2.5. Benzodiazepine receptor system in various structures of the human mature brain in patients with alcoholism

Benzodiazepine receptors in different human mature brain of the alcoholics were performed using autopsy material (postmortem) obtained as a result of an urgent autopsy. Samples of autopsy material of the human brain were obtained during urgent autopsy (no later than 6 hours after the onset of death). Samples of the tissue of the prefrontal cerebral cortex, the cerebellar cortex and the head of the caudate nucleus of the brain in persons who were chronically subjected to alcoholization (based on anamnesis) and control subjects were postmortem. Samples of the brain were frozen and stored in thermoses with liquid nitrogen. A total of 126 samples from different areas of the human brain were obtained for the study of radio-receptor binding, including the basic group and the reference control group. In addition to the data of the anamnesis, the objective biological criteria for chronic alcoholization of man (fatty liver, cirrhosis, etc.) were used to form the main group. The control group included patients who did not have neurological and mental illnesses. Autopsy material was obtained only from males, and the age range was 33–54 years. Alcoholic patients were under the supervision by psychiatrists of Mental Health Research Institute and had a diagnosis according to ICD-10: F10.232; F10.302. Patients

with other psychiatric disorders were not included in this study. The study included only patients whose lethal outcome occurred as a result of acute heart failure and not subjected to resuscitation measures.

reaction with decreasing affinity of receptors. The change in receptor affinity is attributed to neuroplastic changes in the tissue of the developing brain due to the chronic effects of alcohol. In ontogenesis, in the early stages of gestation, the benzodiazepine receptor system of the human brain is normally formed, starting with the 7th week of development. According to the data obtained, the density of BzDR during pregnancy 8–9 – 14-15 weeks increases by almost 200%. During prenatal influence of alcohol, associated with maternal alcoholism, we found that expression of BzDR was higher in comparison with control, at different developmental stages. The data of receptor analysis showed that the density of synaptic BzDR (Bmax) correlates with the morphometric characteristics of the synapses (Table 4). We have shown that the affinity of receptors for the ligand during the development of the brain is somewhat reduced, which indicates the greatest sensitivity of receptors at the earliest stages of development—8–10 weeks of gestation. The prenatal influence of alcohol significantly reduced the affinity of the receptors in the experimental group, which confirms the greatest sensitivity of the BzDR to alcohol at the earliest stage of the formation of the human brain. The results of our study of the human embryonic brain in normal and under the influence of alcohol, which is associated with mother's alcoholism, indicate significant neuroplastic changes in the human

Neuroplastic changes in blood vessels, synapses associated with GABAergic activity and BzDR receptors, in the developing brain under the influence of maternal alcoholism, are aimed at adapting the nervous system of the embryo and fetus to the phenomena of hypoxia, as well as functional failure of GABAergic neurotransmission. However, these adaptive changes in the human embryonic brain differ significantly from the processes of formation of angiogenesis and synaptogenesis and GABAAR neurotransmitter system of the normal human brain, which leads to various somatic disruptions and mental disorders, including the development of FAS

2.5. Benzodiazepine receptor system in various structures of the human mature brain in

Benzodiazepine receptors in different human mature brain of the alcoholics were performed using autopsy material (postmortem) obtained as a result of an urgent autopsy. Samples of autopsy material of the human brain were obtained during urgent autopsy (no later than 6 hours after the onset of death). Samples of the tissue of the prefrontal cerebral cortex, the cerebellar cortex and the head of the caudate nucleus of the brain in persons who were chronically subjected to alcoholization (based on anamnesis) and control subjects were postmortem. Samples of the brain were frozen and stored in thermoses with liquid nitrogen. A total of 126 samples from different areas of the human brain were obtained for the study of radio-receptor binding, including the basic group and the reference control group. In addition to the data of the anamnesis, the objective biological criteria for chronic alcoholization of man (fatty liver, cirrhosis, etc.) were used to form the main group. The control group included patients who did not have neurological and mental illnesses. Autopsy material was obtained only from males, and the age range was 33–54 years. Alcoholic patients were under the supervision by psychiatrists of Mental Health Research Institute and had a diagnosis according to ICD-10: F10.232; F10.302. Patients

brain during the early stages of its growth and development [52, 53].

and PAE.

90 Drug Addiction

patients with alcoholism

The separation of tissue from human brain samples into membrane fractions (synaptosomal and mitochondrial) was carried out by preparative ultracentrifugation. The resulting membrane fractions were frozen and stored at t = 80 C. Investigation of the properties of BzDR "central" type (CBR) and BzDRs "peripheral" type (PBR) was performed by the radioreceptor assay of binding synaptosomal and mitochondrial membranes with selective ligands. We used the parametric method (t test) using Statistika 10.0.

The experimental part of the research was carried out by us in the Laboratory of Neurobiology Mental Health Research Institute (Tomsk) and Laboratory of Clinical Biochemistry Research Center for Mental Health Sciences (Moscow). All ongoing studies were approved by the Ethics Committee.

(I) A study of the binding characteristics of the selective ligand [<sup>3</sup> H]-flunitrazepam with synaptosomal fractions of membranes obtained from various regions of the human brain (postmortem) has shown that the properties of synaptosomal BzDR differ in the structures of the brain studied. The highest affinity of CBR was detected in the caudate nucleus and the lower affinity receptors have been identified in the cerebral cortex (the region of the prefrontal cortex) and in the cerebellar cortex (Figure 17, Table 5).

The density of the receptors in the brain structures studied was also different: the maximum receptor density (Bmax) was detected in the caudate nucleus, in the cerebral cortex (the region of the prefrontal cortex) and in the cerebellar cortex (Figure 18, Table 5). Thus, the results obtained by us testify to the heterogeneity of the CBR in various areas of the human brain in the control group. A comparative analysis of the

Figure 17. Statistical analysis of [<sup>3</sup> H]-flunitrazepam binding parameters [Kd (nM) – constant of dissociation ligandreceptor complex] with synaptosomal membranes in different areas of the human brain in control group (a) and study group (b) (alcoholic patients).


affinity. The largest changes in Kd were found in the cerebral cortex, the caudate nucleus and, to a lesser extent, in the cerebellar cortex (Figures 17 and 18, Table 5). Thus, the changes revealed by us indicate a decrease in the affinity of CBP in the brains of patients under the exposure of chronic alcoholization and an increase in their density in relation

Molecular-Cellular Targets of the Pathogenetic Action of Ethanol in the Human Brain in Ontogenesis…

to the mitochondrial fraction of membranes isolated from various regions of the human brain showed that the degree of manifestation of changes in the PBR properties is not the same in the studied brain structures of patients who had alcoholism according to anamnesis. The greatest changes of PBR in comparison with the control were detected in the caudate nucleus and the cerebellar cortex (Figures 19 and 20, Table 5). The obtained results indicate a heterogeneous change in the properties of BzDR of selective ligands in the human brain under the influence of chronic alcoholization, which confirms the hypothesis of adaptive receptor neuroplasticity and the heterogeneity of the physiological

H]PK-11195

93

http://dx.doi.org/10.5772/intechopen.73333

to the control group, which can be compensatory adaptive in nature [54]. (II) A comparative analysis of the PBR properties in the study of the binding of [3

response in various brain regions to the effect of chronic alcohol exposure [54].

Figure 19. Statistical analysis of [<sup>3</sup>

(alcoholic patients).

The results we obtained are consistent with data from other studies showing a decrease in the function of GABAA/BzDR in the cerebral cortex in patients with alcohol dependence [36, 55]. These data confirm that the low affinity of BzDR can be a neuronal marker of the development of anxiety and conditions associated with chronic alcohol use and AAS. The study of BzDR carried out by us in various areas of the human brain (on postmortal material) showed that the properties of synaptosomal and mitochondrial receptors differ in the brain structures studied: the prefrontal cortex, the caudate nucleus and the cerebellar cortex. CBR are the sites of specific binding of ligands of benzodiazepine series, neurosteroids and alcohol to the GABA receptor, modulating its function allosteric and regulating the processes of inhibition in brain structures that affect the activity of various neurotransmitter systems, including the activity in the structures of the brain associated with the process of natural reinforcement. The higher affinity and

H]PK-11195 binding parameters [Kd (nM) – constant of dissociation ligand-receptor

complex] with mitochondrial membranes in different areas of the human brain in control group (a) and study group (b)

Notes: Bmax<sup>1</sup> , density of binding sites [<sup>3</sup> H]-flunitrazepam with synaptosomal membranes; Kd 1 , constant of dissociation ligand-receptor complex [<sup>3</sup> H]-flunitrazepam with CBR; Bmax<sup>2</sup> , density of binding sites [<sup>3</sup> H]PK-11195 with mitochondrial membranes; Kd 2 , constant of dissociation ligand-receptor complex [<sup>3</sup> H]PK-11195 with PBR; n, the number of cases studied. \* Statistically significant difference indicators binding [<sup>3</sup> H]-flunitrazepam and \*\*[ 3 H]PK-11195 between study and control groups, p < 0.05.

Table 5. Properties of [<sup>3</sup> H]-flunitrazepam and [3 H]PK-11195 binding to the synaptosomal and mitochondrial membranes from different areas of the human brain in alcoholic patients and control.

Figure 18. Statistical analysis of [<sup>3</sup> H]-flunitrazepam binding parameters [Bmax (fmol/mg of protein) – density of binding sites] with synaptosomal membranes in different areas of the human brain in control group (a) and study group (b) (alcoholic patients).

kinetic characteristics of the binding of [<sup>3</sup> H]-flunitrazepam showed a significant increase in the Kd values in the studied brain structures in the patients of the main group as compared to the patients in the control group, which indicates a decrease in receptor

affinity. The largest changes in Kd were found in the cerebral cortex, the caudate nucleus and, to a lesser extent, in the cerebellar cortex (Figures 17 and 18, Table 5). Thus, the changes revealed by us indicate a decrease in the affinity of CBP in the brains of patients under the exposure of chronic alcoholization and an increase in their density in relation to the control group, which can be compensatory adaptive in nature [54].

(II) A comparative analysis of the PBR properties in the study of the binding of [3 H]PK-11195 to the mitochondrial fraction of membranes isolated from various regions of the human brain showed that the degree of manifestation of changes in the PBR properties is not the same in the studied brain structures of patients who had alcoholism according to anamnesis. The greatest changes of PBR in comparison with the control were detected in the caudate nucleus and the cerebellar cortex (Figures 19 and 20, Table 5). The obtained results indicate a heterogeneous change in the properties of BzDR of selective ligands in the human brain under the influence of chronic alcoholization, which confirms the hypothesis of adaptive receptor neuroplasticity and the heterogeneity of the physiological response in various brain regions to the effect of chronic alcohol exposure [54].

The results we obtained are consistent with data from other studies showing a decrease in the function of GABAA/BzDR in the cerebral cortex in patients with alcohol dependence [36, 55]. These data confirm that the low affinity of BzDR can be a neuronal marker of the development of anxiety and conditions associated with chronic alcohol use and AAS. The study of BzDR carried out by us in various areas of the human brain (on postmortal material) showed that the properties of synaptosomal and mitochondrial receptors differ in the brain structures studied: the prefrontal cortex, the caudate nucleus and the cerebellar cortex. CBR are the sites of specific binding of ligands of benzodiazepine series, neurosteroids and alcohol to the GABA receptor, modulating its function allosteric and regulating the processes of inhibition in brain structures that affect the activity of various neurotransmitter systems, including the activity in the structures of the brain associated with the process of natural reinforcement. The higher affinity and

Figure 19. Statistical analysis of [<sup>3</sup> H]PK-11195 binding parameters [Kd (nM) – constant of dissociation ligand-receptor complex] with mitochondrial membranes in different areas of the human brain in control group (a) and study group (b) (alcoholic patients).

kinetic characteristics of the binding of [<sup>3</sup>

Area of the brain

92 Drug Addiction

Prefrontal cortex {М SE}

Cerebellar cortex {М SE}

Notes: Bmax<sup>1</sup>

studied. \*

membranes; Kd

ligand-receptor complex [<sup>3</sup>

control groups, p < 0.05.

Table 5. Properties of [<sup>3</sup>

2

Figure 18. Statistical analysis of [<sup>3</sup>

(alcoholic patients).

N. caudatus {М SE} [ 3

Kd 1 (nM)

Control group (n = 21)

> Bmax<sup>1</sup> (fmol/mg protein)

, density of binding sites [<sup>3</sup>

H]-flunitrazepam binding to synaptosomal membranes

> Kd 1 (nM)

H]-flunitrazepam with CBR; Bmax<sup>2</sup>

, constant of dissociation ligand-receptor complex [<sup>3</sup>

Statistically significant difference indicators binding [<sup>3</sup>

H]-flunitrazepam and [3

from different areas of the human brain in alcoholic patients and control.

Study group (n = 21)

> Bmax<sup>1</sup> (fmol/mg protein)

[ 3

Kd 2 (nM)

1.82 0.07 1772 79 2.12 0.09\* <sup>3165</sup> <sup>565</sup>\* 2.45 0.17 1824 11 3.12 0.13\*\* <sup>2245</sup> <sup>168</sup>\*\*

1.68 0.05 948 112 1.97 0.09\* <sup>2817</sup> <sup>386</sup>\* 1.12 0.09 724 36 2.31 0.16\*\* <sup>1895</sup> <sup>77</sup>\*\*

1.98 0.1 1048 67 2.24 0.21\* <sup>1845</sup> <sup>217</sup>\* 2.61 0.21 1209 98 3.32 0.19\*\* <sup>2479</sup> <sup>123</sup>\*\*

H]-flunitrazepam with synaptosomal membranes; Kd

, density of binding sites [<sup>3</sup>

H]-flunitrazepam and \*\*[

Control group (n = 21)

> Bmax<sup>2</sup> (fmol/mg protein)

H]-PK-11195 binding to mitochondrial membranes

> Kd 2 (nM)

1

H]PK-11195 with PBR; n, the number of cases

3

H]PK-11195 binding to the synaptosomal and mitochondrial membranes

Study group (n = 21)

, constant of dissociation

H]PK-11195 with mitochondrial

H]PK-11195 between study and

Bmax<sup>2</sup> (fmol/mg protein)

H]-flunitrazepam showed a significant increase

H]-flunitrazepam binding parameters [Bmax (fmol/mg of protein) – density of binding

in the Kd values in the studied brain structures in the patients of the main group as compared to the patients in the control group, which indicates a decrease in receptor

sites] with synaptosomal membranes in different areas of the human brain in control group (a) and study group (b)

increase in the number of vessels in the brain [57]. Alcoholization of the mother, leading to prenatal effects of alcohol on the developing fetus, affects the dynamics of embryonic development of the circulatory system in the human brain, which manifests itself in a change in the

Molecular-Cellular Targets of the Pathogenetic Action of Ethanol in the Human Brain in Ontogenesis…

http://dx.doi.org/10.5772/intechopen.73333

95

The effects of ethanol in the early stages of development can disrupt the signaling mechanisms that regulate synaptogenesis. The result was "dilution" of the structure of elementary membranes and damaged membranes are less able to establish strong contact with each other, which is probably due also to a reduced ability of cells that are in constant contact with ethanol, synthesized mediators filling synaptic vesicles. This significantly violated the formation of neuronal mechanisms underlying the susceptibility and processing of information,

The data obtained by us showed a structured picture of synaptogenesis as one of the most significant periods in the formation and development of the brain, providing its functions and determining the adaptive potential in prenatal alcohol influences. The influence of prenatal ethanol on the development of synaptic structures was expressed in reduction of morphometric parameters, namely slowing the formation of synaptic contacts and reducing their formation in the brain of the embryo and fetus in the early stages of development, in contrast to the normally developing brain, which affects synaptogenesis in the developing brain of a person

and can underlie fetal death or serious disorders the child in the future [23, 49, 52–54].

On the background of the decrease in the formation of synaptic structures seen here in the fetal brain during gestation under the influence of maternal alcoholism and the simultaneous decrease in the affinity of synaptosomal BzDR, the tendency to an increase in receptor density can be evaluated as neuroplastic features and compensatory reaction directed to adapting the embryo and fetus nervous system to conditions of functional insufficiency of GABAergic neurotransmission. These new data can broaden the understanding of the molecular basis of predisposition not only to alcoholism but also to various disorders associated with PAE. Children and adolescents who were under the influence of alcohol during the period of prenatal development noted functional disorders of neurocognition, self-regulation and adaptive functioning and various neurobehavioral disorders associated with PAE [58]. Plasticity of ion channels and receptors linked to ion channels regulated by neurotransmitters is significant for the realization of adaptive processes in the brain, providing synaptic plasticity for the formation and development of neural network, physiological and pathophysiological processes. Prenatal alcohol exposure (PAE) can cause irreversible physical, neurological and psychiatric impairments that are present at birth and can have lifelong implications [14, 59]. The relationship between prenatal exposure to alcohol and the frequency of behavioral disorders in children and adolescents is established. [60]. The effect remained significant compared to other variables, including environment, maternal psychopathology and some others, and can cause a different mental dysfunction associated with a violation of brain metabolism in

Similar changes in the benzodiazepine receptor binding were identified by us in the brains of patients with alcoholism also. A decrease in the ability of receptors to bind agonist ligands impairs the ligand:receptor protein ratio, leading to decreased binding of the major neurotransmitter

vascularization of the growing human brain [23].

children and adolescents in the future [61].

which in turn could adversely affect a person's mental activity.

Figure 20. Statistical analysis of [<sup>3</sup> H]PK-11195 binding parameters [Bmax (fmol/mg of protein) – density of binding sites] with mitochondrial membranes in different areas of the human brain in control group (a) and study group (b) (alcoholic patients).

density of CBR in the caudate nucleus and the prefrontal cortex are related to their functional activity in the regulation of emotions and motivated human behavior.

The effect of ethanol causes a change in PBR not associated with GABAAR, localized in the mitochondrial membrane, predominantly in glial cells of the brain, and providing cholesterol transfer into the mitochondria [46], thus affecting the regulation of the synthesis of neurosteroids, which are endogenous modulators of GABAA/BzDR in the CNS [42]. Alcohol carries out some of their effects through PBR, regulating the production of neurosteroids and their metabolites, which are critical components of normal brain function [46]. Thus, PBR indirectly affects GABAergic function in the brain, mainly reacting to neurotoxic effects and various brain damage [36, 55, 56].

The data obtained by us confirm the existence of regulatory mechanisms mediating the relationship between the properties of GABAA/BzDR caused by receptor neuroplasticity and alcohol addiction.
