Age-related changes to ionic currents and excitability in the

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Age-related changes to ionic currents and excitability in the

Transcript Of Age-related changes to ionic currents and excitability in the

Age-related changes to ionic currents and excitability in the Cerebral Giant Cells of
the pond snail, Lymnaea stagnalis.
Greg Scutt
A thesis submitted in partial fulfilment of the requirements of the University of Brighton for the degree of Doctor of Philosophy
October 2012
The University of Brighton

Abstract
One of the major issues we will face over the next 50 years is an 'ageing population', and its associated burden of disease and disability. Included in these are the age-related neurological conditions such as Alzheimer's disease, Parkinson's disease and normal brain ageing. Our understanding of how neurones age in mammalian species is hampered by the complexity of the mammalian brain, and the obvious barriers to biophysical measurements from the neurones of humans.
With a few possible exceptions, the ageing process is universal across the biosphere. This raises the possibility that studying the nervous system of simpler organisms can help to answer wider questions about age-related changes to mammalian neurones. One such organism is the pond snail Lymnaea stagnalis. The well-defined, and relatively simple feeding network of the Lymnaea Central Nervous System contains a pair of serotonergic modulatory interneurones, the cerebral giant cells (CGCs), which are known to be involved in the learning and memory process. These cells are also known to show age-related changes. The work presented in this thesis provides an insight into the mechanisms that underlie some of these changes.
Evidence is provided here that there is a clear reduction in the excitability of the CGCs during ageing: spontaneous firing rate declines, and there is there is an increase in the time course and strength of spike frequency adaptation. Underlying these changes is an age-related increase in a Ca2+-activated K+ current UsAHP), an observation consistent with findings in aged hippocampal neurones. Further evidence is provided which suggests that a disruption to Ca2+ homeostasis may drive these changes. In particular there is a reduction in the time constant of recovery from inactivation of voltage gated Ca2+ currents in aged CGCs, along with an increase in the time course of Ca2+ current activation and deactivation. Furthermore, as animals enter very old age, the amplitude of both a nifedipine insensitive, and nifedipine sensitive current increases, along with a shift in voltage sensitivity. Perhaps the most intriguing age-related change however is a switch in the mode of the Na+/Ca2+ exchanger, which forms part of the CGCs' Ca2+ clearance mechanisms. This may underlie an age-related increase in the time course of spike frequency adaptation. Finally, evidence is presented which shows that injection of the lsAHP enhancing drug l-ethyl-2-benzimidazolinone (1-EBIO) into Lymnaea can negatively affect success in an appetitive-learning paradigm, mimicking the aged behavioural phenotype.
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Table of Contents
Abstract ............................................................................................................................ 2 Table of Figures...........•....•...........................••..•.........•.............•......•........•••..•.......•......_.••8 List of Tables .................................................................................................................. 12 Acknowledgements..•...•...........................•.•..••............................................................... 14 Author's Declaration ..................................................................................................... 15 Definitions ......................................................................................................................................... 16
1 Introduction....................................................................................................................................................................................... 19 1..1 Ageing...........................................................................................................................................................................................19 1.1.1 The ageing process: why we age...............................................................20 1.1.2 How we age: Reactive oxygen species as a cause of ageing .................... 23 1.1.3 The Insulin/Insulin Like Growth Factor-I Pathway and Ageing.............. 35 1.1.4 The mTOR pathway and Ageing .............................................................. 36 1..2 Neuronal Ageing............................................................................................................................39 1.2.1 Age-related neurological disease .............................................................. 39 1.2.2 Normal brain ageing..................................................................................43 1.2.3 The afterhyperpolarisation and ageing......................................................55 1.3 Invertebrate models of neuronal ageing.........................................................58 1.3.1 Lymnaea stagnalis..................................................................................... 62 1.4 Summary .......................................................................................................................67
2 Methods .......................................................................................................................................................................... 70 2.1 Animals.................. ....................................................................................................................................................... 70 2.2 Chemicals and solutions .......................................................................................................................70 2.2.1 Normal saline ............................................................................................ 70
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2.2.2 2.2.3 2.2.4

Zero Na+trEA/4-AP saline ....................................................................... 70 Zero Na+trEA/4-AP Ba2+ substituted saline............................................. 71 Na+ substituted with Lt saline..................................................................71

2.2.5 Pharmacological agents............................................................................. 71

2.3 Preparation of Lymnaea CNS for in vitro electrophysiology........................71 2.4 Current clamp ..................................................................................................74

2.4.1 Equipment and software............................................................................74

2.5 Voltage clamp ...................................................................................................74

2.5.1 Equipment and software............................................................................74

2.6 Behaviour - Appetitive Conditioning.............................................................75

2.7 Data Analysis ....................................................................................................77

2.7.1 Current clamp recordings..........................................................................77

2.7.2 Voltage clamp recordings .........................................................................78

2.7.3 Statistical analysis .....................................................................................78

3 Age-related changes to the CGC afterhyperpolarisation....................................79 3.1 Introduction ...................................................................................................... 79 3.2 Methods.............................................................................................................88 3.2.1 Electrophysiology ..................................................................................... 88 3.2.2 Data analysis ............................................................................................. 93 3.3 Results ...............................................................................................................95 3.3.1 Age-related changes to firing rate in the CGCs ........................................95 3.3.2 Age-related changes to the CGC after-hyperpolarisation.........................96 3.3.3 Relationship between the CGC afterhyperpolarisation and firing rate .....98 3.3.4 Calcium sensitivity of the CGC afterhyperpolarisation during ageing...100 3.3.5 Pharmacology of the CGC afterhyperpolarisation .................................. 116 3.3.6 Learning and memory in Lymnaea, and the after hyperpolarisation ...... 128 4

3.4 Discussion........................................................................................................130

3.4.1 3.4.2

Age-related changes to the firing rate and AHP of the CGCs ................130 Ca2+-activated K+ currents in the CGC during ageing ............................134

3.4.3 Behaviour ................................................................................................144

3.5 Conclusions .....................................................................................................146

4 Age-related changes to HVA-Ca2+ current in the CGCs ..................................149

4.1 Introduction....................................................................................................149

4.2 Methods ........................................................................................................... 153

4.2.1 Electrophysiology ...................................................................................153

4.2.2 Data analysis ...........................................................................................156

4.3 Results .............................................................................................................158 4.3.1 HVA-Ca2+ currents from young and old (10-12 months) CGCs ............158 4.3.2 HVA-Ca2+ currents from young and very old (12-14 months) CGCs ....174

4.4 Discussion........................................................................................................200

4.4.1 4.4.2 4.4.3

A nifedipine sensitive current in the Lymnaea CGCs.............................201 Age-related changes to HVA Ca2+ currents in the CGC.........................205 Relationship between firing rate and Ca2+ current..................................222

4.5 Conclusions ................................................................................ ......................225

4.5.1 Further work............................................................................................226

5 Age-related changes to the firing behaviour of the CGCs ................................ 228

5.1 Introduction ....................................................................................................228

5.1.1 Spike frequency adaptation (SFA) ..........................................................228

5.1.2 Measurements of SFA.............................................................................229

5.2 Methods...........................................................................................................232

5.2.1 Dynamic SFA in response to artificial current injection.........................232

5.2.2

Materials and experimental procedure ....................................................235
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5.2.3 Analysis................................................................................................... 237 5.3 Results ............................................................................................................. 239
5.3.1 SFA in CGCs from young and old animals ............................................239 5.3.2 Dynamics of SFA in young and old CGCs ............................................. 250 5.4 Discussion ........................................................................................................ 257 5.4.1 SFA in the CGCs of young Lymnaea ............................................................ 257 5.4.2 SFA in CGCs from old Lymnaea and comparisons with young.............264 5.5 Conclusions ..................................................................................................... 267

6 Age-related changes to Ca2+ clearance in the CGCs ......................................... 268

6.1 Introduction ......................................................................................................268

6.2 Methods ........................................................................................................... 270

6.2.1 Mathematical determination of tea in young and old Lymnaea CGCs ...270

6.2.2 Determining NCX function in CGCs from young and old Lymnaea .....272

6.2.3 Analysis ................................................................................................... 273

6.2.4 Mathematical determination of tea in young and old Lymnaea CGCs ...273

6.2.5 Measurement of NCX function...............................................................273

6.3 Results .............................................................................................................275

6.3.1 6.3.2

Mathematical determination of tea in young and old Lymnaea .............. 275 Age-related changes to the Na+ / Ca2+ exchanger in the CGCs. .............278

6.4 Discussion........................................................................................................280

6.4.1 6.4.2

Estimation of tea in young and old CGCs...............................................280 Age-related changes to the Na+/Ca2+ exchanger in the CGCs ................ 284

6.4.3 Spike frequency adaptation and the NCX during ageing........................286

6.5 Conclusions ..................................................................................................... 290

7 Discussion .............................................................................................................. 292
7.1 Change to the sAHP of the CGC during ageing .......................................... 292 6

7.2 What underlies the enhanced sAHP in the CGCs of aged animals? ......... 294 7.3 What is driving changes to CGC Ca2+ homeostasis and excitability? ....... 298
7.3.1 ROS .........................................................................................................298 7.3.2 The mTOR pathway ................................................................................ 299 7.3.3 The I/IGF-1 pathway...............................................................................300 7.4 Future work .................................................................................................... 301 7.5 Summary and conclusions............................................................................. 302 8 References.............................................................................................................. 303
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Table of Figures
Figure 1-1 Distribution of individuals in an immortal, self-replicating population by age-group (lifespan is determined by extrinsic hazard).. ................................................22 Figure 1-2 Protein damage / repair / replacement / degradation process and how it is influenced by oxidative stress. ........................................................................................28 Figure 1-3 Schematic representation of the mTOR pathway..........................................38 Figure 1-4 LTP/LTD transition with stimulation frequency...........................................48 Figure 2-1 Diagram of the perfusion bath used for current clamp and voltage clamp experiments. ....................................................................................................................73 Figure 3-1. Age-related changes to basic firing properties of the Lymnaea CGC..........95 Figure 3-2 Age-related changes to components of the CGC after-hyperpolarisation and resting membrane potential. ............................................................................................ 97 Figure 3-3. Scatter plots and correlations between firing frequency and the various components of the AHP for individual young and old CGCs......................................... 99 Figure 3-4. Changes to firing frequency following treatment with 100 µM CdC}z in young and old CGCs.. ...................................................................................................101 Figure 3-5. Effect of treatment with CdC}z on the various components of the CGC AHP in young and old CGCs. ................................................................................................103 Figure 3-6 Action potential characteristics for young and old CGCs in the absence and presence of 100 µM CdC1 104 2............................................................................................ Figure 3-7. I1Imax curves showing the effect of treatment with CdC}z on outward K+ currents..........................................................................................................................106 Figure 3-8. Voltage clamp on the mAHP current .........................................................109 Figure 3-9. Slow tail current (sAHP) and Ca2+ sensitivity............................................111 Figure 3-10. sAHP evoked tail current measured 500ms post voltage step.................. 113 Figure 3-11 I/V plots of outward tail currents measured at 100ms, 250ms and 500ms115

Figure 3-12. Effect of apamin, an SK channel blocker, on firing frequency and the components of the AHP in CGCs of middle aged Lymnaea.........................................118 Figure 3-13. Effect of dequalinium on the mAHP in CGCs from old Lymnaea. ......... 119 Figure 3-14. Effect of dequalinium on voltage clamp measurement of the mAHP and sAHP current in old CGCs............................................................................................120 Figure 3-15. Effect of 1-EBIO on the mAHP and sAHP and firing frequencyin CGCs from young Lymnaea determined in current clamp. .....................................................122 Figure 3-16. Voltage clamp analysis of tail currents in the absence and presence of 50 µM 1-EBIO. ..................................................................................................................123 Figure 3-17. Voltage clamp analysis of tail currents evoked from CGCs of young Lymnaea in the absence or presence of 100 µM 1-EBIO. ............................................125 Figure 3-18. Effect of 1-EBIO in the presence of 8-CPT cAMP on the mAHP and sAHP in young Lymnaea.. ....................................................................................................... 127 Figure 3-19. Effect of 1-EBIO injection on learning in an appetitive conditioning paradigm........................................................................................................................ 129 Figure 4-1. Electrophysiological investigation of peak HVA-Ca2+ currents in young (3-4 months) and old (10-12 month old animals) CGCs. .....................................................159 Figure 4-2. Activation of Ca2+ currents in CGCs of young and old Lymnaea..............161 Figure 4-3 Deactivation of Ca2+ current in CGCs of young and old Lymnaea at high voltages ......................................................................................................................... 163 Figure 4-4. Inactivation of HVA-Ba2+ current in young and old CGCs.. .................... 164 Figure 4-5. Activation/ Steady state inactivation curves of HVA-Ba2+ currents in young and old CGCs. ............................................................................................................... 166 Figure 4-6. Time-course of recovery from Ca2+ and voltage dependent inactivation of HVA-Ca2+ channels in CGCs from young and old Lymnaea. ......................................169
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Figure 4-7. Time-course of recovery from voltage dependent inactivation of HVA-Ba2+ channel current in young and old CGCs. ...................................................................... 172 Figure 4-8. Effect of nifedipine on HVA Ca2+ currents in CGCs from very old (12-14 month) Lymnaea. ..........................................................................................................175 Figure 4-9. Effect of nifedipine treatment on HVA Ca2+ currents in CGCs from young and very old animals (12-14 months)............................................................................ 180 Figure 4-10. Activation curves for HVA Ca2+ currents in CGCs from young and very old animals in the absence and presence of 10 µM nifedipine. .................................... 182 Figure 4-11. Effect of nifedipine on time to peak current values for Ca2+ currents in CGCs from young and very old animals....................................................................... 187 Figure 4-12. Effect of 10 µM nifedipine on slow and fast 'tdeact values for HVA-Ca 2+ current deactivation in CGCs from young and very old animals. ................................191 Figure 4-13. Effect of 10 µM nifedipine on CGC action potential characteristics in young and old animals. ................................................................................................. 193 Figure 4-14. Effect of 8-CPT cAMP on various characteristics of HVA Ca2+ currents in CGCs from young animals............................................................................................194 Figure 4-15. Firing frequency of young and old (10-12 month) CGCs........................197 Figure 4-16. Firing frequency of young and very old (12-14 month) CGCs used in voltage clamp experiments in 4.3.2, and correlations with V50. ................................... 199 Figure 4-17. Proposed state diagram illustrating recovery from inactivation of 2 Ca2+ channel isoforms at hyperpolarised potentials in young animals. ................................ 212 Figure 4-18. Proposed state diagram illustrating recovery from inactivation of 2 Ca2+ channel isoforms at hyperpolarised potentials in old animals. ..................................... 214 Figure 5-1 The kinetics of Spike Frequency Adaptation. .............................................233 Figure 5-2. Effect of age and current injection amplitude on the number of spikes generated during a 5s pulse and action potential width. ...............................................240
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CgcsLymnaeaAnimalsSahpNifedipine