Continuous intracellular recording from mammalian neurons exposed to hyperbaric helium, oxygen, or air

Abstract

We developed a hyperbaric chamber for intracellular recording in rat brain stem slices during continuous compression and decompression of the tissue bath with the inert gas helium. Air, rather than helium, was also used as the compression medium in some cases to increase tissue nitrogen levels. An important feature is the chamber door, which opens or closes rapidly at 1 atmosphere absolute (ATA) for increased accessibility of the microelectrode. The door also closes and seals smoothly without disrupting the intracellular recording. Hyperbaric oxygen was administered during helium compression using a separate pressure cylinder filled with perfusate equilibrated with 2. 3-3.3 ATA oxygen. Measurements of tissue/bath PO(2) and pH confirmed that the effects of compression using helium or air could be differentiated from those due to increased PO(2). One hundred and thirteen neurons were studied during 375 compression cycles ranging from 1 to 20 ATA (mode 3.0 ATA). We conclude that it is technically feasible to record intracellularly from the same mammalian neuron while changing ambient pressure over a physiologically important range. These techniques will be useful for studying how various hyperbaric environments affect neurophysiological mechanisms.

Hyperbaric oxygen and chemical oxidants stimulate CO2/H+-sensitive neurons in rat brain stem slices

“…Slices were prepared from juvenile and adult SpragueDawley rats (body weight Ͼ75 g), as previously described (22). Anesthesia was not used because these agents have a depressant action on neurons (54) and because these agents antagonize neuronal sensitivity to hyperbaric pressure (68).…”

“…However, because a more physiological control level of tissue PO 2 (PtiO 2 ) for the brain slice preparation has not yet been defined (1,23,51), we equilibrated control medium with 95% O 2 at PB of 1 ATA to enable comparison of our data with the results of previous studies. Therefore, to study the effects of further hyperoxia on brain stem slices, as in the present study, requires the use of a hyperbaric chamber, where aCSF PO 2 values in excess of 760 Torr (1 ATA) can be achieved (22,44,51).…”

“…Brain stem slices were incubated in control medium (95% O2-5% CO2) at ϳ25°C for at least 1 h. Brain slices were selected individually and transferred to a tissue chamber inside a hyperbaric chamber customized for brain slice electrophysiology, the details of which are described elsewhere (22). The tissue slice was supported on a nylon mesh and submerged ϳ1 mm deep in a bath of aCSF (ϳ5-ml volume) that was delivered at a constant flow rate of 2 ml/min by using a high-pressure liquid chromatography pump.…”

“…The tissue slice was supported on a nylon mesh and submerged ϳ1 mm deep in a bath of aCSF (ϳ5-ml volume) that was delivered at a constant flow rate of 2 ml/min by using a high-pressure liquid chromatography pump. This hyperbaric chamber enables us to independently change PB, PO2, and PCO2 while maintaining an intracellular recording of membrane potential (Vm) in a single neuron (22). The utility of this novel in vitro model lies in its ability to generate a range of Pti O 2 values in brain slices that are known to alter CNS function in vivo (3,17,46,48,63), but at lower levels of total ambient pressure (PB) (23).…”

“…The electrode, filled with 3 M potassium acetate, was connected to the head stage by a Ag-AgCl wire (Medwire) and had a tip resistance that ranged from 100 to 150 M⍀ (19,21). A low-resistance (Ͻ1 M⍀) combination Ag-AgCl and potassium-gluconate agar bridge reference electrode was placed in the tissue bath to complete the recording circuit (22). All voltage recordings were performed in current-clamp ("bridge") mode.…”