Orexin, c-Fos and co-expressing cell counts in experimental groups 2 hours after NIH arena testing.

#<p>5 sections per animal were bilaterally counted. All values are expressed as group averages, calculated from averages within animals. Results are mean±SEM. Significant results are highlighted in bold.</p><p>*p<0.05, two-way ANOVA with Bonferroni's Multiple Comparison Test.</p

The novelty-induced hypophagia (NIH) arena, and localisation of the lateral hypothalamus.

<p>(<b>a</b>) At 125 cm in diameter with high (50 cm) walls, and painted white, the NIH arena is large, bright and anxiogenic. A bowl of 20 Cheerios® (Nestlé) food treats was placed in the central zone (CZ), defined as a circular area of diameter 30 cm. (<b>b</b>) The lateral hypothalamus (LH) was defined as lateral of the fornix, and bilaterally counted. Orexin cells are stained in black in the bottom panel. Top image taken from Paxinos and Watson (2004).</p

Representative image of orexin cell staining.

<p>Orexin cells co-expressing nuclear c-Fos show characteristic brown (c-Fos) nuclear staining and black (orexin) cytoplasmic staining. Examples of co-expressing cells are indicated by red arrows. The fornix lies to the left of the region shown here, just outside the frame of the image.</p

Acute skin incision causes drops in sensory thresholds that persist over 5 days.

<p>(<b>a</b>) Following the time course of plantar skin incision in the adult over five days post-surgery, both neonatally incised (IN) and control (AN) animals show a significant drop in mechanical withdrawal threshold in the injured paw compared to baseline (p<0.01 and p<0.001) at days 1 and 2 post-surgery. At day 5, only IN animals were still mechanically hypersensitive (p<0.001). IN animals were significantly more sensitive than AN animals (Treatment F_(1,24) = 6.59, p<0.05, 2-way RM ANOVA with Bonferroni post-tests). The contralateral paw showed no differences compared to baseline nor between paws (p>0.05, data not shown). (<b>b</b>) 2 days after adult incision, only neonatally incised (IN+IN, n = 6) animals still showed a decrease in thermal withdrawal threshold (i.e. thermal hypersensitivity) (p<0.01, One-way ANOVA with Bonferroni's Multiple Comparison Test). There was no significant percentage change in threshold in the injured paw of neonatally anesthetized (AN+IN, p>0.05, n = 6) animals 48 hours after an acute adult injury. **p<0.01, ***p<0.001.</p

Summary of experimental groups.

<p>Summary of experimental groups.</p

Acute skin incision affects motivational behaviour that is driven by presence of food treats.

<p>(<b>a</b>) Rearing behaviour decreased (F_(1,56) = 34.83; p<0.001) and (<b>b</b>) percentage of time spent in the central zone increased (F_(1,56) = 13.02); p<0.05) in the NIH arena when animals with no previous pain history were tested 48 hours after an acute adult injury. Graph illustrates AN+IN (n = 14) versus AN (n = 13) for clarity. (<b>c</b>) The increase in time spent in the CZ after acute incision is driven by the presence of treats - when treats were absent, adult incised animals spent less time in the central zone (p<0.05; all groups analysed via one-way ANOVA with Bonferroni's Multiple Comparison Test). *p<0.05, ***p<0.001.</p

Presence of treats affects orexin activation, which is associated with motivational behaviour.

<p>(<b>a</b>) The activation of orexin cells in adult re-injured animals with a ‘pain history’ (IN+IN) is dictated by the presence or absence of food treats in the central bowl of the NIH arena. When treats are present in this group, orexin cell activation increases to 27.2±4.1% compared to when treats are absent (14.8±3.1%) and at baseline (15.5±2.6%). ‘Baseline’ data refers to animals with a history of incision but no adult re-injury (IN), tested in the presence of treats. *p<0.05; one-way ANOVA with Bonferroni's post-tests. (<b>b</b>) There is a strong correlation within individual animals (r = −0.86, p = 0.013) between the percentage of orexin cells co-expressing c-Fos and the number of rears performed by the IN+IN group during testing in the NIH arena.</p

Re-incision to neonatally-injured animals affects motivational behaviours that are driven by presence of food treats.

<p>(<b>a</b>) Rearing behaviour decreased (F_(1,56) = 34.83; p<0.001) and (<b>b</b>) percentage of time spent in the central zone increased (F_(1,56) = 13.02); p<0.05) in neonatally incised animals with an adult re-incision (IN+IN, n = 14) versus neonatally incised alone (IN, n = 19). (<b>c</b>) The presence of treats affected both rearing and time spent in the central zone. When treats were absent (n = 5), animals reared more frequently (p<0.05) and (<b>d</b>) spent significantly less time in the central zone (p<0.01; all groups analysed via one-way ANOVA with Bonferroni's Multiple Comparison Test). *p<0.05, **p<0.01, ***p<0.001.</p

Orexin activation is correlated to motivational behaviour in neonatally-incised and re-injured animals.

<p>n.s. = not significant.</p><p>*p<0.05 (highlighted in bold).</p

EEG, behavioural, and physiological responses to a painful procedure in human neonates with full medical history

<p>We present a dataset combining the cortical, behavioural, and physiological responses of a neonatal population to a single painful stimulus. Responses were recorded from 112 neonates (29–47 weeks corrected age) using a 20 channel electroencephalogram (EEG), which was time-locked to a clinically required heel lance, and a standard behavioral and physiological composite pain score (Premature Infant Pain Profile, PIPP). The dataset also includes the responses to a non-painful control and an auditory stimulus. The infant’s medical history was collected up to the day of the study and collated into an extensive database of variables such as: the condition at birth, diagnoses, medications, previous painful procedures, and injuries, and selected maternal information. This dataset can be used to further investigate the development of cortical, physiological, and behavioural responses to painful stimuli and the impact of various medical conditions and experiences upon these responses, with the potential for understanding complex pain phenotypes. </p