Tryptophan depletion affects compulsive behaviour in rats: strain dependent effects and associated neuromechanisms.

RATIONALE: Compulsive behaviour, present in different psychiatric disorders, such as obsessive-compulsive disorder, schizophrenia and drug abuse, is associated with altered levels of monoamines, particularly serotonin (5-hydroxytryptamine) and its receptor system. OBJECTIVES: The present study investigated whether 5-HT manipulation, through a tryptophan (TRP) depletion by diet in Wistar and Lister Hooded rats, modulates compulsive drinking in schedule-induced polydipsia (SIP) and locomotor activity in the open-field test. The levels of dopamine, noradrenaline, serotonin and its metabolite were evaluated, as well as the 5-HT2A and 5-HT1A receptor binding, in different brain regions. METHODS: Wistar rats were selected as high (HD) or low (LD) drinkers according to their SIP behaviour, while Lister hooded rats did not show SIP acquisition. Both strains were fed for 14 days with either a TRP-free diet (T-) or a TRP-supplemented diet (T+) RESULTS: The TRP depletion diet effectively reduced 5-HT levels in the frontal cortex, amygdala and hippocampus in both strains of rats. The TRP-depleted HD Wistar rats were more sensitive to 5-HT manipulation, exhibiting more licks on SIP than did the non-depleted HD Wistar rats, while the LD Wistar and the Lister Hooded rats did not exhibit differences in SIP. In contrast, the TRP-depleted Lister Hooded rats increased locomotor activity compared to the non-depleted rats, while no differences were found in the Wistar rats. Serotonin 2A receptor binding in the striatum was significantly reduced in the TRP-depleted HD Wistar rats. CONCLUSIONS: These results suggest that alterations of the serotonergic system could be involved in compulsive behaviour in vulnerable populations.

Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using 3H-HOCPCA.

GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (3H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations, 3H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that 3H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using 3H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that 3H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand 3H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

5-HT(2A) and mGlu2 receptor binding levels are related to differences in impulsive behavior in the Roman Low- (RLA) and High- (RHA) avoidance rat strains.

The Roman Low- and High-Avoidance rat strains (RLA-I vs RHA-I) have been bidirectionally selected and bred according to their performance in the two-way active avoidance response in the shuttle-box test. Numerous studies have reported a pronounced divergence in emotionality between the two rat strains including differences in novelty seeking, anxiety, stress coping, and susceptibility to addictive substances. However, the underlying molecular mechanisms behind these divergent phenotypes are not known. Here, we determined impulsivity using the 5-choice serial reaction time task and levels of serotonin transporter (SERT), 5-HT(2A) and 5-HT(1A) receptor binding using highly specific radioligands ((3)H-escitalopram, (3)H-MDL100907 and (3)H-WAY100635) and mGlu2/3 receptor binding ((3)H-LY341495) using receptor autoradiography in fronto-cortical sections from RLA-I (n=8) and RHA-I (n=8) male rats. In the more impulsive RHA-I rats, 5-HT(2A), 5-HT(1A) and SERT binding in the frontal cortex was significantly higher compared to RLA-I rats. In contrast, mGlu2/3 receptor binding was decreased by 40% in RHA-I rats compared to RLA-I rats. To differentiate between mGlu2 and mGlu3 receptor protein levels, these were further studied using western blotting, which showed non-detectable levels of mGlu2 receptor protein in RHA rats, while no differences were observed for mGlu3 receptor protein levels. Collectively, these data show general congenital differences in the serotonergic system and a pronounced difference in mGlu2 receptor protein levels. We suggest that the differences in the serotonergic system may mediate some of the phenotypic characteristics in this strain such as hyper-impulsivity and susceptibility to drug addiction.

The microtubule-associated protein 1A (MAP1A) is an early molecular target of soluble Aß-peptide.

A progressive accumulation of amyloid ß-protein (Aß) is widely recognized as a pathological hallmark of Alzheimer's disease (AD). Substantial progress has been made toward understanding the neurodegenerative cascade initiated by small soluble species of Aß and recent evidence supports the notion that microtubule rearrangements may be proximate to neuritic degeneration and deficits in episodic declarative memory. Here, we examined primary cortical neurons for changes in markers associated with synaptic function following exposure to sublethal concentrations of non-aggregated Aß-peptide. This data show that soluble Aß species at a sublethal concentration induce degradation of the microtubule-associated protein 1A (MAP1A) without concurrently affecting dendritic marker MAP2 and/or the pre-synaptic marker synaptophysin. In addition, MAP1A was found to highly co-localize with the postsynaptic density-95 (PSD-95) protein, proposing that microtubule perturbations might be central for the Aß-induced neuronal dysfunctions as PSD-95 plays a key role in synaptic plasticity. In conclusion, this study suggests that disruption of MAP1A could be a very early manifestation of Aß-mediated synaptic dysfunction-one that presages the clinical onset of AD by years. Moreover, our data support the notion of microtubule-stabilizing agents as effective AD drugs.

Novelty-induced activity-regulated cytoskeletal-associated protein (Arc) expression in frontal cortex requires serotonin 2A receptor activation.

Many psychiatric disorders are characterized by cognitive and emotional alterations that are related to abnormal function of the frontal cortex (FC). FC is involved in working memory and decision making and is activated following exposure to a novel environment. The serotonin 2A receptor (5-HT(2A)R) is highly expressed in the FC where its activation induces hallucinations, while blockade of 5-HT(2A)Rs contributes to the therapeutic effects of atypical antipsychotic drugs. The purpose of the present study was to investigate the involvement of 5-HT(2A)R in FC activation following exposure to a novel environment. As an output of FC activation we measured expression of activity-regulated cytoskeletal-associated protein (Arc). Novelty-exposure (open-field arena) robustly up-regulated FC Arc mRNA expression (∼160%) in mice compared to home-cage controls. This response was inhibited with the 5-HT(2A)R antagonists ketanserin and MDL100907, but not with the selective 5-HT(2C)R antagonist SB242084. Novelty-exposure also induced Arc mRNA expression in hippocampus (∼150%), but not in cerebellum or brainstem. Pretreatment with 5-HT(2A)R antagonist ketanserin did not repress the Arc induction in hippocampus, indicating that the involvement of 5-HT(2A)R in this response is restricted to the FC. Similarly, the novelty-induced stress as determined by increasing levels of plasma corticosterone, was not influenced by 5-HT(2A)R antagonism suggesting that Arc mRNA and stress are activated via distinct mechanisms. Taken together, our results demonstrate that the induction of Arc in the FC following exposure to a novel environment is dependent on the 5-HT(2A)R, and that the simultaneous release of corticosterone is regulated via another system independent of 5-HT(2A)R activation.

Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice.

Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF(2L/2LCk-cre)). A severe impairment specific for the serotonin 2A receptor (5-HT(2A)R) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT(2A)Rs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered by BDNF depletion. 5-HT(2A) ([(3)H]-MDL100907) and 5-HT(1A) ([(3)H]-WAY100635) receptor autoradiography revealed site-specific alterations in BDNF mutant mice. They exhibited lower 5-HT(2A) receptor binding in frontal cortex but increased binding in hippocampus. Additionally, 5-HT(1A) receptor binding was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT(2A) and 5-HT(1A) mRNA expression but normal 5-HT(2C) content in these brain regions in BDNF(2L/2LCk-cre) mice. We investigated whether the reduction in frontal 5-HT(2A)R binding was reflected in reduced functional output in two 5-HT(2A)-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF(2L/2LCk-cre) mutants treated with the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished ESR but no differences in HTR compared to wildtypes. These findings illustrate the context-dependent effects of deficient BDNF signaling on the 5-HT receptor system and 5-HT(2A)-receptor functional output.

Impact of different cell isolation techniques on lymphocyte viability and function.

The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.

Propionic and L-methylmalonic acids induce oxidative stress in brain of young rats.

The in vitro effects of propionic and L-methylmalonic acids on some parameters of oxidative stress were investigated in the cerebral cortex of 21-day-old rats. Chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant capacity (TRAP) were measured in brain tissue homogenates in the presence of propionic or L-methylmalonic acids at concentrations ranging from 1 to 10mM. Both acids significantly increased chemiluminescence and TBA-RS and decreased TRAP, indicating a simulation of lipid peroxidation and a reduction of tissue antioxidant potential. Other organic acids tested which accumulate in some organic acidemias (suberic, sebacic, adipic, 3-methylglutaric and 4-hydroxybutyric acids) did not affect these parameters. This study provides evidence that free radical generation may participate in the neurological dysfunction of propionic and methylmalonic acidemias.

Comparison of spinal mobility and isometric trunk extensor forces with electromyographic spectral analysis in identifying low back pain.

This study compared conventional clinical measurements with electromyographic (EMG) spectral measurements for identification of individuals with low back pain (LBP). Twenty freshman sweep rowers were subjects for this study. Range-of-motion (ROM) measurements were taken for forward bending (FB), backward bending (BB) (double inclinometers), lateral bending (LB) (tape measure), and rotation (double-arm goniometer). Intratester reliability for ROM was also assessed. The Back Analysis System was used to determine static trunk extensor strength (ie, maximal voluntary contraction [MVC]) and to compute EMG spectral parameters from a paraspinal multi-electrode array. A two-group stepwise discriminant-analysis procedure for the ROM and MVC variables correctly identified 57% of the rowers with LBP and 63% of the rowers without LBP. A similar discriminant-analysis procedure for EMG spectral parameters correctly identified either 88% of the rowers with LBP and 100% of the rowers without LBP or 100% of the rowers with LBP and 88% of the rowers with LBP, depending on whether EMG measurements of recovery were calculated at 1 minute or at 2 minutes into the recovery period. Sensitivity (66%) and specificity (71%) results from the more traditional tests suggest that these techniques may be of limited usefulness for LBP screening or diagnosis.