Development of Activity in the Mouse Visual Cortex

Abstract

A comprehensive developmental timeline of activity in the mouse cortex in vivo is lacking. Understanding the activity changes that accompany synapse and circuit formation is important to understand the mechanisms by which activity molds circuits and would help to identify critical checkpoints for normal development. To identify key principles of cortical activity maturation, we systematically tracked spontaneous and sensory-evoked activity with extracellular recordings of primary visual cortex (V1) in nonanesthetized mice. During the first postnatal week (postnatal days P4-P7), V1 was not visually responsive and exhibited long (>10 s) periods of network silence. Activation consisted exclusively of "slow-activity transients," 2-10 s periods of 6-10 Hz "spindle-burst' oscillations; the response to spontaneous retinal waves. By tracking daily changes in this activity, two key components of spontaneous activity maturation were revealed: (1) spindle-burst frequency acceleration (eventually becoming the 20-50 Hz broadband activity caused by the asynchronous state) and (2) "filling-in" of silent periods with low-frequency (2-4 Hz) activity (beginning on P10 and complete by P13). These two changes are sufficient to create the adult-like pattern of continuous activity, alternation between fast-asynchronous and slow-synchronous activity, by eye opening. Visual responses emerged on P8 as evoked spindle-bursts and neuronal firing with a signal-to-noise ratio higher than adult. Both were eliminated by eye opening, leaving only the mature, short-latency response. These results identify the developmental origins of mature cortical activity and implicate the period before eye opening as a critical checkpoint. By providing a systematic description of electrical activity development, we establish the murine visual cortex as a model for the electroencephalographic development of fetal humans.

Significance statement: Cortical activity is an important indicator of long-term health and survival in preterm infants and molds circuit formation, but gaps remain in our understanding of the origin and normal progression of this activity in the developing cortex. We aimed to rectify this by monitoring daily changes in cortical activity in the nonanesthetized mouse, an important preclinical model of disease and development. At ages approximately equivalent to normal human term birth, mouse cortex exhibits primarily network silence, with spontaneous "spindle bursts" as the only form of activity. In contrast, mature cortex is noisy, alternating between asynchronous/discontinuous and synchronous/continuous states. This work identifies the key processes that produce this maturation and provides a normative reference for murine-based studies of cortical circuit development.

Keywords: EEG; mouse; spindle-burst; spontaneous activity.

Figure 1.

Experimental design. A, Experimental setup. Recording from V1 using single-shank multielectrode array (Probe) in head-fixed nonanesthetized mice with the ground (Grd) in cerebellum. EMG wire was inserted into the rear neck muscle. Piezoelectric and temperature sensors are positioned under the belly of the animal (data not shown). Visual stimuli (Light) were 100 ms whole-field flashes to the contralateral eye. B, Representative L4 activity in a P13 mouse. Movement was monitored by EMG and Piezo device and these periods were eliminated from analysis to avoid electrical artifacts and equalize state between animals. Scale bar, 200 μV (LFP), 2 s. C, D, Mean visual-evoked responses were used for layer identification (P8–P24). Mean poststimulus MUA firing rate (C) and LFP (D) show an early response in L4, which was used as a reference to identify other layers. Time of light (100 ms) is shown below. Scale bars: C, 300 spikes/s; D, 300 μV. E, Representative spontaneous LFP from a mouse in C and D. L4 can be identified as the point of diminution for rapid oscillations. Scale bar, 500 μV, 500 ms. F, Representative spontaneous LFP from P5 mouse. L4 is identified by depth and diminution of rapid oscillations. Scale bar is as in E. G, Quantification of eye opening. Population mean and SEM are shown in black circles. Individual animals are shown in gray circles. Gray square shows the period of eye opening used in this study.

Figure 2.

Maturation of spontaneous activity in mouse V1. Representative LFP voltage traces and MUA rasters during quiet wakefulness. LFP is displayed for L4; MUA for L4 and channels 100 and 200 μm above and below L4. At P4, the cortex is mostly quiet and activity consists solely of spindle-shaped (spindle burst) oscillations that result from retinal wave activity (Colonnese and Khazipov, 2010). Spindle bursts become elongated and faster with development, becoming indistinguishable from rapid β-gamma activity by P12 (blue traces show examples). At P10, significant activity between spindle bursts is first observed as high-amplitude, low-frequency modulation (red traces show examples). This low-frequency activity increases in duration, alternating with high-frequency activity, until activity becomes continuous (no silent periods). Therefore, by P14, spontaneous activity is similar to mature (P24). Scale bar, 5 s (1.25 s for expanded traces); 500 μV (250 μV for expanded traces).

Figure 3.

Depth profile of spontaneous activity. Representative trough-triggered (left column) and spike-triggered (right column) mean LFP (red line), current source density (color map) and MUA (lower color map) for an animal at the shown age. Depth is shown relative to L4, the layer of triggering. For spike-triggered MUA, 0 ms spike rate is removed. From P4–P8, both spike-triggered and trough-triggered averages show local oscillation in LFP and MUA with a current sink and maximal spiking in L4. P10 and older shows activity occurring in more isolated events with full columnar involvement.

Figure 4.

Continuous activity emerges between P9 and P13. A, Log of MUA rate for input (L4, top) and deep (L5, bottom) layers by age. Firing rates displayed exponential growth that asymptotes at P13. ANOVA for effect of age (L4, p < 10⁻³; L5, p < 10⁻³). B, Continuity of firing measured as the proportion of time occupied by active periods (not containing network silences >200 ms) for L4 (top) and L5 (bottom). Both layers display rapid acquisition of adult values between P9 and P13, with L5 increasing continuous firing rapidly between P9 and P10. ANOVA for age (L4 and L5, p < 10⁻³). Gray lines show selected ages with significant difference (p < 0.05) by post hoc test. For clarity, not all groups with differences are marked. Gray rectangle indicates the period of eye opening (Fig. 1). Gray circles show individual animals. Black circles are population mean and SEM.

Figure 5.

Maturation of two activity patterns explains spontaneous activity development. A, Mean normalized L4 LFP spectra for each age group. Black lines are population mean, blue lines individual animal spectra. P4–P9 spectra are dominated by 5–20 Hz activity (blue arrow; spindle bursts), the primary frequency of which accelerates with age and merges with the dominant 1/f activity by P10. Low-frequency (2–8 Hz) activity becomes apparent at P10 (red arrow) and is prominent on P14–P15, when the spectra become similar to the spectra of juvenile mice during rest (Hoy and Niell, 2015). B, Whitened mean L4 spectra showing frequencies remaining when dominant 1/f power relationship is removed. Shown are increasing frequency and decreasing amplitude of rapid frequencies (blue arrow) and initiation at P10 followed by increasing amplitude of low-frequency activity (red arrow). C, Mean cross-frequency correlation. Shown is the matrix of correlation coefficients for each frequency band. Note the growth of highly correlated 5–15 Hz frequencies at P4–P5, which gradually shift to become correlated high frequencies by P12. Anticorrelation between high and low-frequency bands is observable by P12, showing the development of aroused and quiescent states. D, NMF of spectral signal reveals two developmental components. Thin lines show individual factors identified in each animal. Factors were sorted into clusters by density-based spatial clustering. Two clusters of factors were identified in the total population, which have been rendered in blue and red. Factor 1 (blue) was identified in all animals except P24 and accelerated with age. Spectra could first be separated into two factors on P10. This second factor (red) remains relatively constant with age. A third factor (pink) was isolated on P24 that appeared to contain rapid rhythms of factor 1 and theta-band activity.

Figure 6.

Evolution of spectral relationships. A, Total spontaneous LFP power (1.5–55 Hz) by age shows increasing amplitude of LFP. ANOVA for effect of age, p < 10⁻³. B, Development of negative cross-frequency correlation. Mean of the lowest 5% of cross-frequency correlations (Fig. 5C) shows the evolution of anticorrelated activity. Rise from P4–P8 is largely due to reduction of very high (30+) frequencies during all activity at young ages. Anticorrelation between low and high frequencies is reflected by drop from P8–P13, when significant anticorrelations emerge and reach mature levels ANOVA p < 10⁻³. C, Occurrence of 2 s windows with significant oscillations as determined by F-variance ratio test. The percentage of periods with activity containing each oscillation are shown by color scale. Note high occurrence and acceleration of oscillations at P4–P9 and the relative paucity of significant oscillations after. In older animals, low-frequency oscillations are observed from P12 to P24. D, Weighted mean frequency of spectral factors identified by NMD (Fig. 5D). Factor 1 occurred in all animals (except P24) and accelerated with age between P9 and P11, reaching plateau values by P13. ANOVA p < 10⁻³. Factor 2 was observed only starting at P10 and its frequency remained constant. ANOVA p = 0.23. Factor 3 (F3) was only observed at P24.

Figure 7.

Visual responses before eye opening are large, of long duration, and oscillatory. A, Representative mean flash-evoked L4 LFP at each age. Red line shows time of 100 ms flash onset. No responses were observed before P8. Scale bar, 100 μV, 500 ms. B, Representative poststimulus time histogram for L4 MUA from the same animal. Scale bar, 100 spikes/s. C, Representative poststimulus increase in spectral power for L4 LFP for the same animal. Color map depicts fold increase over baseline.

Figure 8.

Primary and secondary visual responses develop by different mechanisms. A–C, Representative L4 visual responses at three early ages showing the designations for the parts of the visual response. From top, mean evoked LFP, spectral increase and peristimulus MUA time histogram. The sharp, short-latency negative potential is called N1 and is followed by long-latency, long-duration N2. The MUA responses are called 1° (N1 + 1° = primary) and 2° (N2 + 2° = secondary), respectively. Note that the P8–P9 responses resemble secondary responses in older animals and are thus classified despite occurring first. At later ages, a positive peak is observed (P1), which is analyzed in Figure 9. D–H, Quantification of total (primary + secondary) L4 visual response development by age. For all panels, gray rectangle indicates period of eye opening (Fig. 1). D, Presence or absence of significant visually evoked MUA. E, Summed 1–50 Hz spectral power during the 5 s after stimulation (ANOVA for effect of age p < 10⁻³). Evoked spectral power in the LFP is first significantly different from neonates on P9 and remains stably high until decreasing to mature levels between P12 and P14–P15. F, Total duration of elevated spiking (spike rate >1 SD of baseline mean; p < 10⁻³). Visual responses become very long on P11 before decreasing to adult levels by P14. G, Time-amplitude integrated change in MUA firing rate (total area of gray bars in C and D expressed as the fold increase over baseline; p < 10⁻³). Early responses are very large but shrink rapidly, becoming similar to mature values by P11 despite increasing duration. H, Latency to MUA response (p < 10⁻³). Early responses are long latency, but reach values similar to mature values by P11. I–M, Quantification of N1/1°. I, Presence or absence of N1 component of visual evoked L4 potential. J, Peak negative amplitude of mean visual evoked potential (0–200 ms; p = 0.0053). N1 grew in size steadily during development and did not reach clear asymptote within time period of study. K, Peak 1° MUA (0–200 ms after stimulus; p ≪ 10⁻³). L, Negative slope of N1 (p ≪ 10⁻³). Rapid increases in slope were observed between from P12 and P16–P17. M, Duration of N1 (p < 10⁻³). After rapid sharpening between P9 and P10, duration decreased steadily.

Figure 9.

Immature, large-amplitude secondary responses disappear by eye opening. A, Representative peristimulus MUA time histograms (P10–P14) showing secondary responses. Total mean firing rate for L4 is displayed in gray, with firing elevated (>1 SD) over baseline shown in blue. The 2° duration (C) is the number of seconds of blue signal; the 2° MUA area (D) is the total area of blue. Scale bar, 200 spikes/s, 2 s. B, Fold increase in LFP spectral power during secondary response. Population mean (red line) and SEM (shaded) for animals at indicated age. Scale bar, 4×. Elevated spectral power distribution is similar to the rapid oscillation pattern identified during spontaneous activity (Fig. 4). Evoked oscillation power drops between P12 and P14, similar to the loss of spontaneous rapid frequencies at the same ages. C, Duration of 2° spiking response (ANOVA for effect of age, p < 10⁻³). Secondary spiking responses shorten between P12 and P14. D, Integrated area of 2°spiking (p = 0.014). Evoked spiking peaks at P13 before decaying to adult levels. E, Peak frequency of N2 (p < 10⁻³). Like spontaneous activity, evoked N2 oscillations began as spindle bursts and steadily increased to broadband β-gamma frequencies. F, Peak fold increase in spectral power of N2 (p < 10⁻³) is high through the pre-eye-opening period.

Figure 10.

Development of feedforward inhibition correlated to the loss of secondary response. A, Representative peristimulus MUA time histogram (P10–P14) showing inhibition of spiking between 1° and 2° response (red arrow). The location is determined by lowest spike rate after the 1° response. Scale bar, 100 spikes/s, 50 ms. Black bar indicates stimulus. B, Amplitude of inhibition as change from baseline in spikes/s (ANOVA for effect of age, p < 10⁻³). Visually evoked inhibition of spiking develops between P12 and P14, the same time evoked spindle bursts disappear. C, Latency of inhibition (minimum spike rate after 1° response; p < 10⁻³). Development of light-evoked inhibition shortens latency, which now occurs consistently after the initial response.

Figure 11.

Depth profile of visual evoked responses. A, Left column, Representative single visually evoked response from an animal at key ages. For each animal, the LFP (red line) is overlaid on current source density map (blue is the sink; yellow is the source). Depth of traces is indicated at left; channel number at right. Tetrode configuration for spike recordings is also shown. MUA spike rasters for each tetrode are shown below. A, Right column, Mean visual evoked response for the same animal. MUA spike rates are shown on a color scale. B, Population mean and mean visual response. Depth is aligned to L4 (left).

Figure 12.

Differential timing of SNR for visual response components. A, Representative peristimulus MUA time histogram (P13) showing the calculation of SNRs of primary (1°, top, red) and secondary (2°, bottom, blue) responses. Mean baseline spike rate is used to calculate the expected spike rate (light red/light blue) and SNR, the ratio of the increased spike rate to baseline, is indicated in dark red/dark blue. Values of 7:1 for 1° and 1:1 for 2° are shown. Scale bar, 100 spikes/s. The thick black bar shows the visual response (100 ms). B, SNR by age for 1° (red, ANOVA for effect of age p < 10⁻³), 2° (blue, p < 10⁻³), and inhibitory spike suppression (black, p < 10⁻³). 1° and 2° response development were best fit by an exponential with similar decay but different asymptotes. Inhibition was fitted as a sigmoid with an inflection between P12 and P14–P15. Visual response SNR decreases before total response amplitude decreases around P14 (Fig. 7-8), which is correlated with the emergence of inhibition.

Figure 13.

Two processes describing the developmental origins of cortical activity. Synthesis of evoked and spontaneous cortical activity development in the mouse V1. Activity originates as spindle burst oscillations (6 Hz) produced in response to thalamocortical input, which in V1 are generated by retinal waves during the first postnatal week. Circuit changes cause acceleration of these oscillations until they become the broadband high frequencies caused by asynchronous firing in the cortex in response to visual input and arousal. Before the development of feedforward inhibition at P14, thalamocortical input causes long duration rapid activity that can outlast the stimulus (yellow). A second process leads to the development of ongoing activity in the absence of strong input. This activity begins at P10, taking the form of isolated slow waves that become continuous by P12. By P14 (eye opening), slow activity and rapid activity alternate in cortex with arousal state and sensory input.