Spontaneous neuronal activity was recorded from the monocular region V1 of head-fixed unanesthetized rats between post-natal day (P)5 and 13. Recordings were made during all states of vigilance, though periods of active movement were not routinely analyzed as a caution against movement artefacts. To fully characterize network activity we used DC extracellular recordings of the local field potential (depth EEG) and multiple unit activity (bandpass 0-5000 Hz) from layer 4 (depth 300-500 μm from the cortical surface).
In P10 and P11 rats, the most prominent feature of the depth EEG was the presence of steadily recurring large-amplitude negative infra-slow potentials ( and for Wistar examples, and and for Long-Evans examples). These negative potentials had a median duration of 8.75 s (6-18 s min-max, n = 80 events from 8 pups), and a median interval of 60 s (41-72 s min-max) that were not different between strains. The infra-slow wave was composed of multiple shorter events () that resembled ‘spindle-bursts’ previously described during the first post-natal week (Hanganu et al, 2006
). The leading negative phase contained 3-15 (median = 5) spindle-bursts (duration 0.5- 3 s, median = 0.95 s) of field-potential oscillation in the beta-band (17 – 29 Hz peak frequency, median = 21 Hz). These bursts of beta-band oscillation were separated by short periods (0.5 – 1.5 s) of reduced activity within the larger infra-slow potential. Multi-unit activity (MUA) occured largely during the spindle-bursts, and was strongly correlated with the negative troughs of the beta oscillations ().
Recurring Slow Activity Transients (SATs) in visual cortex of infant rats
Beta-oscillations during SATs synchronize activity in superficial layers
As described above, shorter (0.5-3 s) spindle-shaped bursts of alpha/beta-band oscillation, named ‘spindle-bursts’ have been previously recorded during the first post-natal week in V1 and S1. When DC recordings are made a delta-band component of spindle bursts is observed in S1 (Marcano-Reik and Blumberg, 2008
; Minlebaev et al., 2009
). However these negative potentials are much shorter than the infra-slow waves, described here in an immature animal model for the first time. In fact, these infra-slow events events resemble multiple spindle-bursts occuring in rapid succession. While spiking events with similar characteristics have been described as ‘macro-bursts’ in ferret cortex (Chiu and Weliky, 2001
), for the purposes of this paper we will refer to these events as ‘rat visual cortex (rv)SATs’, or just SATs for short, to emphasize their similarity to the human EEG.
As observed for the first post-natal week (Hanganu et al., 2006
), spontaneous activity was characterized by long-periods of quiescence between SATs, and isolated action potential activity in was rare with the exception of a population of large units in presumptive layer 5a (see methods) which demonstrated persistent tonic activity. Unlike the first post-natal week, however, from P9 we observed spontaneous neuronal activity not associated with spindle bursts/SATs (). These shorter electrographic events consisted of simple field negative shifts with strong multiple-unit activity. Unlike SATs and spindle-bursts, these short bursts did not display rhythmic oscillations of either the field potential or MUA. We have not analyzed these events in depth in the present study, but instead focus on the origins and characteristics of the SATs.
Development of SATs and other cortical activity patterns
To characterize the development of SATs and other activity, we systematically recorded spontaneous activity between P5 and P13 (one day before eye opening). Spontaneous activity between P5-7 (n=50 events from each of 11 animals) was also marked by the presence of recurring SATs (). The appearance and duration of the infra-slow component was more variable at these ages, but we observed many SAT with long duration (>5 s) negative waves similar to those observed during the second week; however, the beta oscillations and MUA activity within these waves was shorter (median 2.2 s) resulting in a long return of the field potential to baseline during which there was no significant neuronal activity. The other primary difference between the P5-7 and P10-13 animals was the absence of short bursts. While shorter events (400 - 1000 ms) were common, these events always had a prominent oscillatory component similar to the longer events, suggesting they are similar network events of varying length ().
Development of SATs and short bursts
We examined the developmental trajectory of this apparent dichotomisation of activity by quantifying the duration of all observed events between P5 and 13 (at least 200 events from 3-4 animals at each age). Duration was defined by the continued negative deflection of the DC field potential occurring in combination with an elevated MUA rate. The most apparent change during this period was a widening of the dynamic range for event duration (). Very long events (>5 s) were first observed on P8 and continued until the end of the recording period. Short events (<400ms) were first commonly observed on P9 and become promient on P10. Their occurrance continued to increase until P13 by which time they were the large majority of spontaneous events (). Another dramatic change in the statistics of spontaneous activity was the establishment of a clear separation of short bursts and SATs, as the number of medium length (2 – 5 s) events was strongly reduced around P10 ().
This splitting of events with maturation was also observed in the relationship between event duration and beta-oscillations (). From P5-7 all events, regardless of duration displayed a strong rhythmic oscillation with a peak frequency between 8 and 31 Hz (n=50 events from each of 11 animals). There were two prominent distributions centered at 22Hz and 10Hz, though duration was not a determining factor in distinguishing events at each frequency, which likely arose as harmonics of the same oscillation. Thus in terms of duration and frequency all events between P5-7 appear to be drawn from a single distribution, quantitatively confirming previous assertion that spindle-bursts constitute a singular event in during the first week in visual cortex (Hanganu et al., 2006
). Between P10-11 the two populations of events were clearly sseparable by both peak frequency and duration, with almost all events greater than 5 s displaying a peak frequency between 18 and 30 Hz, while shorter events almost always had peak frequencies below 10 Hz. Thus by the second post-natal week at least two populations of events can be clearly distinguished.
We further estimated the amount of neuronal activity associated with SATs. Total counts of action potentials over entire recording session revealed that 87 +/- 5% (mean, S.D.; n=8 30 minute recordings P10-11) of layer 4 MUA activity occurred during SATs.
Begining at P12 (at least 100 events from 8 animals) spontaneous activity became much more continuous and the occurence of SATs become less regular. We observed long periods of repeating short events that now resembled the slow waves observed during intermediate and deep sleep. At these ages, while SATs still occurred, they were no longer the dominant activity pattern and could often not be separated from ongoing cortical activity. No SATs were observed in the rats older than P14 (30 min from n=15 P14 – P19).
The clear segregation of SATs from other activity patterns between P10-11 allowed us to study these events in isolation.
Retinal drive of SATs
We used enucleation of the contralateral eye to test the role of retinal activity in generating SATs. Baseline spontaneous activity was first recorded. Then, as a control for surgery, animals were anesthetized, allowed to recover for 30 minutes, and baseline spontaneous activity was again recorded. Following this, animals were enucleated under the same anesthesia and allowed the same recovery time. Transient anaesthesia had no effect on SAT production, but enucleation completely eliminated SATs (). Simultaneous recordings made in V1 ipsilateral to the enucleation showed no change (not shown). The distribution of event duration was quantified for 50 events from each of the 5 pups before and after enucleation (KS-test, control vs. enucleated, p < 0.0001). The elimination of SATs was accompanied by an increase in events of moderate duration (1-3 s; ). Interestingly, these moderate length events sometimes displayed prominent rapid oscillations in the field potential, and where similar to spindle-bursts recorded during the first postnatal week. We examined the effect of enucleation on the generation of rapid oscillations by measuring total frequency power (1-50 Hz) during 30 minute periods before and after enucleation. Enculeation strongly attenuated the frequencies associated with the rapid component of SATs (10-40 Hz; peak attenuation at 22 Hz), but increased in power in below 10 Hz, reflecting the increased occurence of short-duration events. Similar results were obtained after suppression of retinal network driven activity by intraocular injection of urethane (see below). In total these experiments suggest that while oscillatory events of short duration can be generated in visual cortex in the absence of sensory input, the generation of long-duration SATs requires retinal input.
Phase III retinal waves have a similar macro-structure as SATs, but lack beta-oscillations
We examined spontaneous activity in acutely excised retinas in vitro
(n=84 locations from 10 P10-11 retinas) for evidence of SAT-like activity (). Spontaneous retinal activity in the rats at this age was similar to the patterns decribed for phase III retinal waves in mice (Blankenship et al., 2009
; Kerschensteiner and Wong, 2008
). Spiking actvity in the ganglion cell layer was grouped into regularly repeating burst-clusters (median interval 53.5 s; median duration 9.5 s) composed of multiple sub-bursts (median 6). The distributions for the bursts-cluster duration and inter-burst-cluster intervals revealed extensive overlap and no significant differences from V1 SATs (Mann-Whiney, p>0.05; ). We examined the spike rate auto-correlations of the retinal bursts (5 ms bins; 500 ms window) for evidence of rapid oscillations similar to those observed during SATs. Unlike V1 SATs, retinal autocorrelation coefficients revealed only a steady decrease with time, and no evidence of beta-band oscillations ().
We tested the hypothesis that the occurrence of SATs is determined by retinal activity by increasing the occurrence of retinal waves via intra-ocular injection of bicculine (Bic) and strychnine (Str) which increases the rate of wave initiation in vitro (Blankenship et al., 2009
). Contralateral Bic (20μM) + Str (40μM) injection(5-10 μL) massively increased cortical activity, resulting in the constant production of alpha-beta oscillations (spindle-bursts) separated by short (1-5s) silent periods. Well defined SATs could not be resolved, and were replaced by constantly modulating negative potentials (). To quantify the effect we calculated the interval between individual bursts of alpha-beta oscillation (> 3 cycles separated by > 500 ms) following control injection of ACSF and again following Bic + Str injection. In 5/5 P10-11 pups the distribution of event intervals (50 per animal) was significantly shifted (K-S test, p < 0.001 in all cases) from a bimodal distribution to an exponential distribution (). In 4/4 animals in which we made simultaneous recordings from ipsilateral V1, no change was observed (p > 0.05). The increased activity resulted in a selective increase of frequencies between 20 – 30 Hz (30 minute recordings per pup; ).
These results, in combination with the enucleation and intraocular urethane injection experiments, suggest that spontaneous retinal activity is transferred though LGN to cortex to drive SATs. They further predict that the length, sub-burst structuring, and occurrence of V1 SATs (i.e. the macro-structure) are determined by the characteristics of retinal activity, while the prominent rapid oscillations that occur during SATs are generated in thalamus or cortex as a result of the burst excitation provided by retinal input.
We examined the forepaw region of S1 for evidence of SAT-like activity (Supplementary Fig. 1
). DC recordings between P10-11 (30 min from n=3 pups) showed no sign of the regularly recurring SATs observed in V1. The only infra-slow activity observed was infrequent, irregular and of very long duration (> 30 s). Thus retinal wave driven SATs do not appear to propagate to S1, where activity is instead driven by the topographically appropriate somatic receptors.
SATs are eliminated by low levels of anaesthesia
Because SAT-like mega-bursts were observed in unanaesthetized ferret (Chiu and Weliky, 2001
) but not anesthetized mouse V1 (Rochefort et al., 2009
), we examined the effects of isoflurane and urethane anaesthesia on the generation of SATs at P10-11 (). SATs were eliminatd by concentrations of either anaesthetic that reduced spontaneous movement, but did not prevent foot withdrawl to light squeeze. Under either anesthetic short bursts remained, and the event distribution was shifted from two clear peaks, to a single peak below 1s duration (n=50 events for each of 3 pups for each anesthetic, KS-test vs unaenesthetized, p < 0.0001 for both groups). Thus in V1 SATs are more sensitive to anesthesia than short bursts and their examination requires the use of unanesthetized animals.
We examined the locus of the anesthetic effect by intra-ocular injection of urethane (5-10 μL 100mM). Retinal injections rapidly eliminated SATs (<5 min), but not short-bursts, in 4/4 rats (; n=50 events per animal per condition, KS-test ACSF vs injected, p < 0.0001). Animals that received retinal urethane injections had a more frequent short-bursts than I.P. injected rats, suggesting a central locus for short-burst generation but a retinal locus for SATs. These data are complementary to the enucleation experiments as they show that SAT elimination can be accomplished by a less damaging disruption of retinal activity. Furthermore in total this data strongly suggests our short-bursts are the same activity recorded by Rochefort et al (2009)
and determined to be a primitive version of ‘slow-wave’ activity.
Synaptic correlates of SATs
We examined the synaptic composition of SATs with whole-cell voltage clamp recordings made in close proximity (200-400 μm) to the field electrode (). Neurons located 300-600 μm below the cortical surface (n=5 from 3 animals) showed a close corresponence between negative field-potential deflections and whole cell glutamatergic synaptic currents (-80 mV holding potential). Both the infra-slow and rapid components of the SAT could be observed in the single cell currents. The infra-slow potential was associated with a similar slow current flow, while the rapid oscillations were closely locked with fast synaptic currents. Cross-correlation of whole-cell currents and the rapid field oscillation (both signals high-pass filtered above 2 Hz) showed a strong peak correlation (mean correlation coefficient of 0.4 +/- 0.1 S.D., 5 SATs per animal). Cross-correlation during the inter-SATs times showed an equally strong correlation, but spread over a wider time-base. These results together with previous finding in barrel cortex suggest that the rapid oscillations of SATs are generated by rhythmic AMPA receptor mediated synaptic currents at beta-frequency range, while the infra-slow potential is likely generated by summation of NMDA, AMPA and kainate receptor mediated currents (Minlebaev et al., 2009
Beta oscillations of SATs synchronize activity in superficial layers
The depth profile of SAT-related activity was examined with multi-site linear electrode arrays (‘Michigan probes’, 100 μm spacing; ). These recordings showed that MUA was elevated in all cortical layers during SATs, and this firing, as in layer 4, was organized into multiple sub-bursts (). One exception to this was presumptive layer 5a (500-600 μm depth) which often contained units that exhibited tonic firing between bursts as well as between SATs. The beta band field-potential oscillations, by contrast, were localized to superficial layers (L2-4), with a peak 300 - 400 um below the pial surface (). Close examination of the temporal relationship between these beta oscillations and MUA revealed further differences between layers. In superficial layers MUA occurred primarily during the troughs of the beta oscillation, and less frequently during bursts of activity with smaller field deflections. Unit firing in deeper layers however was not strongly coordinated by the beta oscillations, and we did not observe another pattern that organized firing in the deep layers. A depth profile of the beta oscillations was constructed by phase averaging centered on the negative peak of the layer 4 trough (; 1000 events from each of 8 pups). Current source densities (CSD) calculated from these triggered averages show a prominent sink in layer 4 that ascended to superficial layers. Average spike rate (5 ms bins) in layers 2-4 was highest in the trough, and suppressed between phases. Mean spike rate in the deeper layers was much lower than in superficial layers, but showed a small increase following the layer 4 trough.
Because of differences in sampling and baseline spike rate between layers we further examined the synchronization of activity by beta-oscillations by calculating correlation coefficients for normalized spike rates (5 ms bins) between layers (; 10-15 SATs from each of 8 pups; correlation coefficients were generated for each animal and the reported mean is the average of 8 pups). MUA rate changes recorded at electrodes placed in layers 2/3 and 4 were strongly correlated with each other (mean cc 0.52 +/- 0.13 S.D. for adjacent electrodes), but only weakly correlated to sites located in deep layers (mean cc 0.21 +/- 0.14 S.D.;t-test p <0.0001). Even adjacent electrodes in layers 5/6 were less strongly correlated than similarly space electrodes in the superficial layers (mean cc 0.30 +/- 0.08 S.D.; p < 0.0001). The temporal relationship between the rapid (> 2 Hz) component of the field potential and MUA in each layer were further quantified by cross-correlation analysis (). As expected from the trough averaging, spike rates in superficial layers were positively correlated at 0 ms lag with spike-rate modulations on adjacent electrodes, and negatively correlated to the field potential in superficial layers. Peak correlation coefficients for layer 6 occurred with slight delay (5.4 s +/- 2.5 S.D) relative to layer 4, a relationship that was also apparent in the negative correlation to field potentials at the same delay.
In total our data show that the beta-oscillations specifically synchronize activity within a cortical column consistent with data from the somatosensory cortex (Dupont et al., 2006
;Yang et al, 2009
). This effect is strongest for superficial layers, and more modest in deep layers.
Beta oscillations synchronize superficial layers as a spreading wave
Phase III retinal waves co-modulate firing at locations separated by 100's of microns (Blankenship et al, 2009
). We examined how this spread correlates activity patterns within and between cortical hemispheres by recording with horizontal wire electrode arrays placed in both cortices (). Electrodes within the same hemisphere (500 and 1000 μm separation, n=30 SATs from each of 10 animals P10-11) were likely to record SATs with high temporal proximity. On average 59 +/- 8% (S.D.) of SAT starts on one electrode were within 2 s of the start of a SAT on electrodes on the same side, 86 +/- 5% percent occurred within 5 s. In total 79% of SATs occurring on one electrode overlapped by at least 1s SATs 500 μm distance. SATs separated by more than 10 s were very rare, consistent with the clustering of retinal waves observed in vitro
by us and others (Kerschensteiner and Wong, 2008
;Blankenship et al., 2009
). By contrast we observed no correlation in SAT occurrence between hemispheres, consistent with the retinal generation of SATs, poor ipsilateral representation at this age (Smith and Trachtenberg, 2007
) and impaired decussations of albino rats (Lund, 1965
The horizontal synchronization of cortical activity during SATs was examined with 4 shank multi-electrode arrays placed along the rostral-caudal axis (4×4 200 μm separations; ). Recordings of 20 SATs from each of 4 pups each showed the same pattern: SATs often occurred simultaneously on multiple shanks, but were also observed to spread between them consistent with the spread of activity as a random wave-front. Slow spreading activity (>100 ms per 1 mm) was always observed in the horizontal (between shanks) not vertical (between layers) direction. When SATs were recorded simultaneously on multiple shanks, the beta-oscillations in layer 4 were always synchronous (). Sites that became engaged by the spreading SAT developed beta oscillations synchronized to the other electrodes; we never observed non-synchronous oscillations during SATs in the same hemisphere. By contrast SATs in opposite hemispheres were never synchronous (not shown, n=30 SATs from each of 10 animals P10-11). The relative role of this synchrony in coordinating activity laterally and vertically was measured by computing the correlation coefficient between normalized spike rates in layer 4 and layers 5/6 during SATs. In all four pups, electrodes on adjacent shanks in layer 4 were more highly correlated than those in the deeper layers on the same or adjacent shafts (). Population averages showed that for a given separation, layer 4 electrodes were more correlated to other layer 4 electrodes than to deeper electrodes (). The average correlation coefficient of layer 4 electrodes separated by 200 μm was 0.68 +/- 0.04 S.D, but 0.36 +/- 0.02 (t-test, p < 0.0001) to the electrode on the same shaft 200 μm deeper. A similar relationship held for shafts separated by 400 and 600 μm. By contrast electrodes located in layers 5 or 6 had correlation coefficients of 0.38 +/- 0.06 S.D. for electrodes with 200 μm horizontal separation, and 0.39 +/- 0.02 S.D. for the same vertical distance (t-test, p=0.93).
In total the data on horizontal spread are consistent with the hypothesis that SATs are driven by retinal waves and that they synchronize synaptic and spiking activity in superficial layers via the beta oscillations.