Signal Transduction in the Brain: Transcript Part 3
Two Major Neurotransmitters-Dopamine and Glutamate
Dopamine:
This is sort of a summary of the story I am going to tell you, and it turns out that virtually all of these signaling pathways followed the same principles--that one neurotransmitter that we studied most intensively is the neurotransmitter dopamine. And one of the reasons that we studied dopamine so intensively is because abnormality in dopaminergic signaling underlies several major neurological and psychiatric disorders as shown here.
Parkinson's disease is associated with a death of dopaminergic neurons, almost all, well virtually all drugs that are useful in the treatment of schizophrenia work by blocking the ability of dopamine to activate a subclass of dopamine receptors. ADHD, attention deficit hyperactivity disorder, is treated with Ritalin which is an amphetamine-like compound that promotes dopaminergic signaling, and all known drugs of abuse work through the dopamine signaling pathway.
So we did a lot of studies on dopamine and we were very fortunate in these studies to have found a compound, DARPP-32--an acronym for dopamine and cyclic AMP regulated phospho-protein--of 32, 000 molecular weight. DARPP-32 has acted as a Rosetta stone for studying the interaction of dopamine signaling, with signaling through other pathways that interact with the dopamine pathway. So we have been able to dissect things out that way. In fact, in mice, as I will mention shortly, DARPP-32 plays an obligatory role in the actions of dopamine, and in all of the therapeutic drugs and drugs of abuse whose effects are achieved through the dopamine pathway.
This slide shows a coronal section through a monkey brain and immunoperoxidase labeling for DARPP-32. You can see the very intense labeling in the caudatum, in the putamen and in the accumbens and much sparser labeling elsewhere. This is exactly the region of the brain that is most densely innovated by dopaminergic neurons.
So we began to study this part of the brain rather intensively. This is a wiring diagram of the circuitry involved in one of the DARPP-32 pathways. The upper portion is the neuroanatomy, the lower portion, the corresponding neurochemistry. There are nerve cells, the cell bodies of which are in the cortex, the project of the neostriatum, shown here in yellow. They use glutamate as the neurotransmitter and they excite these blue cells. (For those of you who are not scientists these cells are not actually yellow and blue.)
These blue cells contain DARPP-32 and they project from the neostriatum to the substantia nigra. They use GABA as the neurotransmitter and they innovate third order neurons. But they also have branches, axon collaterals, that innervate dopaminergic neurons that feed back on these DARPP- 32 containing neurons to modulate the glutamaturgic transmission.
One of the most interesting cell biological problems at the present time in the brain is to understand how slow synaptic transmission modulates fast synaptic transmission. You can think of it in a way as the fast transmission being the hardware of the brain and the slow synaptic transmission as the software. This is a nice model in which to study how slow the transmission regulates fast transmission, and we found many types of mechanisms, not all of which I will include tonight, but this has been the system that we studied most thoroughly.
How slow synaptic transmission modulates fast synaptic transmission:
Then how does this system work? How does dopamine released from these nerve terminals alter the ability of glutamate released from these nerve terminals to cause nerve impulses to be fired in these DARPP-32 containing nerve cells?
I am formulating the question slightly differently&here is a glutamate nerve terminal activating glutamate receptors. Here is a dopaminergic nerve terminal activating dopamine receptors, and the question is how does this one modify that one?
Well, before telling you this story, let me introduce the cast of characters. There are three major classes of glutamate receptors referred to as NMDA receptors, AMPA receptors and metabotropic receptors. There are two major categories of dopamine receptors referred to as D1 and D2 subclasses. So the question is, how does transmission in this pathway modify transmission in this pathway? And it turns out that both of these receptors modify all three of these receptors and their efficiency in signaling.
Let me go into the story, then. Dopamine, it turns out, activates these D1 receptors. (I am just going to show you most of these schemes and very little substantiating data, but I will show you a little of that, to give you a flavor of the types of experiments that are done.) So Dopamine activates the D1 receptor, and when it does so, it causes the activation of adenyl cyclase, which causes the formation of cyclic AMP, which then activates cyclic AMP-dependent protein kinase (abbreviated PKA), which then phosphorylates DARPP-32, and it phosphorylates it on a specific residue in position 3 and 34.
And I am going to show you three slides that provide some of the evidence for that conclusion. In these experiments we used mouse striatal slices and incubated them for various periods of time in the presence of dopamine. And you can see that there is a transient but very large increase in the state of phosphorylation of the DARPP-32 with a peak at about 2 to 4 minutes. We then ask whether this effective dopamine was due to activation of D1 receptors or D2 receptors.
And it turned out we could mimic dopamine by using an agonist that acts selectively, a D1 receptor compound called SKF82526. So you can see here when this D1 agonist, that is KF, was applied to cells it gives the same affect as dopamine itself. In fact, the D2 receptor agonist had the opposite effect. You can see here that Quinpirole, a D2 selective agonist, caused the dephosphorylation of the DARPP-32.
So we could go back and modify our scheme and say that the dopamine activates the D1 receptor and raises cyclic AMP, and phosphorylates DARPP-32. And to the D2 receptor it does two things: It synergistically causes a decrease in dephosphorylation [by] 1) inhibits the ability of D1 receptors to raise cyclic AMP and 2) by raising ____calcium through activating phospholipase C, it activates a calcium dependent phosphatase called calcineurin or calcium calmodulin-dependent protein phosphatase or PP2B, protein phosphate 2B which dephosphorylates the DARPP-32. So you have a decreased phosphorylation and increased dephosphorylation.
So this is a synergistic affect and a theme that occurs over and over again in these signal transduction pathways. It is beginning to get a little complicated--as a warning to those of you who would like to leave, it is going to get enormously more complex than this. You will wish for these good old days in a very short time.
Glutamate:
So we were quite excited about having shown there was a biochemical affect of dopamine other than making cyclic AMP, and we then asked the question whether, since dopamine can regulate the DARPP-32 phosphorylation, what about glutamate, the other major neurotransmitter that regulates these cells?
And so we did this experiment shown here. Here is phosphorylated DARPP-32 under controlled conditions in the presence of this D1 agonist, SKF. And you have about a 10-fold increase in this experiment. But the D1 agonist is totally abolished by this glutamate agonist, NMDA, showing that there was a dramatic interaction between dopamine and glutamate physiologically as I mentioned, but there is also an interaction here at the biochemical level. So we were more encouraged that we were on the right track by these results.
Direct acting neurotransmitters and their effect on phosphorylation:
Then we began to look at other neurotransmitters which regulate these cells. I should have mentioned in that scheme that I showed you, that these DARPP-32 containing neurons--they are called medium spiny neurons based upon their morphology--they integrate all the information coming into the neostriatum from all over the brain. So there are a whole bunch of nerve cells with different neurotransmitters that all converge on these DARPP-32-containing neurons, and these are the only neurons that can send information out of the neostriatum. That is, the only efferent pathway. So the function of these cells is to integrate a huge amount of information and then send it back out of the neostriatum.
So we looked at every neurotransmitter that was known to have effects on the physiology of these cells and every one of them affected the state of phosphorylation of the DARPP-32. So I forgot something here I wanted to put in the glutamate activating the NMDA receptor also raises calcium, activates the calcium phosphatase and dephosphorylates the DARPP-32.
So now we have looked at all these other neurotransmitters--adenosine, acting on A2A receptors, work through the cyclic AMP-PK pathway to phosphorylate this 3 and 34, serotonin acting on 5HT4 receptors did the same thing. VIP (vasoactive intestinal peptide), which is an important peptide in the brain, raises cyclic AMP and phosphorylates DARPP-32. Nitric oxide, which is diffusing in from nearby neurons, raises the level of cyclic GMP in these cells which activates cyclic GMP-dependent protein kinase, which also phosphorylates 3 and 34 in DARPP-32. This is the first substrate that was found where both PK and PKG could phosphorylate the same residue, and is still the best example. The reason is that these two kinases are mediating the actions of different kinds of neurotransmitters in this part of the brain. So there are all these effects on the phosphorylation side
And then on the dephosphorylation side we found, for example, that glutamate, acting on AMP receptors also brought about dephosphorylation. GABA, the neurotransmitter that DARPP-32-containing cells use, actually has recurrent collaterals which feed back on the nerve cells and inhibit them. And the GABA, by increasing potassium conductances, hyperpolarizes cells, lowers calcium, and thereby shuts off the calcium phosphatase and thereby increases the phosphorylation of the DARPP-32. It is very interesting that both dopamine and GABA which can be inhibitory on these cells both increase DARPP-32 phosphorylation. The dopamine does it by increasing phosphorylation, the GABA does it by decreasing dephosphorylation. As one might have predicted these two affects are synergistic.
Indirect acting neurotransmitters:
So these are all direct acting neurotransmitters, then we have indirectly acting neurotransmitters. Neurotensin, for example, which regulates the physiology of these cells, releases dopamine and causes an increase in phosphorylation of the DARPP-32. CCK (cholecystokinin), by releasing glutamate, brings about the dephosphorylation of DARPP-32.
Therapeutic drugs and their effect on phosphorylation:
We then looked at some therapeutic drugs, for example all of the anti-psychotic drugs. As I mentioned, they all act by blocking the ability of dopamine to activate D2 receptors. One example I shown here is Haldol blocks this activation and thereby increases the state of phosphorylation of DARPP-32, and all of the drugs of abuse seem to do the same thing. For example, opiates acting on "mu" (μ) class of receptors prevent the ability of D1 receptors to increase cyclic AMP. Opiates acting on delta" receptors prevent the ability of A2A receptors to increase cyclic AMP. Cocaine and amphetamine which promote dopaminergic release and increase the amount of dopaminergic signaling also increase phosphorylation DARPP-32. So the CNS depressants decrease DARPP-32 phosphorylation and the CNS stimulates like cocaine and amphetamine, the psychostimulant drugs of abuse, increase phosphorylation.
So one can ask the question as to why so much evolutionary machinery has gone into controlling the state of phosphorylation of DARPP-32. Well, I am going to tell you in just a moment...
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