Details

Autor(en) / Beteiligte

• Uygun, D S
• Yang, C
• Miwa, H
• McKenna, J T
• McNally, J M
• Katsuki, F
• Strecker, R E
• Brown, R E
• Basheer, R

Titel

0028 GABAA Receptors Of The Thalamic Reticular Nucleus Regulate Sleep Spindles: An In Vivo Investigation By CRISPR-cas9 Genetic Abscission

Ist Teil von

• Sleep (New York, N.Y.), 2018-04, Vol.41 (suppl_1), p.A11-A12

Ort / Verlag

US: Oxford University Press

Erscheinungsjahr

2018

Quelle

Oxford Journals 2020 Medicine

Beschreibungen/Notizen

• Abstract Introduction The GABAergic neurons of the thalamic reticular nucleus (TRN) are critical in regulating thalamocortical oscillations, particularly the waxing and waning 10-15Hz spindle-oscillations of non-rapid-eye-movement (NREM) sleep. Spindles are important for sleep-dependent memory consolidation and are impaired in neuropsychiatric disorders such as schizophrenia. Thus, there is considerable interest in understanding their regulation. TRN neurons receive inhibitory inputs from the basal forebrain and hypothalamus, acting on GABAA receptors containing the α3-subunit. However, their functional role remains unclear. Here we use the cutting-edge gene editing CRISPR-Cas9 technique to selectively delete α3-subunits in the parvalbumin (PV)-expressing TRN neurons, and examined the effect on sleep spindles. Methods We crossed lox-stop-lox-Cas9 and PV-Cre mice to generate mice that express the endonuclease Cas9 in PV neurons, and delivered AAV encoding short-guide-RNA (AAV-sgRNA) targeting the α3-subunit into TRN bilaterally via an implanted cannula. A skull electrode implanted above frontal cortex recorded electroencephalogram, and nuchal muscle electrode recorded electromyographic activity. Spindles were detected by a custom-built automated algorithm. Knock down of functional α3-subunits was validated by examining the changes in spontaneous-inhibitory-postsynaptic-currents (sIPSCs) recorded from individual TRN-PV neurons with in vitro electrophysiology. Results NREM spindle density (spindles/min) was reduced 15% from 6.5 ± 2.1 to 5.4 ± 0.8 (n=3) four weeks after AAV-sgRNA administration, compared to pre-AAV condition. TRN-PV neurons with AAV-sgRNA transfection showed a significant reduction in sIPSC (1.01 ± 0.53 Hz, n=5), compared to TRN-PV neurons without AAV transfection (3.47 ± 0.75 Hz, n=6; t-test, p=0.03). Conclusion Our results suggest that in vivo, cell-type and region-specific CRISPR-Cas9 technology is an effective method to probe the role of individual genes in sleep physiology, circumventing the developmental confounds associated with other types of genetic manipulations. Deletion of α3-GABAA receptors from TRN PV neurons reduced NREM sleep spindle density suggesting that selective pharmacological antagonism of α3 receptors may be beneficial in disorders where spindle density is reduced, such as schizophrenia. Support (If Any) VA I01BX001404, I01BX001356, I01BX002774, IK2BX002130; NIMH R01-MH039683, R03-MH107650; T32-HL007901. JTM received partial salary from Merck MISP, no COI with this work.