It was critical that the time from decapitation of the guinea-pig to the start of recording from OHCs was kept as short as possible (ideally < 1 5 minutes), maximizing the time window available to work on the preparation. However, an efficient dissection was not necessarily a prerequisite for healthy basal turn OHCs. Below are a list of alterations made to the standard protocol that significantly improved the quality of T1 OHCs and prolonged their life-span.
a) OHC swelling was reduced by increasing the osmolarity of the
extracellular solution from 325 to 332 mOsm/kg, by the increased addition of D- glucose. Although this did not remove the swelling of the OHCs, it did
significantly slow the process.
b) The concentration of extracellular Ca^^ was reduced from 1 mM to 50
|liM , reducing the electrochemical gradient for Ca^^ movement into the hair cell
either via the transducer channels or via putative Ca^^ channels (Isolation PBS, External Solutions, Table 2.1). This concentration was chosen as it reflects the concentration of C a^ thought to bathe the stereocilia in vivo (Boscher and Warren, 1978). As a ubiquitous second messenger, Ca^^ overloading of the cytoplasm has the potential to activate a whole variety of biochemical cascades that may have deleterious consequences on the OHCs. A low concentration of extracellular Ca^^ may also have helped to reduce swelling by preventing the insertion of synaptic vesicles into the basolateral membrane of the OHC. Ca^^ movement through the transducer channels was further reduced by the addition of 200 |liM DH-streptomycin to the extracellular solution.
c) T1 OHCs survived longer when the extracellular solution was pH
buffered with phosphates rather than HERBS. This effect was also observed in T4 OHCs but over a much longer time course. It is unclear why HERBS is not a favourable pH buffer to use. One hypothesis might be that if phosphate
transporters are expressed in OHCs, phosphates may be able to cross the OHC membrane and effect the intracellular as well as the extracellular environment of the cell. Although there is no evidence for such transporters in OHCs of the cochlea, their existence cannot be ruled out. Phosphate transporters have been
observed in kidney epithelial cells (Miyamoto, Tatsumi, Sonoda, Yamamoto, Minami, Taketani, Takeda, 1995).
d) Supporting cells, that surround OHCs in vivo, may regulate the extracellular environment of the OHC by sequestering or releasing ions. Hensens’s cells that overlie the outer row OHCs were removed in order to improve access to the basolateral membrane of the OHC for patching. As a result of the large, basolateral membrane conductances of T1 OHCs, may accumulate in the extracellular spaces immediately surrounding the OHC causing depolarization. To try and avoid this problem, T1 OHC preparations were dissected and initially bathed in a low K^-concentration extracellular solution (0.4 mM), otherwise identical to the isolation PBS detailed in Table 2.1. This was exchanged for normal extracellular solution ( [K ‘"]o = 4 mM) having
achieved the whole-cell patch-damp configuration.
e) To reduce the metabolic activity of the OHC and thus to slow the degradative processes and the oxygen demand of the tissue, the bulla was placed in ice-cold saline within 1 minute of decapitation and maintained in solution of this temperature throughout the dissection.
Together, the five procedures listed above improved the quality of basal turn OHCs for patching and increased their lifespan from 40-45 minutes to 1-1J hours. Although these modifications eased the time pressures of the
experiment, they clearly did not conquer the problem of rapid cell death, as the survival time of these cells was still significantly less than that of T4 in situ OHCs. Other amendments to the protocol were attempted, but they did not significantly improve the experiment. For completeness, these amendments are listed below;
a) As OHC integrity could be seriously damaged by free radical production within the cell itself (Hirose, Hockenbury and Rubel, 1997), a free radical scavenger, was added to the extracellular solution. However, 10 mM glutathione did not slow down or remove T1 OHC degradation.
b) Substitution of extracellular Na^ for an equal concentration of NMDG did not prolong the lifespan of the preparation. Thus, depolarization of the OHC through Na^ loading does not seem to contribute to T1 OHC death.
c) The quality of the preparation was not improved if either superglue or dental acrylic was used as an alternative to wax for mounting the preparation in the recording chamber.
d) To increase the oxygen saturation of the perfusate and thus minimise OHC hypoxia, the reservoir was bubbled with air. Although this did not improve the quality of the preparation, the possibility of hypoxia being a problem
immediately after decapitation of the guinea-pig cannot be ruled out.
4.2.3 Preventing run-down of T1 OHC currents.
-40 ro 1 B -60
1
3 Y 2 -70 0) -80 N -90 8 10 2 14 0 4 6 12 16 T im e (m ln s)Figure 4.2 The change in zero-current potential of a T1 OHC patched with either standard intracellular solution ( # ) or a potassium fluoride based intracellular solution (■ ). 7 minutes after the start of the experiment, the cell patched with standard intracellular solution had died. For the cell patched with potassium fluoride, the cell was still healthy 15 minutes into the patch, after which time the experiment was terminated.
The zero-current potential of T1 OHCs patched with basic PBS solution (Table 2.1) deteoriated from ~-80 mV to 0 mV over the first 5-7 minutes of a whole-cell recording (Figure 4.2). Without a stable baseline, experimentation on these cells was impossible. The addition of 2.5 mM Mg-ATP to the pipette solution was unable to prevent this run-down. However, when the main
intracellular anion was changed from Cl' to F' (KF, Internal Solutions, Table 2.1), the zero-current potential of the cell remained absolutely steady for upto 15 minutes and often for much longer. The most straightforward explanation for the effect of F" would be to suggest that it relieved the cells of Cl" regulation, a process that was otherwise overloaded, causing the cells to die. Alternatively, as F is known to interact with a number of different proteins such as G-proteins,
phosphatases and phospholipases (Duszyk, Liu, Kamosinska, French and Paul Man, 1995), it may have indirectly influenced the activity of ion channels
perhaps by inhibiting L-type Ca^^ channels and reducing Ca^^ influx into the cell (Todorovic and Lingle, 1998) or by maintaining potassium channels, open around the zero-current potential of the cell, in an open state (Gofa and Davidson, 1996). Finally, F' may inhibit the activation of a non-selective cation conductance, a conductance observed in isolated OHCs (Housley and Ashmore, 1992).