350µm - 450µm Sagittal Brain Sections for LFP and Whole Cell Patch Recordings, & Immunofluorescence

Abstract

(from Multimodal electrophysiological analyses reveal that reduced synaptic excitatory neurotransmission underlies seizures in a model of NMDAR antibody-mediated encephalitis, Wright et al, 2021)

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Seizures are a prominent feature in N-Methyl-D-Aspartate receptor antibody (NMDAR antibody) encephalitis, a distinct neuro-immunological disorder in which specific human autoantibodies bind and crosslink the surface of NMDAR proteins thereby causing internalization and a state of NMDAR hypofunction. To further understand ictogenesis in this disorder, and to test a potential treatment compound, we developed an NMDAR antibody mediated rat seizure model that displays spontaneous epileptiform activity in vivo and in vitro. Using a combination of electrophysiological and dynamic causal modelling techniques we show that, contrary to expectation, reduction of synaptic excitatory, but not inhibitory, neurotransmission underlies the ictal events through alterations in the dynamical behaviour of microcircuits in brain tissue. Moreover, in vitro application of a neurosteroid, pregnenolone sulphate, that upregulates NMDARs, reduced established ictal activity. This proof-of-concept study highlights the complexity of circuit disturbances that may lead to seizures and the potential use of receptor-specific treatments in antibody-mediated seizures and epilepsy.

Method

Local field potential (LFP) recordings

Forty-eight hours after ICV injection or at the end of the recording period in telemetry experiments, rats were anaesthetized using isoflurane followed, after loss of consciousness, by pentobarbital (60 mg/kg, SC) and xylazine (10 mg/kg, IM). Transcardial perfusion was then performed using ice-cold artificial cerebrospinal fluid (cutting aCSF) in which NaCl had been replaced with equimolar sucrose. Animals were decapitated, and the brain was removed and placed in ice-cold cutting aCSF. Sagittal slices at 450μm were cut using a vibratome (Campden Instruments, UK)77. The slices were transferred to a storage chamber containing standard aCSF (in mM: 125 NaCl, 3KCl, 1.6 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 10 glucose) and constantly bubbled with carbogen (95% O2/5% CO2) at room-temperature for at least one hour prior to recording. Slices were then transferred to an interface chamber (Scientific System Design Inc., Canada) and continuously perfused with aCSF (2–3 ml/min) maintained at 30–31 °C and visualised with a SZ51 stereomicroscope (Olympus). A Flaming-Brown micropipette puller (Sutter Instruments, CA) was used to pull borosilicate glass microelectrodes with an open tip resistance of 1–3 MΩ when filled with aCSF. Electrodes were inserted into CA3 and CA1 regions of the hippocampus using Narishige MM-3 (Narishige, Japan) micromanipulators and signals were recorded using an EXT-02 headstage and amplifier (NPI Electronic). HumBugs (Quest Scientific) were used to remove electrical 50 Hz interference from recordings. The signal was then amplified ×100 via an EX10-2F amplifier (NPI Electronics) and filtered (700 Hz low-pass, 0.3 Hz high-pass, sampling rate 5 kHz) and further amplified ×10 using a LHBF-48X amplifier (NPI Electronics). Signals were then digitised at 10 KHz using an analogue to digital converter (Micro-1401-II; CED). LFP recordings were assessed using Spike2 software (CED) for spontaneous epileptiform activity. Spike2 was used to calculate the root mean square (RMS) amplitude of each recording. Epileptiform activity was classified as an event when it displayed an amplitude greater than four-fold the RMS amplitude, providing the event count, while the time difference between these events provided the interevent interval. A custom MATLAB script was used to prevent false event detection, removing those with an interevent interval less than 15 ms. Statistical analysis was conducted using Graphpad Prism 8 (GraphPad Software Inc), statistical tests used to compare groups (Mann-Whitney) were one-tailed. Measurements were expressed as mean (M) ± standard error of the mean (SEM).

Whole-cell patch clamp recordings

For whole-cell patch clamp recordings, brain slices were prepared as above but sliced at a thickness of 350 µm. For recording, the slices were transferred to a submerged chamber (Scientifica, UK) and visualised using Nomarski infra-red optics on a BX51WI Microscope (Olympus). A Flaming-Brown micropipette puller (Sutter Instruments, USA) was used to pull borosilicate glass microelectrodes of 4–6 MΩ for recordings. For voltage clamp recordings, electrodes were filled with an internal solution containing (in mM): 100 CsCl, 40 HEPES, 1 Qx-314, 0.6 EGTA, 5 MgCl2, 10 TEA-Cl, 4 ATP-Na, 0.3 GTP-Na and 1 IEM 1460 (titrated with CsOH to pH 7.25) at 290–295 mOsm for IPSCs. For EPSCs the internal solution contained (in mM): 40 HEPES, 1 Qx-314, 0.6 EGTA, 2 NaCl, 5 Mg-gluconate, 5 TEA-Cl, 10 Phospho-Creatinine, 4 ATP-Na, 0.3 GTP-Na (titrated with CsOH to pH 7.3) at 285 mOsm for EPSCs. The EPSCs and IPSCs were recorded as apparent inward currents at −70 mV using an Axopatch 200B amplifier (Molecular Devices, USA). Signals were low-pass filtered at 5 kHz with an 8-pole Bessel filter and digitised at 10 kHz using a Digidata 1440 A and pClamp software (Molecular Devices). For intrinsic cell excitability experiments, whole-cell current-clamp recordings were made from hippocampal neurons using an Axopatch 700 series amplifier (Molecular Devices, USA) and with a K-gluconate-based intracellular solution containing (in mM) K-gluconate (120) hepes (40) KCL (10) Na2ATP (2) Na-GTP (0.3) MgCl2 (3) EGTA (0.5). Membrane potential was manually adjusted when required to −60 mV by continuous somatic current injection. Membrane response and action potential firing were measured by applying depolarizing steps of current (duration of 1 ms at 10pA increments). For spike/current intensity curves, the number of action potentials was plotted against the injected current. Input resistance was measured from the slope of a linear regression fit to the voltage–current plotted graph between −80 pA to 0 pA. Data were analysed using Axograph and Prism 8, statistical tests used to compare groups (Mann–Whitney and Wilcoxon matched pairs ranked tests) were one-tailed. Measurements are expressed as mean median ± SEM.

For human tissue recordings, brain slices were prepared as above using choline-based artificial cerebrospinal fluid. Briefly, human tissue was obtained with informed parental or guardian consent from paediatric patients prior to them undergoing epilepsy surgery at Birmingham Children’s Hospital. Ethical approval was obtained from the Black Country Local Ethics Committee (10/H1202/23; 30 April 2010), and from Aston University’s ethics committee (Project 308 cellular studies in epilepsy) and through the Research and Development Department at Birmingham Children’s Hospital (IRAS ID12287). Brain tissue was obtained from 7 patients (F:M 4:3), median age 6 years (range 4–16 years). Surgical procedures included frontal resection (three), temporal parietal occipital disconnection (one), temporal lobectomy (two), and right hemispherectomy (one) as described in Supplementary Table 2. Specimens were resected intraoperatively with minimal traumatic tissue damage, and minimal use of electrocautery. Brain tissue was transported in ice-cold choline-based artificial CSF as previously described.