Mutant mice don't become drug addicts
This protein plays a key role in the processes of learning "from positive experience", as well as in the development of drug addiction. The results obtained made it possible to design mutant mice with weakened motivation of behavior by "positive" stimuli, such as food or drugs. Unlike ordinary mice, mutant mice show less persistence in performing actions aimed at obtaining food or a new dose of a drug.
A part of the brain called the nucleus accumbens plays an important role in learning and emotional regulation of behavior. The neurons of the nucleus accumbens carry receptors on their surface that respond to the "pleasure substance" dopamine. This substance is released by other neurons (localized in the midbrain) at those moments when something good happens in our life. As a result, a person (or any other mammal) experiences a feeling of pleasure, and in the future he may form the habit of performing actions aimed at reproducing a situation that leads to the release of dopamine. The effect of many drugs is based on the fact that they either directly stimulate dopamine receptors, or in one way or another increase the concentration of dopamine in the nucleus accumbens (for example, cocaine slows down the reuptake of dopamine by neurons that secrete this substance).
Some key components of the "reward system".
The neurons of the cortex, having received and processed information about something pleasant (a rewarding stimulus), send signals to the "ventral region of the tire" (VTA) – the part of the midbrain whose neurons produce dopamine.
After that, the VTA sends its dopamine signals to the nucleus accumbens, amygdala, prefrontal cortex and other parts of the brain. (MFB, medial forebrain bundle – medial anterior cerebral bundle; septum – septum).
Image from the website thebrain.mcgill.ca .
Learning, habit formation, or drug addiction are forms of long–term memory that require long-term changes in the structure of neurons. Such changes may include the regrowth of new nerve endings and interneuronal contacts – synapses, as well as the strengthening or weakening of existing synapses. Some molecular mechanisms of long-term memory formation are known (see: Neurons compete for the right to participate in the formation of reflexes, "Elements", 04/26/2007). However, very little is known about what happens to the neurons of the "reward system" as a result of stimulation of dopamine receptors. Meanwhile, understanding the molecular mechanisms responsible for learning from positive experiences and for the formation of addictions could be of great practical importance – for example, for the treatment of drug addicts.
A group of researchers from France, the USA and Japan managed to decipher one of these mechanisms, as they reported in the latest issue of the journal Nature. Scientists focused their attention on the protein DARPP-32 (32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein), which was previously known to be synthesized in large quantities by neurons of the nucleus accumbens and participates in the response of neurons to dopamine. Previous studies have shown that as a result of the excitation of dopamine receptors of class D1, a phosphoric acid residue is attached to a certain place of the DARPP-32 protein (namely, to the amino acid threonine in the 34th position, Thr34) (phosphorylation by Thr34 occurs). This leads to a change in the properties of the protein. It acquires the ability to suppress the activity of another protein – PP1 (multifunctional serine/threonine protein phosphatase-1). Suppression of PP1 protein activity, in turn, leads to changes in the degree of phosphorylation of receptors and ion channels, which play a key role in synaptic transmission of nerve impulses and in the plasticity of synapses.
Changes of this kind, however, are of a short-term nature. All this happens in the cytoplasm of the nerve cell. For long-term changes, it is necessary to interfere with the work of the cell nucleus, that is, the regulation of the activity of nuclear genes. The results obtained earlier suggested that DARPP-32 plays some role in this, but no one knew the details. And for the practical use of molecular biology data, it is the details that are important, not the general ideas.
With the help of many complex experiments, scientists have found out that the DARPP-32 protein has another important "phosphorylation site" – the amino acid serine in the 97th position (Ser97). The phosphorylation of this amino acid residue serves as a signal for molecular systems transporting proteins through the nuclear membrane. It turned out that if the DARPP-32 protein is phosphorylated by Ser97, it accumulates in the cytoplasm, and if not, it is transported to the nucleus.
Experiments have shown that cocaine, morphine, as well as the process of learning some actions with positive reinforcement - all this leads to dephosphorylation of DARPP–32 by Ser97 and, as a consequence, to the accumulation of this protein in the nucleus.
What does DARPP-32 do when it gets into the core? As it turned out, it does the same thing as in the cytoplasm: it suppresses the activity of the multifunctional protein PP1 (which was already mentioned above). A direct consequence of PP1 inactivation in the nucleus is the phosphorylation of histone H3 (histones are proteins on which DNA molecules in the nucleus are "wound"). Phosphorylation of histone H3, in turn, leads to activation of many different genes. Here they are, the long-term effects! It was previously known that histone H3 phosphorylation is an important component of long-term memory formation. Thus, the picture as a whole has developed (see diagram).
A diagram illustrating the mechanism of influence of the DARPP-32 protein on the activity of genes in the nucleus of a neuron.
Dopamine stimulates dopamine receptors (D1R). The consequence of this (through a number of intermediates) is phosphorylation of the DARPP-32 protein by threonine in the 34th position and dephosophorylation by serine in the 97th position (phosphoric acid residues are depicted as pink circles with the letter P). As a result, the protein DARPP-32 phosphorylated by Thr34 accumulates in the nucleus, where it suppresses the activity of the PP1 protein. This leads to an increase in the level of phosphorylation of histone H3, as a result of which a number of genes are activated. Histones are depicted in the form of blue coils on which a DNA strand is wound. Fig. from the discussed article in Nature.Having deciphered this mechanism, scientists naturally wanted to see what would happen if they interfered with its work.
Is it possible, by changing the structure of the DARPP-32 protein, to reduce the probability of its entering the nucleus and, accordingly, to reduce the risk of drug addiction?
Scientists have made a small change (mutation) in the DARPP-32 protein gene in mice, as a result of which the amino acid serine in the 97th position was replaced by alanine. Unlike serine, alanine cannot be phosphorylated. Thus, in mutant mice, the excitation of dopamine receptors should not lead to the accumulation of DARPP-32 in the nuclei of neurons. This was confirmed: even under the influence of strong drugs in the neurons of mutant mice, the DARPP-32 protein remained in the cytoplasm and did not go to the nucleus.
Since the change made to the structure of the protein was very small, it theoretically should not have affected all its other functions. This was also confirmed: the behavior of mutant mice in general looked quite normal. They even retained the ability to learn. The differences were revealed only in special tests designed to assess the degree of motivation of behavior.
Two variants of motivation were tested: food and narcotic. In the first experiment, mutant mice and control mice were trained to press a button with their noses to get something tasty. Both groups of mice were trained equally successfully, even if the reward was not given for each button press, but only sometimes, in a small percentage of cases.
After that, the trained mice were given the opportunity to press the button as much as they liked, without giving any reward for it. Scientists watched when the unfortunate animals would get tired of this pointless activity. They tested how persistent the mice would be in their actions aimed at obtaining food. This is how the degree of "food motivation" is assessed.
And here, for the first time, mutant mice behaved differently from their "normal" relatives. Mutants are tired of pressing the button much faster than ordinary mice. They were less motivated.
Similar results were obtained in another experiment, where the "reward" was not food, but a dose of cocaine. Mutant mice already familiar with the effects of the drug showed less persistence in trying to get a new dose than ordinary mice in the same situation.
It seems that scientists have discovered a very important component of the most complex system of emotional regulation and motivation of behavior. It is amazing how complex and subtle this system turned out to be (about many components of which science still knows very little). Who would have guessed that by replacing an amino acid in a single protein, one can radically change one of the character traits key to the formation of drug addiction, without affecting the rest of the properties of the psyche?
Of course, here we are talking about such basic neurobiological mechanisms that cannot be unique to mice. Humans also have the DARPP-32 protein, and its work is probably based on the same principles. However, the authors are modestly silent about the possible practical applications of their discovery.
Source: Alexandre Stipanovic, et al. A phosphatase cascade by which rewarding stimuli control nucleosomal response // Nature. Advance online publication, 21 May 2008 (doi:10.1038/nature06994).
Portal "Eternal youth" www.vechnayamolodost.ru26.05.2008