In this work, we describe the fabrication and working of a modular microsystem that recapitulates the functions of the "Neurovascular Unit". The microdevice comprised a vertical stack of a poly(dimethylsiloxane) (PDMS) neural parenchymal chamber separated by a vascular channel via a microporous polycarbonate (PC) membrane. The neural chamber housed a mixture of neurons (~4%), astrocytes (~95%), and microglia (~1%). The vascular channel was lined with a layer of rat brain microvascular endothelial cell line (RBE4). Cellular components in the neural chamber and vascular channel showed viability (>90%). The neural cells fired inhibitory as well as excitatory potentials following 10 days of culture. The endothelial cells showed diluted-acetylated low density lipoprotein (dil-a-LDL) uptake, expressed von Willebrand factor (vWF) and zonula occludens (ZO-1) tight junctions, and showed decreased Alexafluor™-conjugated dextran leakage across their barriers significantly compared with controls (p < 0.05). When the vascular layer was stimulated with TNF-α for 6 h, about 75% of resident microglia and astrocytes on the neural side were activated significantly (p < 0.05 compared to controls) recapitulating tissue-mimetic responses resembling neuroinflammation. The impact of this microsystem lies in the fact that this biomimetic neurovascular platform might not only be harnessed for obtaining mechanistic insights for neurodegenerative disorders, but could also serve as a potential screening tool for central nervous system (CNS) therapeutics in toxicology and neuroinfectious diseases.

The basolateral amygdala (BLA) is critical for associating initially neutral cues with appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ACh) projections. We measured BLA ACh signaling and activity of neurons expressing CaMKIIα (a marker for glutamatergic principal cells) in mice during cue-reward learning using a fluorescent ACh sensor and calcium indicators. We found that ACh levels and nucleus basalis of Meynert (NBM) cholinergic terminal activity in the BLA (NBM-BLA) increased sharply in response to reward-related events and shifted as mice learned the cue-reward contingency. BLA CaMKIIα neuron activity followed reward retrieval and moved to the reward-predictive cue after task acquisition. Optical stimulation of cholinergic NBM-BLA terminal fibers led to quicker acquisition of the cue-reward contingency. These results indicate BLA ACh signaling carries important information about salient events in cue-reward learning and provides a framework for understanding how ACh signaling contributes to shaping BLA responses to emotional stimuli.

31The basolateral amygdala (BLA) is critical for associating initially neutral cues with 32 appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ACh) 33 projections. We measured BLA ACh signaling and principal neuron activity in mice during cue-34 reward learning using a fluorescent ACh sensor and calcium indicators. We found that ACh 35 levels and activity of nucleus basalis of Meynert (NBM) cholinergic terminals in the BLA (NBM-36 BLA) increased sharply in response to reward-related events and shifted as mice learned the 37 tone-reward contingency. BLA principal neuron activity followed reward retrieval and moved to 38 the reward-predictive tone after task acquisition. Optical stimulation of cholinergic NBM-BLA 39 terminal fibers during cue-reward learning led to more rapid learning of the cue-reward 40 contingency. These results indicate that BLA ACh signaling carries important information about 41 salient events in cue-reward learning and provides a framework for understanding how ACh 42 signaling contributes to shaping BLA responses to emotional stimuli. 43 44 1991; Zaborszky et al., 2012). Optical stimulation of BLA-projecting cholinergic terminal fibers 62 (NBM-BLA) during fear conditioning is sufficient to strengthen fear memories (Jiang et al., 2016) 63and may support appetitive behavior (Aitta-aho et al., 2018). Cholinergic NBM neurons increase 64 their firing in response to both rewarding and aversive unconditioned stimuli (Hangya et al., 65 2015). A recent study has also demonstrated that NBM cells fire in response to a conditioned 66 stimulus during trace fear conditioning, indicating that ACh signaling may be involved in learning 67 about cues that predict salient outcomes . 68 5We hypothesized that ACh signaling in the BLA is a critical neuromodulatory signal that 69 responds to both unconditioned stimuli and cues that gain salience, thereby coordinating activity 70 in circuits necessary for learning cue-reward contingencies. To test this hypothesis, we 71 measured relative levels of BLA ACh (ACh signaling), cholinergic NBM-BLA terminal fiber 72 activity (BLA ACh signal origin), and the activity of BLA principal neurons (BLA output) across all 73 phases of learning in an appetitive operant learning task to evaluate how BLA output and ACh 74 signaling are related to behavioral performance in this paradigm. We then optically stimulated 75 cholinergic NBM fibers locally in the BLA while mice learned to nose poke in response to an 76 auditory cue to receive a food reward to determine if accelerating the increase in ACh signaling 77 that occurs as mice learn the task would enhance performance. We also pharmacologically 78 blocked different ACh receptors during the learning task to determine the subtypes involved, 79and varied the timing of optical stimulation of cholinergic NBM-BLA terminal fibers to determine 80 whether time-locked ACh release with the reward-predictive cue is necessary for the 81 improvement of the task performance. These studies provide a novel framewo...

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as co-culture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary hepatocyte culture systems suffer from reproducibility issues due to phenotypic variation and expensive, limited supplies of donor lots. Here we describe a microfluidic bilayer device that sustains primary human hepatocyte phenotypes, including albumin production, factor IX production, cytochrome P450 3A4 drug metabolism and bile canaliculi formation for at least 14 days in a simple monoculture format with static media. Using a variety of channel architectures, we describe how primary cell phenotype is promoted by spatial confinement within the microfluidic channel, without the need for perfusion or co-culture. By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRG™-KO) humanized mouse model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-014-9877-x) contains supplementary material, which is available to authorized users.