In this segment, we are going to now discuss the more conventional role of GABA, and that is in the generation of NREM sleep. This paper published by Ron Szymusiak and Dennis McGinty in 1986 was a really important finding that they published. Because they found a population of neurons in the basal forebrain that had a NREM-on discharge pattern. And what you can see by looking at the middle panel on the right that I've highlighted in red, this cell is firing clearly at its fastest rate during NREM sleep, and it's pretty quiet during waking and REM sleep. And the reason why this was such an important finding is that no one had ever recorded NREM-on neurons before, and this suggested that these neurons in the basal forebrain may be the ones that are driving the generation of NREM sleep. It was later shown that these neurons are GABAergic that project and they project to the cortex, and we'll get to that. And we'll get to that in this slide. So Hassani working with Barbara Jones showed that GABAergic neurons in the basal forebrain that have a NREM-on discharge pattern project to the cerebral cortex. So he worked with identified neurons and these are likely the same neurons that Szymusiak and McGinty recorded from. This piece of data from Giancarlo Vanini shows that the GABA levels in the cerebral cortex are what would be expected based on the discharge pattern and projections that we just viewed. In other words, GABA levels in cortex which are indicated on the y-axis are greatest during NREM sleep, and significantly lower during wakefulness and during REM sleep. So we don't really know the functional relationships between GABA levels and cortical activity during sleep. But we do know that this is what the ambient GABA tone in the cortex is during NREM sleep, that its ambient GABAergic tone in the cortex is high during NREM sleep. We know a little bit more about the role of GABAergic neurons in the anterior hypothalamus in the generation of REM sleep, and so that's what we're going to focus on now. This is a coronal drawing of the rat brain, and up in the top left you see a sagittal view. The red line indicates the level at which this coronal section in the main view is taken, and so that's this red line. And what you see here are these GABAergic nuclei, the ventrolateral preoptic nucleus, the preoptic area, and the median preoptic nucleus. We're going to talk about all these nuclei contained GABAergic neurons, and we're going to talk about them and their role in the generation of NREM sleep. So one, let's begin with the ventrolateral preoptic area, otherwise known as VLPO. So here's another very important study that was published in 1996 from Cliff Saper's group showing that neurons in the ventrolateral preoptic area increase c-fos expression during the recovery sleep that follows deprivation. So let me unpack that a little bit. There are many ways to look at neuronal activity, and we have focused in this presentation on cell discharge. The advantage of cell discharge in optogenetics is that you record from single cells, you can record from single cells, and you can identify those neurons and you can know exactly what those neurons are doing across time. The limitation of course is that there are many, many neurons in the brain and recording from neurons one at a time is laborious, and you won't get the complete picture that way. So the neuron discharge is the gold standard, but it's great to have other techniques that can corroborate or refute data we see from cell discharge. And c-fos expression is that kind of data. What c-fos is an immediate early gene and its activation is correlated with cell activation. And so what c-fos expression allows a person to do is an immunohistochemical reaction that you can look at one moment in time only, but you can look across the entire brain and see where the activation is. So again for cell discharge, we can look across time and look at identified cells and know exactly what they're doing in phase relationship to changes in states or any dependant measure. What c-fos expression gives us is one point in time, but across the entire brain, so we can look at lot more areas and a lot more cells. What we're looking at on the right are coronal sections and here's the optic chiasm, so the ventrolateral preoptic area is located here in this box. And they showed that during the recovery sleep that follows deprivation, those VLPO neurons are activated, and that you can see by seeing all those dots which indicates c-fos expression. They had to study recovery sleep because one of the limitations of c-fos is it takes about 30 or 45 minutes to be expressed. So in a rodent model you can't look at specifically REM sleep or NREM sleep, you have to enhance sleep by depriving, and then let the animals recover. Nonetheless it gives us valuable information, and it fit and it shows us that this area is firing, these cells are active during recovery sleep. There are also neurons in the median preoptic nucleus and the preoptic area, and in the basal forebrain that express Fos during recovery sleep. So it's not just the VLPO neurons in these three areas that I showed you that are identified as being GABAergic, all are activated during recovery sleep. This study in the next couple of slides I'm going to show you, again, from Szymusiak's group, looks at the discharge rate of neurons in the ventrolateral preoptic areas, so it's a follow up to that c-fos study. And I want to show you that this is the percent change in firing rate from wakefulness plotted against four states. Wakefulness. The transition from wakefulness into sleep. The early part of NREM sleep and the late part of NREM sleep, so once the animal is really deeply into NREM sleep. And if we look at the red line, the baseline discharge rate in these cells in normal non-sleep deprived animals, you see that as the animal gets sleepier and enters into sleep, these cells increase their discharge rates. All right, so that fits with the c-fos data. These investigators also showed that during the recovery sleep that follows deprivation, a similar pattern to the control non-deprived state occurs, but that in every state measured, discharge rate was higher during recovery sleep than during the physiological nonrecovery sleep. What this finding suggests is that the discharge of these neurons may in fact contribute to the intensity of sleep. And so we think that some sleep is deeper than other sleep and the intensity may be coded in the firing rate of these neurons. Looking at the discharge rate of the median preoptic nucleus neurons, we see that some of them increase their discharge rates prior to sleep onset. We've talked previously about these phasic relationships to state changes and how important they are. These cells fire faster during NREM sleep. And they fire in fact, at their fastest rates during REM sleep. An example of such a neuron is shown in this slide. And at the very top we see this plotting of what state the animal is in, so the first column here shows wakefulness. And you can see that in all by the cell discharge is fast during wakefulness, that the EEG is activated and the animal is active, moving around with a lot of muscle tone. There's a break in the recording, and then you see the entry into NREM sleep which occurs right here. What you see here is an increase in discharge rate of this neuron just prior to the onset of NREM sleep. And that's indicated by this rate, this summarizes discharge rate. And what you see is more than a two-to-one increase in discharge rate at this point as this animal is preceding into NREM sleep. This finding is compatible with the median preoptic nucleus neurons driving the onset of NREM sleep. And again, this phasic relationship to the state change is key. You can see just with the naked eye that as the animal gets deeper into NREM sleep and then transitions into REM, there is an increase in the firing rate, each one of these black lines represents, again, one action potential, and the summary of the rate discharge cells peaks during REM sleep. The EEG, just to remind you, is activated during REM as it is during wakefulness. The main difference here comes in the muscle tone where you see the motor atonia and then here, right here, as the animal wakes up, the burst in muscle tone and is still, momentarily, stops firing. So these sleep-on GABAergic neurons in the ventrolateral preoptic area and in the median preoptic nucleus project to all the major arousal-promoting, monoaminergic nuclei at the dorsal raphe, the locus coeruleus, and the tuberomammillary nucleus that we've reviewed, now you're familiar with these nuclei. And they release GABA which is inhibitory, and the discharge of these sleep-on neurons inhibits the discharge of the wakefulness promoting neurons. If we look at transmitter release in the next two slides now, what I want to do is, we've talked about the role of acetylcholine, we've talked about the role of GABA, and we've mentioned their interaction at the level of the pons. I also want to talk about the interaction in the forebrain of GABAergic and cholinergic transmission and what this might mean for sleep state control. So as I showed you, this is the transmitter level pattern for GABA in the cortex, highest during NREM, we see the opposite pattern for acetylcholine in the cortex, lowest during NREM. If we put these together and I'll show you the ratio data, one of the key points I want to make here which is really a key take home message from this entire lecture. And that is that the state specific changes in transmitter release are also brain regions specific, okay. So GABA, this pattern of GABA levels across states is different in the cortex than what we saw in the ponds, the same for acetylcholine, different in the cortex than what we saw in the pons. So different in the same brain region between transmitters, different within transmitters across brain regions, key point. Now if I put these two data together and plot it as ratios as we did for the brainstem data, looking at transmitter levels as percent of wakefulness. And we make waking 1, since it's the newest in the normal baseline state. What you can see then is GABAergic transmission during NREM sleep goes up, acetylcholine goes down, and the ratio of GABA-to-ACh is 3. Interestingly, the ratio of GABA to acetylcholine in REM sleep in the cortex is very similar to the ratio during waking. And so the real difference at the level of the cortex between GABAergic and cholinergic transmission happens during NREM sleep when the cortex is relatively inactive with low acetylcholine and high GABA. And now as a final comparison to show you the difference, again, in these transmitter ratios between the cortex and the pons pontine reticular formation, how different the neurochemical milieu is. In the cortex, we have similar GABA-to-ACh ratios during waking and during REM sleep, where as in the pontine reticular formation, we have similar GABA-to-acetylcholine ratios during waking and NREM sleep. So in the cortex, the differences in NREM. And the brainstem, the differences in REM sleep. So it suggests that this GABAergic-to-cholinergic ratio might be an indicator of the neurochemical milieu that characterizes these states. Our next segment will be on another NREM related neuromodulator, and that is adenosine.
