Probing neural circuit mechanisms in Alzheimer's disease using novel technologies.

The study of Alzheimer's Disease (AD) has traditionally focused on neuropathological mechanisms that has guided therapies that attenuate neuropathological features. A new direction is emerging in AD research that focuses on the progressive loss of cognitive function due to disrupted neural circuit mechanisms. Evidence from humans and animal models of AD show that dysregulated circuits initiate a cascade of pathological events that culminate in functional loss of learning, memory, and other aspects of cognition. Recent progress in single-cell, spatial, and circuit omics informs this circuit-focused approach by determining the identities, locations, and circuitry of the specific cells affected by AD. Recently developed neuroscience tools allow for precise access to cell type-specific circuitry so that their functional roles in AD-related cognitive deficits and disease progression can be tested. An integrated systems-level understanding of AD-associated neural circuit mechanisms requires new multimodal and multi-scale interrogations that longitudinally measure and/or manipulate the ensemble properties of specific molecularly-defined neuron populations first susceptible to AD. These newly developed technological and conceptual advances present new opportunities for studying and treating circuits vulnerable in AD and represent the beginning of a new era for circuit-based AD research.

Anatomical and molecular characterization of parvalbumin-cholecystokinin co-expressing inhibitory interneurons: implications for neuropsychiatric conditions.

Inhibitory interneurons are crucial to brain function and their dysfunction is implicated in neuropsychiatric conditions. Emerging evidence indicates that cholecystokinin (CCK)-expressing interneurons (CCK+) are highly heterogenous. We find that a large subset of parvalbumin-expressing (PV+) interneurons express CCK strongly; between 40 and 56% of PV+ interneurons in mouse hippocampal CA1 express CCK. Primate interneurons also exhibit substantial PV/CCK co-expression. Mouse PV+/CCK+ and PV+/CCK- cells show distinguishable electrophysiological and molecular characteristics. Analysis of single nuclei RNA-seq and ATAC-seq data shows that PV+/CCK+ cells are a subset of PV+ cells, not of synuclein gamma positive (SNCG+) cells, and that they strongly express oxidative phosphorylation (OXPHOS) genes. We find that mitochondrial complex I and IV-associated OXPHOS gene expression is strongly correlated with CCK expression in PV+ interneurons at both the transcriptomic and protein levels. Both PV+ interneurons and dysregulation of OXPHOS processes are implicated in neuropsychiatric conditions, including autism spectrum (ASD) disorder and schizophrenia (SCZ). Analysis of human brain samples from patients with these conditions shows alterations in OXPHOS gene expression. Together these data reveal important molecular characteristics of PV-CCK co-expressing interneurons and support their implication in neuropsychiatric conditions.

Psychedelics and Neural Plasticity: Therapeutic Implications.

Psychedelic drugs have reemerged as tools to treat several brain disorders. Cultural attitudes toward them are changing, and scientists are once again investigating the neural mechanisms through which these drugs impact brain function. The significance of this research direction is reflected by recent work, including work presented by these authors at the 2022 meeting of the Society for Neuroscience. As of 2022, there were hundreds of clinical trials recruiting participants for testing the therapeutic effects of psychedelics. Emerging evidence suggests that psychedelic drugs may exert some of their long-lasting therapeutic effects by inducing structural and functional neural plasticity. Herein, basic and clinical research attempting to elucidate the mechanisms of these compounds is showcased. Topics covered include psychedelic receptor binding sites, effects of psychedelics on gene expression, and on dendrites, and psychedelic effects on microcircuitry and brain-wide circuits. We describe unmet clinical needs and the current state of translation to the clinic for psychedelics, as well as other unanswered basic neuroscience questions addressable with future studies.

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

OBJECTIVES: Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. METHODS: In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. RESULTS: Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. CONCLUSIONS: These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment.

Intranasal Delivery of Ketamine Induces Cortical Disinhibition.

Our previous studies find that subcutaneously administered (s.c.) subanesthetic ketamine promotes sustained cortical disinhibition and plasticity in adult mouse binocular visual cortex (bV1). We hypothesized that intranasal delivery (i.n.) of subanesthetic ketamine may have similar actions. To test this, we delivered ketamine (10 mg/kg, i.n.) to adult mice and then recorded excitatory pyramidal neurons or PV+ interneurons in L2/3 of bV1 slices. In pyramidal neurons the baseline IPSC amplitudes from mice treated with ketamine are significantly weaker than those in control mice. Acute bath application of neuregulin-1 (NRG1) to cortical slices increases these IPSC amplitudes in mice treated with ketamine but not in controls. In PV+ interneurons, the baseline EPSC amplitudes from mice treated with ketamine are significantly weaker than those in control mice. Acute bath application of NRG1 to cortical slices increases these EPSC amplitudes in mice treated with ketamine but not in controls. We also found that mice treated with ketamine exhibit increased pCREB staining in L2/3 of bV1. Together, our results show that a single intranasal delivery of ketamine reduces PV+ interneuron excitation and reduces pyramidal neuron inhibition and that these effects are acutely reversed by NRG1. These results are significant as they show that intranasal delivery of ketamine induces cortical disinhibition, which has implications for the treatment of psychiatric, neurologic, and ophthalmic disorders.

Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium.

Hephaestin (Heph) is a ferroxidase protein that converts ferrous to ferric iron to facilitate cellular iron export by ferroportin. Many tissues express either Heph or its homologue, ceruloplasmin (Cp), but the retina expresses both. In mice, a combined systemic mutation of Heph and systemic knockout of Cp (Cp(-/-), Heph(sla/sla)) causes retinal iron accumulation and retinal degeneration, with features of human age-related macular degeneration; however, the role of Heph and Cp in the individual retinal cells is unclear. Herein, we used conditional knockout mice to study Heph's role in retinal pigment epithelial (RPE) and photoreceptor cells. Loss of both Heph and Cp from RPE cells alone results in RPE cell iron accumulation and degeneration. We found, however, that RPE iron accumulation in these conditional knockout mice is not as great as in systemic knockout mice. Photoreceptor-specific Heph knockout indicates that the additional iron in the RPE cells does not result from loss of ferroxidases in the photoreceptors, and Cp and Heph play minor roles in photoreceptors. Instead, loss of ferroxidases in other retinal cells causes retinal iron accumulation and transfer of iron to the RPE cells. Cp and Heph are necessary for iron export from the retina but are not essential for iron import into the retina. Thus, our studies, revise how we think about iron import and export from the retina.

Tracking longitudinal population dynamics of single neuronal calcium signal using SCOUT.

In vivo calcium imaging enables simultaneous recording of large neuronal ensembles engaged in complex operations. Many experiments require monitoring and identification of cell populations across multiple sessions. Population cell tracking across multiple sessions is complicated by non-rigid transformations induced by cell movement and imaging field shifts. We introduce SCOUT (Single-Cell spatiOtemporal longitUdinal Tracking), a fast, robust cell-tracking method utilizing multiple cell-cell similarity metrics, probabilistic inference, and an adaptive clustering methodology, to perform cell identification across multiple sessions. By comparing SCOUT with earlier cell-tracking algorithms on simulated, 1-photon, and 2-photon recordings, we show that our approach significantly improves cell-tracking quality, particularly when recordings exhibit spatial footprint movement between sessions or sub-optimal neural extraction quality.

Beyond t test and ANOVA: applications of mixed-effects models for more rigorous statistical analysis in neuroscience research.

In basic neuroscience research, data are often clustered or collected with repeated measures, hence correlated. The most widely used methods such as t test and ANOVA do not take data dependence into account and thus are often misused. This Primer introduces linear and generalized mixed-effects models that consider data dependence and provides clear instruction on how to recognize when they are needed and how to apply them. The appropriate use of mixed-effects models will help researchers improve their experimental design and will lead to data analyses with greater validity and higher reproducibility of the experimental findings.

Single-cell epigenome analysis reveals age-associated decay of heterochromatin domains in excitatory neurons in the mouse brain.

Loss of heterochromatin has been implicated as a cause of pre-mature aging and age-associated decline in organ functions in mammals; however, the specific cell types and gene loci affected by this type of epigenetic change have remained unclear. To address this knowledge gap, we probed chromatin accessibility at single-cell resolution in the brains, hearts, skeletal muscles, and bone marrows from young, middle-aged, and old mice, and assessed age-associated changes at 353,126 candidate cis-regulatory elements (cCREs) across 32 major cell types. Unexpectedly, we detected increased chromatin accessibility within specific heterochromatin domains in old mouse excitatory neurons. The gain of chromatin accessibility at these genomic loci was accompanied by the cell-type-specific loss of heterochromatin and activation of LINE1 elements. Immunostaining further confirmed the loss of the heterochromatin mark H3K9me3 in the excitatory neurons but not in inhibitory neurons or glial cells. Our results reveal the cell-type-specific changes in chromatin landscapes in old mice and shed light on the scope of heterochromatin loss in mammalian aging.

Neuregulin signaling mediates the acute and sustained antidepressant effects of subanesthetic ketamine.

Subanesthetic ketamine evokes rapid antidepressant effects in human patients that persist long past ketamine's chemical half-life of ~2 h. Ketamine's sustained antidepressant action may be due to modulation of cortical plasticity. We find that ketamine ameliorates depression-like behavior in the forced swim test in adult mice, and this depends on parvalbumin-expressing (PV) neuron-directed neuregulin-1 (NRG1)/ErbB4 signaling. Ketamine rapidly downregulates NRG1 expression in PV inhibitory neurons in mouse medial prefrontal cortex (mPFC) following a single low-dose ketamine treatment. This NRG1 downregulation in PV neurons co-tracks with the decreases in synaptic inhibition to mPFC excitatory neurons for up to a week. This results from reduced synaptic excitation to PV neurons, and is blocked by exogenous NRG1 as well as by PV targeted ErbB4 receptor knockout. Thus, we conceptualize that ketamine's effects are mediated through rapid and sustained cortical disinhibition via PV-specific NRG1 signaling. Our findings reveal a novel neural plasticity-based mechanism for ketamine's acute and long-lasting antidepressant effects.

HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements, Production, and Applications.

Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

Autistic-like behavior, spontaneous seizures, and increased neuronal excitability in a Scn8a mouse model.

Patients with SCN8A epileptic encephalopathy exhibit a range of clinical features, including multiple seizure types, movement disorders, and behavioral abnormalities, such as developmental delay, mild-to-severe intellectual disability, and autism. Recently, the de novo heterozygous SCN8A R1620L mutation was identified in an individual with autism, intellectual disability, and behavioral seizures without accompanying electrographic seizure activity. To date, the effects of SCN8A mutations that are primarily associated with behavioral abnormalities have not been studied in a mouse model. To better understand the phenotypic and functional consequences of the R1620L mutation, we used CRISPR/Cas9 technology to generate mice expressing the corresponding SCN8A amino acid substitution. Homozygous mutants exhibit tremors and a maximum lifespan of 22 days, while heterozygous mutants (RL/+) exhibit autistic-like behaviors, such as hyperactivity and learning and social deficits, increased seizure susceptibility, and spontaneous seizures. Current clamp analyses revealed a reduced threshold for firing action potentials in heterozygous CA3 pyramidal neurons and reduced firing frequency, suggesting that the R1620L mutation has both gain- and loss-of-function effects. In vivo calcium imaging using miniscopes in freely moving RL/+ mutants showed hyperexcitability of cortical excitatory neurons that is likely to increase seizure susceptibility. Finally, we found that oxcarbazepine and Huperzine A, a sodium channel blocker and reversible acetylcholinesterase inhibitor, respectively, were capable of conferring robust protection against induced seizures in RL/+ mutants. This mouse line will provide the opportunity to better understand the range of clinical phenotypes associated with SCN8A mutations and to develop new therapeutic approaches.

Neuregulin and ErbB expression is regulated by development and sensory experience in mouse visual cortex.

Neuregulins (NRGs) are protein ligands that impact neural development and circuit function. NRGs signal through the ErbB receptor tyrosine kinase family. NRG1/ErbB4 signaling in parvalbumin-expressing (PV) inhibitory interneurons is critical for visual cortical plasticity. There are multiple types of NRGs and ErbBs that can potentially contribute to visual cortical plasticity at different developmental stages. Thus, it is important to understand the normal developmental expression profiles of NRGs and ErbBs in specific neuron types in the visual cortex, and to study whether and how their expression changes in PV inhibitory neurons and excitatory neurons track with sensory perturbation. Cell type-specific translating ribosome affinity purification and qPCR was used to compare mRNA expression of nrg1,2,3,4 and erbB1,2,3,4 in PV and excitatory neurons in mouse visual cortex. We show that the expression of nrg1 and nrg3 decreases in PV neurons at the critical period peak, postnatal day 28 (P28) after monocular deprivation and dark rearing, and in the adult cortex (at P104) after 2-week long dark exposure. In contrast, nrg1 expression by excitatory neurons is unchanged at P28 and P104 following sensory deprivation, whereas nrg3 expression by excitatory neurons shows changes depending on the age and the mode of sensory deprivation. ErbB4 expression in PV neurons remains consistently high and does not appear to change in response to sensory deprivation. These data provide new important details of cell type-specific NRG/ErbB expression in the visual cortex and support that NRG1/ErbB4 signaling is implicated in both critical period and adult visual cortical plasticity.

Subanesthetic Ketamine Reactivates Adult Cortical Plasticity to Restore Vision from Amblyopia.

Subanesthetic ketamine evokes rapid and long-lasting antidepressant effects in human patients. The mechanism for ketamine's effects remains elusive, but ketamine may broadly modulate brain plasticity processes. We show that single-dose ketamine reactivates adult mouse visual cortical plasticity and promotes functional recovery of visual acuity defects from amblyopia. Ketamine specifically induces downregulation of neuregulin-1 (NRG1) expression in parvalbumin-expressing (PV) inhibitory neurons in mouse visual cortex. NRG1 downregulation in PV neurons co-tracks both the fast onset and sustained decreases in synaptic inhibition to excitatory neurons, along with reduced synaptic excitation to PV neurons in vitro and in vivo following a single ketamine treatment. These effects are blocked by exogenous NRG1 as well as PV targeted receptor knockout. Thus, ketamine reactivation of adult visual cortical plasticity is mediated through rapid and sustained cortical disinhibition via downregulation of PV-specific NRG1 signaling. Our findings reveal the neural plasticity-based mechanism for ketamine-mediated functional recovery from adult amblyopia.

Neuregulin directed molecular mechanisms of visual cortical plasticity.

Experience-dependent critical period (CP) plasticity has been extensively studied in the visual cortex. Monocular deprivation during the CP affects ocular dominance, limits visual performance, and contributes to the pathological etiology of amblyopia. Neuregulin-1 (NRG1) signaling through its tyrosine kinase receptor ErbB4 is essential for the normal development of the nervous system and has been linked to neuropsychiatric disorders such as schizophrenia. We discovered recently that NRG1/ErbB4 signaling in PV neurons is critical for the initiation of CP visual cortical plasticity by controlling excitatory synaptic inputs onto PV neurons and thus PV-cell mediated cortical inhibition that occurs following visual deprivation. Building on this discovery, we review the existing literature of neuregulin signaling in developing and adult cortex and address the implication of NRG/ErbB4 signaling in visual cortical plasticity at the cellular and circuit levels. NRG-directed research may lead to therapeutic approaches to reactivate plasticity in the adult cortex.

Development of Local Circuit Connections to Hilar Mossy Cells in the Mouse Dentate Gyrus.

Hilar mossy cells in the dentate gyrus (DG) shape the firing and function of the hippocampal circuit. However, the neural circuitry providing afferent input to mossy cells is incompletely understood, and little is known about the development of these inputs. Thus, we used whole-cell recording and laser scanning photostimulation (LSPS) to characterize the developmental trajectory of local excitatory and inhibitory synaptic inputs to mossy cells in the mouse hippocampus. Hilar mossy cells were targeted by visualizing non-red fluorescent cells in the dentate hilus of GAD2-Cre; Ai9 mice that expressed tdTomato in GAD+ neurons, and were confirmed by post hoc morphological characterization. Our results show that at postnatal day (P)6-P7, mossy cells received more excitatory input from neurons in the proximal CA3 versus those in the DG. In contrast, at P13-P14 and P21-P28, the largest source of excitatory input originated in DG cells, while the strength of CA3 and hilar inputs declined. A developmental trend was also evident for inhibitory inputs. Overall inhibitory input at P6-P7 was weak, while inhibitory inputs from the DG cell layer and the hilus predominated at P13-P14 and P21-P28. The strength of local DG excitation and inhibition to mossy cells peaked at P13-P14 and decreased slightly in older P21-P28 mice. Together, these data provide new detailed information on the development of local synaptic connectivity of mossy cells, and suggests mechanisms through which developmental changes in local circuit inputs to hilar mossy cells shape their physiology and vulnerability to injury during postnatal periods.