THE INFLUENCE OF THE ANTERIOR LEFT AMYGDALA COMPLEX IN THE DEVELOPMENT OF ALCOHOL DEPENDENCE IN FEMALE RATS

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The almond complex (MC, corpus amygdaloideum) is a part of the limbic system of the brain that acts as a polysensory centre. The information analysed in it is transmitted to the visceral brainstem and its higher sections - the optic tuberosity and neocortex [1, 2].

The MC of the human brain is located close to the medial surface of the temporal lobe. Since the MC nuclei are close to the ancient cortex, this area is called pereamygdaloidal. Thus the MC of a human being is located on border with the inferior and medial surfaces of pereamygdaloid and entorhinal cortex area. Outside, the amygdaloid complex is in contact with the white matter of the temporal lobe, and above with the lenticular nucleus and the lower edge of the fence. It reaches posteriorly to the lower horn of the lateral ventricle, and at the apex of this horn it comes close to the hippocampus [3]. (Figure 1).

Figure 1 - Location of the amygdala complex in the human brain [4]

The formation of the amygdala complex occurs in several stages simultaneously with the corticalisation of the vertebrate brain. In this regard, the ancient, old and new cortex are distinguished, which explains the separation of the palaeoamygdala, archamygdala and neoamygdala complexes [4, 5].

The functions of the MC are related to the provision of defensive behaviour, autonomic, motor and emotional reactions, motivation of conditioned reflex behaviour. [5]. 

Joint work of MC nuclei can play a protective function - for example, an unpleasant taste or odour makes a person experience negative emotions and stay away from what causes them - spoiled food, which can be poisoned, or waste products, which may contain dangerous bacteria.  Depending on its location, the almond-shaped body forms different emotions. Electrical stimulation of the right amygdala produces negative emotions, especially fear and sadness. In contrast, stimulation of the left amygdala can evoke both pleasant (happiness) and unpleasant (fear, anxiety, sadness) emotions [6].

The amygdala also plays a role in reward and anxiety processes. When the central nucleus is damaged, the animal loses interest in reward. A completely damaged amygdala reduces the ability to respond to changes in reward value and leads to inappropriate behaviour [7].

As a result of MC removal, the effects of increasing the sensitivity of GABA receptors to GABA under the influence of benzodiazepines are observed to increase the frequency of opening of chloride channels, resulting in a greater number of negatively charged chloride ions entering the interior of the neuron, leading to hyperpolarisation of the neuronal membrane and the development of inhibitory processes. Benzodiapine facilitates transmission through the GABAergic system involved in memory formation. GABA plays an important role in the amygdala, being one of the components of inhibitory circuits, carries out the balance between excitation and inhibition. Disruption of GABAergic inhibition in the basolateral nucleus can lead to increased anxiety and depression as well as seizure activity [7, 8].

Alcohol is the most common xenobiotic in the world. Despite many years of research on different models of experimental alcoholism, today we cannot speak of any unambiguous relationship between biological or social factors and the development of addiction. The phenomenon of mental dependence on alcohol (1st stage of alcoholism) is a disturbance of biochemical processes in the CNS induced by excessive concentrations of acetaldehyde. Acetaldehyde is 30 times more toxic than ethanol (its minimum lethal dose is 30 times lower than that of ethyl alcohol). However, acetaldehyde reacts extremely easily with amino groups of amines in general and biogenic amines in particular. In the latter case, the formation of tetrahydroisoquinoline derivatives (TGIQ) - salsolinol and tetrahydropaverine (THP), which belong to the group of endogenous morphine-like substances (endorphins), is possible. These substances have morphine-like activity and, in addition, are substrates for the formation of other, even more active morphine-like compounds. It is these substances that affect opiate receptors of the hypothalamic ‘pleasure centre’, inducing euphoria when taking alcohol and forming a pathological attachment to it [9].

The development of physical dependence on ethanol, which begins to manifest itself from the second stage of chronic alcoholism, is caused by the progression of two closely interrelated processes: the increase in tolerance to ethanol and the development of withdrawal syndrome. For example, the level of anxiety can influence the development of alcohol dependence. In the course of a lot of work with experimental alcoholism, scientists have learnt that it is not anxiety itself that causes a preference for alcohol, but the response to a stress reaction (excitation or inhibition). Therefore, depending on the response, for example, as a result of arousal, rats accompanied with increased levels of anxiety are more likely to develop alcohol dependence [9].

Conditions and methods of research

 The study was carried out in accordance with the norms of bioethical attitude to laboratory animals, the protocol of the experiment was approved at the meeting of the Commission on Scientific Ethics of the Faculty of Biology of Samara University. The experiment involved 10 sexually mature female rats. The animals were divided into two groups: experimental and control, 5 individuals in each group and placed in different cages. For the duration of the experiment, water in drinkers was replaced with 5% alcohol solution free access. In determining the concentrations of ethanol and the timing of forced alcoholisation we relied on the data of the authors [6], who showed that 6% aqueous ethanol solution consumed by rats for a fortnight leads to changes in the metabolism of dopamine and serotonin in the amygdala and prefrontal cortex of the brain. The operation was performed under urethane anaesthesia (1.0 g/kg) administered intraperitoneally. The animal's head was placed in the head holder of the stereotactic device (fixation of three points: maxilla and ear canals). The hair on the dorsal surface of the head was clipped with Cooper scissors. Next, a skin incision was made and soft tissues were removed. Having anaesthetised the epicondyle and adjacent tissues with lidocaine application, the epicondyle was removed and the bregma point was marked. Then the animal was placed in the stereotactic device and the skull was marked according to the atlas coordinates P= 2.4 mm; L= 5.0 mm; V= 8.5 mm from bregma - for the anterior left part of the amygdaloid complex (Paxions, Watson, 1986).

After establishing the required location, the trepanation hole was drilled using a burr. An isolated electrode was inserted into the hole along its entire length. The indifferent electrode was fixed on the ear of the animal. The central part of the tonsil was destroyed with 1μA current for 10 seconds using a direct current stimulator B5-44. The active electrode was connected to the anode and the cathode to the rat skin. After electrocoagulation, the electrode was carefully removed from the rat brain. The trepanation hole was covered with dental cement, ligature was applied to the skin, processed and additionally fixed with BF-6 glue. Then each rat was monitored until the moment of awakening and several hours afterwards to make sure that the animal tolerated the operation normally. During 7 days we carried out postoperative observations of the animal.Statistical processing of the data was carried out with the help of SigmaPlot, Microsoft Excel software package using Student's criteria. Differences at p < 0.05 were considered statistically significant.

Results of the study and their discussion

 Figures 2-5 show the overall results of the ‘Open Field’ test of rats before the consumption of 5% alcohol solution and afterwards for 4 weeks. To assess motor activity, exploratory activity and anxiety, the animals were tested in the Open Field facility. The rats of the experimental group after amygdalectomy surgery showed greater motor and exploratory activity and exhibited the least anxiety compared to the control group. This difference was clearly seen at week 4, when the animals of both groups of rats were given drinkers with water and alcohol with free access.

Based on the results of this table (Table 1), we can conclude that the rats of the control group, at the week with free access to drinkers with 5% alcohol solution and ordinary water, more readily preferred alcohol to water, namely almost 2 times. However, the experimental group, after amygdalectomy surgery, behaved the opposite way. Animals with destroyed amygdala were almost 2.5 times more willing to choose water over alcohol.

Table 1

 Preference of alcohol and water consumption in control and experimental groups

There was a significant decrease in vertical activity in the control group at week 4 of the experiment (p≤0.001) compared to weeks 1 and 2 (by 99%). Thus, the exploratory activity in the experimental group at week 4 was found to be 9.4 on average, while that of the control group was 0.2. This means that the exploratory activity of the experimental group was almost 97% (p=0.01) higher than that of the control group (Table 2, Figure 2).

Table 2

 Vertical motor activity in control and experimental groups 4 weeks after amygdalectomy

Figure 2 - Change in the number of vertical stands in the Open Field test of the control and experimental groups at different periods of the study

The symbol (#) marks statistically significant differences within the group at ## P≤0.01; ### p≤0.001.

Asterisks (*) indicate statistically significant differences between control and experimental groups at *** p≤0.001.

At week 4 of the experiment, animals in the experimental group showed a rather sharp decline in exploratory activity by 77% compared to week 1 and 73% compared to week 2. The control also significantly decreased compared to week 1 and 2 by 91%. However, we can note that the mean number of exploratory holes by the experimental group (2.4) at week 4 was significantly higher than that of the control (0.8), namely by 67% (p=0.035) (Table 4, Figure 4).

Table 4

 Number of investigated openings in control and experimental groups 4 weeks after amygdalectomy in the ‘Open Field’

Figure 4 - Number of holes explored in the Open Field test of the control and experimental groups at different periods of the study

The symbol (#) marks statistically significant differences within the group at ## P≤0.01; ### p≤0.001.

Asterisks (*) indicate statistically significant differences between control and experimental groups at ** p≤0.01.

A significant jump in grooming episodes is clearly seen in the control group at week 4, indicating an increase in anxiety level.Namely by 91% as compared to week 1 and 86% as compared to week 2. Also compared to the experimental group which has about single number of grooming episodes, the control is 91% higher at week 4 (Table 5, Figure 5).

Table 5

 Change in the number of anxiety grooming episodes in the control and experimental groups 4 weeks after amygdalectomy in the Open Field

Figure 5 - Change in the number of manifestations of anxious grooming in the Open Field test of the control and experimental groups in different periods of the study

The symbol (#) marks statistically significant differences within the group at ### p≤0.001.

Asterisks (*) indicate statistically significant differences between control and experimental group at *** p≤0.001.

Conclusions

Thus, in the paper, based on the analysis of the results of the conducted experiment with laboratory rats in the ‘Open Field’ test, it can be concluded that long-term alcohol consumption leads to the formation of addictive behaviour towards ethyl alcohol. Also on the above-mentioned data withdrawal syndrome in rats of the control group at the 4th week is clearly seen, namely increase of anxiety and decrease of motor and research activity. In animals of the experimental group the withdrawal syndrome was not observed, as well as there was no preference for alcohol after the operation on destruction of the left amygdala complex. Research and motor activity increased in the operated animals, and the manifestation of anxiety was not noted. This is due to changes in the metabolism of dopamine and serotonin in the destroyed amygdala and prefrontal cortex. Given the function of serotonin in regulating impulsive behaviour, and dopamine with positive reinforcement, it is likely that the destruction of the amygdala has disrupted the regulation of these two neurotransmitters in the prefrontal cortex, which affects the nigrostriatal pathway. This pathway, runs in the brain and connects the substantia nigra compacta in the midbrain to the dorsal striatum (i.e., caudate nucleus and putamen) in the forebrain. The nigrostriatal pathway affects motor activity by stimulating it, as well as cognition, reward, and dependence processes. These changes may have resulted in compensatory mechanisms that promote more rational water selection and increased motor activity in animals of the experimental group.

About the authors

Eugenia Metlina

Author for correspondence.
Email: mozartello6@gmail.com
Russian Federation, 443086, Russia, Samara, Moskovskoe shosse str. 34

References

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