Rapid Postnatal Expansion of Neural Networks Occurs in an Environment of Altered Neurovascular and Neurometabolic Coupling

Abstract

In the adult brain, increases in neural activity lead to increases in local blood flow. However, many prior measurements of functional hemodynamics in the neonatal brain, including functional magnetic resonance imaging (fMRI) in human infants, have noted altered and even inverted hemodynamic responses to stimuli. Here, we demonstrate that localized neural activity in early postnatal mice does not evoke blood flow increases as in the adult brain, and elucidate the neural and metabolic correlates of these altered functional hemodynamics as a function of developmental age. Using wide-field GCaMP imaging, the development of neural responses to somatosensory stimulus is visualized over the entire bilaterally exposed cortex. Neural responses are observed to progress from tightly localized, unilateral maps to bilateral responses as interhemispheric connectivity becomes established. Simultaneous hemodynamic imaging confirms that spatiotemporally coupled functional hyperemia is not present during these early stages of postnatal brain development, and develops gradually as cortical connectivity is established. Exploring the consequences of this lack of functional hyperemia, measurements of oxidative metabolism via flavoprotein fluorescence suggest that neural activity depletes local oxygen to below baseline levels at early developmental stages. Analysis of hemoglobin oxygenation dynamics at the same age confirms oxygen depletion for both stimulus-evoked and resting-state neural activity. This state of unmet metabolic demand during neural network development poses new questions about the mechanisms of neurovascular development and its role in both normal and abnormal brain development. These results also provide important insights for the interpretation of fMRI studies of the developing brain.

Significance statement: This work demonstrates that the postnatal development of neuronal connectivity is accompanied by development of the mechanisms that regulate local blood flow in response to neural activity. Novel in vivo imaging reveals that, in the developing mouse brain, strong and localized GCaMP neural responses to stimulus fail to evoke local blood flow increases, leading to a state in which oxygen levels become locally depleted. These results demonstrate that the development of cortical connectivity occurs in an environment of altered energy availability that itself may play a role in shaping normal brain development. These findings have important implications for understanding the pathophysiology of abnormal developmental trajectories, and for the interpretation of functional magnetic resonance imaging data acquired in the developing brain.

Keywords: GCaMP imaging; fMRI; flavoprotein fluorescence; functional hyperemia; neurovascular coupling; oxygen consumption; postnatal neural development.

Figure 1.

GCaMP expression is observed in neurons of layers 2/3 and 5 of Thy1-GCaMP3 mice in all three age groups. Images of coronal slices (10 μm) stained with DAPI taken from P7, P10, and adult (P120) mice. White boxes on images on left indicate regions magnified in images on right .

Figure 2.

Spatiotemporal analysis of GCaMP fluorescence responses in the adult brain to three types of somatosensory stimulation. A, Left, Grayscale images of a representative adult Thy1-GCaMP3 mouse. Bregma suture lines are marked with white lines; boxes represent expected regions of neural responses based on stimulation type and side. Right, Time sequences of GCaMP responses during the initial stimulation period for three different types of unilateral, 4 s stimuli (note, electrical and tactile hindpaw stimulation were performed on the right and left hindpaws respectively to permit randomized collection of all data in the same animal). B, Analysis of neural response onset times. Onset time calculated as the time of the peak of the first derivative of the time course of each pixel. Left, Grayscale image from representative GCaMP3 mouse. Right, Map depicting the onset timing of the GCaMP response for each pixel that reached ≥40% of the peak response. Numbers indicate the sequence of propagations of the neural response: 1, contralateral hindpaw region; 2, ipsilateral hindpaw region; 3, close to the contralateral visual cortex. C, Relative onset time comparing regions 2 and 3 to the origin of the response (region 1: hindpaw region of the contralateral somatosensory cortex and region of peak neural response).

Figure 3.

Spatiotemporal progression of neural network development. A, The developing neural response to unilateral hindpaw stimulation. Top, Grayscale images of representative P7, P11, P13, and adult mice. Bregma suture lines are marked with white lines. Bottom, GCaMP response maps comparing 0–2 s poststimulus onset to baseline. B, Normalized GCaMP responses to 4 s hindpaw stimulation for P7–P8, P10–P13, and adult age groups (n = 5, 6, 6 mice) extracted from the contralateral hindpaw region of the somatosensory cortex. Error bounds show SEM. C, Analysis of neural response onset times. Top, Grayscale images of representative animals. Bottom, Maps depicting the onset timing of the GCaMP response for each pixel that reached ≥50% of the peak response. Numbers and arrows indicate the sequence and directions of propagations of the neural response, all indicating initial onset in the hindpaw region of the somatosensory cortex contralateral to stimulation. D, GCaMP response onset time (measured as the peak of the first derivative of the time courses) decreases significantly between the P7–P8 and P10–P13 age groups (p < 0.005, Student's t test). E, Spatial spread of the neural response increases with age (p < 0.005 comparing the P7–P8 and P10–P13 age groups to the adult), calculated as the percentage of pixels that reach ≥50% of the peak response pixel. All error bounds and bars show SEM.

Figure 4.

Neural responses are not accompanied by functional hyperemia in the neonatal mouse brain. A, Averaged hemodynamic and GCaMP time courses from the somatosensory cortex of Thy1-GCaMP3 mice in P7–P8, P10–P13, and adult age groups (n = 5, 6, 6 mice respectively). HbO, HbR, total hemoglobin (HbT), and GCaMP fluorescence (Ca²⁺) time courses were extracted from the region of maximal neural responses to unilateral hindpaw stimulation in each mouse (B, white boxes). Error bounds show SEM. B, Grayscale images of representative mice from each age group. Bregma suture lines are marked with white lines. C, GCaMP response maps for the same mice comparing the time period 0–2 s poststimulus onset to baseline. D, Δ[HbT] functional maps for the same mice comparing the time period 2–4 s poststimulus onset to baseline (averages of n = 9, 12, 51 trials respectively). E, F, Peak amplitude (E) and area under the curve (F) of GCaMP and Δ[HbT] responses (for both the raw [HbT] signal and residual responses after subtraction of the global Δ[HbT] component; n = 5, 6, 6 mice respectively). Error bars show SEM, p values use Student's t test. Responses were extracted from the region of peak GCaMP response. G, H, Full-width half-maximum (FWHM) of GCaMP responses (G) and peak values of (raw) Δ[HbT] responses (H) to a 4 s hindpaw stimulus across age groups, compared with FWHM of adult GCaMP response to a 2 s stimulus (n = 5, 6, 6, 4 mice respectively).

Figure 5.

Local cerebral blood flow does not increase in response to stimulation in the neonatal mouse brain. A–C, Averaged time courses of Δ[HbO], Δ[HbR], Δ[HbT], and %Δ cerebral blood flow from laser speckle-flow measurements from the contralateral hindpaw region of the somatosensory cortex of mice in P7–P8 (A), P10–P13 (B), and adult (C) age groups (n = 5, 4, 3 respectively).

Figure 6.

Awake and anesthetized postnatal mice exhibit robust neural responses to tactile hindpaw stimulus with minimal functional hyperemia. A–C, Left, Grayscale images of representative urethane-anesthetized, isoflurane-anesthetized, and awake mice. Bregma suture lines are marked with white lines. Right, GCaMP response maps for the same mice comparing the time period 0–0.2 s, 0.2–0.4 s, and 0.4–0.6 s poststimulus onset to baseline (n = 9, 6, 2 trials respectively). Note: GCaMP6 mice were used for these experiments. D, Peak amplitude of GCaMP and Δ[HbT] responses (n = 5, 3, 4, 3 mice respectively) in P7–P8 mice to electrical and tactile stimulus. Error bars show SEM; p values use Student's t test comparing HbT responses to electrical stimulation. Responses were extracted from the region of peak GCaMP response. E, Comparison of peak amplitude of GCaMP and Δ[HbT] responses in a single adult mouse to electrical and tactile stimuli.

Figure 7.

Age dependence of stimulus-evoked changes in oxidative metabolism. A, Averaged hemodynamic and FAD response time courses in wild-type mice during unilateral hindpaw stimulation (n = 5, 6, 6 mice respectively), extracted from region of peak FAD response (B, white boxes). Error bounds show SEM. B, Grayscale images of the cortex for a representative mouse from each age group. Bregma suture lines are marked with white lines. C, D, FAD functional response maps for the same mice comparing the time period 1.5–2.5 (C; light phase) and 3.5–4.5 s poststimulus onset (D; dark phase) to baseline. E, Δ[HbT] functional map for the same mice comparing the time period 2–4 s poststimulus onset to baseline.

Figure 8.

Quantitative comparison between neural and metabolic responses. A, Overlaid GCaMP and FAD time courses for each age group (different cohorts of mice). B, Comparisons between the onset delay and duration timing of FAD compared with GCaMP responses for each age. Onset delay time was calculated as the time from the start of the stimulus (O s) to the peak of the first derivative of the response. Response width was calculated as the full-width half maximum of the calcium or positive FAD responses respectively. C, Area under the curve of FAD responses increases significantly with age (p < 0.005 between P7–P8 and P10–P13 mice and p < 0.05 between P10–P13 and adult mice; Student's t test), while the area under the curve of the GCaMP response is approximately constant with age. All error bars show SEM.

Figure 9.

Subtraction of global signals reveals local oxygen consumption with minimal localized hyperemia. A, P8 global response. Left, Grayscale image, Bregma suture lines are marked with white lines. Center, Maps of the global components of the Δ[HbT] and Δ[HbR] responses (score of the first principal component). Right, Time courses of the global components. B, P8 local (residual) response. Left, GCaMP functional map for the time period 0–2 s following stimulus onset. Center, Residual Δ[HbT] and Δ[HbR] functional maps after subtraction of the global component for the period 4–6 s poststimulus onset. Right, Residual time courses of the response in regions which reached ≥50% of the maximum GCaMP response. C, D, Same as A and B for a representative P13 mouse. E, Average residual response time courses across animals after global subtraction for the younger two age groups with the GCaMP responses overlaid. Adult time courses are shown for reference (note the 15× expanded scale).

Figure 10.

Spontaneous neural activity increases with age. A, Right, Time courses of spontaneous (no stimulus) Δ[HbT], Δ[HbR], Δ[HbO], and GCaMP activity over a 3 min period for four representative animals extracted from the regions indicated by white boxes at left as either solid or dashed lines respectively (mean values, low pass filtered at 3 Hz for hemodynamics, maximum values for GCaMP high pass filtered at 5 Hz). Left, RGB merge maps (>50% peak) of three spontaneous GCaMP events (peaks indicated by arrow colors on the time course plots). The number and spatial spread of spontaneous events increases with age. Bregma suture lines are marked with white lines. B, The number of spontaneous events per second averaged across all animals within each age group, demonstrating a significant increase in spontaneous activity with age (p < 0.005, Student's t test, error bars show SEM). C, Time courses of neural events and corresponding hemodynamic activity (P7–P8: n = 5 mice, 15 trials, 161 events; P10–P13: n = 6 mice, 24 trials, 490 events; adult: n = 5 mice, 28 trials, 934 events). Note that the nonzero pre-event baseline in adult hemodynamics reflects the high frequency of neighboring events.