The amplitude and extent of PCP-induced brain activation increased dose-dependently. The inspection of statistical activation maps suggested that at PCP 1.0 mg/kg, there were no significant differences between PCP and control groups. At PCP 2.0 mg/kg, activation was localized in mPFC, nucleus accumbens, minor striatal regions, and discrete cortical regions.
At PCP 3.0 mg/kg, activation covered the whole striatum and large parts of thalamus.
Figure 14. Spearman´s rank correlation coefficients (ρ) between mean increases in blood oxygenation level dependent (BOLD) signal intensity during forepaw stimulation period and preceding functional connectivity, both measured from primary somatosensory cortex. Data were obtained from both paws of six rats (four stimulation sessions per paw at 22-min intervals).
Additionally, the cortical activation was remarkably enhanced compared to PCP 2.0 mg/kg.
At PCP 5.0 mg/kg, as compared to PCP 3.0 mg/kg, enhanced activation was observed only in posterior cortical regions, indicating that a maximum spatial response had been reached already at PCP 3.0 mg/kg. The findings were similar in the group-level time series and AUC calculations; time courses and AUC values at mPFC were similar at PCP doses 2.0, 3.0, and 5.0 mg/kg, while a clear dose-dependent increase was seen in the visual cortex, suggesting that certain frontal brain regions are fully activated already by a low PCP dose but that the activation of posterior regions requires higher PCP doses.
The PCP-induced acute fMRI signal changes were fully supported by PCP and DA levels in the brain extracellular fluid (in vivo microdialysis experiments). Unbound PCP levels increased dose-dependently in both mPFC and striatum. However, PCP was found to increase extracellular dopamine levels in mPFC already at the 2.0 mg/kg dose, while PCP 3.0 mg/kg was required to increase dopamine levels in striatum. A good temporal correlation was observed between extracellular dopamine levels and BOLD signal changes (R2~0.6, p<0.001).
The FC analysis indicated that brain networks were disturbed by PCP; the global connectivity decreased as a function of PCP dose (Figure 15). Several pathways originating from key regions associated with SCZ pathophysiology were affected in dose-dependent manner, such as connections originating from thalamus, striatum, mPFC, nucleus accumbens, and hippocampus (logistic regression analysis, p<0.05). Additionally, the integration and connectivity of cortex was severely affected (logistic regression analysis, p<0.001).
Mesocortical and mesolimbic connections, which are dysfunctional in SCZ, were disturbed in a dose-dependent manner (logistic regression analysis, p<0.05), similarly to hippocampal connections (logistic regression analysis, p<0.05). The analysis of interhemispheric connectivity indicated that the integration of hippocampus was significantly decreased already by PCP 2.0 mg/kg (one-way ANOVA, Dunnett´s multiple comparison, p<0.05).
Figure 15. Changes in global functional connectivity (ΔCorrelation) 1h after treatment. Correlation values were averaged from 12 regions of interest from each subject (n=7/group).
The effects of different PCP doses on both AUC and FC in four representative regions are shown in Figure 16, which implies that a region-specific increase in AUC is not a necessity for disturbed connectivity. For example, the AUC changes in hippocampus were only minor throughout the dose range, but there was a considerable effect of PCP on FC (Δcorrelation) in hippocampus. In contrast, AUC appeared to increase in primary somatosensory cortex after the administration of PCP 1.0 mg/kg, but there was no clear decrease in connectivity.
In behavioral tests, high PCP doses increased locomotor activity (3.0 and 5.0 mg/kg against saline, t-test p<0.001 2 h after treatment). Additionally, the locomotor activity exhibited excellent temporal cross-correlation with the decreased FC in the motor cortices (R2=0.8, p<0.001). In the novel object recognition test, rats treated with 2.0 mg/kg PCP had lower (t-test, p<0.001) discrimination between familiar and novel objects than saline-treated rats. In the social interaction test, rats treated with 1.5 mg/kg PCP investigated each other significantly less (t-test, p<0.05) than control rats.
Acute subcutaneous administration of PCP 5.0 mg/kg increased peripheral MABP only mildly (~15 mmHg). The increase in MABP was observed at ~5 min after the injection, while BOLD signal only reached its maximum amplitude around 30-45 min. Therefore, the low correlation between MABP and BOLD signal (R2~0.2) suggests that the PCP-induced increase in MABP does not make any major contribution to the PCP-induced fMRI signal changes in brain.
Figure 16. The effects of phencyclidine on blood oxygenation level dependent signal (measured as area under curve, AUC) and averaged functional connectivity in four regions of interest. Color of marker indicates grouping, and shape of marker region of interest.
6 Discussion and Conclusions
The present work compared the effects of different anesthesia protocols on acute nicotine-induced BOLD signal changes (I). Additionally, the relationship between baseline FC and stimuli-induced BOLD signal changes under different anesthetics was investigated (II).
Studies I and II were intended to improve the preclinical phMRI methodology by providing a means to minimize some of the confounding effects induced by anesthetics. The results, together with the literature review, provide a basis for the selection of anesthetic for phMRI experiments (I), and a simple tool for measuring the brain hemodynamic responsiveness to external stimuli (II). Therefore, the findings of studies I and II can significantly improve the quality of preclinical phMRI studies, when the use of anesthesia is unavoidable.
In the third part of this thesis, an optimized preclinical fMRI protocol was implemented into the multimodal characterization of induced SCZ-like symptoms in rats (III). PCP-induced dose-dependent changes in biomarkers in neural activity were detected for different SCZ-like symptom classes, and the alterations detected in fMRI and in vivo microdialysis experiments paralleled the changes in selected behavioral tests. By exploiting the results of study III, the existing PCP-model can be applied better for inducing different SCZ-like symptom classes, and be incorporated into drug development programs seeking improved drugs to treat SCZ.
6.1 THE IMPACT OF ANESTHESIA PROTOCOL ON PHMRI RESPONSES