The first pHFOs, reported almost a decade ago in patients with mesial temporal lobe epilepsy and rat models of this disorder (Bragin et al., 1999a, 1999b), were FRs in the frequency range of 250–600 Hz. An important distinction between these pHFOs and normal ripples is that the former are readily recorded from dentate gyrus, where the latter never occur under normal conditions. FRs typically occur on interictal electroencephalography (EEG) spikes and are of particular interest because they appear to be uniquely associated with regions capable of generating spontaneous seizures (Bragin et al., 1999c; Staba et al., 2002a; Jacobs et al., 2008), and could reflect the basic neuronal events underlying epileptogenicity (Bragin et al., 2000a; Engel et al., 2003). The fact that FRs occur not only interictally, but at the onset of ictal events in rodents, strongly suggests that FRs are associated with mechanisms of seizure generation and are not merely a consequence of injury (Bragin et al., 1999c, 2005). In rodents, FR-generating neurons are not homogeneously distributed throughout the epileptogenic tissue, but occur in small discrete clusters that are spatially stable over long periods of time (Bragin et al., 2002a, 2003). In patients with mesial temporal lobe epilepsy (MTLE), there is a strong association between the occurrence of FRs and hippocampal atrophy on magnetic resonance imaging (MRI) (Staba et al., 2007; Ogren JA, Wilson CL, Bragin A, Lin JJ, Salamon N, Dutton RA, Luders E, Fields TA, Toga AW, Thompson PM, Engel J Jr, Staba RJ, unpublished manuscript). Patient studies also have revealed pHFOs in neocortex that bear the same relationship to areas of ictal onset as mesial temporal pHFOs (Worrell et al., 2004; Urrestarazu et al., 2007).
The conclusion from unit and field potential recordings in rodents and humans is that FRs represent field potentials of population spikes from clusters of abnormal synchronously bursting neurons, in contrast to ripples, which represent field potentials of summated IPSPs (Bragin et al., 2002a, 2007). Whereas synchronously bursting neurons have long been considered the hallmark of epileptogenic tissue (Schwartzkroin, 1983), and can be easily identified in acute animal models, such as the neocortical penicillin focus where up to 90% of principal neurons participate in interictal EEG events (Matsumoto & Ajmone Marsan, 1964), less than 10% do so in chronic experimental cortical foci (Ishijama, 1972; Wyler et al., 1975), and fewer than 5% in hippocampus of patients with MTLE (Babb et al., 1981, 1987). Consequently, although it can be difficult to identify, and study, chronically epileptogenic tissue using microelectrode identification of bursting units (Colder et al., 1996a, 1996b); (however, see Staba et al., 2002b), FR field potentials, even though generated by widely spaced small clusters of neurons, can be recorded much more easily and serve as a much more robust marker of epileptogenicity (Engel et al., 2003).
Are fast ripples a variant of normal ripples?
Recent research has been directed at understanding whether FRs represent a variation of normal HFOs, or reflect entirely different pathologic neuronal interactions. In addressing this question, investigators (including ourselves) have tended to focus only on the frequency difference between ripples and FRs when, in fact, we now know that frequency is not a reliable differential feature. For instance, Foffani et al. (2007) compared in vitro slice recordings from area CA3 of hippocampus from normal and epileptic rats. HFOs were initiated by lowering calcium and raising potassium concentrations in the medium. They elegantly demonstrated that in epileptic rats, synchronicity was actually reduced during HFOs in the FR frequency range compared to synchronicity during HFOs in the ripple frequency range, and suggested that pathologic FRs are a harmonic of normal ripples. There are numerous reasons, however, that this is not likely to be the case.
Spectral frequency alone is not sufficient for separating normal from pathologic oscillations (Bragin et al., 2007). Within days after intrahippocampal kainic acid (KA)–induced status epilepticus, HFOs in both the ripple and FR frequency range can be recorded in the dentate gyrus of rats that later develop recurrent spontaneous seizures, but not in similarly treated rats that do not develop spontaneous seizures (Bragin et al., 2004). In this case, the ripple frequency HFOs are pathologic because ripple oscillations never occur in dentate gyrus under normal conditions, although some granule cells can discharge at ripple frequency rates during sharp waves (Penttonen et al., 1997). Furthermore, whereas Foffani et al. (2007) based their conclusions, in part, on the fact that the amplitude of FR frequency oscillations was decreased compared to that of ripple oscillations, in chronic in vivo recordings, the amplitude of oscillations in the FR frequency band in dentate can be much higher than the amplitude of oscillations in the ripple frequency band recorded from the same microelectrode (Fig. 1). This suggests, at least for pHFOs, that FRs are not necessarily harmonics of ripple frequency oscillations. Note, however, that both ripple and FR frequency pHFOs are also seen in electrodes 2 and 3 in Fig. 2, where the former could be a harmonic of the latter. The weakness of power analysis for separation of pathologic and normal HFOs is also illustrated in the power histograms presented in Figs. 2 and 3, where bursts of population spikes occur with frequency peaks at 180 Hz, 250 Hz, and 350 Hz.
Figure 1. An example of ripple and fast ripple (FR) oscillations in wide bandwidth (top trace, 0.1 Hz–5 kHz) recording from dentate gyrus. Bottom trace band pass filtered between 80 and 500 Hz. Normalized power spectrogram computed from underlined segments in wide bandwidth recording corresponding to ripple (red line) and fast ripple (blue line). Note the larger amplitude and greater power centered at 270 Hz associated with the fast ripple compared to the ripple power centered at 170 Hz.
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Figure 2. Voltage-depth profiles of dentate gyrus potentials evoked by perforant-path stimulation (EP), and spontaneous pathologic high-frequency oscillations (pHFOs) recorded at the same position of the array of recording microelectrodes separated by 200 μm. pHFOs with both fast ripple (FR) and ripple frequencies are recorded by the microelectrodes located within the granular cell layer. Here the FR frequency oscillations could be harmonics of the ripple frequency oscillations, which have the shape of population spikes, whereas in the neighboring microelectrodes the shape of the electrical signal changes and appears more as oscillations than as population spikes. Ripple frequency oscillations occur in the CA3 area (dashed box) before FRs in the dentate gyrus granular layer. The bottom (red) line represents electrical activity recorded in a bipolar montage between recording sites 2 and 6 (between dentate gyrus and CA3 area), with power histogram on the left. GrL and CA3 indicate correspondingly the dentate gyrus granular layer and CA3c areas of hippocampus. Diamonds indicate the location of recording sites reconstructed based on analysis of histologic sections and the shape of evoked potentials.
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Figure 3. Synchronization of multiunit discharges before and during fast ripples (FRs) recorded from an epileptic KA rat. (A) The red line at the top is the field activity recorded within the granular layer. Lines 1–7 are single traces of multiunit activity recorded by seven microelectrodes located within the dentate gyrus. Bottom—perievent histogram of 466 FRs triggered by the peak of the first population spike of each FR. (B) Shows an expansion of the first population spike indicating that it consists of small individual spikes. (C) Power histogram of the bursts of population spikes presented in A.
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An argument for the hypothesis that pHFOs in the dentate gyrus reflect synchronous bursts of population spikes is that the maximum amplitude is within the granule cell layer and the shape has similar voltage depth profiles as potentials evoked by perforant-path stimulation (Fig. 2). In contrast, ripple oscillations are more prominent in the hilus–CA3 area (Fig. 2). Analysis of these oscillations without knowledge of the exact location of recording sites could also lead to misinterpretation of the results, as illustrated by the bipolar recording at the bottom of Fig. 2 (Bipolar 2–6). Here ripple oscillations appear to precede FR oscillations in the same area, when the monopolar recordings show that they actually are generated by different neuronal populations. Evidence that pHFOs reflect bursts of population spikes also derives in part from multiunit recordings demonstrating that neurons increase their discharge frequency before pHFOs appear. At the onset of pHFOs it is impossible to separate unit activity because all neuronal events collapse into the field population spike (Fig. 3). When the recording microelectrode is in the granule cell layer, however, brief “mini spikes” are visible on the descending part of the population spikes, supporting the idea that the field oscillation is the result of spatial summation of action potentials of many synchronously discharging neurons (Fig. 3, dashed box). In fact, the shape of pHFO oscillations varies significantly from one recording site to another. Within the granule cell layer, pHFO activity can have a shape resembling discrete population spikes but takes on the appearance of oscillations at more remote recording sites (Fig. 2). The identification of pathologic oscillations in other brain areas where ripple oscillations occur under normal conditions is more problematic. In these areas, oscillations in the FR frequency band (250–600 Hz) should be considered as pathologic, while oscillations in the ripple frequency band (100–200 Hz) could be normal or pathologic.
When are ripple frequency oscillations abnormal?
Rather than ask whether pHFOs may be a variant of ripples, an alternative, perhaps more relevant, question is whether some ripple frequency oscillations outside the dentate gyrus could in fact be pHFOs. Whereas it is easy to identify the pathologic nature of HFOs that occur in the dentate gyrus, where ripples are normally absent, it is not yet possible to definitively conclude that all ripple frequency oscillations recorded in epileptic hippocampus and parahippocampal structures outside of the dentate gyrus are normal. Indeed, it is more reasonable to assume, given the data that the frequency of pHFOs in epileptic dentate gyrus can range from 80–600 Hz, that not only FRs, but some oscillations in the ripple frequency range, outside of dentate gyrus, also reflect epileptogenicity. It is then possible to suggest that at least some, and perhaps all, ripple frequency HFOs recorded in slice preparations in vitro reflect pathologic rather than normal mechanisms, as a result of neuronal disconnections and artificial environmental conditions. This might then explain why some authors have concluded that ripple frequency oscillations recorded in vitro reflect a brief series of population spikes, with maximum amplitude in the cellular layer (Draguhn et al., 1998; Dzhala & Staley, 2004; D’Antuono et al., 2005). An alternative interpretation of the data presented by Foffani et al. (2007), therefore, would be that oscillations in all frequency ranges described in their paper reflect bursts of population spikes and are pathologic, although some of them are in the ripple and others in the FR frequency range. FRs could then occur as a result of out-of-phase firing in part of the generating neuronal network, owing to a reduction of spike-timing reliability (as might be the case for the recordings from electrodes 2 and 3 in Fig. 2). In these conditions, both frequency bands of oscillations would be pathologic.
Because invasive electrode recordings are performed to delineate epileptogenic tissue, it is not possible to characterize HFO occurrence in normal human brain. The existence of neuronal clusters generating pHFOs outside of the seizure onset zone (Jirsch et al., 2006; Jacobs et al., 2008), however, is consistent with the view that epilepsy is a disorder involving diffuse neuronal networks (Spencer, 2002).
The most significant parameter that separates normal from pathologic HFOs is the evidence that the latter represent abnormal bursts of population spikes. The fact that the shape of population spikes changes dependent on the distance between the recording electrodes and the area of generation can cause pHFOs to look like normal oscillations. Therefore, there is a need for additional means to differentiate pathologic from normal oscillations.