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The “dorsal pons”, or “dorsal pontine tegmentum” (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study’s translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data. The dorsal pons in the brainstem is packed with clusters of neurons, including the parabrachial nucleus, that are involved in many vital functions. Here, authors use single nucleus RNA sequencing and MERFISH to create a spatially defined transcriptional atlas of this region.

The blood-brain barrier (BBB), formed by specialized endothelial cells (ECs), regulates the extracellular composition of the central nervous system (CNS). Little is known about whether there are regional specializations of the BBB that may control the function of specific neural circuits. We use single cell RNA-seq to characterize ECs from nine CNS regions in male mice: cortex, hippocampus, cerebellum, spinal cord, striatum, thalamus, hypothalamus, midbrain, and medulla/pons. Although there is a core BBB transcriptional profile, there are significant regional specializations. Stra6, a retinoid transporter, is highly enriched in the BBB of the nucleus accumbens shell (ShNAc) and ventral cochlear nucleus, and is controlled by dietary vitamin A, through endothelial RARƔ. EC Stra6 regulates the deposition of retinoids specifically in the ShNAc and cochlear nucleus, and is required for the function of the ShNAc, in a retinoid-dependent manner. Thus regional specializations of the BBB can regulate the function of local brain regions. The blood-brain barrier (BBB) regulates the extracellular composition of the central nervous system (CNS), but it is not known whether its properties differ across CNS regions. Here, the authors show in mice that the BBB exhibits regional specializations, and that such specializations can be important for the function of specific neural circuits.

In order to fulfill their evolutionary role as support cells, astrocytes have to tolerate intense oxidative stress under conditions of brain injury and disease. It is well known that astrocytes exposed to mild oxidative stress are preconditioned against subsequent stress exposure in dual hit models. However, it is unclear whether oxidative stress leads to stress tolerance, stress exacerbation, or no change in stress resistance in astrocytes. Furthermore, it is not known whether reactive astrocytes surviving intense oxidative stress can still support nearby neurons. The data in this Brief Report suggest that primary cortical astrocytes surviving high concentrations of the oxidative toxicant paraquat are completely resistant against subsequent oxidative challenges of the same intensity. Inhibitors of multiple endogenous defenses (e.g., glutathione, heme oxygenase 1, ERK1/2, Akt) failed to abolish or even reduce their stress resistance. Stress-reactive cortical astrocytes surviving intense oxidative stress still managed to protect primary cortical neurons against subsequent oxidative injuries in neuron/astrocyte co-cultures, even at concentrations of paraquat that otherwise led to more than 80% neuron loss. Although our previous work demonstrated a lack of stress tolerance in primary neurons exposed to dual paraquat hits, here we show that intensely stressed primary neurons can resist a second hit of hydrogen peroxide. These collective findings suggest that stress-reactive astroglia are not necessarily neurotoxic, and that severe oxidative stress does not invariably lead to stress exacerbation in either glia or neurons. Therefore, interference with the natural functions of stress-reactive astrocytes might have the unintended consequence of accelerating neurodegeneration.

Background α-synucleinopathy emerges quite early in olfactory structures such as the olfactory bulb and anterior olfactory nucleus (OB/AON) in Parkinson's disease. This may contribute to smell impairments years before the commencement of motor symptoms. We tested whether α-synucleinopathy can spread from the OB/AON to regions of the limbic telencephalon that harbor connections with olfactory structures. Findings α-synuclein fibrils were infused into the OB/AON. Inclusions containing pathologically phosphorylated α-synuclein (pSer129) were observed three months later in the piriform and entorhinal cortices, amygdala, and hippocampal formation. The retrograde tract-tracer FluoroGold confirmed the existence of first-order afferents at these sites. Some sites harbored FluoroGold + neurons but no inclusions, suggestive of selective vulnerabilities. Multiple areas close to the injection site but not connected with the OB/AON remained free of inclusions, suggesting a lack of widespread uptake of fibrils from interstitial diffusion. Two independent pSer129 antibodies revealed the same labeling patterns and preadsorption control experiments confirmed a loss of pSer129 staining. Dense total α-synuclein (but not pSer129) staining was apparent in the OB/AON 1.5 h following fibril infusions, suggesting that pSer129 + staining did not reflect exogenously infused material. Waterbath sonication of fibrils for 1 h improved α-synucleinopathy transmission relative to 1 min-long probe sonication. Electron microscopy revealed that longer sonication durations reduced fibril size. The Thioflavin stain labeled cells at the infusion site and some, but not all inclusions contained ubiquitin. Three-dimensional confocal analyses revealed that many inclusions ensconced NeuN + neuronal nuclei. Young and aged mice exhibited similar topographical spread of α-synucleinopathy. Conclusions 1) α-synucleinopathy in this model is transmitted through some, but not all neuroanatomical connections, 2) pathology is largely confined to first-order afferent sites at three months and this is most parsimoniously explained by retrograde transport, and 3) transmission in aged animals is largely similar to that in young control animals at three months post-infusion.

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.

Dopamine loss and motor deficits in Parkinson’s disease typically commence unilaterally and remain asymmetric for many years, raising the possibility that endogenous defenses slow the cross-hemispheric transmission of pathology. It is well-established that the biological response to subtoxic stress prepares cells to survive subsequent toxic challenges, a phenomenon known as preconditioning, tolerance, or stress adaptation. Here we demonstrate that unilateral striatal infusions of the oxidative toxicant 6-hydroxydopamine (6-OHDA) precondition the contralateral nigrostriatal pathway against the toxicity of a second 6-OHDA infusion in the opposite hemisphere. 6-OHDA-induced loss of dopaminergic terminals in the contralateral striatum was ablated by cross-hemispheric preconditioning, as shown by two independent markers of the dopaminergic phenotype, each measured by two blinded observers. Similarly, loss of dopaminergic somata in the contralateral substantia nigra was also abolished, according to two blinded measurements. Motor asymmetries in floor landings, forelimb contacts with a wall, and spontaneous turning behavior were consistent with these histological observations. Unilateral 6-OHDA infusions increased phosphorylation of the kinase ERK2 and expression of the antioxidant enzyme CuZn superoxide dismutase in both striata, consistent with our previous mechanistic work showing that these two proteins mediate preconditioning in dopaminergic cells. These findings support the existence of cross-hemispheric preconditioning in Parkinson’s disease and suggest that dopaminergic neurons mount impressive natural defenses, despite their reputation as being vulnerable to oxidative injury. If these results generalize to humans, Parkinson’s pathology may progress slowly and asymmetrically because exposure to a disease-precipitating insult induces bilateral upregulation of endogenous defenses and elicits cross-hemispheric preconditioning.

Abstract Disclosure: N. Neamonitaki: None. T. Thaweethai: None. D. Pant: None. K. James: None. M.D. Soffer: None. E.A. Rosenberg: None. C.E. Powe: Consulting Fee; Self; Mediflix, Inc. Other; Self; Wolters Kluwer, Up to Date Author. Background: Epidemiologic studies show a reduction in type 2 diabetes incidence with increased lactation duration and intensity, but physiologic mechanisms underlying this association are incompletely characterized. We examined associations between lactation and glycemic physiology in postpartum women. ​ Methods: We conducted a secondary analysis of postpartum participants in a prospective longitudinal cohort study that examined glucose and insulin physiology via serial 2-hr 75-gram oral glucose tolerance tests during pregnancy and postpartum in women with diabetes risk factors. We used linear regression to examine associations between exclusive breastfeeding and fasting glucose, adjusting for age, race/ethnicity, education, body mass index, and number of weeks postpartum. Secondary analyses examined associations with post-load glucose and insulin physiology. Additional analyses considered exclusive breastfeeding compared to exclusive formula feeding and a combination of formula and breastfeeding separately using a categorical exposure variable. Results: Of N=108 women at a mean (SD) of 11 (5.0) weeks postpartum, 56 reported exclusively breastfeeding and 52 reported not exclusively breastfeeding (15 were exclusively formula feeding and 37 were both breast- and formula feeding). Mean (SD) fasting glucose was 83 (7.0) mg/dl in those exclusively breastfeeding and 85 (8.9) mg/dl in those who were not. Fasting glucose was not significantly different in those who were exclusively breastfeeding compared to those who were not after confounder adjustment (β [95% CI] =-1.3 [-4.6, 2.0] mg/dl, P=0.45). The 60- minute post-load glucose was nominally higher in those exclusively breastfeeding, with a mean (SD) of 132 (37.5) mg/dl versus 118 (31.6) mg/dl in those who were not; this difference did not reach statistical significance in the adjusted model (β [95% CI] =12.6 [-2.1, 27] mg/dl, P=0.09). In additional analyses, fasting glucose was not significantly different between women exclusively breastfeeding, exclusively formula feeding, or both breast- and formula feeding; however, post-load glucose at 30 and 60 minutes was higher in the exclusively breastfeeding group compared to the exclusively formula feeding group (30-minute: β [95% CI]= 16 [0.36,32] mg/dl, P=0.045); 60-minute: 26 [4.0,47] mg/dl, P=0.02) in adjusted models. We did not find significant associations with insulin physiology measures. Conclusion: We did not observe lower fasting glucose with exclusive breastfeeding. Instead, glucose levels at 30 and 60 minutes after an oral glucose load were higher in exclusively breastfeeding participants compared to exclusively formula feeding participants. Presentation: 6/2/2024