CHAPTER 3 – AGE DIFFERENCES IN ASTROCYTES 50
3.5 CONCLUSIONS 83
This chapter set out to investigate the changes that occur in electrophysiological profiles of cortical astrocytes from somatosensory cortex during the process of non-‐‑pathological ageing. Table 2 summarises the results, covered in this chapter.
Potassium
current
Change/effect in aged mice
Cell capacitance ↑
Current density Total ↑
Kir -‐‑
KD ↑
KA ↑
4-‐‑aminopyridine block Total -‐‑
Kir -‐‑
KD -‐‑
KA -‐‑
Barium chloride block Total -‐‑
Kir ↓
KD -‐‑
KA -‐‑
Combined block Total -‐‑
Kir -‐‑
KD -‐‑
KA ↑
Table 2: Summary of effects, described in Chapter 3.
Firstly, the size of the cell somas was compared between the two age groups. It was found to be significantly greater in older mice. Since it is known that there is an increase in the GFAP production around the 40 -‐‑ 44 weeks old mice (Amenta et al. 1998) so the increased capacitance in Group III was expected. Current densities
on average were higher in isolated cells from Group III, when measured with all but one electrophysiological protocol. The exceptions were the data collected for the lowest voltage steps of the protocol for recording inwardly rectifying
potassium currents. There was no significant change in the current density of inwardly rectifying potassium current between young mature and old mice. It is possible that this lack of increase in the current density can lead to astrocytes being unable to effectively remove extracellular potassium following action
potential (Rossi 2015; Finch 2003). However, it can also be a consequence of ageing as opposed to a cause.
The proportions of currents were described by their blocker-‐‑sensitivities. Proportion of current sensitive to both 4-‐‑aminopyridine and barium were
measured in separate experiments and some experiments were performed using both blockers together. All experiments using 40 µμM of 4-‐‑aminopyridine showed no significant difference in the proportion of the overall current susceptible to blocker between young mature and old mice. When taken together with the data collected for current density, it can be concluded that the since the current density rises but the proportion of 4-‐‑aminopyridine sensitive current remains the same, there is in fact an increase in the proportion of 4-‐‑aminopyridine sensitive current from young mature to old mice.
When a blocker for inwardly rectifying potassium current – barium chloride, was applied the isolated astrocytes from young mature mice showed a greater
inhibition of the total current than astrocyte isolated from old mice. The proportion of the current that was barium chloride -‐‑ sensitive at 100 µμM was reduced by 50 per cent or more. It appears that the inwardly rectifying potassium channels expression or functioning reduces with non-‐‑pathological ageing. These data point to similar processes, underlying non-‐‑pathological ageing and various pathological settings, for instance ischemia-‐‑like conditions (Pannicke et al. 2000).
However, this reduction was only seen at the protocol aimed for recording the inwardly rectifying potassium current; the trend was similar in other protocols, but high error bars coupled with only a small fraction of this current type at other protocols.
When a cocktail of both blockers at the same concentrations was used, the data appeared in disagreement with the previous experiments. The overall trend displayed an increase in the proportion of current, which was sensitive to both of the blockers. Some of the protocols (namely the protocol for recording total potassium current, negative voltage steps of protocol for recording the inwardly rectifying potassium current and protocol for recording the voltage-‐‑gated
potassium current) showed no significant difference, whereas the proportion of the total current blocked by the mixture of both blockers increased in some voltage steps, including the most positive voltage steps in the protocol for recording
inwardly rectifying potassium current and some of the voltage steps, at which the rapidly desensitizing potassium current was measured. It also appeared that in young mature mice the proportion of the current blocked by the addition of both 4-‐‑aminopyridine and barium chloride together was smaller than what was expected from the experiments where the blockers were applied separately. The proportion of the total current that should be blocked by the blocker cocktail was 69 per cent for -‐‑70 mV (from holding potential -‐‑80 mV) but instead it was only 40 per cent. Therefore it is possible that the simultaneous blockage of multiple potassium channels leads to increased build up of potassium ions inside the
patched cells and an increased conductance via the leaky potassium channel types, unaffected by the blockers used (L. Y. Kucheryavykh et al. 2009; Zhou et al. 2009).
In conclusion, it has been shown that in non-‐‑pathological ageing prominent changes in cortical astrocyte take place. The capacitance of the cells increase, showing that on average astrocytes increase in size, potentially signifying that the
proportion of reactive astrocytes increases. Current densities of voltage-‐‑dependent potassium currents (total, KD and KA or slowly deactivating and rapidly
desensitizing potassium current types) increase with age, whereas the current density of inwardly rectifying potassium current does not. The fractions of the overall current that are sensitive to 4-‐‑aminopyridine blocked (specific blocker for voltage gated potassium currents at the concentration used) (Tse et al. 1992). The Barium-‐‑sensitive fraction of the current was significantly diminished in old mice. These changes shift the overall balance from the healthy state towards more reactive and hyperpolarised astrocytes, similar to those brought about by age-‐‑ related diseases, such as Alzheimer disease.