Toxoplasma gondii infection reduces predator aversion in rats through epigenetic modulation in the host medial amygdala

Male rats (Rattus novergicus) infected with protozoan Toxoplasma gondii relinquish their innate aversion to the cat odours. This behavioural change is postulated to increase transmission of the parasite to its definitive felid hosts. Here, we show that the Toxoplasma gondii infection institutes an epigenetic change in the DNA methylation of the arginine vasopressin promoter in the medial amygdala of male rats. Infected animals exhibit hypomethylation of arginine vasopressin promoter, leading to greater expression of this nonapeptide. The infection also results in the greater activation of the vasopressinergic neurons after exposure to the cat odour. Furthermore, we show that loss of fear in the infected animals can be rescued by the systemic hypermethylation and recapitulated by directed hypomethylation in the medial amygdala. These results demonstrate an epigenetic proximate mechanism underlying the extended phenotype in the Rattus novergicus–Toxoplasma gondii association.     

Infection and immunity with the RH strain of Toxoplasma gondii in rats and mice.

Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.     

Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.     

Cannabinoid modulation of electrically evoked pH and oxygen transients in the nucleus accumbens of awake rats

Cannabinoid receptors have been implicated in the regulation of blood flow in the cerebral vasculature. Because the nucleus accumbens (NAc) shows high levels of central cannabinoid receptor 1 (CB1) expression we examined the effects of cannabinoids on the local transient alkaline shifts and increases in extracellular oxygen induced by electrical stimulation of the medial forebrain bundle (MFB) in conscious animals. These changes result from increases in cerebral blood flow (CBF) and metabolism in the NAc that are evoked by the stimulation. Oxygen and pH changes were monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the NAc of freely moving rats. Administration of the cannabinoid receptor agonist WIN55,212-2 potently inhibited extracellular oxygen and pH changes, an effect that was reversed and prevented by pre-treatment with the CB1 receptor antagonists SR141716A and AM251. The effects on pH following WIN55,212-2 were similar to those following nimodipine, a recognized vasodilator. When AM251 was injected alone, the amplitude of electrically evoked pH shifts was unaffected. Administration of AM404 and VDM11, endocannabinoid transport inhibitors, partially inhibited pH transients in a CB1 receptor-dependent manner. The present findings suggest that CB1 receptor activation modulates changes in two well-established indices of local blood flow and metabolism resulting from electrically evoked activation of ascending fibers. Although endogenous cannabinoid tone alone is not sufficient to modify these responses, uptake blockade demonstrates that the system has the potential to exert CB1-specific effects similar to those of full agonists.     

Effects of reserpine on dopamine metabolite in the nucleus accumbens and locomotor activity in freely moving rats

The effect of reserpine (2 mg/kg i.p.) on both locomotor activity and the turnover of dopamine metabolite in the rat nucleus accumbens was estimated by using an activity monitor (Animex) and by in vivo brain microdialysis. Three to five hours after reserpine administration locomotor activity was reduced and there was a concomitant increase in the level of the dopamine metabolite, homovamillic acid. These findings suggest that depletion of dopamine from the nucleus accumbens may result in decreased locomotor activity. The data support the notion that dopamine in this tissue contributes to the control of locomotion.     

Catecholamine mapping within nucleus accumbens: differences in basal and amphetamine-stimulated efflux of norepinephrine and dopamine in shell and core

The nucleus accumbens is believed to play a critical role in mediating the behavioral responses to rewarding stimuli. Although most studies of the accumbens focus on dopamine, it receives afferents from many other nuclei, including noradrenergic cell groups in the brainstem. We used in vivo microdialysis to measure extracellular levels of both norepinephrine and dopamine in the accumbens shell and core. Regional analysis of shell and core and border regions demonstrated that norepinephrine was high in shell and decreased from medial shell to lateral core, where baseline levels were low or undetectable. Conversely, extracellular dopamine in core was twice the level seen in shell. Both catecholamines increased following a single injection of amphetamine (2 mg/kg, i.p.). The norepinephrine response was greater and long-lasting in shell compared with core. The maximal dopamine response was higher in core than in shell, but the duration of the effect was comparable in both regions. The distinct neurochemical characteristics of shell and core are likely to contribute to the functional heterogeneity of the two subregions. Furthermore, norepinephrine may be involved in many of the functions generally attributed to the accumbens, either directly or indirectly via modulation of extracellular dopamine.     

Repeated cocaine administration changes the function and subcellular distribution of adenosine A1 receptor in the rat nucleus accumbens

Adenosine A1 receptor (A1) protein and mRNA is increased in the nucleus accumbens following repeated cocaine treatment. In spite of this protein up-regulation, A1 agonist-stimulated [35S]GTPgammaS binding was attenuated in accumbens homogenates of rats withdrawn for 3 weeks from 1 week of daily cocaine injections. Cellular subfractionation revealed that the discrepancy between total A1 protein and G protein coupling resulted from a smaller proportion of receptors in the plasma membrane. The decrease in functional receptor in the plasma membrane was further indicated by diminished formation of heteromeric receptor complex consisting of A1 and dopamine D1A receptors. To explore the functional significance of the altered distribution of A1 receptors, at 3 weeks after discontinuing repeated cocaine or saline, animals were injected with cocaine and 45 min later the subcellular distribution of A1 receptors quantified. Whereas a cocaine challenge in repeated saline-treated animals induced a marked increase in membrane localization of the A1 receptor, the relative distribution of receptors in repeated cocaine rats was not affected by acute cocaine. These data suggest that the sorting and recycling of A1 receptors is dysregulated in the nucleus accumbens as the consequence of repeated cocaine administration.     

Monoamine Changes in the Brain of BALB/c Mice Following Sub-Lethal Infection with Nocardia asteroides (GUH-2)

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included head-shake, stooped posture, bradykinesia, and hesitation to forward movement (6). The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.     

Deep brain stimulation of the accumbens increases dopamine,serotonin, and noradrenaline in the prefrontal cortex

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is effective in treatment‐refractory obsessive‐compulsive disorder and major depressive disorder. However, little is known about the neurobiological mechanisms underlying the rapid and effective changes of DBS. One of the hypotheses is that DBS modulates activity of monoamine neurotransmitters. In this study, we evaluated the effects of DBS in the NAc core on the extracellular concentration of monoaminergic neurotransmitters in the medial (mPFC) and orbital prefrontal cortex (OFC). Freely moving rats were bilaterally stimulated in the NAc core for 2 h while dopamine, serotonin, and noradrenaline were measured using in vivo microdialysis in the mPFC and the OFC. We report rapid increases in the release of dopamine and serotonin to a maximum of 177% and 127% in the mPFC and an increase up to 171% and 166% for dopamine and noradrenaline in the OFC after onset of stimulation in the NAc core. These results provide further evidence for the distal effects of DBS and corroborate previous clinical and pre‐clinical findings of altered neuronal activity in prefrontal areas.     

Differences in dopamine responsiveness to drugs of abuse in the nucleus accumbens shell and core of Lewis and Fischer 344 rats

The use of inbred rat strains provides a tool to investigate the role of genetic factors in drug abuse. Two such strains are Lewis and Fischer 344 rats. Although several biochemical and hormonal differences have been observed between Lewis and Fischer 344 strains, a systematic comparison of the effect of different drugs of abuse on dopamine (DA) transmission in the shell and core of the nucleus accumbens of these strains is lacking. We therefore investigated, by means of dual probe microdialysis, the effect of different doses of morphine (1.0, 2.5, and 5.0 mg/kg), amphetamine (0.25, 0.5, and 1.0 mg/kg) and cocaine (5, 10, and 20 mg/kg) on DA transmission in the shell and in the core of nucleus accumbens. Behavior was monitored during microdialysis. In general, Lewis rats showed greater DA responsiveness in the NAc core compared to F344 rats except after 2.5 mg/kg of morphine and 20 mg/kg of cocaine. In the NAc shell, different effects were obtained depending on drug and dose: after 1.0 mg/kg of morphine no strain differences were observed, at 2.5 and 5.0 mg/kg Lewis rats showed greater increase in DA in the NAc shell. Following amphetamine and cocaine challenge, Lewis rats showed greater DA increase in the shell after 0.25 mg/kg of amphetamine and 20 mg/kg of cocaine. Behavioral activation was greater in Lewis rats in response to the lowest dose of morphine (1.0 mg/kg), to the highest dose of amphetamine (1.0 mg/kg) and to all doses of cocaine. These differences might be the basis for the different behavioral responses of these strains to drugs of abuse.     

Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopamine levels in the nucleus accumbens shell in rats

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.     

A decrease in the addition of new cells in the nucleus accumbens and prefrontal cortex between puberty and adulthood in male rats

Adolescence involves shifts in social behaviors, behavioral flexibility, and adaptive risk‐taking that coincide with structural remodeling of the brain. We previously showed that new cells are added to brain regions associated with sexual behaviors, suggesting that cytogenesis may be a mechanism for acquiring adult‐typical behaviors during adolescence. Whether pubertal cell addition occurs in brain regions associated with behavioral flexibility or motivation and whether these patterns differ between pubertal and adult animals had not been determined. Therefore, we assessed patterns of cell proliferation or survival in the prefrontal cortex and nucleus accumbens. Pubertal and adult male rats were given injections of bromo‐deoxyuridine (BrdU). To assess cell proliferation, half of the animals from each group were sacrificed 24 h following the last injection. The remaining animals were sacrificed at Day 30 following the last injection to evaluate cell survival. Adult animals had significantly lower densities of BrdU‐immunoreactive (ir) cells in the prefrontal cortex, irrespective of post‐BrdU survival time, whereas in the nucleus accumbens, adult animals had a lower density of BrdU‐ir cells at the short survival time; however, the density of BrdU‐ir cells was equivalent in pubertal and adult animals at the longer survival time. These data provide evidence that cell addition during puberty may contribute to the remodeling of brain regions associated with behavioral flexibility and motivation, and this cell addition continues into adulthood, albeit at lower levels. Higher levels of cell proliferation or survival in younger animals may reflect a higher level of plasticity, possibly contributing to the dynamic remodeling of the pubertal brain. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 633–642, 2014     

The role of the cannabinoid type 1 receptor and down-stream cAMP/DARPP-32 signal in the nucleus accumbens of methamphetamine-sensitized rats

Blockade of the cannabinoid type 1 (CB(1)) receptor could suppress methamphetamine self-administration; however, the cellular mechanism remains unclear. In this study, we intended to investigate the significance of brain CB(1) receptors on the development of behavioral sensitization to methamphetamine. Male Sprague-Dawley rats treated with chronic methamphetamine (4 mg/kg, i.p.) for either 7 or 14 days developed behavioral sensitization to methamphetamine (1 mg/kg) at withdrawal day 7. A progressive decrease in numbers of CB(1) receptor (both Bmax and mRNA) but increase in binding affinity (Kd) was noticed during withdrawal days 3 to 7. Microinjection of CB(1) antagonist [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] into the nucleus accumbens (NAc) at withdrawal day 7, significantly suppressed the behavioral sensitization to methamphetamine. In NAc brain slices preparation, acute incubation with CB(1) agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP 55940) dose-dependently enhanced cAMP accumulation in sensitized rats; no change was noticed in control groups. Consequently, treatment of CP 55940 induced a dose-dependent (10 nmol/L-10 micromol/L) phosphorylation on down-stream dopamine and cAMP-regulated phosphoprotein of Mr 32 000 (DARPP-32)/Thr34 in sensitized rats, while only 10 micromol/L CP 55940 was able to enhance the phosphoDARPP-32/T34 in control groups. Alternatively, both basal activity of calcineurin (PP-2B) and CP 55940-induced changes in the amount of PP-2B in the NAc were both decreased in sensitized rats, but not in controls. Overall, we demonstrated that brain CB(1) receptor and its down-stream cAMP/DARPP-32/T34/PP-2B signaling are profoundly altered in methamphetamine-sensitized animals.     

Histological and Ultrastructural Studies of the Interaction of Toxoplasma gondii Tachyzoites with Mouse Omentum in Experimental Infection1

Mouse omentum was studied after intraperitoneal challenge with tachyzoites of Toxoplasma gondii. Parasites inhabit omental histiocytes, fibroblasts, mesothelial cells, and free peritoneal macrophages. Recently infected cells showed enhanced metabolic and functional activity. Villous projections of the parasitophorous vacuole wall appeared, usually opposite the anterior pole of the parasite. In mesothelial cells, projections formed terminal swellings not observed in other infected cells. Activation of host cells was followed by reduction of the density of the cytoplasmic matrix, autophagosome formation, and intracellular edema, indicating the damage. The wall of the parasitophorous vacuole loses the supporting host cell endoplasmic reticulum that was attached to the vacuole just after entrance of the parasite into the cell. Then lysis of the parasitophorous vacuole and complete cell destruction occurs. The growth of parasites in undamaged cells does not coincide with the inflammatory response. Inflammation of the peritoneum develops only after the start of mass destruction of infected cells. Thus tachyzoites of Toxoplasma exert significant pathogenic effects by their ability to activate the host cell, causing lysis of the parasitophorous vacuole and subsequent destruction of the entire cell.     

Effect of operant self-administration of 10% ethanol plus 10% sucrose on dopamine and ethanol concentrations in the nucleus accumbens

Although operant ethanol self-administration can increase accumbal dopamine activity, the relationship between dopamine and ethanol levels during consumption remains unclear. We trained Long-Evans rats to self-administer escalating concentrations of ethanol (with 10% sucrose) over 7 days, during which two to four lever presses resulted in 20 min of access to the solution with no further response requirements. Accumbal microdialysis was performed in rats self-administering 10% ethanol (plus 10% sucrose) or 10% sucrose alone. Most ethanol (1.6 +/- 0.2 g/kg) and sucrose intake occurred during the first 10 min of access. Sucrose ingestion did not induce significant changes in dopamine concentrations. Dopamine levels increased within the first 5 min of ethanol availability followed by a return to baseline, whereas brain ethanol levels reached peak concentration more than 40 min later. We found significant correlations between intake and dopamine concentration during the initial 10 min of consumption. Furthermore, ethanol-conditioned rats consuming 10% sucrose showed no effect of ethanol expectation on dopamine activity. The transient rise in dopamine during ethanol ingestion suggests that the dopamine response was not solely due to the pharmacological properties of ethanol. The dopamine response may be related to the stimulus properties of ethanol presentation, which were strongest during consumption.