Endogenous noise of neocortical neurons drives atypical sensory response variability in autism data recordings

Experimental DesignWe performed <i>in vivo</i> whole–cell patch–clamp recordings of neocortical neurons of the primary somatosensory cortex to examine tactile stimulus–evoked sensory processing in anesthetized mice and to probe the causal role of endogenous noise sources and parameters for atypical sensory information processing in autism. Throughout the text, the terms autism and autistic people/individuals are used, in line with recent evidence suggesting that these terms are preferred in the autistic community and are less stigmatizing ⁸⁷.Ethical statementAll experimental procedures were performed in accordance with the EU directive 2010/63/EU and French law following procedures approved by the Bordeaux Ethics Committee (CE2A50) and Ministry for Higher Education and Research. Mice were maintained under controlled conditions (temperature 22-24°C, humidity 40-60%, 12h/12h light/dark cycle, light on at 07:00) in a conventional animal facility with <i>ad libitum</i> access to food and water. All experiments were performed during the light cycle.MiceSecond generation <i>Fmr1</i> knockout (<i>Fmr1</i>^−/y) ⁴⁶ and wild-type littermate mice at P26-42 were used in our study. Mice were maintained in a mixed 129/Sv/C57Bl/6J/FVB background (backcrossed 6 generations into C57Bl/6J) as described in ⁴⁶. Male wildtype and <i>Fmr1</i>^−/y littermates were generated by crossing <i>Fmr1</i>^+/− females with <i>Fmr1</i>^+/y male mice from the same production, and the resulting progeny used for our experiments was either <i>Fmr1</i>^+/y (wild-type) or <i>Fmr1</i>^−/y (KO). Mice were maintained in collective cages following weaning (3–5 litter males per cage). Cages were balanced for genotype and supplemented with minimal enrichment (cotton nestlets). Number of mice are given in the figure captions. The genotype of experimental animals was re-confirmed <i>post-hoc</i> by tail-PCR.SurgeryMice (P26–42) were anaesthetized with a mixture of ketamine (100 mg.kg^{− 1}) and xylazine (10 mg.kg^{− 1}) injected intraperitoneally and supplemented as necessary throughout the procedure. Proper depth of anesthesia was monitored by testing the absence of a foot-pinch reflex and whisker movement. Mice were head-fixed using non-puncture ear-bars and a nose-clamp (SR-6M, Narishige). Body temperature was maintained at 37°C. Prior to making an incision on the skin to expose the skull, 0.1 ml of a 1:4 Lidocaine to saline solution was administered subcutaneously and waited for 2 to 5 minutes to induce local analgesia. Following a careful removal of the scalp, and the remaining tissue on the skull, a small craniotomy was made above the S1 hindpaw region (1 mm posterior and 1.5 mm lateral from Bregma, confirmed with intrinsic imaging coupled with hind paw stimulation) using a dental drill (World Precision Instruments).In vivo whole-cell patch-clamp recordingsBlind, <i>in vivo</i> whole-cell recordings were performed from layer 2/3 pyramidal neurons of the hindpaw region of S1 in anaesthetized mice, as described previously ⁴⁴^,⁴⁶. Neurons were identified by their electrophysiological properties, and in some cases by their <i>post-hoc</i> morphology. Depth of neurons was on average 263 µm from pia, ranging from 175 µm to 374 µm. There was no genotype difference in the depth of recording (WT = 261.69 ± 34.91 µm; <i>Fmr1</i>^{<em>−/y</em>} = 259.72 ± 49.12 µm; p &gt; 0.05, unpaired student t-test). Data were acquired at 20 kHz sampling rate and low-pass filtered at 3 kHz using Dagan BVC-700A amplifier (Dagan, Minneapolis, USA), Digidata 1320A and Clampex 10.4 software (Axon Instruments). Recording pipettes with an open-tip resistance of 4–6 MΩ were pulled from borosilicate glass using a PC-10 puller (Narishige) and filled with intracellular solution containing (in mM): 130 K-methanesulphonate, 10 Hepes, 7 KCl, 0.05 EGTA, 2 Na₂ATP, 2 MgATP, 0.5 Na₂GTP (all products from Sigma Aldrich); pH 7.28 (adjusted with KOH); osmolarity was 280–295 osm. In a subset of experiments, biocytin (1.5–2.5 mg/ml) was added to the recording solution for post-hoc neuronal identification and anatomical comparison. The intracellular solution was filtered using a 0.22-µm pore-size centrifuge filter (Costar Spin-X). Cells were excluded from the analysis if the pipette access resistance exceeded 50 MΩ or the neuron was depolarized more than − 50 mV.Neocortical application of the specific BK _Cachannel agonist, BMS191011To pharmacologically target BK_Ca channels, we used the specific channel agonist, BMS191011 (3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3<i>H</i>)-one, 100 µM; Tocris). A stock solution with a concentration of 50 mM BMS191011 was prepared in DMSO and stored at − 20°C. For direct neocortical application the drug was diluted to a final concentration of 100 µM in PBS (final concentration of DMSO in PBS: 0.2%). Cortical application of BMS191011 (~ 1 ml) was performed at least 30 minutes prior to the whole-cell patch-clamp experiments. Drug allocation was semi-randomized and balanced for cage composition.