Developmental stress elicits preference for methamphetamine in the spontaneously hypertensive rat model of attention-deficit/hyperactivity disorder.

BACKGROUND: Developmental stress has been hypothesised to interact with genetic predisposition to increase the risk of developing substance use disorders. Here we have investigated the effects of maternal separation-induced developmental stress using a behavioural proxy of methamphetamine preference in an animal model of attention-deficit/hyperactivity disorder, the spontaneously hypertensive rat, versus Wistar Kyoto and Sprague-Dawley comparator strains. RESULTS: Analysis of results obtained using a conditioned place preference paradigm revealed a significant strain × stress interaction with maternal separation inducing preference for the methamphetamine-associated compartment in spontaneously hypertensive rats. Maternal separation increased behavioural sensitization to the locomotor-stimulatory effects of methamphetamine in both spontaneously hypertensive and Sprague-Dawley strains but not in Wistar Kyoto rats. CONCLUSIONS: Our findings indicate that developmental stress in a genetic rat model of attention-deficit/hyperactivity disorder may foster a vulnerability to the development of substance use disorders.

Effect of cocaine on striatal dopamine clearance in a rat model of developmental stress and attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) and developmental stress are considered risk factors for the development of drug abuse. Though the physiological mechanisms underlying this risk are not yet clear, ADHD, developmental stress and drug abuse are known to share underlying disturbances in dopaminergic neurotransmission. Thus, we hypothesized that clearance of cocaine-induced elevations in striatal dopamine would be prolonged in a rat model of ADHD and that this would be further increased by exposure to developmental stress. In the current study, male spontaneously hypertensive rats (SHRs), a well-validated model of ADHD, and control Wistar-Kyoto (WKY) rats were exposed to either standard rearing (nMS) or a maternal separation (MS) paradigm involving removal of the pups from the dam for 180 min/day over 13 days. This produced a 2 × 2 factorial design (SHR/WKY × nMS/MS) with 5-6 rats/group. Striatal clearance of exogenously applied dopamine was measured via in vivo chronoamperometry, and the difference in dopamine uptake parameters before and after cocaine administration was compared between experimental groups. Cocaine, a potent dopamine transporter inhibitor, reliably increased the clearance time of dopamine though no difference in this parameter was found between SHR and WKY strains. However, developmental stress elevated the cocaine-induced increase in time to clear 50% of exogenously applied dopamine (T50) in SHR but had no effect in WKY rats. These findings suggest that a strain × environment interaction prolongs elevated levels of dopamine thereby potentially increasing the rewarding properties of this drug in SHR.

Application of quartz crystal microbalance with dissipation monitoring technology for studying interactions of poxviral proteins with their ligands.

Poxviruses are one of the most complex of animal viruses and encode for over 150 proteins. The interactions of many of the poxviral-encoded proteins with host proteins, as well as with other proteins, such as transcription complexes, have been well characterized at the qualitative level. Some have also been characterized quantitatively by two hybrid systems and surface plasmon resonance approaches. Presented here is an alternative approach that can enable the understanding of complex interactions with multiple ligands. The example given is that of vaccinia virus complement control protein (VCP). The complement system forms the first line of defense against microorganisms and a failure to appropriately regulate it is implicated in many inflammatory disorders, such as traumatic brain injury, Alzheimer's disease (AD), and rheumatoid arthritis. The complement component C3 is central to the complement activation. Complement regulatory proteins, capable of binding to the central complement component C3, may therefore effectively be employed for the treatment and prevention of these disorders. There are many biochemical and/or immunoassays available to study the interaction of proteins with complement components. However, protocols for many of them are time consuming, and not all assays are useful for multiple screening. In addition, most of these assays may not give information regarding the nature of binding, the number of molecules interacting with the complement component C3, as well as kinetics of binding. Some of the assays may require labeling which may induce changes in protein confirmation. We report a protocol for an assay based on quartz crystal microbalance with dissipation monitoring (QCM-D) technology, which can effectively be employed to study poxviral proteins for their ability to interact with their ligand. A protocol was developed in our laboratories to study the interaction of VCP with the complement component C3 using Q-sense (D-300), equipment based on QCM-D technology. The protocol can also be used as a prototype for studying both proteins and small-sized compounds (for use as anti-poxvirals) for their ability to interact with and/or inhibit the activity of their ligands.

Central nervous system distribution of the poxviral proteins after intranasal administration of proteins and titering of vaccinia virus in the brain after intracranial administration.

Poxviral proteins are known to interact with the immune system of the host. Some of them interact with the transcription factors of the host, whereas others interact with the components of the immune system. Vaccinia virus secretes a 28.8-kDa complement control protein (VCP), which is known to regulate the complement system. This protein helps the virus to evade the immune response of the host. Such viral proteins might also prove beneficial in the treatment and prevention of the progression of the disorders, where up-regulation of the complement system is evident. VCP has been shown experimentally to be effective in protecting tissues from inflammatory damage in the rodent models of Alzheimer's diseases (AD), spinal cord injury, traumatic brain injury, and rheumatoid arthritis. Not only VCP, but also other poxviral proteins could be used therapeutically to treat or prevent the progression of the brain disorders, where the immune system is inadequately controlled. However, being a protein that cannot traverse the brain barrier because of its size, delivery of such proteins to the central nervous system (CNS) could be a limiting factor in their usefulness as CNS therapeutics. In this chapter, we show methods for the intranasal route of administration of a protein and show ways to detect its distribution in the cerebrospinal fluid (CSF) and to the different parts of the brain. These protocols can be extended to examine the distribution of viral antigens in the brain. A protocol is also included to quantitate vaccinia virus in different segments of the brain after intracranial administration of the virus.

Effect of maternal separation on mitochondrial function and role of exercise in a rat model of Parkinson's disease.

Early life stress, such as maternal separation, causes adaptive changes in neural mechanisms that have adverse effects on the neuroplasticity of the brain in adulthood. As a consequence, children who are exposed to stress during development may be predisposed to neurodegenerative disorders in adulthood. A possible mechanism for increased vulnerability to neurodegeneration may be dysfunctional mitochondria. Protection from neurotoxins, such as 6-hydroxydopamine (6-OHDA), has been observed following voluntary exercise. The mechanism of this neuroprotection is not understood and mitochondria may play a role. The purpose of this study was to determine the effects of maternal separation and exercise on mitochondrial function in a rat model of Parkinson's disease. Maternally separated (pups separated from the dam for 3 h per day from postnatal day (P) 2-14) and non-separated rats were placed in individual cages with or without attached running wheels for 1 week prior to unilateral infusion of 6-OHDA (5 µg/4 µl, 0.5 µl/min) into the left medial forebrain bundle at P60. After 2 h recovery, rats were returned to their cages and wheel revolutions recorded for a further 2 weeks. On P72, the rats' motor function was assessed using the forelimb akinesia test. On P74, rats were sacrificed for measurement of mitochondrial function. Exercise increased the respiratory control index (RCI) in the non-lesioned hemisphere of 6-OHDA-lesioned rats. This effect was evident in the striatum of non-separated rats and the prefrontal cortex of maternally separated rats. These results suggest that early life stress may reduce the adaptive response to exercise in the striatum, a major target of dopamine neurons, but not the prefrontal cortex in this model of Parkinson's disease.

Maternal separation affects dopamine transporter function in the spontaneously hypertensive rat: an in vivo electrochemical study.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR) is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT) in ways that distinguish SHR from control rat strains. METHODS: SHR and control Wistar-Kyoto (WKY) rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function. RESULTS: Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (k-1) in SHR striatum. Consistent with this observation, the dopamine clearance time (T100) was increased in SHR. These results suggest that the chronic mild stress of maternal separation impaired the function of striatal DAT in SHR. CONCLUSIONS: The present findings suggest that maternal separation failed to alter the behaviour of SHR in the open field and elevated plus maze. However, maternal separation altered the dopaminergic system by decreasing surface expression of DAT and/or the affinity of DAT for dopamine, increasing the time to clear dopamine from the extracellular fluid in the striatum of SHR.

Investigation of interaction of vaccinia virus complement control protein and curcumin with complement components c3 and c3b using quartz crystal microbalance with dissipation monitoring technology.

C3 and C3b, the components central to the complement activation, also play a damaging role in several inflammatory disorders. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are natural compounds with different biological origins reported to regulate complement activation. However, both VCP and Cur have not been investigated for their interaction with the third component (C3) prior to it being converted to its activated form (C3b). These two compounds have also not been compared to each other with respect to their interactions with C3 and C3b. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a novel technology used to study the interaction of biomolecules. This technology was applied to characterize the interactions of VCP, Cur and appropriate controls with the key complement components. Cur as well as VCP showed binding to both C3 and to C3b, Cur however bound to C3b to a lesser extent.

Effect of exercise on synaptophysin and calcium/calmodulin-dependent protein kinase levels in prefrontal cortex and hippocampus of a rat model of developmental stress.

Stress affects the brain differently depending on the timing, duration and intensity of the stressor. Separation from the dam for 3 h per day is a potent stressor for rat pups which causes activation of the hypothalamic-pituitary-adrenal (HPA) axis, evidenced by increased plasma levels of adrenocorticotropin (ACTH) and glucocorticoids. Behaviourally, animals display anxiety-like behaviour while structurally, changes occur in neuronal dendrites and spines in the hippocampus and prefrontal regions involved in emotion and behaviour control. The aim of the present study was to determine whether maternal separation alters expression of synaptic markers, synaptophysin and calcium/calmodulin-dependent protein kinase II (CaMKII), in rat hippocampus and prefrontal cortex. A second aim was to determine whether voluntary exercise had a beneficial effect on the expression of these proteins in rat brain. Maternal separation occurred from postnatal day 2 (P2) to P14 for 3 h per day. Exercised rats were housed in cages with attached running wheels from P29 to P49. At P65, the prefrontal cortex and hippocampus were removed for protein quantification. Maternal separation did not have any effect while exercise increased synaptophysin and CaMKII in the ventral hippocampus but not in the dorsal hippocampus or prefrontal cortex. Since the ventral hippocampus is associated with anxiety-related behaviour, these findings are consistent with the fact that voluntary exercise increases anxiety-like behaviour and improves learning and memory.

Effect of exercise on learning and memory in a rat model of developmental stress.

Adverse life events occurring in early development can result in long-term effects on behavioural, physiological and cognitive processes. In particular, perinatal stressors impair neurogenesis in the hippocampus which consequently impairs memory formation. Exercise has previously been shown to have antidepressant effects and to increase cognitive functioning by increasing neurogenesis and neurotrophins in the hippocampus. The current study examined the effects of maternal separation, which has been shown to model anxiety in animals, and the effects of exercise on learning and memory. Forty-five male Sprague-Dawley rats were divided into four groups, maternally separated / non-runners, maternally separated / runners, non-separated / runners and non-separated / non-runners. Maternal separation occurred from postnatal day 2 (P2) to 14 (P14) for 3 h per day. Exercised rats were given voluntary access to individual running wheels attached to their cages from P29 to P49. Behavioural testing (Morris water maze (MWM) and object recognition tests) took place from P49 to P63. Maternally separated rats showed no significant difference in anxiety levels in the elevated plus maze and the open field compared to the normally reared controls. However, rats that were allowed voluntary access to running wheels showed increased levels of anxiety in the elevated plus maze and in the open field. Maternal separation did not have any effect on memory performance in the MWM or the object recognition tasks. Exercise increased spatial learning and memory in the MWM with the exercised rats displaying a decreased latency in locating the hidden platform than the non-exercised rats. The exercised rats spent significantly less time exploring the most recently encountered object in the temporal order task in comparison to the non-exercised controls, therefore showing improved temporal recognition memory. All groups performed the same on the other recognition tasks, with all rats showing intact memory performance. Results indicate that maternal separation had little effect on the rats whereas exercise enhanced both spatial and recognition memory.

Effect of exercise on dopamine neuron survival in prenatally stressed rats.

Prenatal stress has been associated with increased vulnerability to psychiatric disturbances including schizophrenia, depression, attention-deficit hyperactivity disorder and autism. Elevated maternal circulating stress hormones alter development of neural circuits in the fetal brain and cause long-term changes in behaviour. The aim of the present study was to investigate whether mild prenatal stress increases the vulnerability of dopamine neurons in adulthood. A low dose of 6-hydroxydopamine (6-OHDA, 5 microg/4 microl saline) was unilaterally infused into the medial forebrain bundle of nerve fibres in the rat brain in order to create a partial lesion of dopamine neurons which was sufficient to cause subtle behavioural deficits associated with early onset of Parkinson's disease without complete destruction of dopamine neurons. Voluntary exercise appeared to have a neuroprotective effect resulting in an improvement in motor control and decreased asymmetry in the use of left and right forelimbs to explore a novel environment as well as decreased asymmetry of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta and decreased dopamine cell loss in 6-OHDA-lesioned rats. Prenatal stress appeared to enhance the toxic effect of 6-OHDA possibly by reducing the compensatory adaptations to exercise.

Intracranial administration of vaccinia virus complement control protein in Mo/Hu APPswe PS1dE9 transgenic mice at an early age shows enhanced performance at a later age using a cheese board maze test.

One of the key pro-inflammatory mediators activated by amyloid protein in neurodegenerative disorders of the brain, such as Alzheimer's disease is the complement system. Vaccinia virus complement control protein secreted by vaccinia virus, commonly known as VCP, was found to inhibit amyloid protein mediated up-regulation of complement system in vitro. In the current research investigation, VCP was administered twice (First dose at 3 weeks and the second dose at 6-7 months) intracranially into the parietal cortical area of Mo/Hu APPswe transgenic mice. At the age of 2 years or more, the same mice were subjected to cued-learning, spatial learning, probe and reverse probe trial paradigms of cheese board maze tasks for cognitive assessment. A significant difference was observed between VCP treated mice and the transgenic controls on days two and three of the cued trials and probe trials. The VCP treated group showed a similar trend as revealed during the spatial learning trial and reverse probe trial. A differential pattern of thioflavine S staining was observed in the VCP treated group. These results suggest that administration of VCP at an early age in transgenic mice may be effective in regulating the progression to the familial form of Alzheimer's disease at a later age.

Stress reduces the neuroprotective effect of exercise in a rat model for Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease of nigrostriatal dopamine (DA) neurons that project from the substantia nigra pars compacta (SNc) to the striatum. To further understand PD, researchers have developed standardized animal models of PD. In this study, Long Evans (LE) rats were unilaterally lesioned by injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), into the medial forebrain bundle (MFB) of the left hemisphere. The rats were divided into three groups randomly; group 1 (runners) were housed in individual cages with attached running wheels, group 2 (stressed-runners) had access to individual free running wheels, except post-lesion when the rats were subjected to immobilization of the running wheel for 1 h per day for 14 days, as well as one session of 24-h food deprivation and a 7-h shift in the light/dark cycle. Group 3 (non-runnners) were housed individually in cages with attached running wheels that were permanently immobilized. Subcutaneous injection of the DA agonist, apomorphine, caused stressed-runners and non-runners to rotate vigorously away from the side of the lesion (contralaterally). Apomorphine-induced rotations provide a behavioural measure of the extent of the lesion, a depletion of more than 80% of DA neurons is required to produce vigorous contralateral rotations in response to apomorphine injection. Runners rotated significantly less than non-runners and stressed-runners. The number of rotations performed by stressed-runners was not significantly different from non-runners, suggesting that stress had cancelled the neuroprotective effect of running. Immunohistochemical staining for tyrosine hydroxylase in the SNc revealed slightly less destruction of DA neurons in the runners than in stressed-runners or non-runners, although these differences did not achieve statistical significance. The behavioural results confirm a previous finding suggesting that voluntary exercise is neuroprotective. A novel finding is that mild stressors cancel the neuroprotection afforded by voluntary exercise.

Voluntary running distance is negatively correlated with striatal dopamine release in untrained rats.

This study examined the relationship between voluntary running distance and glutamate- and K+-stimulated dopamine release in the striatum (nucleus accumbens and caudate-putamen) of male Long-Evans rats. Twenty-one rats were housed individually in cages with attached running-wheels for 1 week. There was a 19-fold variability between rats in voluntary running distances over this period (range = 2.3-44.6 km). The average distance completed during the week was 16 +/- 2.8 km. There was a strong positive correlation between the running distances completed during the first 24 h (day 1) and the last 24 h. Certain rats were therefore inclined to run from the start. The average daily running distance (2.4 +/- 0.4 km per day) was negatively correlated with the weight of the rat (r = -0.82). Glutamate-stimulated release of dopamine was not a significant predictor of voluntary running distance. However, the average daily running distance was negatively correlated with K+-stimulated dopamine release in the nucleus accumbens core and caudate-putamen but not the nucleus accumbens shell. The present findings suggest that decreased depolarization-induced release of striatal dopamine may be a predictor of hyperactivity. The results show, in a normal population of Long-Evans rats, that there are, at the end of the continuum, rats that display some of the neurochemical and behavioral characteristics of a rat model for attention-deficit hyperactivity disorder.