Microcontact Printing of Cholinergic Neurons in Organotypic Brain Slices

Alzheimer's disease is a severe neurodegenerative disorder of the brain, characterized by beta-amyloid plaques, tau pathology, and cell death of cholinergic neurons, resulting in loss of memory. The reasons for the damage of the cholinergic neurons are not clear, but the nerve growth factor (NGF) is the most potent trophic factor to support the survival of these neurons. In the present study we aim to microprint NGF onto semipermeable 0.4 μm pore membranes and couple them with organotypic brain slices of the basal nucleus of Meynert and to characterize neuronal survival and axonal growth. The brain slices were prepared from postnatal day 10 wildtype mice (C57BL6), cultured on membranes for 2–6 weeks, stained, and characterized for choline acetyltransferase (ChAT). The NGF was microcontact printed in 28 lines, each with 35 μm width, 35 μm space between them, and with a length of 8 mm. As NGF alone could not be printed on the membranes, NGF was embedded into collagen hydrogels and the brain slices were placed at the center of the microprints and the cholinergic neurons that survived. The ChAT+ processes were found to grow along with the NGF microcontact prints, but cells also migrated. Within the brain slices, some form of re-organization along the NGF microcontact prints occurred, especially the glial fibrillary acidic protein (GFAP)+ astrocytes. In conclusion, we provided a novel innovative microcontact printing technique on semipermeable membranes which can be coupled with brain slices. Collagen was used as a loading substance and allowed the microcontact printing of nearly any protein of interest.

Altered Functional Connectivity of the Basal Nucleus of Meynert in Subjective Cognitive Impairment, Early Mild Cognitive Impairment, and Late Mild Cognitive Impairment

Background: The spectrum of early Alzheimer's disease (AD) is thought to include subjective cognitive impairment, early mild cognitive impairment (eMCI), and late mild cognitive impairment (lMCI). Choline dysfunction affects the early progression of AD, in which the basal nucleus of Meynert (BNM) is primarily responsible for cortical cholinergic innervation. The aims of this study were to determine the abnormal patterns of BNM-functional connectivity (BNM-FC) in the preclinical AD spectrum (SCD, eMCI, and lMCI) and further explore the relationships between these alterations and neuropsychological measures.Methods: Resting-state functional magnetic resonance imaging (rs-fMRI) was used to investigate FC based on a seed mask (BNM mask) in 28 healthy controls (HC), 30 SCD, 24 eMCI, and 25 lMCI patients. Furthermore, the relationship between altered FC and neurocognitive performance was examined by a correlation analysis. The receiver operating characteristic (ROC) curve of abnormal BNM-FC was used to specifically determine the classification ability to differentiate the early AD disease spectrum relative to HC (SCD and HC, eMCI and HC, lMCI and HC) and pairs of groups in the AD disease spectrum (eMCI and SCD, lMCI and SCD, eMCI and lMCI).Results: Compared with HC, SCD patients showed increased FC in the bilateral SMA and decreased FC in the bilateral cerebellum and middle frontal gyrus (MFG), eMCI patients showed significantly decreased FC in the bilateral precuneus, and lMCI individuals showed decreased FC in the right lingual gyrus. Compared with the SCD group, the eMCI group showed decreased FC in the right superior frontal gyrus (SFG), while the lMCI group showed decreased FC in the left middle temporal gyrus (MTG). Compared with the eMCI group, the lMCI group showed decreased FC in the right hippocampus. Interestingly, abnormal FC was associated with certain cognitive domains and functions including episodic memory, executive function, information processing speed, and visuospatial function in the disease groups. BNM-FC of SFG in distinguishing eMCI from SCD; BNM-FC of MTG in distinguishing lMCI from SCD; BNM-FC of the MTG, hippocampus, and cerebellum in distinguishing SCD from HC; and BNM-FC of the hippocampus and MFG in distinguishing eMCI from lMCI have high sensitivity and specificity.Conclusions: The abnormal BNM-FC patterns can characterize the early disease spectrum of AD (SCD, eMCI, and lMCI) and are closely related to the cognitive domains. These new and reliable findings will provide a new perspective in identifying the early disease spectrum of AD and further strengthen the role of cholinergic theory in AD.

Involvement of the basal nucleus of Meynert on regional cerebral cortical vasodilation associated with masticatory muscle activity in rats

We examined the neural mechanisms for increases in regional cerebral blood flow (rCBF) in the neocortex associated with mastication, focusing on the cortical vasodilative system derived from the nucleus basalis of Meynert (NBM). In pentobarbital-anesthetized rats, parietal cortical rCBF was recorded simultaneously with electromyogram (EMG) of jaw muscles, local field potentials of frontal cortex, multi-unit activity of NBM neurons, and systemic mean arterial pressure (MAP). When spontaneous rhythmic EMG activity was observed with cortical desynchronization, an increase in NBM activity and a marked rCBF increase independent of MAP changes were observed. A similar rCBF increase was elicited by repetitive electrical stimulation of unilateral cortical masticatory areas. The magnitude of rCBF increase was partially attenuated by administration of the GABAergic agonist muscimol into the NBM. The rCBF increase persisted after immobilization with systemic muscle relaxant (vecuronium). rCBF did not change when jaw muscle activity was induced by electrical stimulation of the pyramidal tract. The results suggest that activation of NBM vasodilator neurons contributes at least in part to the rCBF increase associated with masticatory muscle activity, and that the NBM activation is induced by central commands from the motor cortex, independently of feedback from brainstem central pattern generator or contracting muscles.