The memory impairment associated with Alzheimers disease has long been associated with a loss in acetylcholine function and pathophysiology of the entorhinal/perirhinal, or rhinal cortex. In monkeys, lesions of this region as well as cholinergic deafferentation lead to severe memory deficits. We recently compared the effects of selective infusion of the (cholinergic receptor) m1 antagonist pirenzepine, and the m2 antagonist methoctramine directly into the rhinal cortex. Our findings suggest that m1 and m2 receptor subtypes in the perirhinal cortex have functionally dissociable roles, and that visual recognition memory is critically dependent on the m1 receptor subtype. To examine more about the m1 muscarinic-dependent intracellular signaling pathways that underlie critical synaptic changes important for memory we used a proteomics approach to uncover the molecular signaling pathways activated during memory formation in a region-specific manner using two techniques: laser capture microdissection and reverse phase protein microarrays. Sections of snap-frozen perirhinal tissue from Rhesus monkeys were prepared and stained for Nissl substance. Tissue subregions of perirhinal cortex (Layers III and V/VI), as well as hippocampus (cell body layers of CA1, CA3, and dentate gyrus and their corresponding dendritic fields), were isolated using laser capture microdissection. Lysed tissue samples were then printed onto reverse phase protein arrays (RPPA). These arrays were probed with antibodies against phosphorylated, as well as total, proteins involved in muscarinic signaling, synaptic transmission, and neuronal activity. We focused on the muscarinic acetylcholine receptor (M1AChR) and the mechanistic target rapamycin (mTOR) signaling pathways to determine the baseline biological variability of the phosphoprotein signature within monkey brain cortical layers. The ventral portion of the anterior TE (TEav), the perirhinal cortex (PRh), the entorhinal cortex (ERh) and the rostral hippocampus (HF) were dissected from both hemispheres of four rhesus monkeys, and key laminae of TEav, PRh, and ERh, as well as key strata of HF within each subregion, were microdissected using laser capture microdissection. Phosphoprotein and total protein levels of 19 endpoints within the m1AChR or mTOR signaling pathways were quantified using reverse phase protein microarrays. Comparing the variability of phosphoprotein levels between (a) the local microenvironment at the mid rostro-caudal level of the PRh within a single monkey, (b) along the rostro-caudal axis of the PRh within a single monkey, and (c) at the mid rostro-caudal level of the PRh between monkeys, revealed a different pattern for the m1AChR signaling pathway compared to mTOR signaling. As expected, m1AChR pathway signaling variability increased step-wise from the local mid-PRh microenvironment to the entire PRh between monkeys. In contrast, mTOR signaling showed a significantly higher variability that did not follow the same pattern, with only a slight increase of variability across monkeys compared to within monkey PRh. Memory formation and retrieval depends on the translaminar and layer-specific inter-area flow of information within a highly complex neural network. We therefore wanted to establish whether baseline m1AChR and mTOR signaling differed between individual layers of the cortical subregions of visual association area TE and the perirhinal cortex. Overall, m1AChR pathway activation was not different between individual layers within anterior TEav, perirhinal cortex or the entorhinal cortex. However, mTOR pathway activation was increased in PRh layers 5/6 compared to layer 3. In addition, mTOR signaling was increased in ERh layers 2/3 versus layers 5/6. Interestingly, this complements the known anatomical connections, where PRh layers 5/6 feed into ERh layers 2/3. Using the workflow we established, we were able to quantify mTOR and m1AChR signaling pathway activation within specific laminae of the monkey PRh, as well as critical brain regions feeding into PRh (area TEav) and those receiving input from the PRh (ERh and hippocampus). The pattern of mTOR pathway activation followed the physiologic route of neuronal signaling between the PRh and the ERh. This sets the stage to determine the role of key cell signaling pathways on visual recognition memory in behaviorally trained non-human primates. This project has now been terminated.
