-Independent Mechanisms

Mitochondria release proteins that propagate both caspase-dependent and caspase-independent cell death pathways. AIF (apoptosis-inducing factor) is an important caspase-independent death regulator in multiple neuronal injury pathways. Presently, there is considerable controversy as to whether AIF is neuroprotective or proapoptotic in neuronal injury, such as oxidative stress or excitotoxicity. To evaluate the role of AIF in BAX-dependent (DNA damage induced) and BAX-independent (excitotoxic) neuronal death, we used Harlequin (Hq) mice, which are hypomorphic for AIF. Neurons carrying double mutations for Hq/Apaf1-/- (apoptosis proteases-activating factor) are impaired in both caspase-dependent and AIF-mediated mitochondrial cell death pathways. These mutant cells exhibit extended neuroprotection against DNA damage, as well as glutamate-induced excitotoxicity. Specifically, AIF is involved in NMDA- and kainic acid- but not AMPA-induced excitotoxicity. In vivo excitotoxic studies using kainic acid-induced seizure showed that Hq mice had significantly less hippocampal damage than wild-type littermates. Our results demonstrate an important role for AIF in both BAX-dependent and BAX-independent mechanisms of neuronal injury.

Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Lipidomics era: Accomplishments and challenges

Lipid mediators participate in signal transduction pathways, proliferation, apoptosis, and membrane trafficking in the cell. Lipids are highly complex and diverse owing to the various combinations of polar headgroups, fatty acyl chains, and backbone structures. This structural diversity continues to pose a challenge for lipid analysis. Here we review the current state of the art in lipidomics research and discuss the challenges facing this field. The latest technological developments in mass spectrometry, the role of bioinformatics, and the applications of lipidomics in lipid metabolism and cellular physiology and pathology are also discussed.

Clusterin Biogenesis Is Altered during Apoptosis in the Regressing Rat Ventral Prostate

Clusterin was first characterized as an apoptosis-associated transcript after it was identified as testosterone-repressed prostate message (TRPM-2) that is expressed in the epithelial cells of the regressing rat ventral prostate. Increases in clusterin mRNA and protein have been consistently detected in apoptotic cell death paradigms, establishing clusterin gene expression as a prominent marker of apoptotic cell loss. However, enhanced protein expression has also been reported in surviving cells. This ambiguity makes it difficult to define the contribution of clusterin to apoptosis. To address this problem, a panel of polyclonal and monoclonal antibodies were raised against the clusterin α-chain, β-chain, and mixed α/β epitopes. These antibodies detect changes in the biogenesis of clusterin during apoptosis by Western analysis and immunohistochemistry. A 42-kDa glyco/isoform of clusterin appears to be up-regulated in dying epithelial cells. This glyco/isoform is apparently generated as a result of apoptosis-induced stimulation of a normal but under-utilized, synthetic pathway. These data demonstrate that clusterin synthesized by apoptotic cells can be immunologically distinguished from clusterin synthesized by surviving cells in damaged tissue.

Heroin self-administration in rats under a progressive ratio schedule of reinforcement

Heroin self-administration behavior under a progressive ratio (PR) schedule of reinforcement was evaluated in rats. The schedule was designed to restrict drug intake, minimize opiate dependency, and quantify the number of responses emitted (final response ratio) in order to receive a limited number of heroin infusions. Final ratios were found to be stable and did not increase with chronic (31 days) PR reinforcement. The ability of the PR schedule to detect changes in heroin reinforcement was demonstrated by evaluating the effect of naltrexone pretreatment and unit dose alteration on final ratios. Naltrexone (0.4 mg/kg) reduced final ratios and an inverted U dose-response relationship was established for the unit heroin doses 12.5–100 µg/injection. Maximal final ratios occurred with 50 µg/injection heroin reinforcement. This PR schedule may provide a useful method for evaluating the effects of pharmacological manipulations or lesions on opiate reinforcement.

Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells

The survival, proliferation, self-renewal and differentiation of human pluripotent stem cells (hPSCs, including human embryonic stem cells and human induced pluripotent stem cells) involve a number of processes that require cell-cell and cell-matrix interactions. The cell adhesion molecules (CAMs), a group of cell surface proteins play a pivotal role in mediating such interactions. Recent studies have provided insights into the essential roles and mechanisms of CAMs in the regulation of hPSC fate decisions. Here, we review the latest research progress in this field and focus on how E-cadherin and several other important CAMs including classic cadherins, Ig-superfamily CAMs, integrins and heparin sulfate proteoglycans control survival and differentiation of hPSCs

Cleavage of amyloid precursor protein elicited by chronic cerebral hypoperfusion

In the present study, we sought to determine whether low-grade, chronic vascular insufficiency induced in a rodent model of chronic cerebrohypoperfusion is sufficient, in and of itself, to trigger cleavage of the amyloid precursor protein (APP) into beta A-sized fragments. We report that chronic two vessel occlusion (2VO) results in progressive accumulation of beta A peptides detected by Western analysis in aged rats correlating with a shift in the immunohistochemical localization of APP from neurons to extracellular deposits in brain parenchyma. These data indicate that the 2VO paradigm reproduces features of beta A biogenesis characteristic of sporadic Alzheimers disease.

Chronic cerebral hypoperfusion elicits neuronal apoptosis and behavioral impairment

CHRONIC reductions in cerebral blood flow associated with aging and progressive neurodegenerative disorders can precipitate cognitive failure. To assess whether chronic cerebrovascular insufficiency elicits neuronal apoptosis, apoptotic cell death in the hippocampus was quantitated in a rat model of permanent carotid occlusion. Bilateral carotid artery occlusion (2VO) was shown to induce apoptotic morphology and DNA strand breaks in hippocampal neurons 2 and 27 weeks after ligation. The rate of pyramidal cell apoptosis was higher at chronic (27 weeks) compared to sub-chronic (2 weeks) time points. 2VO-induced apoptosis resulted in a decrease in total pyramidal cell number at 27 weeks but not at earlier time points, indicating progressive neuronal loss. Working and reference memory errors in the radial arm maze were strongly correlated with the number of apoptotic neurons in CA1 but not CA3 pyramidal cell fields. These data provide the first indication that apoptotic loss of pyramidal neurons may play a role in memory impairment associated with clinical conditions of chronic cerebrovascular insufficiency.

Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor

Inhibitors of HIV protease have been shown to have antiapoptotic effects in vitro, yet whether these effects are seen in vivo remains controversial. In this study, we have evaluated the impact of the HIV protease inhibitor (PI) nelfinavir, boosted with ritonavir, in models of nonviral disease associated with excessive apoptosis. In mice with Fas-induced fatal hepatitis, Staphylococcal enterotoxin B-induced shock, and middle cerebral artery occlusion-induced stroke, we demonstrate that PIs significantly reduce apoptosis and improve histology, function, and/or behavioral recovery in each of these models. Further, we demonstrate that both in vitro and in vivo, PIs block apoptosis through the preservation of mitochondrial integrity and that in vitro PIs act to prevent pore function of the adenine nucleotide translocator (ANT) subunit of the mitochondrial permeability transition pore complex.