Effects of posterior hypothalmic lesions on formalin induced pain behaviours 2

lesioned area in SuML was either ipsilateral or contralateral to the formalin injected paw. It is important to note that the damaged area in ventral lesion was not confined within one restricted area of the SuM but sometimes encroached upon adjacent areas as well. For example, lesion that covered major part of mSuM might encroach upon minor portion of SuML. To eliminate the ambiguity, the assignment of the groups was carried out using the numerical method described below: mSuM lesion: the lesioned area in mSuM was more than 40% (ranging from 40%100%), with lesion in unilateral SuML being relatively minor, generally ranging from 0%-27% (e.g. Table 1). Unilateral SuML lesion: the lesioned area in unilateral SuML was more than 40% (ranging from 42%-100%), with lesion in mSuM being relatively minor, generally ranging from 0%-20% (e.g. Table 1). Out of the 17 animals with ventral lesions, there were 4 animals with lesions that encroached upon both mSuM and SuML. In these animals the extent of the mSuM and the unilateral SuML that were damaged ranged from 38%-100% and 78-100%, respectively. These animals were not included in the analysis to determine site specific effects, if any, of the lesion. The remaining animals were distributed into mSuM and SuML groups. The number of the animals used and the extent of lesion in each group are summarized in various 66 A Bregma -4.20 B Bregma -4.44 C Bregma -4.68 Figure 12. Digitized images (500 dpi) of the lesion at each plane of section in an animal with mSuM lesion. The damaged area occupied the full extent of mSuM region and encroached on SuML laterally especially in the posterior sections (Bregma -4.68) 67 Groups mSuM n=6 SuML n=7 Lesion volume in mSuM (mm3) Volume of mSuM (mm3) % of lesion of mSuM against mSuM Lesion volume in unilateral SuML (mm3) Volume of unilateral SuML (mm3) % of lesion of SuML against unilateral SuML 0.044±0.011 0.063±0.0175 70.84±17.50 (40~92) 0.0094±0.0054 0.048±0.0102 17.66±13.67 (0~27) 0.0029±0.004 0.063±0.0175 4.69±7.70 (0~20) 0.030±0.0137 0.048±0.0102 63.34±28.94 (20~100) Table 1. Extent of lesion within medial supramammillary and lateral supramammillary against whole mSuM and the whole unilateral SuML region, respectively. Data is presented as mean ± SEM. It should be noted that all SuML lesion animals (n=7) were included to evaluate the lesion extent in Table 1. Six were used in open field, and the same five animals in open field test plus another animal were used subsequently in formalin test. Therefore, there were total seven animals used to calculate the overall lesion profile for SuML lesion group. 68 tables (See section 3.2, 3.3, and 3.4). The SuML group comprised animals with either left or right SuML lesions. Two tailed unpaired t-test showed no difference between left and right SuML lesion animals. The statistics are as follows: (1) ambulation in open field test [n=3 (left); n=3 (right)], P=0.540; (2) flinches in formalin test, P=0.978 [n=2 (left); n=4 (right)]; (3) duration of licks, P=0.169 [n=2 (left); n=4 (right)]; (4) ambulation in formalin test, P=0.290 [n=2 (left); n=4 (right)]. Thus, these animals were grouped indiscriminately to form the SuML group. The detailed description of the estimation of the size of the lesion is described below (Figs. 13-15, Table 1-3). It should be noted that all SuML lesion animals (n=7) were included to evaluate the lesion extent in Table 1. Six were used in open field, and the same five animals in open field test plus another animal were used subsequently in formalin test. Therefore, there were total seven animals used to calculate the overall lesion profile for SuML lesion group. 69 A B C D Figure 13. Illustration of the calculations to determine the extent of the lesion. The coronal sections represented are adapted from Paxinos and Watson (2007) and corresponds to P 4.44 mm from Bregma. The vertical lines demarcate the supramammillary (SuM) region into medial (mSuM) and lateral (SuML) regions. Hatched area represents lesion that encroaches upon both mSuM and SuML and areas slightly dorsal to SuM region. The example used in this illustration is taken from the mSuM group. (A) The region within the red broken lines represents ‘lesion within mSuM’. (B) The region within the red broken line represents whole ‘mSuM’. The area within the broken lines in A and B were calculated using computer software and represented the area of the lesion and the area of the whole mSuM, respectively, for those sections. The corresponding volume was calculated by multiplying the area with the thickness of the section, which is 0.06 mm in this case. Similar calculations for area and volume were performed at different anteriorposterior levels (bregma -4.20 to bregma -P4.92 mm) and the values were added together to give the total area (volume) of lesion and total area (volume) of the mSuM. The percentage of lesion of mSuM against whole mSuM is calculated using the formula as follows: % of lesion of mSuM against whole mSuM = Total volume of ‘lesion within mSuM’ x100% Total volume of ‘mSuM’ Areas highlighted in (C) and (D) represent ‘lesion within SuML’ and ‘unilateral SuML’ respectively. The total area (volume) were calculated as described above and 70 the percentage of lesion of SuML against whole unilateral SuML is calculated as follows: % of lesion of SuML against Whole unilateral SuML = Total volume of ‘lesion within SuML’ Total volume of ‘unilateral SuML’ 71 x100% A B C D Figure 14. Illustration of the calculations to determine the extent of the lesion against total volume of the supramammillary region (SuM). The coronal section represented is adapted from Paxinos and Watson (2007) and correspond to P 4.44 mm from Bregma. The example used in this illustration is taken from mSuM group. The figure is developed as for Fig. 10 except that the area within the red broken line in B and D represents the ‘whole SuM’ at the level of the section. The calculations were performed as described in Fig. 10. Percentage of lesion of mSuM against whole SuM is calculated using the formula as follows: % of lesion of mSuM against whole SuM = Volume of ‘lesion within mSuM’ Volume of ‘whole SuM’ x100% Percentage of lesion of SuML against whole SuM is calculated as follows: % of lesion of SuML against whole SuM = Volume of ‘lesion within SuML’ Volume of ‘whole SuM’ 72 x100% A B C D Figure 15. Illustration of the calculations to determine the extent of the lesion in the medial and lateral supramammillary regions (mSuM and SuML, respectively) against total volume of the lesion. The coronal section represented is adapted from Paxinos and Watson (2007) and correspond to P 4.44 mm from Bregma. The example used in this illustration is taken from mSuM group. The figure is developed as for Fig. 10 except that the area within the red broken line in B and D represents the ‘whole lesion’ at the level of the section. The lesion covered the area within the SuM and area dorsal to it. The calculations were performed as described in Fig. 10. Percentage of lesion of mSuM against whole lesion is calculated using the formula as follows: % of lesion of mSuM against whole lesion = Volume of ‘lesion within mSuM’ Volume of ‘whole lesion’ x100% Percentage of lesion of SuML against whole lesion is calculated as follows: % of lesion of SuML against whole lesion = Volume of ‘lesion within SuML’ Volume of ‘whole lesion’ 73 x100% Lesion volume in mSuM (mm3) Lesion volume in SuML (mm3) Volume of whole SuM (mm3) % of lesion of mSuM against whole SuM % of lesion of SuML against whole SuM % of lesion of SuM against whole SuM mSuM n=6 0.044±0.0110 0.0094±0.00540 0.15±0.0330 42.13±17.69 5.4±2.99 34.77±6.53 SuML n=7 0.0029±0.00481 0.030±0.0137 0.15±0.0330 0.88±2.17 28.33±9.80 30.01±9.79 Groups Table 2. Extent of lesion of medial supramammillary and lateral supramammillary against whole supramammillary. Data was presented as mean ± SEM. It should be noted that all SuML lesion animals (n=7) were included to evaluate the lesion extent in Table 1. Six were used in open field, and the same five animals in open field test plus another animal were used subsequently in formalin test. Therefore, there were total seven animals used to calculate the overall lesion profile for SuML lesion group. 74 Lesion volume in mSuM (mm3) Lesion volume in SuML (mm3) Volume of whole lesion (mm3) % of lesion of mSuM against whole lesion % of lesion of SuML against whole lesion mSuM n=6 0.044±0.0110 0.0094±0.00540 0.14±0.0592 38.35±20.14 4.50±2.31 30.18±6.57 SuML n=7 0.0029±0.00481 0.030±0.0137 0.39±0.196 2.08±4.75 12.24±13.26 14.06±17.89 Dorsal n=9 -- -- 0.51±0.406 -- Groups % of lesion of SuM against whole lesion -- Table 3. Extent of lesion of medial supramammillary and lateral supramammillary against whole lesion. Data was presented as mean ± SEM. It should be noted that dorsal lesions avoided areas in supramammillary. One-way ANOVA followed by post-hoc analyses indicated that there is no difference in lesion size among all groups (Groups, F3, 21=3.06, P=0.071). It should be noted that all SuML lesion animals (n=7) were included to evaluate the lesion extent in Table 1. Six were used in open field, and the same five animals in open field test plus another animal were used subsequently in formalin test. Therefore, there were total seven animals used to calculate the overall lesion profile for SuML lesion group. 75 3.2 Effects of PH-SuM lesion on animal exploratory behavior An open field test to evaluate exploratory behavior was performed on animals that were, 10 days later, also subjected to formalin test and c-Fos immunohistochemistry (see sections 3.3 and 3.4). The various groups of animals that were evaluated for exploration are shown in Table 4. Figure 16 illustrates the time course of the distance traveled by the animals in the open field test. Ambulation in the first 2-min block was the highest, and then decreased with time for all the groups of animals. One-way ANOVA indicated no significant difference in the distance traveled by the 4 groups of animals, namely vehicle, dorsal, mSuM and the SuML groups (Time, F 3, 38 = 2.50, p =0.074; Fig 17). Interestingly, the total distance traveled in the mSuM group tended to be high while that in the SuML tended to be low. Indeed, a two tail unpaired t-test revealed a significant difference between the two (P < 0.05; Fig 18). Distance traveled by the animals in the central and peripheral region of the open field was also analyzed (data not shown). The pre-set central area was 35.6 X 35.6 cm in the center of the open field with the remaining area considered as the periphery. Ambulation in the periphery showed a similar pattern as seen with the overall ambulation in the open filed test. Again, a significant difference was observed between mSuM group and SuML group (Time, F 3, 38 =2.90, p[...]... lesion of SuM against whole lesion Groups Number of animals % of lesion of mSuM against mSuM Vehicle 8 - - - - Dorsal 9 - - - - mSuM 4 SuML 6 78.63±10.89 12. 90±14.85 36 .21 ±5.81 5.48±8.13 63.88±31. 42 25.4±14.77 33 .24 ±3.91 15.38±19 .22 Table 5 Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus (PH) - supramammillary (SuM) region on indices of formalin. .. injection of formalin (1 .25 %, 0.1ml) (A) Note the large number of cells on the right as compared to left of the spinal cord (B) Diagrammatic representation of the L4 spinal cord, illustrating the laminar subdivisions 87 Groups Number of animals % of lesion of mSuM against mSuM % of lesion of SuML against unilateral SuML % of lesion of SuM against whole SuM % of lesion of SuM against whole lesion Vehicle... whole lesion Groups Number of animals % of lesion of mSuM against mSuM Vehicle 21 - - - Dorsal 9 - - - mSuM 6 70.84±17.50 17.66±13.67 34.77±6.53 30.18±6.57 SuML 6 2. 12 3.90 59 .23 27 .33 24 .46±14. 12 7.38 2. 98 Table 4 Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus (PH) - supramammillary (SuM) region on exploration The lesion extent was calculated... in formalin test among all groups 4.1 Lesions Previous studies have explored the effect of lesion of the SuM region on animal behaviors related to emotion, cognition and motivation (Pan and McNaughton, 20 02; Aranda et al., 20 06) The SuM was lesioned either electrolytically (Aranda et al., 20 06) or with neurotoxins (Pan and McNaughton, 20 02) However, electrolytic destruction also affects fibers of passage... lesion Vehicle 7 - - - - Dorsal 9 - - - - mSuM 3 81.41±11.47 8 .21 ±14.11 35.46±6.88 34. 52 3.63 SuML 5 2. 54±4 .2 57 .25 ±30.08 23 .3±15.40 7. 62 3 .26 Table 6 Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus (PH) - supramammillary (SuM) region on spinal c-Fos in the formalin test The lesion extent was calculated as explained in Fig 13-15 It should be... whole SuM % of lesion of SuM against whole lesion Vehicle 7 - - - - Dorsal 4 - - - - mSuM 4 78.63±10.89 12. 90±14.85 36 .21 ±5.81 33 .24 ±3.91 SuML 4 3.17±4.56 60.88±33.44 18.43± 12. 84 7.89±3.70 Table 7 Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus (PH) - supramammillary (SuM) region on hippocampal c-Fos in the formalin test The lesion extent was... dorsal CA1 comprised of the c-Fos positive neurons in the pyramidal cell layer of anterior CA1 and the posterior- dorsal subdivision of the posterior CA1, while the count for ventral CA1 included the c-Fos positive neurons in the pyramidal cell layer of the was the ventral subdivision of posterior CA1 (see Figs 23 and 24 ) Each bar is mean ± SEM 95 A B Figure 26 The lack of effect of lesion of the medial and... expression is illustrated in Figs 19 - 22 The various groups and the extent of lesion are shown in Tables 5 and 6 The size of lesion for the dorsal, mSuM and SuML groups were 0.51±0.406 mm3, 0.14±0.05 92 mm3, and 0.39±0.196 mm3, respectively One-way ANOVA followed by post-hoc 77 % of lesion of SuML against unilateral SuML % of lesion of SuM against whole SuM % of lesion of SuM against whole lesion Groups... of flinches, duration of licking and ambulation distance in the 2nd phase (11-60 min) is illustrated in Fig 20 No significant effect of AMPA treatment was observed on licks (Groups, F3, 23 = 0. 92, P=0.451) and ambulation (Groups, F3, 23 = 0.77, P=0. 521 ) However, an overall significant effect of lesion was observed on flinches (Groups, F 3, 23 =3.03, P ... 81.41±11.47 8 .21 ±14.11 35.46±6.88 34. 52 3.63 SuML 2. 54±4 .2 57 .25 ±30.08 23 .3±15.40 7. 62 3 .26 Table Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus... 63.88±31. 42 25.4±14.77 33 .24 ±3.91 15.38±19 .22 Table Summary of the groups of animals that were evaluated for the effects of lesion of the posterior hypothalamus (PH) - supramammillary (SuM) region on. .. Groups Number of animals % of lesion of mSuM against mSuM % of lesion of SuML against unilateral SuML % of lesion of SuM against whole SuM % of lesion of SuM against whole lesion Vehicle - -