Sistema Económico (SE)

Median frequency (F50), spectral edge frequency (F95) and total power (Ptot)

Means and standard error of the means are presented in Table 2.9. Statistical analyses were not performed due to the small number of animals.

Table 2.9: Mean and standard error of the mean (SEM) for F50, F95 and Ptot (five EEG periods each of 30 sec duration) for non-anaesthetised joeys (n = 1 for each age).

Graphical Comparison

Visual inspection showed that there were differences in the EEG power of frequency

Wallaby age F50 (Hz) F95 (Hz) Ptot (arbitrary

units) 137 days Mean 4.97 24.74 19.72 SEM 0.11 0.09 0.33 155 days Mean 6.08 24.67 34.03 SEM 0.19 0.16 0.86 189 days Mean 4.95 25.44 59.69 SEM 0.06 0.05 0.38 193 days Mean 3.73 23.80 51.34 SEM 0.06 0.98 0.50 F50 = median frequency F95 = spectral edge frequency Ptot = total power

spectra between the three joeys (Figure 2.10). The power of all frequencies in the joey aged 137 days was lower than that of the other two joeys. The EEG power of the joey aged 189 days was higher than of the joey aged 193 days for the majority of frequencies.

Comparison between joeys anaesthetised and not anaesthetised during EEG studies

Means and standard error of the means for the four groups are presented in Table 2.10. Median frequency and Ptot were higher in both younger and older joeys that were anaesthetised compared to those that were not. The reverse was true for F95 for both younger and older joeys. The differences in F50 and Ptot between anaesthetised and not anaesthetised joeys were greater at the older ages, while that of F95 was greater between anaesthetised and not anaesthetised joeys at the younger ages.

Eigenvalues and component scores of the component matrix are presented in Table 2.11. Multivariate analysis of the frequency spectra identified five Principal Components with Eigenvalues >1 that together contributed 61.4% of the overall variation in the data. Component loadings of Principal Component 1 (PC1) were high for all frequencies (1-30Hz) (majority of loadings >0.5). Hence, this factor could be seen to represent overall power of EEG activity. The loadings of PC2 were high for frequencies of 23-30Hz, those for PC3 were high for frequencies of 1-4, 6, 10-12 and 30Hz, those for PC4 were high for frequencies of 6-8, 11, 12 and 28-30Hz and those for PC5 were high for frequencies of 4-6, 18, 23 and 24Hz.

Figure 2.10: Means of log-transformed power in frequencies (1-30Hz) of the EEG power spectrum for the three non-anaesthetised joeys. Standard errors of the means are shown as vertical bars.

Contrasts were present in all PCs apart from PC1. In PC2 there was a contrast between frequencies of 23-30Hz (positive) and 1, 6-10 and 12-15Hz (negative), In PC3 there was a contrast between frequencies of 1-4, 6, 10-12 and 30Hz (positive) and 13, 14, 16-23 and 26Hz (negative), in PC4 there was a contrast between frequencies of 6-8, 11, 12 and 28-30Hz (positive) and 1, 3, 4, 9, 17, 20, 21, 24 and 25Hz (negative) and in PC5 there was a contrast between frequencies of 4-6, 18, 23 and 24Hz (positive) and 1, 8, 12, 13, 20, 21, 26, 28 and 29Hz (negative).

Table 2.10: Mean, standard error of the mean (SEM) and number of data points used (n) for F50, F95 and Ptot calculations for younger (137-145 days) and older (189-196 days) joeys that were anaesthetised (baseline – 1.0% endtidal halothane) or not anaesthetised.

Wallaby age F50 (Hz) F95 (Hz) Ptot (arbitrary units)

Young joeys Mean 4.39 25.02 23.89

Not anaesthetised SEM 0.38 0.36 1.00

(n=1) n 20 20 20

Young joeys Mean 4.98 23.52 27.34

Anaesthetised SEM 0.21 0.39 1.26

(n=2) n 40 40 40

Old joeys Mean 3.92 24.57 53.82

Not anaesthetised SEM 0.28 0.35 1.74

(n=2) n 40 40 40

Old joeys Mean 5.37 24.22 88.18

Anaesthetised SEM 0.33 0.25 3.17

(n=2) n 40 40 40

F50 = median frequency F95 = spectral edge frequency Ptot = total power

Table 2.11: Eigenvalues and component scores of the component matrix of all frequencies for Principal Components (PCs) 1 to 5 calculated by Principal Component Analysis for comparison of anaesthetised and non-anaesthetised joeys.

Eigenvalues PC 1 PC 2 PC 3 PC 4 PC 5 Total 13.23 2.09 1.32 1.13 1.03 Percentage of variance 44.1% 7.0% 4.4% 3.8% 3.4% Frequency PC 1 PC 2 PC 3 PC 4 PC 5 1Hz 0.559 0.122 0.377 -0.408 -0.214 2Hz 0.422 0.259 0.631 -0.238 0.262 3Hz 0.546 0.312 0.464 -0.079 -0.165 4Hz 0.39 0.472 0.207 0.044 -0.366 5Hz 0.462 0.48 0.035 0.547 0.044 6Hz 0.382 0.497 -0.014 0.025 0.545 7Hz 0.507 0.458 -0.293 0.002 0.256 8Hz 0.57 0.396 -0.241 -0.182 -0.094 9Hz 0.61 0.309 -0.119 -0.116 -0.044 10Hz 0.73 0.176 -0.026 -0.040 0.025 11Hz 0.653 0.069 0.010 -0.165 0.254 12Hz 0.717 0.072 -0.070 -0.231 0.141 13Hz 0.672 -0.045 -0.271 -0.161 -0.289 14Hz 0.752 0.036 -0.217 -0.146 -0.057 15Hz 0.744 0.034 -0.063 -0.17 -0.154 16Hz 0.736 -0.185 -0.175 -0.138 -0.055 17Hz 0.749 0.015 0.016 0.057 -0.185 18Hz 0.753 -0.084 -0.032 0.172 -0.073 19Hz 0.77 -0.099 -0.142 0.075 -0.032 20Hz 0.805 -0.0120 -0.075 0.001 -0.153 21Hz 0.805 -0.092 0.037 0.012 -0.091 22Hz 0.769 -0.095 -0.011 -0.009 -0.022 23Hz 0.796 -0.203 -0.039 0.056 0.049 24Hz 0.736 -0.253 0.126 0.012 0.008 25Hz 0.682 -0.082 -0.043 0.028 0.031 26Hz 0.682 -0.3 -0.058 0.042 0.084 27Hz 0.636 -0.188 0.158 0.159 0.098 28Hz 0.589 -0.404 0.040 0.077 0.234 29Hz 0.685 -0.433 0.205 0.112 0.156 30Hz 0.677 -0.219 0.215 0.073 0.104

Figure 2.11 shows the separation of PC scores along the 1st and 2nd Principal Component axes according to age and whether joeys were anaesthetised or not. The PC analysis was able to distinguish between the two age groups (young and old), with the younger joeys scoring low (low EEG power) and the older joeys scoring higher (higher EEG power) on PC1 (overall EEG power). In addition, the PC analysis was able to distinguish the older joeys according to whether they were anaesthetised or not, also along PC1, where joeys that were not anaesthetised scored lower (lower EEG power) than those that were anaesthetised. The younger joey that was not anaesthetised, and to some extent the older joeys that were not anaesthetised, scored relatively higher on PC2 (frequencies of 23-30Hz) than the joeys that were anaesthetised. Due to the contrast present in this component, joeys that were not anaesthetised showed a higher proportion of frequencies between 23-30Hz present in their EEGs, while those joeys that were anaesthetised showed a tendency to an increase in frequencies of 1, 6-10 and 12-15Hz and lower proportion of frequencies between 23-30Hz. PC1 (power of the EEG) contributed 44.5% to the overall variation, while PC2 (frequencies of 23-30Hz) contributed only 5.3%. Separation according to PCs 3,4 and 5 did not yield any distinctions between ages or whether or not animals had been anaesthetised.

Figure 2.11: Factor scores calculated for frequencies (1-30Hz) by Principal Component Analysis plotted against the 1st and 2nd Principal Component axes for both age group (137-145 days = young; 189-196 = old) and treatments (anaesthesia or no anaesthesia) are shown and include 20 data points per joey per treatment.

2.4 Discussion

The present study aimed to investigate the cerebrocortical (EEG) responses of lightly anaesthetised tammar wallaby joeys of varying ages to toe clamping and the responsiveness of the EEG to different halothane concentrations. As far as the candidate is aware this is the first investigation of this type in any metatherian species. The main findings were as follows. First, there was little or no EEG activity in joeys aged up to about 100 days of in-pouch age, but EEG epochs of sufficient duration for FFT analysis were present by about 140 days. Second, the percentage of time occupied by isoelectric periods decreased, and that occupied by EEG activity increased, with age. Third, during each observation period of each individual joey aged up to about 190 days, the percentage of time occupied by isoelectric EEGs in joeys increased with increasing endtidal halothane. Fourth, EEG power in all frequencies significantly increased with in-pouch age. Fifth, for the parameters investigated there was an age-related progression in EEG responses to clamping, although overall the responses were marginal to moderate. The responses ranged from no effects on EEG parameters in animals between 95-130 days due to the absence of EEG activity through a marginal response in joeys aged 140-181 days to a moderate response in those aged 187-260 days. Sixth, changes in halothane concentrations led to only marginal changes in EEG parameters in the intermediate and older age groups. Seventh, in a small number of joeys there were differences in EEG parameters between anaesthetised and non-anaesthetised joeys according to age.

Postnatal developmental changes of in-pouch tammar wallaby joeys

Before examining the results obtained in the present study on anaesthetised and non- anaesthetised in-pouch joeys, it would be beneficial to describe the normal progression of cerebrocortical development, brain electrical activity and behaviour of tammar wallaby joeys after birth. This knowledge will be essential for an integrated analysis of the development of pain perception per se, and conscious perception in general, in the tammar wallaby pouch young. Such an analysis may allow inferences to be drawn regarding the welfare status of joeys involved in pest control operations or during road accidents in Australia and New Zealand.