Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called “cFork (see fork)”, includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.

Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC(swell)) in the Brain

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl⁻- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC(swell)) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC(swell) in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl⁻ conductance (I(Cl,swell)) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I(Cl,swell) with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I(Cl,swell) observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC(swell) in the brain.

Cortical Axonal Secretion of BDNF in the Striatum Is Disrupted in the Mutant-huntingtin Knock-in Mouse Model of Huntington's Disease

Deficient BDNF signaling is known to be involved in neurodegenerative diseases such as Huntington's disease (HD). Mutant huntingtin (mhtt)-mediated disruption of either BDNF transcription or transport is thought to be a factor contributing to striatal atrophy in the HD brain. Whether and how activity-dependent BDNF secretion is affected by the mhtt remains unclear. In the present study, I provide evidence for differential effects of the mhtt on cortical BDNF secretion in the striatum during HD progression. By two-photon imaging of fluorescent BDNF sensor (BDNF-pHluorin and -EGFP) in acute striatal slices of HD knock-in model mice, I found deficient cortical BDNF secretion regardless of the HD onset, but antisense oligonucleotide (ASO)-mediated reduction of htts only rescues BDNF secretion in the early HD brain before the disease onset. Although secretion modes of individual BDNF-containing vesicle were not altered in the pre-symptomatic brain, the full-fusion and partial-fusion modes of BDNF-containing vesicles were significantly altered after the onset of HD symptoms. Thus, besides abnormal BDNF transcription and transport, our results suggest that mhtt-mediated alteration in activity-dependent BDNF secretion at corticostriatal synapses also contributes to the development of HD.