Acute focal 4-AP seizures
Acute focal seizures were elicited with injection of 4-AP. These seizures typically began with a large negative spike followed by a low amplitude fast activity which evolved into rapid spike-and-wave activity that gradually increased in periodicity and decreased in amplitude until the seizure terminated ().
Light scattering and CBV imaging of center-surround dynamics
Optical recording at 800 nm demonstrated an antagonistic center-surround dynamic (). The complex, dynamic relationship between the positive signal in the focus and the negative signal in the surround evolved over the course of the seizure. For the purposes of data analysis, we chose two moments in time that were representative of the predominant interaction, namely the timepoint at which the positive signal in the seizure focus was at its maximum, an average of 36.5±8.6 s after seizure initiation and the timepoint at which the negative signal in the seizure surround was at its maximum, an average of 11.6±9.3 s after seizure initiation. Average data over all experiments revealed that the positive signal diminished in amplitude with increasing distance from the seizure focus (, n=5 rats, 11 seizures). The maximum amplitude of the positive signal in the focus was 6.2±1.3%. Statistical testing revealed a significant decline in the positive signal within each ring to a value of 0.14 ±1.0% in the last ring at 2.5 mm (ANOVA: p<0.001). The inverted, negative signal in the surrounding cortex, on the other hand, was largest at a distance of 2.0 mm from the focus with a maximal amplitude of −2.1±0.2%, which was also statistically different from the values in other rings (ANOVA; p<0.001, ). Although not a direct measure of neuronal activity, the signal indirectly represents increases in neuronal activity in the focus and decreases in neuronal activity in the surround by measuring mostly changes in light scattering in the tissue (see Methods).
Optical recording at 570 nm provided a map of CBV during seizure activity (), nearly identical to those previously published by our group (Zhao et al., 2009
). The 570 nm map revealed a similar center-surround effect as seen with the 800 nm data but the activity was less focal and had more vascular artifact, which was not seen in the 800 nm data (). The maximal increase in CBV in the focus was 11.5±1.9 %, which decreased with distance but did not reach baseline, even 2.5 mm from the focus (ANOVA; p <0.001; , n=5 rats, 18 seizures). A decrease in CBV in the surround was also identified with a maximum change of −2.3±0.4% peaking at 2.0 mm from the focus (ANOVA; p<0.01, ). Hence, CBV maps provide an indirect and less spatially precise measurement of excitatory and inhibitory pattern of neuronal activity, as measured by changes in blood volume, as compared with those measured with light scatter changes (), and these maps clearly show a decrease in CBV surrounding a seizure focus. The etiology of this negative signal in the surround and its timing with respect to the seizure onset was then investigated in further detail. Of note, using intrinsic signal imaging in this acute seizure model, we did not find any pre-ictal increase or decrease in either light reflection or CBV during a 20 s window before seizure onset.
Metabolic mapping of center-surround dynamics
In a previous paper using laser Doppler flowmetry (LDF), we showed that the decrease in CBV surrounding the 4-AP focus was accompanied by a transient decrease in CBF, followed by a later increase in CBF as the seizure propagated horizontally (Zhao et al., 2009
). Tissue oxygenation in the surround, on the other hand, increased throughout the duration of the seizure (Zhao et al., 2009
). Although we assumed that a drop in blood flow and an increase in tissue oxygenation would be associated with a decrease in oxygen metabolism from neuronal inhibition, having not directly measured metabolism, the possibility remained that the increase in activity in inhibitory interneurons would result in an increase in metabolism, or that only a fraction of the pyramidal cells would be inhibited while others would increase their activity, resulting in a net increase or no change in metabolism. To address this question, we first investigated metabolism using autofluorescence imaging (AFI) of flavoproteins, so as to sample large areas of the cortex simultaneously including both the seizure focus and the surround (n=5 rats). With this technique, we could also determine if the resulting maps of seizure onset were more focal than hemodynamic maps, since the signal arises purely from local mitochondria rather than non-local vascular supply (Chance et al., 1979
; Shibuki et al., 2003
; Husson et al., 2007
; Reinert et al., 2007
AFI measured changes in the redox state of mitochondrial flavoproteins, primarily flavin adenine dinucleotide (FAD), since its oxidized form (FAD+) is more fluorescent than the reduced form FADH (Chance et al., 1979
). Increases in neuronal activity result in increases in intracellular Ca+
and depletion of ATP and production of ADP, which leads to a reduction in the proton gradient across the inner mitochondrial membrane and an increase in flavoprotein fluorescence (Reinert et al., 2007
; Llano et al., 2009
). Similar to AFI of normal sensory processing, we found that the signal was biphasic, having an early oxidation phase i.e. “light phase” (), arising from neuronal oxidative metabolism, followed by a later, more prolonged reduction phase i.e. “dark phase” ( ), presumably arising from a combination of glycolysis in astrocytes and contamination from increased CBV and the intrinsic signal (Kasischke et al., 2004
; Sirotin and Das, 2010
). However, certain aspects of the AFI maps showed that epileptic events differ from normal physiologic sensory responses. In the early light phase (0~2 sec), the maximal positive AFI peak occurred in the focus with an amplitude of 1.5±0.3 % and decreased towards the periphery (ANOVA; p<0.001, ). Unlike normal physiologic responses to sensory stimulation in the neocortex, we identified an inhibited surround with a peak negative AFI of −0.9 ±0.3% (ANOVA; p<0.01, ). The strong positive AFI signals were centered between 0 and 1.0 mm, while the smaller negative AFI signal were between 1.5 and 2.5 mm. As the seizure progressed, the signal inverted and a decrease in AFI of −3.3±0.9% was recorded in the focus and a peak increase of 1.9±0.3% (ANOVA; p<0.001) was recorded in the surround (). In the late part of the dark phase (> 5 s), the negative signal in the focus reached −6.2±1.4% while the positive peak in the surround decreased to a maximum of 0.9±0.2% (ANOVA; p<0.001, ). These findings confirm an early increase in metabolism in the epileptic focus, consistent with the dip in hemoglobin and tissue oxygenation, or “initial dip” previously demonstrated in this model. However, the decrease in AFI during the early light phase surrounding the ictal focus is consistent with a transient decrease in oxidative metabolism in the surround, a newly described phenomenon that is consistent with a decrease in neuronal activity. At later timepoints, interpretation of the etiology of the AFI signal is less clear, resulting from a combination of changes in CBV, which make up a significant proportion of the optical signal at the emission wavelength of AFI, as well as glycolysis in astrocytes, which is the primary component of the later “dark” phase of the AFI signal.
Figure 2 Cellular and tissue metabolic changes in the ictal focus and surround. (A) Flavoprotein autofluorescence images (AFI) at selected time points after seizure onset in a single animal reveal biphasic signal with a center-surround effect during the early (more ...)
In order to overcome these temporal limitations of AFI, namely overlap between the emission spectrum of AFI and the intrinsic CBV signal at timepoints > 2 s, we directly derived tissue oxygen metabolism using simultaneous measurements of CBF and tissue oxygenation in the focus (n=9 rats) and the surround (n=8 rats) and calculated cortical oxygen consumption (CMRO2
) using Gjedde’s method (Gjedde, 2006
; Thomsen et al., 2009
). In the focus, CMRO2
was significantly increased 2.2 s after seizure onset (ANOVA; p<0.05) and reached a maximum increase of 14.7 ± 3.8% (ANOVA; p<0.001) compared with the pre-ictal baseline (n=9 rats, 54 seizures, , top). In the surround region, two different phenomena were identified. In most of the rats, CMRO2
showed a significant sustained decrease by an average of −8.3±2.5% (5 of 8 rats; n=43 seizures; ANOVA; p<0.05). In the rest of the animals, we found a transient increase of 6.3±1.3 % (1.3 to 3.2 s; ANOVA; p<0.05; , middle) followed by a sustained decrease to −5.7± 1.4% (3 of 8 rats; n=39 seizures; ANOVA; p<0.05; , bottom). These results show the expected dramatic increase in metabolism in the focus and confirm the AFI results showing an overall decrease in metabolism in the surround consistent with a net decrease in neuronal activity. However, unlike the AFI results, the measurements of tissue metabolism persist for a longer timeperiod, supporting the correlation between only the early light phase of the AFI data and neuronal metabolism.
Two-photon measurements of arteriolar diameter
In order to determine the etiology of the transient drop in CBF and CBV in the surround at seizure onset, we measured arteriolar diameter using 2-photon imaging (n=4 rats). Low magnification images were first used to navigate the surface vasculature and determine spatial distances of specific vessels relative to the injection site of the 4-AP (). High magnification movies of individual arterioles allowed for tracking diameter changes during seizure activity near () and far () from the seizure focus. We found that arterioles dilated in response to the seizure, with a decreasing amount of dilation with increasing distance from the 4-AP injection site () (n= 4 rats, 71 vessels, 45 seizures, 143 measurements). 97% of the measured arterioles within 2.5 mm of the seizure focus dilated (), with little response for vessels further away. For vessels in a 1 mm ring centered on the 4-AP injection site (focus) arterioles dilated by an average of 63±5% of their baseline diameter, (). In the ring 1.5–2.5 mm from the 4-AP injection site (surround), we observed early vascular constriction followed by delayed dilation in all seizures measured (). In this ring, 69% of the vessels displayed this early constriction, with a diminished fraction of vessels dilating for vessels closer or farther from the seizure focus (). On average, the vessels in this ring constricted by 7%±1% of their baseline diameter during this constriction phase, with smaller constrictions for vessels closer or farther from the seizure focus ().
Figure 3 Seizures induce spatially-dependent vascular changes. (A) Two-photon image of fluorescently-labeled surface vasculature (Blue arrow, implanted electrode. boxed areas, near and far regions from the seizure focus highlighted in parts B–E). L↔M, (more ...)
Temporal characteristics of vascular reactivity
Having demonstrated for the first time that active vascular constriction in the ictal surround occurs, we sought to determine the timing of this event with respect to seizure onset. Plotting the temporal profile of vasodilation compared with vasoconstriction (), we determined that vasodilation in the focus occurred 0.5 ± 0.1 s after seizure onset whereas vasoconstriction in the surround occurred 5.3 ± 0.5 s prior to seizure onset () (p < 1.0E-7, Mann-Whitney U test). Note that all vasoconstriction was observed to occur prior to seizure onset. There was no significant correlation between the onset time either of constriction or dilation and either seizure duration, arteriole diameter, and maximum LFP amplitude ().
Figure 5 Constriction and dilation onset time as a function of (A and D) seizure duration, (B and E) arteriole diameter, and (C and F) maximum LFP value, respectively. The plots indicate that both onset times show a poor correlation with the investigated parameters. (more ...)