Results
The sensory and multisensory properties of neurons in PPr (Fig. 1A) were electrophysiologically recorded in 18 male ferrets that ranged in age from P80 to P300. A total of 538 PPr neurons were tested with visual, tactile and combined visual-tactile stimulation presented in a random, interleaved manner. Fig. 1B depicts the experimental setup. Recorded neurons were grouped by their response patterns into the following categories: unimodal visual (V), unimodal tactile (T), bimodal (VT) and subthreshold multisensory (S). Fig. 1C shows representative examples of PPr neurons from each of these categories. Neuronal responses defined as multisensory integration (MSI) showed a statistically significant response change (either depression or enhancement) evoked by a combined visual-tactile stimulus when compared to that elicited by the most effective unimodal cue (Meredith & Stein, 1983). Examples of responses showing MSI are illustrated in the raster/histograms shown in the middle row of Fig. 1C. In addition, subthreshold multisensory responses are defined as a statistically significant response change elicited by combined visual-tactile stimulation when a neuron was activated by only one sensory modality presented alone. Examples of such subthreshold multisensory responses are shown in Fig. 1C (bottom row).
To evaluate the incidence and distribution of the different neuronal categories during postnatal development, the sampled PPr neurons were sorted by age-group. As defined in Methods, animals were divided by age in the following groups: Infancy, (P80-P90; Neurons/Animals=49/2), Early adolescence (P120-P155; 241/6), Mid adolescence (P160-P200; 179/6) and Late adolescence (P240-P300; 69/2). As shown in Fig. 2, these results show that, although each neuronal response type was observed in each age group, their relative proportions systematically changed. Chi-square tests (with Bonferroni corrections for multiple comparisons) showed that the Infant group exhibited more unimodal V but fewer bimodal VT cells than all the other groups (P<0.008). All other comparisons did not reach significance. Figure 2B replots this same data to emphasize the progressive change in the proportions of neurons in each response category. From this it is evident that the proportions of unimodal V cells markedly reduce from infancy to early adolescence and decrease even further through late adolescence. In contrast, the number of bimodal VT cells dramatically increase from infancy to early adolescence and these numbers remain high through late adolescence. Likewise, the incidence of the other form of multisensory neuron, the subthreshold category, also proportionally increases steadily during development.
Next, the level of neuronal responsiveness across postnatal development was examined. The average number of spikes evoked by sensory stimulation was collated by age group, as illustrated in Fig. 3 and summarized in Table 1. In general, response activity tended to increase from infant to early adolescence but then decreased through late adolescence.
For unimodal neurons, responses were only significantly different between early and mid-adolescence stages (univariate ANOVA, f = 4.7, p = 0.004; followed by post hoc Bonferroni test, p = 0.002). In bimodal neurons, the early-adolescence group showed significantly higher responses than all other age groups (univariate ANOVA, f = 7.2, p < 0.001; followed by post hoc Bonferroni test; Early vs Infancy, p = 0.03; Early vs Mid, p = 0.02; Early vs Late, p = 0.001). No significant difference in responsiveness across ages was observed in subthreshold neurons.
Next, we evaluated the rates of spontaneous activity over the course of development. This was calculated by recording the number of spikes per second fired during the 500ms interval before the onset of the sensory stimulus for each age group. These results are illustrated in Fig. 4 and summarized in Table 2.
No significant differences in spontaneous activity were observed between age groups in unimodal and subthreshold neurons. In bimodal neurons, the Late-adolescence group showed significantly higher spontaneous activity (univariate ANOVA, f = 4, p = 0.008; followed by post hoc Bonferroni test) than Early (p = 0.03) and Mid-adolescence (p = 0.004). That spike rates are elevated in bimodal PPr neurons has been reported previously (Foxworthy et al., 2014).
Next, we quantified the proportion of bimodal neurons that exhibited significant MSI. No bimodal neurons showed integration during infancy but, as shown in Figure 5A, the proportions of bimodal neurons that exhibit MS enhancement gradually increase during development (Infancy=none, Early=23%, Mid=28%, Late 67%). Only a single example of multisensory depression was observed among the bimodal neurons sampled, which occurred in the early adolescence group. In the context of the levels of MSI, an increase in the magnitude of response change was observed from early to mid-adolescence and remained constant after that. Figure 5B shows that the magnitude of enhancement was significantly higher for bimodal neurons during mid- and late-adolescence when compared to early. Kruskall-Wallis non-parametric ANOVA showed significant differences (KW-H= 10.07, p <0.001). Post Hoc Mann Whitney tests comparisons between groups showed significant differences between Early vs Mid (Z= -2.87, p = 0.004) and Early vs Late (Z= -4.21, p <0.001) but not Mid vs Late (Z = -0.71, p = 0.48). Median response changes in these groups: Early= 32.7%; Mid= 49.9%; Late= 51.3%.
Not all bimodal neurons exhibit MSI in response to effective multisensory stimulation. Neurons presenting small changes in firing in response to combined visual-tactile stimulation may fail to achieve the statistical significance that defines MSI. Nevertheless, these non-integrative responses contribute to the population response of an area and small changes in response can collectively produce a major change at the population level. Therefore, it seems conceivable that population responses can reveal differences not observed when only neurons that meet MSI criteria are considered (Keum et al. , 2023). Fig 5C shows response changes in bimodal neurons that failed to show MSI. Importantly, these non-integrative response changes tended to increase during development, such that the median value of multisensory response change was: Infancy= 9.8%; Early= 6.4%; Mid= 13%; Late= 15%. Kruskall-Wallis non-parametric ANOVA showed that the differences observed were significant (KW-H= 10.07, P<0.001). Post Hoc Mann Whitney tests comparisons between groups showed significant differences between Infancy Vs Mid (Z= -2, p = 0.037); Infancy Vs Late (Z= -2, p= 0.036); Early Vs Mid (Z=-2.4, p =0.016); Early Vs Late (Z= -2.4, p = 0.018).
A similar, progressive increase in magnitude of multisensory response change is shown in Fig. 5D which displays cumulative probability curves of the multisensory response change measured for all bimodal neurons (integrative and non-integrative) from the different age groups. In this figure, a value of zero indicates that the combined visual-tactile stimulation resulted in a number of spikes that was equal to the most effective unimodal stimulus, while positive and negative values indicate increases or decreases in response changes, respectively, regardless of statistical significance. The cumulative response curves shown in Figure 5D consistently shift upwards from infancy to late adolescence. A Kruskall-Wallis non-parametric ANOVA (KW) followed by post-hoc Kolmogorov-Smirnov tests showed that all groups were significantly different from each other (p<0.05 for all comparisons).
Last, we evaluated the development of subthreshold multisensory neurons (Fig. 6). Only a single subthreshold neuron was observed within the infant age-group (which showed MS enhancement), while progressively more examples of subthreshold responses were observed during the adolescent periods (Early, 3.3% of PPr neurons; Mid, 7.6%; Late 12.5%; also illustrated in Figure 2B). For all subthreshold responses to combined VT stimulation, the median magnitude of multisensory response change during the adolescent periods are shown in Figure 6A: Early (41%), Mid (64%), Late (54%) with no statistical significance observed between these groups. Overall, subthreshold multisensory responses were most frequently enhanced while only a few showed response depression (Early: 3 out of 14 neurons with an average depression of -29%±4.2; Mid: 1 out of 14 neurons that showed a depression of -22%). Figures 6B and C summarize the levels of multisensory enhancement and multisensory depression, respectively, observed in subthreshold neurons; no statistical differences in proportion or effects were observed between these age groups.