bioRxiv Subject Collection: Neuroscience's Journal
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Sunday, August 18th, 2024
| Time |
Event |
| 12:19a |
Quantitative 3D histochemistry reveals region-specific amyloid-β reduction by the antidiabetic drug netoglitazone
A hallmark of Alzheimer's disease (AD) is the extracellular aggregation of toxic amyloid-beta (A{beta}) peptides in form of plaques. Here, we identify netoglitazone, an antidiabetic compound previously tested in humans, as an A{beta} aggregation antagonist. Netoglitazone improved cognition and reduced microglia activity in a mouse model of AD. Using quantitative whole-brain three-dimensional histology (Q3D), we precisely identified brain regions where netoglitazone reduced the number and size of A{beta} plaques. We demonstrate the utility of Q3D in preclinical drug evaluation for AD by providing a high-resolution brain-wide view of drug efficacy. Applying Q3D has the potential to improve pre-clinical drug evaluation by providing information that can help identify mechanisms leading to brain region-specific drug efficacy. | | 1:30a |
Overcoming off-target optical stimulation-evoked cortical activity in the mouse brain in vivo
Genetic engineering of exogenous opsins sensitive to a wide range of light wavelengths allows the interrogation of brain circuits to an unprecedented temporal and spatial precision. In particular, red-shifted opsins offer access deeper within the brain tissue. It is however crucial to consider the potential unintended back-activation of endogenous opsins due to laser light striking the back of the retina. Here, we found that in complete darkness and with no expression of exogenous opsins, optical fiber laser stimulation at wavelengths of 637 nm (red), 594 nm (orange), or 473 nm (blue) from within the ipsilateral mouse visual cortex resulted in a strong neuronal response in its contralateral counterpart. This neuronal activation occurred even at low laser intensities (1 mW at the fiber tip, ~31.8 mW/mm2) and was most pronounced using red wavelengths. We therefore took advantage of retinal light adaptation using external illumination with a relatively dim ambient light source (20 lux) which was found to completely abolish orange and blue laser-evoked neuronal activation from within the brain, even at high laser intensities (15 mW, ~477.3 mW/mm2). To prevent red laser-evoked retinal activation, however, only much lower intensities (2.5 mW, ~79.6 mW/mm2) combined with external illumination (20 lux) could be used. These findings demonstrate the critical need for careful selection of light wavelengths and intensities for laser stimulation during optogenetic experiments in the mouse brain in vivo. Additionally, light adaptation of the retina through ambient light exposure offers an effective solution to minimize unintended retinal activation. | | 1:30a |
Electrical Synapse Rectification Enables Dual-Network Activity in the crab Cancer borealis
Flexibility of rhythmic networks includes neuromodulator-elicited changes in neuronal participation between networks. We are examining the role of rectifying electrical synapses in this neuronal switching. Electrical synapses can have complex, non-intuitive effects on network output. However, it is often difficult to measure and manipulate rectification across conditions to determine their functional contributions. Here, we use the Jonah crab Cancer borealis to investigate rectification in well-described rhythmic networks. In an established modulatory state, stimulating the projection neuron MCN5 or bath applying its neuropeptide Gly1-SIFamide causes the two LPG neurons to switch from pyloric rhythm-only (food filtering, 1 Hz) activity to dual pyloric and gastric mill rhythm (chewing, 0.1 Hz) activity. Typically, LPG is co-active with the two PD and single AB pyloric pacemaker neurons due to rectifying electrical coupling. In Gly1-SIFamide, LPG continues to burst in pyloric time with AB/PD but periodically escapes and generates intrinsic longer-duration gastric mill-timed bursts, decreasing its overall synchrony with AB/PD, while AB/PD retain their synchronous pyloric timing. Using two-electrode voltage clamp recordings, we find that Gly1-SIFamide does not alter electrical coupling strength or the rectification between LPG and AB/PD. However, in a computational model, rectification is necessary for LPG to escape AB/PD electrical coupling and generate longer, gastric mill-timed bursts. This was confirmed in the biological system by adding a dynamic clamp non-rectifying electrical synapse between LPG and PD, which decreased the LPG escape from AB/PD and its gastric mill-timed activity. Thus, rectification between electrically coupled oscillators can underlie modulator-elicited changes in their synchrony. | | 2:46a |
Integrative genomics approach identifies glial transcriptomic dysregulation and risk in the cortex of individuals with Alcohol Use Disorder
Alcohol use disorder (AUD) is a prevalent neuropsychiatric disorder that is a major global health concern, affecting millions of people worldwide. Past molecular studies of AUD used underpowered single cell analysis or bulk homogenates of postmortem brain tissue, which obscures gene expression changes in specific cell types. Here we performed single nuclei RNA-sequencing analysis of 73 post-mortem samples from individuals with AUD (N=36, Nnuclei= 248,873) and neurotypical controls (N=37, Nnuclei= 210,573) in both sexes across two institutional sites. We identified 32 clusters and found widespread cell type-specific transcriptomic changes across the cortex in AUD, particularly affecting glia. We found the greatest dysregulation in novel microglial and astrocytic subtypes that accounted for the majority of differential gene expression and co-expression modules linked to AUD. Analysis for cell type-specific enrichment of aggregate genetic risk for AUD identified subtypes of glial and neuronal cell types as potential key players not only affected by but causally linked to the progression of AUD. Most of the enrichments for genetic risk for AUD were in glial cell types. These results highlight the importance of cell-type specific molecular changes in AUD and offer opportunities to identify novel targets for treatment. | | 7:52a |
A nanobody-based proximity ligation assay detects constitutive and stimulus-regulated native Arc/Arg3.1 oligomers in hippocampal neuronal dendrites
Activity-regulated cytoskeleton-associated (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state, however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody. | | 7:52a |
C9orf72 repeat expansion-carrying iPSC-microglia from FTD patients show increased phagocytic activity concomitantly with decreased number of autophagosomal-lysosomal vesicles
C9orf72 hexanucleotide repeat expansion (HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The role of microglia in these C9orf72 HRE-associated diseases is understudied. To elucidate effects of C9orf72 HRE on microglia, we have characterized human induced pluripotent stem cell-derived microglia (iMG) from behavioral variant frontotemporal dementia (bvFTD) patients carrying the C9orf72 HRE. C9orf72 HRE iMG were compared to iMG from healthy controls and sporadic bvFTD patients. The phenotypes of iMG were analyzed using bulk RNA sequencing, biochemical and immunofluorescence analyses, and live cell imaging. C9orf72 HRE-carrying iMG showed nuclear RNA foci and poly-GP dipeptide repeat proteins but no decreased C9orf72 mRNA or protein expression. TDP-43 pathology was absent from all bvFTD iMG. As compared to healthy control iMG, quantitative immunofluorescence analyses indicated that all bvFTD iMG had reduced number, size, and intensity of LAMP2-A-positive vesicles. C9orf72 HRE-carrying iMG additionally showed decreased number, size, and intensity of p62/SQSTM1-positive vesicles. These changes were accompanied by increased phagocytic activity of the C9orf72 HRE-carrying iMG. Serum starvation increased phagocytic activity also in the iMG of sporadic bvFTD patients. RNA sequencing revealed that iMG of C9orf72 HRE-carrying bvFTD patients as compared to the iMG of sporadic bvFTD patients showed differential gene expression in pathways related to RNA and protein regulation and mitochondrial metabolism. Our data suggest potential alterations in the autophagosomal/lysosomal pathways in bvFTD patient iMG, which are further reinforced by the C9orf72 HRE and functionally manifest as increased phagocytic activity. | | 8:19a |
A triple dissociation across the medial, ventral, and lateral orbitofrontal cortex in rats making sequential foraging decisions
The orbitofrontal cortex (OFC) is functionally heterogeneous across its medial-to-lateral axis, though the nature of this heterogeneity is unclear. The present study leveraged high-density neural recording across the medial-to-lateral span of the OFC using a neuroeconomic task in rats to clarify how functional heterogeneity within the OFC participates in sequential cost-benefit foraging. Lateral and ventral OFC contained opposing representations of an encountered reward's subjective benefit and its associated opportunity costs, respectively. These representations directly competed to make accept-reject decisions during reward evaluation. If a poor-quality reward was mistakenly accepted, ventral and medial OFC participated in reevaluation processes during change-of-mind decisions. Collectively, these results suggest a triple dissociation in representational content and timing across the medial-to-lateral OFC during sequential foraging. | | 8:19a |
Dynamic coding and sequential integration of multiple reward attributes by primate amygdala neurons
The value of visual stimuli guides learning, decision-making and motivation. Although stimulus values often depend on multiple attributes, how neurons extract and integrate distinct value components from separate cues remains unclear. Here we recorded the activity of amygdala neurons while monkeys viewed sequential cues indicating the probability and magnitude of expected rewards. Amygdala neurons frequently signalled reward probability in an abstract, stimulus-independent code that generalized across cue formats. While some probability-coding neurons were insensitive to magnitude, signalling pure probability rather than value, many neurons showed biphasic responses that signalled probability and magnitude in a dynamic (temporally-patterned) and flexible (reversible) value code. Specific neurons integrated these reward attributes into risk signals that quantified the uncertainty of expected rewards, distinct from value. Population codes were accurate, mutually transferable between value components and expressed differently across amygdala nuclei. Our findings identify amygdala neurons as a substrate for the sequential integration of multiple reward attributes into value and risk. | | 11:45p |
Iron chelation by oral deferoxamine treatment decreased brain iron and iron signaling proteins
Background: Deferoxamine (DFO) and other iron chelators are clinically used for cancer and stroke. They may also be useful for Alzheimers disease (AD) to diminish iron from microbleeds. DFO may also stimulate antioxidant membrane repair which is impaired during AD. DFO, and other chelators do enter the brain despite some contrary reports. Objective: Low dose, oral DFO was given in lab chow to wildtype (WT) C57BL/6 mice to evaluate potential impact on iron levels, iron-signaling and storage proteins, and amyloid precursor protein (APP) and processing enzymes. Young WT mice do not have microbleeds or disrupted blood-brain barrier of AD mice. Methods: Iron was measured by MRI and chemically after two weeks of dietary DFO. Cerebral cortex was examined for changes in iron metabolism, antioxidant signaling, and APP processing by Western blot. Results: DFO decreased brain iron by 18% (MRI) and decreased seven major proteins that mediate iron metabolism by at least 25%. The iron storage proteins ferritin light and heavy chain decreased by at least 30%. APP and secretase enzymes also decreased by 30%. Conclusions: WT mice respond to DFO with decreased APP, amyloid processing enzymes, and antioxidant repair. Potential DFO treatment for early-stage AD by DFO should consider the benefits of lowered APP and secretase enzymes. |
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