Whole-cell Patch from Pyramidal Cells in mPFC; Coronal 300µm Slices Prepared with 7000smz-2

Abstract

(from https://doi.org/10.1101/2021.03.16.435686)

Post-traumatic stress disorder (PTSD) is a chronic, debilitating mental illness marked by abnormal fear responses and deficits in extinction of fear memories. The pathophysiology of PTSD is linked to decreased activation of the ventromedial prefrontal cortex (vmPFC). This study aims to investigate underlying functional changes in synaptic drive and intrinsic excitability of pyramidal neurons in the rodent homolog of the vmPFC, the infralimbic cortex (IL), following exposure to single prolonged stress (SPS), a paradigm that mimics core symptoms of PTSD in rats. Rats were exposed to SPS and allowed one week of recovery following which brain slices containing the PFC were prepared for whole-cell patch clamp recordings from layer V pyramidal neurons in the IL. Our results indicate that SPS reduces spontaneous excitatory synaptic drive to pyramidal neurons. In addition, SPS decreases the intrinsic membrane excitability of IL PFC pyramidal cells, as indicated by an increase in rheobase, decrease in input resistance, hyperpolarization of resting membrane potential, and a reduction in repetitive firing rate. Our results suggest that SPS causes a lasting reduction in PFC activity, supporting a body of evidence linking traumatic stress with prefrontal hypoactivity.

Method

Brains were quickly isolated and dura matter carefully removed before removing the cerebellum. The brain was then immediately glued to a cutting stage and immersed in NMDG solution (92 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4, and 0.5 mM CaCl2) at a temperature of 34-36°C and continuously bubbled with 95% oxygen and 5% carbon-dioxide. Coronal slices containing the mPFC were sectioned at 300 μm thickness using a vibrating microtome (Vibratome 7000smz-2; Campden Instruments, Lafayette, IN) with ceramic blades (Campden Instruments) at an advance speed of 0.03 mm/s. Vertical vibration of the blade was manually tuned in accordance with the user manual, and was set to 0.1 – 0.3 μm. Bath temperature was kept within the desired range of 34-36°C, by adding warm or cold water into the external chamber of the Vibratome, and was monitored throughout the cutting procedure with a conventional mercury/glass thermometer. The slices were allowed to recover for 1 hour in oxygenated NMDG solution at 34-36°C. At the end of recovery, slices were transferred to a chamber containing oxygenated artificial CSF solution (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1 mM NaH2PO4, 25 mM glucose, 1 mM MgCl2, 2 mM CaCl2) for at least 30 minutes at room temperature after which the slices were ready for in vitro patch clamp recordings.