Fluorescein Isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC-labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels

Protracted effects of chronic stress on serotonin-dependent thermoregulation

Chronic stress is known to affect serotonin (5HT) neurotransmission in the brain and to alter body temperature. The body temperature is controlled in part, by the medial preoptic area (mPOA) of the hypothalamus. To investigate the effect of chronic stress on 5HT and how it affects body temperature regulation, we examined whether exposure to a chronic unpredictable stress (CUS) paradigm produces long-term alterations in thermoregulatory function of the mPOA through decreased 5HT neurotransmission. Adult male Sprague-Dawley rats underwent 21 d of CUS. Four days after the last stress exposure, basal body temperature in the home cage and body temperature in a cold room maintained at 10 °C were recorded. The CUS rats had significantly higher subcutaneous basal body temperature at 13:00 h compared to unstressed (NoStress) rats. Whereas the NoStress rats were able to significantly elevate body temperature from basal levels at 30 and 60 min of exposure to the cold room, the CUS rats showed a hypothermic response to the cold. Treatment during CUS with metyrapone, a corticosterone synthesis inhibitor, blocked stress-induced decrease in body temperature in response to the cold challenge. CUS also decreased 5HT transporter protein immunoreactivity in the mPOA and 5HT2A/C agonist injection into the mPOA after CUS exposure caused stressed rats to exhibit a sensitized hyperthermic response to cold. These results indicate that the CUS induced changes to the 5HTergic system alter mPOA function in thermoregulation. These findings help us to explain the mechanisms underlying chronic stress-induced disorders such as chronic fatigue syndrome wherein long lasting thermoregulatory deficits are observed

Chronic-Stress-Induced Behavioral Changes Associated with Subregion-Selective Serotonin Cell Death in the Dorsal Raphe

The current study examined the neurochemical mechanisms and neuroanatomical changes underlying coexisting behavioral effects associated with chronic-stress-induced alterations in serotonin (5HT) neurons. Chronic unpredictable stress (CUS) to adult male rats produced depression-like changes with cognitive dysfunction and selective cell death in the interfascicular nucleus of the dorsal raphe (DRif), resulting in decreased 5HTergic innervation of medial prefrontal cortex (mPFC). Twenty-one days of CUS decreased basal plasma levels of corticosterone and produced a shorter latency to immobility and longer durations of immobility in the force-swim test that persisted for 1 month after CUS. Deficits in acquisition, recall, perseveration, and reversal learning were evident 1 month after CUS. MK801 treatment during CUS blocked the changes in the forced-swim test and deficits in memory recall. These behavioral changes were associated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive soma and the eventual loss of 5HT neurons in the DRif and its projections to the mPFC as evidenced by fewer labeled cells in the DRif after retrograde tracer injections into the mPFC of stressed rats. Similar to the effects of MK801 on behavior, MK801 pretreatment during stress blocked the CUS-induced decreases in 5HT soma within the DRif and its projections to the mPFC. Finally, the depression-like behaviors were blocked by acute injection of the 5HT2A/C agonist (−)-2,5-dimethoxy-4-iodoamphetamine hydrochloride into the mPFC before forced-swim testing. These results identify a cause and mechanism of 5HTergic dysfunction of the mPFC and associated mood and cognitive behaviors., SIGNIFICANCE STATEMENT Chronic stress causes persistent mood and cognitive changes typically associated with dysregulated serotonin (5HT) transmission in the medial prefrontal cortex (mPFC), but the cause of this dysregulation is unknown. Prior studies have focused on 5HTergic terminals in this region, but this study shows that chronic stress causes NMDA-receptor-dependent and subregion-specific cell death of 5HT neurons in the dorsal raphe. The consequent decreased 5HT innervation of the mPFC was associated with mood and cognitive changes that persisted long after the termination of stress. These findings identify a mechanism of subregion-selective death of 5HT neurons in the dorsal raphe, a defined neuroanatomical pathway, and a behavioral phenotype that mirror stress-associated diseases such as major depressive disorder

Cerebrovascular Injury After Serial Exposure to Chronic Stress and Abstinence from Methamphetamine Self-Administration

Cerebrovascular damage caused by either exposure to stress or the widely abused drug, methamphetamine (Meth) is known but stress and drug abuse frequently occur in tandem that may impact their individual cerebrovascular effects. This study examined their co-morbid cerebrovascular effects during abstinence from self-administered Meth after the exposure to chronic unpredictable stress (CUS). Exposure to CUS prior to unrestricted Meth self-administration had no effect on Meth intake in rats; however, the pro-inflammatory mediator cyclooxygenase-2 (COX-2) and the breakdown of cell-matrix adhesion protein β-dystroglycan in isolated cerebral cortical capillaries were increased after 3 days of abstinence and persisted for 7 days. These changes preceded decreases in occludin, a key structural protein component of the blood-brain barrier. The decrease in occludin was blocked by the COX-2 specific inhibitor nimesulide treatment during abstinence from Meth. The changes in COX-2, β-dystroglycan, and occludin were only evident following the serial exposure to stress and Meth but not after either one alone. These results suggest that stress and voluntary Meth intake can synergize and disrupt cerebrovasculature in a time-dependent manner during abstinence from chronic stress and Meth. Furthermore, COX-2 inhibition may be a viable pharmacological intervention to block vascular changes after Meth exposure

Did they report it to stop it? A realist evaluation of the effect of an advertising campaign on victims’ willingness to report unwanted sexual behaviour

Tackling unwanted sexual behaviour (USB) on public transport is a concern for transit authorities across the world. However, high rates of underreporting mean a lack of reliable information about USB, presenting a key barrier to prevention. This paper presents a realist evaluation of an initiative called ‘Report It To Stop It’ (RITSI) implemented in London, UK, to tackle underreporting. RITSI aimed to encourage victims to report details of USB incidents to police and transit authorities through media campaigns. Results show that the initiative did increase reporting of USB and, that this increase was not due to a rise in the prevalence of USB. Crucially, there was no evidence of any increase in passengers’ fear of crime during the campaign activity. However, the impacts of this campaign were more pronounced in earlier waves, and on certain modes of transport. These findings demonstrate the importance of the context in motivating reporting behaviour change

The involvement of matrix metalloproteinases in nicotine conditioned place preference in adolescent female rats

The process of learning new information or modifying existing information requires synaptic plasticity in the brain. In order for these plasticity events to occur, the extracellular matrix (ECM) is reconfigured and reorganized by their primary modulators-- the matrix metalloproteinases (MMP). MMPs are a class of proteinases that are known to be involved in the learning and memory process by modulating synaptic plasticity. Our hypothesis is that MMP dependent synaptic restructuring takes place during the acquisition of drug dependent learning. We used a nicotine conditioned place preference (CPP) model to understand the underlying synaptic events that drives drug preference. There has been some debate regarding the ability of nicotine to elicit CPP in rats. Therefore the goal of the studies described in chapter 2 was to first establish that nicotine induced CPP. Our results show that nicotine produces robust, reproducible CPP at 0.03mg/kg dose using a 5 day drug administration protocol. We also observed that higher doses of nicotine produced conditioned place aversion. In chapter 3 we noted changes in the activity of MMPs - 2, 3, and 9 in the hippocampus and prefrontal cortex after conditioning. Inhibition of MMPs during nicotine conditioning interfered with the development of CPP. Elevation in MMP-3, but not MMP-2 and MMP-9 expression, accompanied re-activation of previously learnt drug related memory in both the hippocampus and the prefrontal cortex. Changes in the activity of cortactin, an actin bound cytoskeletal marker protein, were also observed during the acquisition of CPP but not following re-exposure to the drug context. These results suggest that MMPs - 2, 3, and 9 are involved in facilitating intracellular and extracellular events required for synaptic plasticity and memory consolidation while MMP-3 is uniquely involved in the reconsolidation process

A Role for Matrix Metalloproteinases in Nicotine-Induced Conditioned Place Preference and Relapse in Adolescent Female Rats

Reconfiguration of extracellular matrix proteins appears to be necessary for the synaptic plasticity that underlies memory consolidation. The primary candidates involved in controlling this process are a family of endopeptidases called matrix metalloproteinases (MMPs); however, the potential role of MMPs in nicotine addiction-related memories has not been adequately tested. Present results indicate transient changes in hippocampal MMP-2, -3, and -9 expression following context dependent learning of nicotine-induced conditioned place preference (CPP). Members of a CPP procedural control group also indicated similar MMP changes, suggesting that memory activation occurred in these animals as well. However, hippocampal MMP-9 expression was differentially elevated in members of the nicotine-induced CPP group on days 4 and 5 of training. Inhibition of MMPs using a broad spectrum MMP inhibitor (FN439) during nicotine-induced CPP training blocked the acquisition of CPP. Elevations in hippocampal and prefrontal cortex MMP-3 expression—but not MMP-2 and -9—accompanied reactivation of a previously learned drug related memory. Decreases in the actin regulatory cytoskeletal protein cortactin were measured in the HIP and PFC during the initial two days of acquisition of CPP; however, no changes were seen following re-exposure to the drug related environment. These results suggest that MMP-9 may be involved in facilitating the intracellular and extracellular events required for the synaptic plasticity underlying the acquisition of nicotine-induced CPP. Furthermore, MMP-3 appears to be important during re-exposure to the drug associated environment. However, rats introduced into the CPP apparatus and given injections of vehicle rather than nicotine during training also revealed a pattern of MMP expression similar to nicotine-induced CPP animals

A Role for Matrix Metalloproteinases in Nicotine-Induced Conditioned Place Preference and Relapse in Adolescent Female Rats

Reconfiguration of extracellular matrix proteins appears to be necessary for the synaptic plasticity that underlies memory consolidation. The primary candidates involved in controlling this process are a family of endopeptidases called matrix metalloproteinases (MMPs); however, the potential role of MMPs in nicotine addiction-related memories has not been adequately tested. Present results indicate transient changes in hippocampal MMP-2, -3, and -9 expression following context dependent learning of nicotine-induced conditioned place preference (CPP). Members of a CPP procedural control group also indicated similar MMP changes, suggesting that memory activation occurred in these animals as well. However, hippocampal MMP-9 expression was differentially elevated in members of the nicotine-induced CPP group on days 4 and 5 of training. Inhibition of MMPs using a broad spectrum MMP inhibitor (FN439) during nicotine-induced CPP training blocked the acquisition of CPP. Elevations in hippocampal and prefrontal cortex MMP-3 expression—but not MMP-2 and -9—accompanied reactivation of a previously learned drug related memory. Decreases in the actin regulatory cytoskeletal protein cortactin were measured in the HIP and PFC during the initial two days of acquisition of CPP; however, no changes were seen following re-exposure to the drug related environment. These results suggest that MMP-9 may be involved in facilitating the intracellular and extracellular events required for the synaptic plasticity underlying the acquisition of nicotine-induced CPP. Furthermore, MMP-3 appears to be important during re-exposure to the drug associated environment. However, rats introduced into the CPP apparatus and given injections of vehicle rather than nicotine during training also revealed a pattern of MMP expression similar to nicotine-induced CPP animals