As summarized in the introduction, several studies used microdialysis to assess glutamate levels across the sleep/waking cycle, but the results were inconsistent. Other studies used indirect markers to assess the effects of sleep and waking on glutamatergic signaling. Some reports measured tissue glutamate content in whole brain after total or REM sleep deprivation, with mixed results: no change (Karadzic et al., 1971
; Himwich et al., 1973
) or an increase in glutamate after sleep loss (Davis et al., 1969
; Bettendorff et al., 1996
). REM sleep deprivation was also shown to increase glutamine turnover (Mark et al., 1969
), and protein levels of glutamine synthetase (Sallanon-Moulin et al., 1994
). Transcript levels of genes coding for enzymes involved in glutamate synthesis (glutamine synthase, glutaminase), glutamate receptor subunits (GluR2, GluR3, (Cirelli and Tononi, 2000
)) and intracellular proteins implicated in glutamate receptor clustering (Homer/Vesl, Narp) are also upregulated during waking (Cirelli et al., 2004
). Finally, the number of GluR1-containing AMPA receptors increases after waking relative to sleep in cortex and hippocampus (Vyazovskiy et al., 2008b
). Thus, there is evidence that changes in glutamatergic signaling occur across behavioral states, but the link with glutamatergic neurotransmission is indirect.
Here we used in vivo
amperometry to measure cortical extracellular glutamate continuously for several days with second-by second resolution. This allowed us to reach several novel conclusions. First, within each sleep-wake cycle (<30 min in rats) glutamate levels increased rapidly and progressively during waking and REM sleep, and decreased progressively during NREM sleep. These dynamic within-state changes could not be detected with microdialysis due to its poor temporal resolution. Supporting our finding, a recent study focusing on the posterior hypothalamus used glutamate biosensors similar to those employed here and found rapid increases in glutamate levels in waking and REM sleep during a 7-hour window in the middle of the day (John et al., 2008
Second, the minute-by-minute glutamate levels reflected the sleep-wake history of the previous several hours: glutamate levels were low in an awake rat if it had been asleep for several hours, and high in a sleeping rat if it had been mostly awake for the previous hours. Thus, the previous sleep-wake history contributes to the absolute levels of glutamate at any given time, while the direction of these changes is determined by the current behavioral state. Glutamatergic activity is associated with high energy demand (Attwell and Laughlin, 2001
), and brain energy consumption is lower during NREM sleep relative to waking and REM sleep (Madsen et al., 1991
; Maquet, 1995
). Strikingly, cerebral metabolism in mice is also contingent upon the previous sleep-waking history (Vyazovskiy et al., 2008a
). Specifically, the uptake of 2-deoxyglucose during quiet waking is 15–20% lower in mice that had slept for ~ 2.5h prior to the injection of the tracer, compared to mice that had been awake prior to the injection (Vyazovskiy et al., 2008a
Third, the previous sleep-waking history not only affected the absolute concentration of glutamate, but also modulated its rate of change. SWA is a reliable indicator of sleep pressure, and increases as a function of the duration of prior waking (Achermann and Borbely, 2003
). Glutamate levels decreased at a faster rate during NREM sleep periods with high SWA than during those with low SWA. Overall, the rate of glutamate decline during NREM sleep was positively correlated with SWA. Also, the rate of decrease in glutamate during sleep following sleep deprivation, when sleep pressure is high, was roughly twice as fast as the average decrease during spontaneous NREM sleep. Hence, high sleep pressure enhances the NREM-related decline in glutamate levels.
Another factor modulating the changes in glutamate concentration during waking appears to be sleepiness. During sleep deprivation the frequency of sleep attempts increased, indicating an increase in sleepiness. Meanwhile, glutamate levels increased during the first hour of sleep deprivation, but by the third hour they started to decrease. Thus, towards the end of the deprivation, when rats attempted to sleep the most, the concentration of glutamate declined despite continued wakefulness. Changes in glutamate levels during spontaneous waking may also be affected by sleepiness, because most “atypical” waking epochs (associated with decreasing glutamate concentrations), occurred during short waking periods within long, consolidated NREM sleep periods.
The second-by-second concentration of glutamate in extrasynaptic space is the result of two opposite processes: extracellular release of glutamate from neurons and astrocytes, and intracellular uptake by high-affinity transporters. The fluctuations in extrasynaptic glutamate with behavioral state could therefore be due to changes in release or in transporter activity. Most glutamate uptake in cortex is mediated by the glutamate transporter-1, GLT-1 and the glutamate/aspartate transporter, GLAST (Tzingounis and Wadiche, 2007
). Their expression/activity is modulated by many factors downstream of neuronal activity, including cAMP and kinases (Duan et al., 1999
), and their block results in increased extracellular glutamate levels and neurodegeneration (Rothstein et al., 1996
). The changes in glutamate levels observed in our study could be accounted for by decreased uptake during waking and REM sleep, and increased uptake during NREM sleep. There is no evidence, however, that GLT-1 and GLAST activity is reduced during waking. GLAST mRNA expression in rat cortex increases by 30% following 8h of waking (Cirelli and Tononi, 2000
), and GLAST expression increases following exposure to glutamate, resulting in increased uptake (Duan et al., 1999
). Thus, if present, changes in glutamate transporter expression may serve to oppose the progressive increases in glutamate during waking and REM sleep, rather than contribute to it. The latter changes, instead, may reflect changes in glutamate release.
Under physiological conditions, glutamate is released into the extrasynaptic space by spillover from synapses (Asztely et al., 1997
; Biro and Nusser, 2005
; Sem'yanov, 2005
), exocytosis from astrocytes (Santello and Volterra, 2008
), and active transport by the cystine-glutamate antiporter (Baker et al., 2002
). Extrasynaptic glutamate as measured by our microelectrodes is affected by block of action potentials by tetrodotoxin, indicating that it reflects neuronal activity (Day et al., 2006
; Hascup et al., 2008
). Astrocytic release of glutamate can be coupled with neuronal activity (Santello and Volterra, 2008
), but the relative contribution of synaptic spillover and astrocytic release cannot be determined in our experimental conditions. Nevertheless, activity-induced release of glutamate is likely to underlie the changes in extrasynaptic glutamate concentration across the sleep-wake cycle.
Initial studies suggested that firing rates in cortex are higher during waking and REM sleep and lower during NREM sleep (Desiraju, 1972
; Noda and Adey, 1973
). Recent reports indicate that neuronal firing rates during NREM sleep are similar to waking during the up state of the slow oscillation, but are close to zero during the down state (Steriade et al., 2001
). Therefore, the progressive rise of glutamate during waking and REM sleep could result from the inability of glutamate transporters to maintain a constant extrasynaptic glutamate concentration during high levels of neuronal activity. Meanwhile, during NREM sleep, the occurrence of down states could allow transporters to gradually reduce the extrasynaptic concentration of glutamate. Given that glutamate transporters complete a full cycle in ~70 ms (Wadiche et al., 1995
) and that down states typically last 80–300 ms (Ji and Wilson, 2007
), each transporter could conceivably remove 1–4 molecules of glutamate per down state. Large slow waves, indicative of high sleep pressure, are associated with even longer silent periods of neuronal activity (Vyazovskiy V, unpublished observations; (Calvet et al., 1973
)). This could account for the faster decline in the concentration of glutamate during NREM sleep with high SWA. It is tempting to speculate that NREM sleep may be a propitious time for replenishing presynaptic glutamate reserves depleted by tonic glutamate release during wakefulness, though there is so far little indication that the presynaptic glutamate pool may constitute a limited resource over long periods of time.
The progressive changes in extracellular glutamate reported here shows that, for whatever reason, glutamate release and clearance mechanisms are not kept in full balance in the short term. Instead, glutamate levels systematically increase during EEG activated states, raising the question whether they may become neurotoxic. In vitro
studies of cultured neurons report glutamate-induced excitotoxicity at concentrations below those observed here (1–12 µM; (Rosenberg et al., 1992
; Cheung et al., 1998
). However, astrocyte-rich cultures are much less sensitive to glutamate excitotoxicity (EC50
= 205 µM; (Rosenberg et al., 1992
= 100–200 µM; (Regan and Choi, 1991
)), and high glutamate levels are required to produce excitotoxicity in vivo
(Mangano and Schwarcz, 1983
; Choi, 1988
). While it remains unclear whether the progressive increase of glutamate concentrations in waking can approach excitotoxic conditions, the present results show that NREM sleep effectively opposes glutamate accumulation in the extrasynaptic space. Thus, thanks to the alternation of wakefulness and NREM sleep, glutamate levels are kept within a homeostatic range, remaining relatively constant across the 24-hour period. Moreover, as shown by the positive correlation between the rate of glutamate decline during NREM sleep and the levels of SWA, the homeostasis of extracellular glutamate in the cortex may be linked to sleep homeostasis. At this point, it is unknown whether NREM sleep is in any way critical to glutamate homeostasis, and it is likely that, in the absence of sleep, other compensatory mechanisms come into play. Perhaps the glutamate-decreasing effect of NREM sleep is especially relevant in pathological conditions, such as ischemia, that can lead to glutamate-induced excitotoxicity.