Well, in this segment, we're now going to talk about the role of GABA in promoting wakefulness and inhibiting REM sleep. And that is as I mentioned, an unconventional role for GABA. And may spend some time on it because it's a great illustration of the concept that the same transmitter can have different effects on states depending where in the brain the transmitter is acting, and that's illustrated by this unconventional of GABA in promoting wakefulness. Further on, in the future segment, we'll talk about the more conventional of GABA in generating REM sleep. GABA is the major inhibitory transmitter in the brain. Most of the drugs that are used clinically to produce a loss of wakefulness, whether it be sleep, sedation, or general anesthesia, act by enhancing transmission at GABA A receptors. Activation of GABA_B receptors also causes sleep. Today, we're going to focus on GABA A receptors, and in this table, I've summarized the drugs that are FDA indicated for the treatment of insomnia. And the key point of this table is that all, but two of these drugs act by enhancing transmission at GABA A receptors. The last two drugs, Ramelteon and Doxeplin, are Ramelteon is a melatonin receptor agonist, and Doxepin is an H-1 histamine receptor antagonist. But the other drugs all act by enhancing GABAergic transmission. I'll also show you in the next segment a drug that was added to this list in February of 2015, a new class of drug FDA indicated for the treatment of insomnia. Point here is the vast majority of these drugs is a Benzodiazepine or benzodiazepine receptor agonist that enhances GABAergic transmission. However, in preclinical studies, it's been found that the effects of GABA on sleep and wakefulness are brain-region specific. For example, in the pontine reticular formation which ELC abbreviated as PRF or PNO, GABAergic transmission actually increases wakefulness and inhibits sleep, and in particular, GABA is inhibitory to REM sleep. So I'm going to show you some data now for this effect of GABA. Here we see a mouse that's been implanted with electrodes for recording the EEG and a guide tube for making a micro injection of a drug into the pontine reticular formation. And so, I wanted to show this example again as a way of saying because the brain has no pain receptors, under surgical anesthesia, we can implant these animals with these electrodes, and do these studies to test specific hypotheses about the roles of particular receptors and transmitters in specific brain regions on causing sleep and wakefulness. So in this study, Flint injected a GABA receptor agonist called Muscimol into the pontine reticular formation of awake mice. And what she found was that by activating GABAergic transmission, she got an increase in wakefulness, and a concentration dependent decrease in non-REM sleep, so beautiful inhibition of non-REM sleep, and a rather abrupt elimination of REM sleep. The fact that this response in particular is inhibition of non-REM sleep is concentration dependent, supports the interpretation that this is a receptor mediated response. So, I want to spend a little time now focusing on GABAergic transmission and a refresher on the GABAergic synapse because I'm going to go on to show you different ways of manipulating GABAergic transmission in the pontine reticular formation by altering this, the components of this in synapse. So here's a schematic of the GABAergic synapse, showing a presynaptic terminal, a postsynaptic cell neuron and then the glia component. So the so-called tripartite synapse which is typical of GABAergic transmission. Muscimol, the drug we just saw data for, acts by enhancing transmission at the postsynaptic GABA A receptor. So within the synapse. There are also now known extra synaptic GABA A receptors. So, these Receptors are located outside the synapse and they detect volume conduction, ambient levels of GABA, and they are important for general anesthesia, many general anesthetics act by enhancing transmission at extra synoptic GABA receptors. These are also the GABA that's measured, for example, using in vivo microdialysis, is the GABA that's acting at these Extrasynaptic GABA receptors. So Jean Carl Vanini showed using a drug called Gaboxadol, which activates extrasynaptic GABA receptors. Vanini also showed a concentration appended increase in wakefulness, a decrease in non-REM sleep, and a complete inhibition of REM sleep by promoting GABAergic transmission at extrasynaptic GABA receptors. Great. These drugs are Gaboxadol and Muscimol, they're very useful tools, they're exogenous. Normally, the brain never sees these drugs. How can we look at the effects of endogenous GABA, and the effects on sleep and wakefulness? We can do that by manipulating GABAergic transmission. So here's again, our normal synapse, and the dots here reflect the normal basal amounts of GABA within the synapse. You can see the GABA being packaged into vesicles and being released. So we have normal ambient levels of GABA. One thing one can do to pharmacologically manipulate GABA, is to block the GABA transporter. The GABA transporter takes GABA that's been released, and takes it back up into the presynaptic terminal, or into the glia to terminate the action of GABA. So if you block that transporter, you get levels of GABA in synapse and outside the synapse. So you promote GABAergic transmission and you increase extracellular levels of GABA. So if one does this within the pontine reticular formation, what do you think happens to sleep? Well- Sleep is inhibited just like it is when we give a exogenous agonist at GABA receptors. So you increase in wakefulness and a decrease in non-REM sleep and REM sleep. Likewise, one can also decrease the levels of GABA in the synapse and in the extracellular space by blocking the synthesis of GABA with a drug that inhibits the synthetic enzyme GAD. So the neuron makes less GABA, less GABA is released. GABA levels go down, and if GABA levels in the pontine reticular formation go down, wakefulness goes down and there's a compensatory increase in both non-REM sleep and REM sleep. And you can see in this photograph that this animal has assumed a normal sleep posture and the EEG demonstrates that this looks very much like normal sleep. So we have evidence from these studies of manipulating levels of GABA that endogenous GABA in the pontine reticular formation, plays a role in regulating sleep and wakefulness, and endogenous GABA seems to promote wakefulness and inhibits sleep. Another way to test the effects of an endogenous GABA, is to block the receptors, and those data are shown in this slide. Bicuculline is a drug that blocks GABA A receptors. And if one puts Bicuculline into the pontine reticular formation, again you see a concentration appended inhibition of wakefulness and a real concentration appended enhancement of REM sleep and also an increase in non-REM sleep. Here's the real dramatic effect. Concentration made an increase in REM sleep. So how might this be happening? I mean these findings together clearly imply that GABAergic transmission in the pontine reticular formation inhibits REM sleep, because if you block GABAergic transmission you get REM enhancement. Well by what mechanisms, is the obvious next question. So we have already reviewed data showing that cholinergic transmission in the pontine reticular formation promotes REM sleep since GABA is an inhibitory transmitter might GABA inhibit the release of acetylcholine in the pontine reticular formation. And so we are shown that Flint asked that question and she blocked GABA-A receptors in the pontine reticular formation using the drug Bicuculline, and she measured simultaneously the release of acetylcholine. So by using in-vivo microdialysis, she delivered Bicuculline just to this area of the pontine reticular formation and simultaneously measured acetylcholine and found a beautiful concentration appended increase in the release of acetylcholine when the GABA A receptors were blocked. So this finding means that GABA does inhibit the release of acetylcholine in the pontine reticular formation. So GABA inhibits REM and it inhibits the release of acetylcholine, and we know that acetylcholine causes REM. Yet, how do we know whether these findings are just true-true but unrelated or really causally related? And the way to do that is using another drug, and this study was done by Jerry Marks and his colleagues. Jerry Marks showed that the increase in REM sleep that occurs by blocking GABA receptors is blocked by inhibiting muscarinic receptors. So what that means is that when you give this GABA antagonist and get an increase in acetylcholine and an increase in REM sleep, that increase in REM sleep is mediated by acetylcholine because if you block the acetylcholine receptors, there is no increase in REM sleep. So, as a nice pharmacological study showing that GABA receptors in the pontine reticular formation promote wakefulness and inhibit REM sleep, the mechanism by which involves inhibition of acetylcholine release. Well one then wants to say, is there a metric by which we can quantify the relationship between GABAergic and cholinergic transmission across the sleep cycle within a particular brain area? So I'm going to show you that metric in the next three slides, two of which you've already seen. One shows GABA levels in the pontine reticular formation, endogenous GABA levels which decrease across the sleep wake cycle and are at their lowest during REM sleep. I remember GABA is inhibitory to REM sleep when it's acting in the pontine reticular formation. Likewise, endogenous acetylcholine increases during REM sleep in the pontine reticular formation and GABA inhibits this release. If we put these two transmitter release patterns together and express them as a percent of waking levels so that the waking level of each transmitter is 100%, shown here, and we plot the ratio of GABAergic to cholinergic transmission in the pontine reticular formation across the sleep wake cycle, we see that actually the ratio is very similar during waking and non-REM, but differs quite significantly during REM sleep. And so this finding supports the interpretation that REM sleep occurs in a neurochemical milieu where there is reduced GABAergic transmission and enhanced cholinergic transmission. So low GABAergic tone and high cholinergic tone in the pontine reticular formation contribute to the generation of REM sleep. How is this wake-promoting, sleep-inhibiting effect of GABA within the pontine reticular formation relevant? How is it not just some sort of odd pre-clinical curiosity? Well one reason could be that we know in clinical situations, that first of all, benzodiazepines which act at GABA receptors, many of them although they increase total sleep time, they actually inhibit REM sleep and they increase REM latency. So they inhibit REM sleep, not totally, but they push it back certainly. And so we think that increasing GABAergic transmission in the pons which is what these drugs do, may be one of the mechanisms by which benzodiazepines decrease REM sleep. When we give these drugs clinically, we bathe the entire brain in these drugs. So we take them systemically, either intravenously or orally, and the drugs distributes throughout the brain in contrast to what we do when we give them in specific brain regions in animals. So what we see is the integrated output of these drugs acting at many different levels of the brain, when we look at behavior after taking a drug systemically. So clearly the sleep promoting effects of the benzodiazepines are the overall response. But the sleep is not exactly normal and the REM suppressant effects may be due to the actions of the benzodiazepines at the level of the pontine brainstem. Benzodiazepines have also been known to sometimes produce paradoxical excitation. And one case in which this is true is in children. Many children who are sedated with benzodiazepines, to go in the scanner for example, in the hospital, have a paradoxical response and become very agitated and almost uncontrollable in which case. So it's very unpleasant experience and the case has to be canceled and so forth. We don't know why that is, but the fact that enhancing the enhancement of wakefulness and wake promoting systems by GABAergic transmission, may underlie at least some of the mechanism by which benzodiazepines can sometimes produce a paradoxical response. So that's an exciting area for future study. And I'm going to end our discussion of GABA's role in wakefulness here. In the next segment, we'll turn to another wake-promoting transmitter, Hypocretin, also known as Orexin, and that Orexin segment will be followed then by a discussion of the sleep-promoting effects of GABA.
