ROLE OF GABA/GLYCINE DEPOLARIZATION AND HYPERPOLARIZATION IN NEONATAL CIRCUIT DEVELOPMENT

During development, GABA/glycinergic responses switch from depolarizing to hyperpolarizing due to the gradual decrease in chloride equilibrium potential (ECl) to a more negative value than the resting membrane potential. Depolarizing GABA/glycinergic responses and the developmental switch to hyperpolarization are believed to play a key role in neuronal circuit development, yet a clear demonstration of how and to what degree they are important has not been investigated. In my dissertation studies, I investigated the functional significance of the developmental switch to hyperpolarizing GABA/glycinergic responses in circuit development. To this end, I compared synaptic strength in a brain slice preparation containing the intact topographic pathway of GABA/glycinergic projections from the Medial Nucleus of Trapezoid Body (MNTB) to the lateral superior olive (LSO) between wild type (WT) and KCC2-knockdown (KD) mice. In KCC2-KD mice, the developmental switch to GABA/glycinergic hyperpolarization was prevented due to reduced expression of the potassium chloride co-transporter 2 (KCC2) (KCC2-KD mice). I found that the GABA/glycinergic MNTB to LSO synapses in KCC2-KD mice undergo normal refinement through strengthening and elimination during development. Furthermore, the glutamatergic cochlear nucleus (CN) inputs to LSO neurons maintained their normal strength even when depolarizing MNTB synaptic inputs were strengthened, resulting in an abnormally high amount of depolarization. Based on these results, I concluded that the hyperpolarizing switch of GABA/glycinergic responses is not a necessary condition for the refinement of inhibitory synapses during development. Furthermore, homeostatic regulation of excitability in LSO neurons seemed to be impaired, due to the normal strengthening of depolarizing MNTB synapses together with the unaltered CN synaptic strength in KCC2-KD mice. In addition, my results suggest that GABA/glycinergic synapses can regulate their synaptic strength independently of ECl, emphasizing the importance of chloride homeostasis when investigating the strength of inhibition.However, the strength of the CN inputs to the MNTB, the calyx of Held, was reduced in MNTB neurons in KCC2-KD mice at hearing onset, suggesting that the developmental switch to hyperpolarizing GABA/glycine responses is necessary to maintain the normal strength of the calyx of Held synapse. I discuss possible mechanisms of the reduced strength of calyx of Held synapses in the absence of hyperpolarizing GABA/glycinergic responses. Finally, in immature cortical neuronal cells in vitro, I demonstrated that KCC2 overexpression is sufficient to terminate the GABAergic excitatory period earlier than normal development. Based on these results, I generated KCC2OVER mice in which KCC2 can be overexpressed in a temporally regulated, neuronal-specific manner (Appendix) in vivo. Overexpression of KCC2 both in vitro and in vivo will help us to understand the role of excitatory (or depolarizing) GABA and glycine responses in neuronal circuit development

Synapse elimination and learning rules co-regulated by MHC class I H2-Db.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Synapse elimination and learning rules co-regulated by MHC class I H2-Db.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons

A Lactate-Induced Response to Hypoxia

SummaryOrganisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

Therapies and vaccines for COVID-19, caused by the SARS-CoV-2 viral pandemic, are urgently needed. Here the authors establish and screen an antibody library from a convalescent COVID-19 patient to isolate a neutralizing antibody with the ability to reduce viral titer and alleviate symptoms in ferret, hamster, and rhesus monkey infection models