Faculty Bibliography

Spreading depolarization (SD) is a sudden, large, and synchronous depolarization of principal cells which also involves interneurons and astrocytes. It is followed by depression of neuronal activity, and it slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury (TBI), but there are many questions about its basic neurophysiology and spread. Research into SD in hippocampus using slices is often used to gain insight and SD is usually triggered by a focal stimulus with or without an altered extracellular buffer. Here, we optimize an in vitro experimental model allowing us to record SD without focal stimulation, which we call spontaneous. This method uses only an altered extracellular buffer containing 0 mM Mg²⁺ and 5 mM K⁺ and makes it possible for simultaneous patch and extracellular recording in a submerged chamber plus intrinsic optical imaging in slices of either sex. We also add methods for quantification and show the quantified optical signal is much more complex than imaging alone would suggest. In brief, acute hippocampal slices were prepared with a chamber holding a submerged slice but with flow of artificial cerebrospinal fluid (aCSF) above and below, which we call interface-like. As soon as slices were placed in the chamber, aCSF with 0 Mg²⁺/5 K⁺ was used. Most mouse slices developed SD and did so in the first hour of 0 Mg²⁺/5 K⁺ aCSF exposure. In addition, prolonged bursts we call seizure-like events (SLEs) occurred, and the interactions between SD and SLEs suggest potentially important relationships. Differences between rats and mice in different chambers are described. Regarding optical imaging, SD originated in CA3 and the pattern of spread to CA1 and the dentate gyrus was similar in some ways to prior studies but also showed interesting differences. In summary, the methods are easy to use, provide new opportunities to study SD, new insights, and are inexpensive. They support previous suggestions that SD is diverse, and also suggest that participation by the dentate gyrus merits greater attention.

Flipping through social media feeds, viewing exhibitions in a museum, or walking through the botanical gardens, people consistently choose to engage with and disengage from visual content. Yet, in most laboratory settings, the visual stimuli, their presentation duration, and the task at hand are all controlled by the researcher. Such settings largely overlook the spontaneous nature of human visual experience, in which perception takes place independently from specific task constraints and its time course is determined by the observer as a self-governing agent. Currently, much remains unknown about how spontaneous perceptual experiences unfold in the brain. Are all perceptual categories extracted during spontaneous perception? Does spontaneous perception inherently involve volition? Is spontaneous perception segmented into discrete episodes? How do different neural networks interact over time during spontaneous perception? These questions are imperative to understand our conscious visual experience in daily life. In this article we propose a framework for spontaneous perception. We first define spontaneous perception as a task-free and self-paced experience. We propose that spontaneous perception is guided by four organizing principles that grant it temporal and spatial structures. These principles include coarse-to-fine processing, continuity and segmentation, agency and volition, and associative processing. We provide key suggestions illustrating how these principles may interact with one another in guiding the multifaceted experience of spontaneous perception. We point to testable predictions derived from this framework, including (but not limited to) the roles of the default-mode network and slow cortical potentials in underlying spontaneous perception. We conclude by suggesting several outstanding questions for future research, extending the relevance of this framework to consciousness and spontaneous brain activity. In conclusion, the spontaneous perception framework proposed herein integrates components in human perception and cognition, which have been traditionally studied in isolation, and opens the door to understand how visual perception unfolds in its most natural context.

Background: Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH) is a rare monogenic disorder caused by SLC34A3 pathogenic variants, characterized by renal phosphate wasting, hypophosphatemia, hypercalciuria (HC), elevated 1,25-dihydroxyvitamin D, nephrocalcinosis (NC), and urinary stone disease (USD). Previously we reported a high prevalence of kidney cysts in CYP24A1 deficiency. Thus, in the current study, we characterized cyst presence in HHRH, another monogenic cause of HC, NC, and USD.
Method(s): Medical records from Mayo Clinic and Rare Kidney Stone Consortium research results were queried for all patients with genetically confirmed HHRH diagnosis. Clinical characteristics and imaging data are summarized in table 1.
Result(s): Among 12 patients with SLC34A3 pathogenic variants (7 monoallelic, 5 biallelic), 42% (5/12) were males. Median age at clinical presentation was 17 yrs (range 8-46) and at genetic confirmation 42 yrs (range 9-66). None had a family history of cystic kidney disease. Kidney cysts (Figure 1) were present in 75% (9/12), among whom median age at first kidney imaging and first cyst detection was 41 yrs (range 9-64). Median number of cysts per patient was 3 (range 1-23). The number of cysts >=5 mm in size was above the 97.5th percentile of an age-and sex-matched control population in 6/9 (67%). At least 2 cysts >=5 mm in size were found in 100% of children.
Conclusion(s): We found a strong association between HHRH and kidney cysts. Similarities in the biochemical profiles of HHRH and CYP24A1 deficiency suggest elevated active vitamin D, and/or HC may be potential factors in cyst formation. Further studies are needed to evaluate the role of the SLC34A3 gene in cyst formation. (Figure Presented)

Protein kinase B (PKB/AKT) is a central kinase involved in many neurobiological processes. AKT is expressed in the brain as three isoforms, AKT1, AKT2, and AKT3. Previous studies suggest isoform-specific roles in neural function, but very few studies have examined AKT isoform expression at the cellular level. In this study, we use a combination of histology, immunostaining, and genetics to characterize cell-type-specific expression of AKT isoforms in human and mouse brains. In mice, we find that AKT1 is the most broadly expressed isoform, with expression in excitatory neurons and the sole detectable AKT isoform in gamma-aminobutyric acid ergic interneurons and microglia. By contrast, we find that AKT2 is the sole isoform expressed in astroglia and is not detected in other neural cell types. We find that AKT3 is expressed in excitatory neurons with AKT1 but shows greater expression levels in dendritic compartments than AKT1. We extend our analysis to human brain tissues and find similar results. Using genetic deletion approaches, we also find that the cellular determinants restricting AKT isoform expression to specific cell types remain intact under Akt deficiency conditions. Because AKT signaling is linked to numerous neurological disorders, a greater understanding of cell-specific isoform expression could improve treatment strategies involving AKT.

Down syndrome (DS) is the primary genetic cause of intellectual disability (ID), which is due to the triplication of human chromosome 21 (HSA21). In addition to ID, HSA21 trisomy results in a number of neurological and physiological pathologies in individuals with DS, including progressive cognitive dysfunction and learning and memory deficits which worsen with age. Further exacerbating neurological dysfunction associated with DS is the concomitant basal forebrain cholinergic neuron (BFCN) degeneration and onset of Alzheimer's disease (AD) pathology in early mid-life. Recent single population RNA sequencing (RNA-seq) analysis in the Ts65Dn mouse model of DS, specifically the medial septal cholinergic neurons of the basal forebrain (BF), revealed the mitochondrial oxidative phosphorylation pathway was significantly impacted, with a large subset of genes within this pathway being downregulated. We further queried oxidative phosphorylation pathway dysregulation in Ts65Dn mice by examining genes and encoded proteins within brain regions comprising the basocortical system at the start of BFCN degeneration (6 months of age). In select Ts65Dn mice we demonstrate significant deficits in gene and/or encoded protein levels of Complex I-V of the mitochondrial oxidative phosphorylation pathway in the BF. In the frontal cortex (Fr Ctx) these complexes had concomitant alterations in select gene expression but not of the proteins queried from Complex I-V, suggesting that defects at this time point in the BF are more severe and occur prior to cortical dysfunction within the basocortical circuit. We propose dysregulation within mitochondrial oxidative phosphorylation complexes is an early marker of cognitive decline onset and specifically linked to BFCN degeneration that may propagate pathology throughout cortical memory and executive function circuits in DS and AD.

The striatum integrates sensorimotor and motivational signals, likely playing a key role in reward-based learning of goal-directed behavior. However, cell type-specific mechanisms underlying reinforcement learning remain to be precisely determined. Here, we investigated changes in membrane potential dynamics of dorsolateral striatal neurons comparing naïve mice and expert mice trained to lick a reward spout in response to whisker deflection. We recorded from three distinct cell types: (i) direct pathway striatonigral neurons, which express type 1 dopamine receptors; (ii) indirect pathway striatopallidal neurons, which express type 2 dopamine receptors; and (iii) tonically active, putative cholinergic, striatal neurons. Task learning was accompanied by cell type-specific changes in the membrane potential dynamics evoked by the whisker deflection and licking in successfully-performed trials. Both striatonigral and striatopallidal types of striatal projection neurons showed enhanced task-related depolarization across learning. Striatonigral neurons showed a prominent increase in a short latency sensory-evoked depolarization in expert compared to naïve mice. In contrast, the putative cholinergic striatal neurons developed a hyperpolarizing response across learning, driving a pause in their firing. Our results reveal cell type-specific changes in striatal membrane potential dynamics across the learning of a simple goal-directed sensorimotor transformation, helpful for furthering the understanding of the various potential roles of different basal ganglia circuits.

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although nonamplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the neocortex of wild-type C57BL/6J mice. While we observed weak but consistent nonamplified off-target double inverted open reading frame (DIO) expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed notable Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype, or promoter. We also evaluated whether Cre-independent expression had functional effects via designer receptors exclusively activated by designer drugs (DREADDs). The DREADD agonist C21 (compound 21) had no effect on contextual fear conditioning or c-Fos expression in DIO-hM3Dq-mCherry⁺ cells of C57BL/6J mice. Together, our results indicate that DIO constructs have off-target expression in wild-type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.Significance StatementAdeno-associated viruses (AAVs) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study, we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 (compound 21) had no detectable effect on double inverted open reading frame (DIO)-hM3Dq-mCherry-infected neurons in C57BL/6J mice. Given the widespread use of DIO recombinant AAVs by the neuroscience community, our data support careful consideration when using DIO constructs in control animals.