On the role of forebrain cholinergic innervation for phencyclidine-induced behaviors and gene expression patterns

Abstract: The basalo - cortical cholinergic innervation reaches neural targets in cortex cerebri where it modulates the incoming sensory information, regulating arousal, attention, v igilance, memory and cognition. P rogressive de generation of the cholinergic neurons in the basal forebrain is considered a hallmark of Alzheimer ’s disease re lated to cognitive impairment in this condition. Dysregulation of cholinergic neurotransmission has also been implicated in schizophrenia, a chronic and debilitating neurodevelopmental disorder characterized by positive and negative symptoms and cognitive impairments, with deficits in thought processes, perceptions and emo tional responsiveness. Modeling specific disruption of cholinergic function allows studies of its behavioral and molecular consequences and may contribute to the development of future thera pies for Alzheimer’s disease and Schizophrenia. To investigate the role of the cholinergic system, we carried out uni - and bilateral choliner gic denervation of cortex cerebri in male Lister hooded rats using the selective immu notoxin 19 2 IgG - saporin to specifically target the cholinergic neurons of nucleus basalis of magnocellularis (NBM). IgG - saporin effectively removed cholinergic afferents to the cortical mantle. Intact and denervated rats were challenged with phencyclidine (PCP), a n on - competi tive NMDA receptor antagonist, which is known to produce schizophrenia - like psychosis in humans and hence used to model aspects of schizophrenia in rats. Experiments were conducted to determine the effects on behavior and to shed light on the un derlying molecular mechanisms. Negative symptoms of schizophrenia include social withdrawal, which is studied in animal models using social interaction tests. We found that cortical cholinergic denervation lead to a significant reduction in the duration o f active social interaction in pairs of lesioned animals versus pairs of sham - operated controls. After an acute dose low of PCP (1 mg/kg, s.c.), there was a marked decrease in active social interaction for both groups, such that there was no longer a diffe rence between lesioned and control animals. To evaluate cognitive impairments, particularly those reflecting declarative memory, a novel object recognition test was used. Neither cholinergic denervation alone, nor an acute PCP dose (1 mg/kg, s.c.) alone, b locked the ability of rats to recognize a novel object. However, animals lacking cortical cholinergic inner vation and chal lenged with PCP were no longer able to recognize the novel object (Paper I). Behavioral paradigms have typically been analyzed manually, though automated hard ware and software systems have been introduced to speed up the process and eliminate subjec tive errors. How - ever, the reliability and accuracy of computerized scoring nee ds to be verified for each specific paradigm. For novel object recognition, the program scoring reduced analysis time for the task, but needed manual corrections to avoid erroneous data points. Manual for scoring of social interaction revealed that this la bor intensive approach was more nuanced and allowed to discern complex behaviors, whereas the automated scoring system registered more global interaction patterns. It is concluded that manual and computerized techniques for scoring social interaction offer complimentary information on various aspects of this complex behavioral task (Paper II). The next studies were aimed to examine the bases of the behavioral responses to cortical cholinergic denerva tion and PCP challenge at the molecular level. PCP - induc ed neuronal activation was mapped using quantitative in situ hybridization of the neuronal immediate early gene c - Fos mRNA levels. Transcription of c - Fos responds to many different forms of activation, including stress and toxins. We found that two doses o f PCP used (2 and 3 mg/kg, s.c.) caused a marked increase in neuronal c - Fos mRNA expression at 30 and 60 min after PCP administration, though these doses did not alter the levels of BDNF or Nogo receptor mRNA. Importantly, PCP responses were markedly dampe ned in cholinergically denervated regions of cortex cerebri (Paper III). RNA - Sequencing was then employed to understand gene regulatory processes at a global level in somatosensory cortex with regard to effects of chronic denervation and/or a PCP challen ge (1 and 3 mg/kg, s.c.). A first round of analysis focused on the genes most significantly altered by denervation (Egr1, Dusp6, Ier2, Nr4a1), PCP treatment (Cyr61, Bcl6b, Apold1, Dusp1) and those altered over time (Sox9, Coq10b, Zfp189, Rnf39) (Paper IV). The most common response was for mRNA levels to increase in response to PCP and for cholinergic de nervation to dampen these increases. The findings presented in this thesis support a role for the cholinergic system in assigning significance to incoming stimuli as exemplified by regulating the response of cortical neurons to a PCP challenge. The findings are also compatible with a proposed involvement of cholinergic dysfunction in schizophrenia.