The Mechanism Of Long-term Potentiation In Basolateral Amygdala And Its Role In Drug Addiction

Abstract In recent years, a large body of experimental results on synaptic plasticity has been accumulated in neuroscience. Long-term potentiation (LTP) and Long-term depression are the main forms of synaptic plasticity, which are generally regarded as potential cellular mechanisms underlying learning and memory. LTP in hippocampus is most studied and complicated in cellular mechanism, which is induced by various ways such as drugs or electric stimulus. In behavior experiments the learning capacity decreased significantly and LTP in hippocampus was failed to be induced when the NMDA receptor function was blocked by the application of NMDA receptor antagonist in hippocampus, indicating LTP in hippocampus is closely linked with learning and memory. As an important part of limbic system, Amygdala is mainly involved in emotion-related learning and memory. The research of synaptic plasticity such as LTP in amygdala will help to explore the functions of the amygdala.The amygdala is closely connected with the common reward pathway of ventral tegmental area - nucleus accumbens in drug addiction. Furthermore, behavioral experiments showed that the amygdala was involved in the process of drug addiction. The core character of drug addiction is compulsive drug-abuse: the loss of control over drug-taking and drug seeking. During the period of drug addiction and withdrawal, the memory about drug-related psychological dependence is solid and lasting. Especially in the morphine-type drugs addicts, the special memories about drug-related information is hard to weaken with time and the extraction of the information is closely related with the environment. Drug addiction is widely considered to be an enhanced memory and the role of LTP in drug addiction is well-focused as a cellular mechanism of learning and memory. Drug abuse affected LTP in drug addiction-related brain areas such as the ventral tegmental area, nucleus accumbens and hippocampus, suggesting that these changes were caused by the abuse of drug and enhanced by the form of memory. Amygdala is involved in the occurrence and development of drug addiction, however, the role of amygdala LTP in drug addiction is unclear.In our experiments we compared the effects of different patterns of titanic stimulus on the induction of LTP in basolateral amygdala (BLA) by recording field potentials in BLA in rat brain slices in vitro. We explored the cellular process of BLA LTP by bath application of receptor antagonists and cell pathway inhibitors. Then we established the animal models of morphine addiction, extinction and reinstatement groups by using conditioned place preference. Studies were done to compare the differences of BLA LTP in brain slices from different groups to explore the role and mechanism of BLA LTP in drug addiction.Brain slices were prepared after the decapitation of rats. The sharpened tungsten bipolar stimulating electrodes and recording microelectrode tips were visually positioned in external capsule (EC) and in the basolateral region of the amygdala individually using a dissecting microscope. The tips of field potential electrodes were located about 250μm under the surface of the slices and the distance of stimulating electrode and recording electrode was about 2 mm. Stimuli were delivered using a RM6240 programmable stimulator. Single 0.1-ms monophasic square pulses were applied continuously throughout the experiment at 0.1 Hz. The field potentials were amplified with an SWF-2W amplifier. On- and off-line data acquisition and analysis was carried out using RM6240. Different parameters were applied to induce LTP in BLA to understand the relationship between stimulating pattern and the induction of LTP. To explore the possible molecular mechanism of BLA LTP, N-methyl-D-Aspartate (NMDA) receptor antagonist, the inhibitors of protein kinase C (PKC) and tyrosine protein kinase were added to the perfusion and then LTP was induced. In our experiments in the prerequisite of slices in good activity and recording system noise level under 0.01mv, field potentials in BLA were evoked stably with stimulating electrodes placed in the external capsule (EC) using domestic-made microelectrode amplifier and recording system. The amplitudes of field potentials in BLA were about one-tenth of that in hippocampus CA1. The fielding potentials in BLA were almost totally blocked in the presence of AMPA antagonist CNQX (10μM) and NMDA receptor antagonist APV (100μM), indicating excitatory post synaptic potentials in BLA are glutamate receptor-dependent. LTP in BLA was induced by application of two high frequency stimulations (HFS, 100 Hz, 1s each train) of internal 10 min to external capsule. The theta burst stimulation protocol was applied because it mimics the typical firing mode of pyramidal cells during learning.The slope of field potentials 30 min after HFS was 146.1±6.9%(n=9,p<0.01)) of the initial baseline values. High-frequency stimulation of 100Hz is the common pattern to induce LTP. Theta burst stimulation (TBS) is another model being often used to induce LTP because it mimics the typical firing mode of pyramidal cells during learning and is closer to physiological stimulus. The parameter of TBS is a brief, high-frequency pulse train (5 pulses at 100 Hz) given at the theta-rhythm (5Hz) for 4 sec. Experiments were done to compare the effects of different intervals of two TBS on the expression of LTP in BLA. Two TBS of 10 s interval failed to induce LTP in BLA. The interval of two TBS increased to 10 min and 30 min, individually, both types of stimulations enhanced f-EPSPs in BLA and the enhanced f-EPSPs lasted more than 30 min. Two TBS of 10 min interval is better in the induction of LTP in BLA, however, there is no significant difference in statistics between the interval of 10 min and 30 min. LTP in BLA was input specific and was blocked by N-methyl-D-Aspartate (NMDA) receptor antagonist APV. The effect of protein kinase C (PKC) on LTP was then determined using PKC inhibitor chelerythrine chloride.Bath application of chelerythringe chloride had no effect on basic field potentials and paired-pulse ratio (PPR). However, in the presence of chelerythrine chloride, two TBS failed to induce LTP. In contrast, bath application of chelerythrine chloride 10 min after the second TBS didn't affect the maintenance of LTP in BLA. Furthermore, LTP in BLA induced by two TBS of 10 min and 30 min interval was also blocked by bath application of tyrosine protein kinase (TPK) inhibitor genistein.We established the animal models of morphine addiction by using conditioned place preference. Brain slices were prepared two to four days after the establishment of morphine addiction (before the extinction) and BLA LTP was induced by using two 10 s-interval HFS of 100 Hz in slices. Then we compared the differences of BLA LTP between addiction group and control. In control, application of two HFS only induces a short-term synaptic potentiation (STP). The field potential almost returned to normal and the slope was 114.0±6.3% (n=9, P>0.05) of the initial baseline values 30 min after HFS.In addiction group, the field potential increased significantly after two HFS and the enhanced field potential lasted more than 30 min. The slope of field potential was 154.8±5.3% (n=14, P<0.01) of the baseline 30 min after HFS. To observe the role of protein kinase A (PKA) in BLA LTP in addiction group, the PKA inhibitor PKI-(6-22)-amide Tocris(1μmol/L) was added to the perfusion. The PKA inhibitor had no effects on basal synaptic potential and the field potential was still 99.4±3.8% (n=6 P>0.05) of the basal values after 15 min perfusion. However, in the presence of PKI-(6-22)-amide Tocris, the BLA LTP induced by two HFS attenuated obviously and the slope of field potential was 122.2±4.6% (n=6) of baseline.The extinction model of morphine addiction was established by application of CPP. Two 10 s-interval HFS induced LTP in slices from extinction model and the slope of field potential was 128.0±9.3% (n=8 P<0.01) of baseline 30 min after HFS. However, BLA LTP attenuated in extinction group compared to the addiction group. To establish the relapse model, we adapted two ways of semi-dose morphine and forcing-swimming for 5 min in icy-cold water to reinstate extinguished CPP. Two HFS again obviously induced BLA LTP in slices from the relapse model and there was no significant difference between two ways of reinstatement. The field potential was 142.7±8.0% (n=7, P<0.01) of baseline in semi-dose morphine reinstatement group and 143.3±6.0% (n=5 P<0.01) in forcing-swimming reinstatement group individually.The results showed that the 10 min-interval stimulation of two TBS or 100 Hz was the better pattern of inducing BLA LTP in slices. BLA LTP is input-specific and NMDA receptor-dependent. Furthermore, intracellular protein kinase C and tyrosine protein kinase were involved in the induction and maintenance of BLA LTP. LTP in amygdala changed with the occurrence and development of morphine addiction. BLA LTP enhanced in morphine addiction model but attenuated in extinction group (which was potentiated compared to control) and strengthened again in relapse model, suggesting that the change of BLA LTP caused by addictive drugs is not durable, simple one-way but two-way trend. The changes in basolateral amygdala in the process of drug addiction include the adaptive changes of neurons and synaptic plasticity. Amygdala is involved in the process of learning and memory. The study of LTP in amygdala in the character, nature, pattern of manifestation and cellular mechanism will help to understand the function of amygdala and its role in learning and memory. The research of the role and mechanism of LTP of amygdala and learning and memory in drug addiction and relapse will prodive a scientific basis for the evaluation and intervention of addiction development and relapse. On the other hand, drug addiction may be a model to study synaptic plasticity in nervous system and the process of learning and memory, helping to illuminating the brain mechanism of learning and memory.