2.4. Formation of benzodiazepine receptors of the developing human brain of the fetus in conditions of prenatal exposure to alcohol

To study the formation of benzodiazepine receptors of the synaptic structures of the brain of the developing fetus in normal and prenatal influences of alcohol, BzDR were investigated by radio-receptor binding with [<sup>3</sup> H]-flunitrazepam using synaptosomal fraction obtained from the brain of fetuses and human embryos. Radioanalysis was performed in a Rack-beta scintillation β-counter. The dissociation constant (Kd) and number of specific binding sites (Bmax) were determined by analysis of saturation curves in Scatchard coordinates. Linear Scatchard blots were analyzed in all cases which confirm the presence of only a specific population of binding sites. Distributions of parameters did not deviate from the normal, so statistical analysis of the data was performed by parametric variational statistics (Student's test) on Statistika 10.0; differences were regarded as significant at p < 0.05. Correlational relationships were assessed by Spearman analysis. Experimental work was carried out in the Department of Clinical Neuroimmunology and Neurobiology of Mental Health Research Institute, Tomsk National Research Medical Center RASci (Tomsk) and in the Laboratory of Clinical Neuromorphology and Laboratory of Clinical Biochemistry of Mental Health Research Center RASci (Moscow). All the studies were approved by the Ethics Committee of the Mental Health Research Institute.

Studies of the properties of human brain BzDR at 8–9 weeks of development showed that specific [<sup>3</sup> H]-flunitrazepam binding site density (Bmax) was greater in the study group than the control group (Figure 15, Table 3). There was a decrease in receptor affinity for the [<sup>3</sup> H] flunitrazepam, in the main study group, related to the increase in the value of Kd (Figure 16, Table 3). The dissociation constant—Kd is inversely proportional to the receptor affinity for their ligand, that is affinity corresponds—1/Kd. The observed increases in Kd indicate a decrease in the affinity of the receptors. The data obtained indicate an increase in the expression of receptors with a decrease in their affinity for the ligand in human embryo brains under the prenatal alcohol exposure.

Figure 14. Morphometric values for postsynaptic density lengths in the control and study groups at different weeks of

7–8 Weeks 9 Weeks 10 Weeks 11 Weeks

C M SE N = 210

25.21 3.0 23.56 2.4 36.21 1.56 32.45 1.23\* 42.37 1.70 35.80 2.37\* 63.33 2.51 51.90 2.88\*

– – 54.521 2673 48.861 <sup>6773</sup>\* 66.964 3833 63.178 <sup>3168</sup>\* 75.742 3207 66.750 <sup>4436</sup>\*

– – 896.28 63.7 798.90 40.09\* 948.19 58.2 941.56 64.44 1276.02 73.08 1129 86.87\*

Table 2. Morphometric parameters of synapses in the human brain at different stages of embryonic development.

S M SE N = 210 C M SE N = 210 S M SE N = 210

development.

86 Drug Addiction

Measure C

M SE N = 90

S M SE N = 90

C M SE N = 210

Notes: C, control group; S, study group (materials from alcoholic mothers).

Significant differences between study and control groups (p < 0.01).

S M SE N = 210

Stage of development

Length of postsynaptic density

Area of postsynaptic terminals

\*

Perimeter of postsynaptic terminals

At 10 weeks of gestation, there were not expressive changes in [3 H]-flunitrazepam-binding parameters (Kd and Bmax) in compared groups. However, it should be noted that the dynamics of changes in receptor density is discrete, nonlinear. At this period, slight changes in the binding parameters in the control and experimental groups were noted. Density of receptors increases slightly between the 9th and 10th weeks of fetal development. There is some inhibition of growth in receptor density (Figure 16, Table 3), especially in the main group. This correlated with morphometric evaluation of synapses: decreases in presynaptic terminal area and postsynaptic density length in the main experimental group relative to the control group (Table 4).

Alcohol in the early stages of pregnancy, according to the data, negatively affects the formation of synaptic contacts and benzodiazepine receptors in the human brain, reducing the functional activity of the brain and its development. We found that from the 12–13 weeks of pregnancy, a significant increase in receptor expression (Bmax) was observed, and this trend of increasing prescription density continued during the gestation period of 14–15 weeks (Figures 15 and 16,

Table 3). However, in the experimental group, with prenatal exposure to alcohol, the affinity of the receptors decreased at all stages of the human brain development, and the increase in expression and density of receptors can be considered as compensatory adaptive brain

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

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receptor complex] with synaptosomal membranes of human embryonic and fetuses brain in the control (a) and basic

54.521 2673 r = 0.79 p = 0.0003 \*\*

66.964 3833 r = 0.62 p = 0.0002 \*\*

75.742 3207 r = 0.76 p = 0.0001 \*\*

groups between Bmax and P (\*), Bmax and S (\*\*) and Bmax and L (\*\*\*); p, level of significance of correlational relationships.

Notes: L, postsynaptic density length; S, presynaptic terminal area; P, presynaptic terminal perimeter; r, correlation between control and study

Table 4. Correlation analysis of morphometric parameters of synapses (presynaptic terminal area perimeter and area,

H]-flunitrazepam binding parameters [Kd (nM) – constant of dissociation ligand-

1210.00 32.79

1367.40 30.38

1824.13 47.28

H]-flunitrazepam specific binding site density (BzDR) at different developmental

798.90 40.09 r = 0.78 p = 0.0004 \*

941.56 64.44 r = 0.82 p = 0.0006 \*

1129 86.87 r = 0.79 p = 0.0004 \*

48.861 6773 r = 0.64 p = 0.0002 \*\*

63.178 3168 r = 0.71 p = 0.0001 \*\*

66.750 4436 r = 0.70 p = 0.0003 \*\*

32.45 1.23 r = 0.85 p = 0.0007 \*\*\*

35.80 2.37 r = 0.88 p = 0.0005 \*\*\*

51.90 2.88 r = 0.83 p = 0.0008 \*\*\*

Control group (M SE) Study group (M SE) Bmax P S LBmax P SL

> 36.21 1.56 r = 0.89 p = 0.0004 \*\*\*

> 42.37 1.70 r = 0.87 p = 0.0008 \*\*\*

> 63.33 2.51 r = 0.91 p = 0.0003 \*\*\*

Figure 16. Statistical analysis of [<sup>3</sup>

8–9 984.22

10–11 1156.00

12–13 1456.29

postsynaptic density length) and [3

11.64

15.22

24.17

896.28 63.7 r = 0.80 p = 0.0006 \*

948.19 58.2 r = 0.77 p = 0.0004 \*

1276.02 73.1 r = 0.83 p = 0.0008 \*

groups (b) in dynamics.

Developmental period.

weeks

stages.

Figure 15. Statistical analysis of [<sup>3</sup> H]-flunitrazepam binding parameters [Bmax (fmol/mg of protein) – density of binding sites] with synaptosomal membranes of human embryonic and fetuses brain in the control (a) and study (b) groups in dynamics.


Notes: Bmax, [<sup>3</sup> H]-flunitrazepam binding density with synaptosomal BzDR; Kd, ligand-receptor complex dissociation constant ([<sup>3</sup> H]-flunitrazepam with synaptosomal BzDR). \* Statistically significant differences between study and control groups, p < 0.01.

Table 3. [ 3 H]-flunitrazepam binding properties with synaptosomal membranes from human embryo and fetus brains (8–15 weeks of development).

Table 3). However, in the experimental group, with prenatal exposure to alcohol, the affinity of the receptors decreased at all stages of the human brain development, and the increase in expression and density of receptors can be considered as compensatory adaptive brain

activity of the brain and its development. We found that from the 12–13 weeks of pregnancy, a significant increase in receptor expression (Bmax) was observed, and this trend of increasing prescription density continued during the gestation period of 14–15 weeks (Figures 15 and 16,

Developmental period, weeks Control group Study group

sites] with synaptosomal membranes of human embryonic and fetuses brain in the control (a) and study (b) groups in

Kd nM n Bmax fmol/mg

H]-flunitrazepam binding density with synaptosomal BzDR; Kd, ligand-receptor complex dissociation

H]-flunitrazepam binding properties with synaptosomal membranes from human embryo and fetus brains

protein

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

r = 0.47 p = 0.0001

r = 0.50 p = 0.0001

r = 0.23 p = 0.0001

r = 0.73 p = 0.005

Statistically significant differences between study and control

Kd nM n

9

10

8

6

<sup>1591</sup> 0.023\* r = 0.22 p = 0.014

<sup>1792</sup> 0.019\* r = 0.49 p = 0.04

<sup>1982</sup> 0.018\* r = 0.19 p = 0.014

<sup>2450</sup> 0.068\* r = 0.56 p = 0.0027

Bmax fmol/mg protein

<sup>8</sup>–<sup>9</sup> 984.22 11.64 1500 0.024 9 1210.00 32.79\*

<sup>10</sup>–<sup>11</sup> 1156.00 15.22 1700 0.019 8 1367.40 30.38\*

<sup>12</sup>–<sup>13</sup> 1456.29 24.17 1900 0.023 7 1824.13 33.51\*

<sup>14</sup>–<sup>15</sup> 1712.00 35.24 2120 0.031 5 1938.17 47.28\*

H]-flunitrazepam with synaptosomal BzDR). \*

Notes: Bmax, [<sup>3</sup>

groups, p < 0.01.

(8–15 weeks of development).

Figure 15. Statistical analysis of [<sup>3</sup>

constant ([<sup>3</sup>

dynamics.

88 Drug Addiction

Table 3. [ 3

Figure 16. Statistical analysis of [<sup>3</sup> H]-flunitrazepam binding parameters [Kd (nM) – constant of dissociation ligandreceptor complex] with synaptosomal membranes of human embryonic and fetuses brain in the control (a) and basic groups (b) in dynamics.


Notes: L, postsynaptic density length; S, presynaptic terminal area; P, presynaptic terminal perimeter; r, correlation between control and study groups between Bmax and P (\*), Bmax and S (\*\*) and Bmax and L (\*\*\*); p, level of significance of correlational relationships.

Table 4. Correlation analysis of morphometric parameters of synapses (presynaptic terminal area perimeter and area, postsynaptic density length) and [3 H]-flunitrazepam specific binding site density (BzDR) at different developmental stages.

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.

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

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

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 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

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

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

H]-flunitrazepam binding parameters [Kd (nM) – constant of dissociation ligand-

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

region of the prefrontal cortex) and in the cerebellar cortex (Figure 17, Table 5).

H]-flunitrazepam with

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91

resuscitation measures.

Committee.

Figure 17. Statistical analysis of [<sup>3</sup>

group (b) (alcoholic patients).

the parametric method (t test) using Statistika 10.0.

(I) A study of the binding characteristics of the selective ligand [<sup>3</sup>

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

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 and PAE.
