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.