Adult participants reported increased pain with increasing stimulus intensity (r = 0.48; p < 0.0001), and most frequently described the pain as pricking (n = 8 of 10) and sharp (n = 6 of 10). In infants, application of the stimuli evoked visible withdrawal of the stimulated leg, which could be observed at all stimulus intensities, whereas in adults, reflex withdrawal of the leg or foot was not observed at any stimulus intensity. While low threshold stimuli can also evoke reflex withdrawal in infants (Cornelissen et al., 2013), this observation confirms that the stimuli applied in this study were detected by the peripheral nervous system and transmitted to the central nervous system. Although noxious stimulation can elicit reflex limb withdrawal in adults, supraspinal modulation of the input means this activity is often suppressed in experimental studies.

In adults, noxious stimulation evoked significant increases in BOLD activity in cortical and subcortical brain regions, including primary somatosensory cortices, anterior cingulate cortex (ACC), bilateral thalamus, and all divisions of the insular cortices (Figure 1). All brain regions that had a significant increase in BOLD following noxious stimulation are identified in Table 1, and are consistent with previous literature (Tracey and Mantyh, 2007). In infants, the increases in BOLD activity evoked by the noxious stimulation were extremely similar to that seen in adults, and all but two of the 20 regions that were active in the adults were active in infants (Table 1 and Figure 1). While in adults the parietal lobe, pallidum, and precuneus cortex were only active in the brain regions contralateral to the site of stimulation, in infants these brain regions were also active on the ipsilateral side to the stimulus. Additional brain regions that were only active in the infants included the bilateral auditory cortices, hippocampus, and caudate (Table 1). The increased bilateral activity and greater number of active regions in infants are likely due to the immature cortico-cortical and interhemispheric pathways (Kostovic and Jovanov-Milosevic, 2006). Major reorganisation of the cortical circuitry occurs after the first postnatal month when there is a striking retraction of exuberant axons in the corpus callosum and there is a cessation of growth of the long cortico-cortical afferent pathways (Jovanov-Milosevic et al., 2006; Kostovic and Jovanov-Milosevic, 2006).

Figure 1 Download asset Open asset Comparison of nociceptive-evoked brain activity in selected brain regions that are active in both adults and infants. Significantly, active voxels across each stimulus intensity level are presented for (A) adult and (B) infant participants (applied force: adults 32–512 mN; infants 32–128 mN). Each colour represents activity in a different anatomical brain region. (A) Adult activity is overlaid onto a standard T1 weighted MNI template and (B) infant activity is overlaid onto a standard T2 weighted neonatal template, corresponding to a 40-week gestation infant. ACC: anterior cingulate cortex; S1: primary somatosensory cortex: PMC: primary motor cortex; SMA: supplementary motor area. https://doi.org/10.7554/eLife.06356.003

Table 1 Identification of all active brain regions in adults and infants following acute noxious stimulation at all stimulus intensities (applied force: adults 32–512 mN; infants 32–128 mN) https://doi.org/10.7554/eLife.06356.004 Adults Infants Anatomical area Region Peak Z within cluster MNI coords Rank Slope of regression (*E-03) P val* Peak Z within cluster Neonate template coords Rank Slope of regression (*E-03) P val* x y z x y z Active regions in both adults and infants Intensity encoding regions (in adults) Temporal gyrus Contra 3.92 64 −34 20 1 1.01 0.0002 3.05 32 −32 12 1 2.46 0.0083 Cingulate gyrus Anterior 4.11 6 4 40 2 0.65 0.0005 2.58 −1 1 26 11 1.01 0.3971 Opercular cortex Contra 5.60 40 6 10 3 0.63 0.0001 3.38 32 −13 19 2 2.23 0.0391 Insula Contra 4.18 34 14 6 4 0.61 0.0001 3.04 19 −22 23 3 2.15 0.0207 Supramarginal gyrus Contra 4.33 64 −38 20 5 0.60 0.0008 3.29 25 −23 39 9 1.08 0.1749 Postcentral gyrus Contra 4.28 58 −18 22 6 0.60 0.0012 3.85 15 −22 52 10 1.01 0.2667 Visual cortex Contra 3.62 44 −62 4 7 0.59 0.0004 3.25 21 −52 34 6 1.41 0.0814 Putamen Contra 3.68 22 6 6 8 0.55 0.0001 3.30 17 −17 18 8 1.20 0.1656 Thalamus Contra 3.51 14 −14 0 9 0.50 0.0010 3.58 6 −16 15 4 1.91 0.0592 Insula Ipsi 4.67 −38 −18 14 10 0.49 0.0001 2.59 −26 −14 14 5 1.69 0.1015 Supplementary motor area Contra 3.91 8 4 46 11 0.39 0.0008 3.50 6 −18 48 7 1.23 0.2315 Non intensity encoding regions (in adults) Cerebellum Ipsi 3.88 −20 −66 −44 0.35 0.0029 3.53 −3 −46 −6 3.57 0.0164 Temporal gyrus Ipsi 3.72 −52 −56 10 0.18 0.5487 3.41 −32 −22 14 2.90 0.0196 Supramarginal gyrus Ipsi 4.59 −64 −28 20 0.51 0.0035 3.13 −31 −24 30 2.79 0.0055 Cerebellum Contra 3.36 20 −70 −50 0.31 0.0246 3.16 2 −44 −6 2.72 0.1634 Opercular cortex Ipsi 5.23 −50 −28 26 0.50 0.0018 2.69 −27 −12 13 2.23 0.0710 Postcentral gyrus Ipsi 4.71 −62 −18 24 0.44 0.0375 3.52 −31 −15 41 2.12 0.0845 Thalamus Ipsi 3.52 −12 −14 10 0.42 0.0018 3.48 −1 −20 13 1.67 0.1009 Angular gyrus Ipsi 3.59 −58 −50 18 0.53 0.0107 2.98 −23 −39 33 1.56 0.0528 Precentral gyrus Ipsi 4.01 −58 0 10 0.43 0.0578 3.46 −23 −17 48 1.53 0.1247 Frontal gyrus Contra 3.88 58 12 0 0.56 0.0212 3.11 11 −12 48 1.42 0.0646 Cingulate gyrus Posterior 3.71 −14 −28 38 0.08 0.2480 3.18 −9 −23 35 1.42 0.1101 Angular gyrus Contra 3.71 60 −46 18 0.54 0.0080 3.12 22 −51 35 1.42 0.0407 Precuneous cortex Contra 3.60 16 −68 40 0.38 0.0714 3.70 5 −30 52 1.19 0.1623 Visual cortex Ipsi 3.82 −52 −70 10 −0.09 0.3758 2.59 −7 −40 11 1.17 0.1657 Brainstem 3.86 10 −26 −8 0.33 0.1710 2.99 −3 −27 −10 1.11 0.4350 Parietal lobule Contra 3.10 20 −44 68 0.61 0.1097 3.10 27 −24 46 1.09 0.1271 Putamen Ipsi 3.63 −16 10 −2 0.45 0.0023 3.13 −14 −14 19 0.92 0.2813 Supplementary motor area Ipsi 3.55 −6 4 44 0.40 0.0219 3.16 −4 −10 46 0.91 0.3903 Precentral Gyrus Contra 4.05 58 4 8 0.44 0.0276 3.76 6 −20 53 0.88 0.2672 Frontal gyrus Ipsi 3.57 −8 22 32 −0.24 0.1954 2.79 −13 −9 50 0.70 0.4820 Pallidum Contra 3.40 16 −4 −4 0.49 0.0071 2.84 13 −13 13 0.64 0.4863 Active regions in adults only Amygdala Contra 3.49 20 −2 −14 0.69 0.0160 Amygdala Ipsi 4.28 −20 −2 −12 0.43 0.0860 Orbitofrontal cortex Ipsi 3.40 −18 4 −16 0.42 0.0157 no activity Orbitofrontal cortex Contra 3.57 34 30 −2 0.44 0.0460 Active regions in infants only Precuneous cortex Ipsi 3.80 −1 −26 52 1.26 0.1699 Pallidum Ipsi 3.16 −8 −5 14 0.59 0.4787 Parietal lobule Ipsi 3.31 −28 −23 33 0.99 0.2711 Auditory cortex Contra 2.89 26 −14 18 3.07 0.0119 Auditory cortex Ipsi 3.34 −17 −29 19 2.56 0.0304 Caudate Contra no activity 3.61 13 −17 22 0.59 0.5822 Caudate Ipsi 3.47 −7 −8 18 1.05 0.3415 Hippocampus Contra 2.61 21 −25 9 1.84 0.1288 Hippocampus Ipsi 2.77 −15 −31 9 1.00 0.3326 Parahippocampus Contra 3.02 11 −23 0 1.53 0.3740 Parahippocampus Ipsi 2.99 −7 −24 −8 0.19 0.9013

Although the infant brain activity was widespread, the specificity of the response was demonstrated, as it was not present across all brain regions. For example, brain regions not commonly associated with the cerebral processing of nociceptive stimulation in the adult, such as the olfactory cortex, cuneus, and fusiform gyrus, were also not active in the infants. 14% of voxels across the whole brain were active following the application of the 128 mN stimuli in infants compared with 9% of voxels following the 512 mN stimuli in adults (Figure 2). In contrast, the 128 mN stimulus activated less than 1% of voxels in the adult brain. This demonstrates that the coverage and distribution of brain activity evoked by the 128 mN stimulus in infants was most similar to that evoked by the 512 mN stimulus in adults (Figure 2). This suggests that infants have increased sensitivity to nociceptive stimuli compared with adults, which is supported by previous data that show spinal nociceptive reflex withdrawal activity has greater amplitude and duration in infants compared with adults (Andrews and Fitzgerald, 1999; Skljarevski and Ramadan, 2002; Cornelissen et al., 2013). These data strongly imply that the threshold for evoking widespread nociceptive brain activity in infants is substantially lower than in adults. It is, however, not known whether the increased brain activity observed at a lower threshold in the infants is due to increased peripheral drive, for example due to differences in skin thickness between the adult and infant populations, or due to differences in transduction or subsequent central processing of the nociceptive input.

Figure 2 with 1 supplement with 1 supplement see all Download asset Open asset Noxious-evoked brain activity in response to the maximal presented stimulus in adults (512 mN) and infants (128 mN). Red-yellow coloured areas represent active brain regions (threshold z ≥ 2.3 with a corrected cluster significance level of p < 0.05). An image of a midline sagittal brain slice (right panel) identifies the location of each example slice in the horizontal plane. (A) Adult activity is overlaid onto a standard T1 weighted MNI template and (B) infant activity is overlaid onto a standard T2 weighted neonatal template, corresponding to a 40-week gestation infant. https://doi.org/10.7554/eLife.06356.005

Noxious stimulation in infants did not evoke activity in the amygdala or orbitofrontal cortex (OFC) (Table 1), and in contrast to the adults, where activity was present across all divisions of bilateral insular cortices, activity in the anterior division was not present (Figure 1). A recent white matter tractography study of the adult brain shows that the anterior insula has dominant connections with the OFC (Wiech et al., 2014). Based on many imaging studies spanning a range of stimuli and tasks, it is thought that activation in the anterior insula reflects the net evaluation of the affective impact of an impending situation. Similarly, the OFC is sensitive to stimuli with an emotional valence, however, it primarily responds to the reward value of the stimulus (including negative value) rather than its sensory features. Importantly, the OFC also encodes the anticipation of future outcomes, which makes it well suited for guiding subsequent decisions (Kahnt et al., 2010). It is likely that the infants are too immature and inexperienced to evaluate and contextualise the nociceptive stimulus into a coordinated decision and response, which might account for the lack of activity within these regions. Similarly, in adults the amygdala is thought to attach emotional significance to the nociceptive inputs it receives, and to play a role in fear and anxiety (Simons et al., 2014), which may reflect affective qualities that the newborn infant does not yet ascribe to the stimulus.

In light of these observations, it is plausible that infants do not experience the full range of aversive qualities that adults associate with nociceptive input. Indeed, this hypothesis is supported by evidence from rat pups, which shows that avoidance behaviour in a fear-conditioning paradigm does not manifest until postnatal day 10, and is associated with the enhancement of neural activity within the amygdala (Sullivan et al., 2000; Sullivan, 2001). Nevertheless, the observation that brain structures involved in affective processing, such as the anterior cingulate cortex, are activated following noxious stimulation suggests that infants do have the capacity to experience an emotionally relevant context related to incoming sensory input. Indeed, in adults the modulation of pain-related activity in the anterior cingulate cortex closely parallels a selective change in perceived unpleasantness (Rainville et al., 1997).

11 brain regions significantly encoded stimulus intensity in adults, whereas none of the active regions in infants exhibited significant intensity encoding (Table 1). Although the trend for intensity encoding in infants is clearly evident in some brain regions, these data suggest that infants do not discriminate stimulus intensity as well as adults (Figure 2—figure supplement 1). As only three stimulus intensities were applied to the infants it is plausible that if the intensity range were increased, significant intensity encoding may be observed. Nevertheless, when considering adult brain regions that did significantly intensity encode, three of the four highest ranked brain regions (ranked according to the degree of intensity encoding, and identified as the contralateral temporal gyri, opercular cortex, and all divisions of the insular cortex), were ranked in the same order within the top three regions in infants, highlighting the remarkable similarity in how the immature infant brain and adult brain encode nociceptive information (Table 1). Intensity encoding has been reported following low intensity von Frey hair stimulation (Williams et al., 2015).

Inferences about the subjective experience of pain are highly speculative, whether based on brain imaging data, behavioural responses or other autonomic or physiological observations. In most adults, where the pain experience can be communicated verbally, it is not always necessary to rely on surrogate measures when attempting to quantify an individual's pain experience or when assessing the need for analgesic provision. However, where verbal report is not possible as in the infant population or in those who are cognitively impaired, reliance on surrogate measures is essential when making inferences about pain perception. As cortical activation is a fundamental requirement for an experience to be interpreted as painful, inferences based on patterns of brain activity may provide the most reliable surrogate measure of pain compared with alternative approaches based on behavioural and physiological indicators that may not be reliably linked to central sensory or emotional processing in the brain (Oberlander et al., 2002; Ranger et al., 2007). This does not, however, negate the importance of taking a multidimensional approach to infant pain assessment by considering measures of brain activity in the context of other well-characterised behavioural and physiological indicators. Indeed, some researchers have argued that reverse inference based on brain imaging results should be used merely as a guide to direct further enquiry rather than a direct means to interpret results (Poldrack, 2008). Nevertheless, it has been shown using multivariate pattern analysis that pain-related brain activity can be classified and discriminated from other psychological states, suggesting a neural state for pain perception that is distinct from other sensory modalities and affective experiences (Yarkoni et al., 2011; Wager et al., 2013). Although we cannot necessarily infer an infant's subjective experience based on a given pattern of brain activity, these results make certain conclusions more likely. The closer the pattern of brain activity mimics activity observed in adults—who can report their subjective experience—the stronger the inference. The patterns of brain activity observed in this study make it likely that the infant experience is similar to that described by adults.

Pain is defined as an unpleasant sensory and emotional experience. This study provides the first demonstration that many of the brain regions that encode pain in adults are also active in full-term newborn infants within the first 7 days of life. This strongly supports the hypothesis that infants are able to experience both sensory and affective aspects of pain, and emphasizes the importance of effective clinical pain management.