**2. Methods**

Investigations were performed on Wistar rats from the Rappolovo supplier, Russian Academy of Medical Sciences (Leningradskaya Oblast). Several series of experiments were

(Lichtensteiger et al., 1988; Dani & Heinemann, 1996). Prenatal exposure to nicotine predominantly damages cholinergic, noradrennergic, and dopaminergic projections in the brain during postnatal life, with later cognitive and behavioral dysfunction in adult offspring (Naeye & Peters, 1984; Milberger et al., 1996; Fergusson et al., 1998; Orlebeke et al.,

The neurobehavioral teratogenic actions of barbiturates are also mediated by disrupted functioning of septohippocampal cholinergic conducting pathways, which is accompanied by a deficiency of synaptic transmission and accompanying hippocampus-related behavioral deficit (Wallace, 1984; Smith et al, 1986; Yanai, 1984, 1996; Steingart et al,, 2000; Azmitia, 2001; Beer et al. 2005; Beer et al., 2005). Phenobarbital, previously used for the prophylaxis of neonatal hyperbilirubinemia and bleeding in neonatal children, is a teratogenic factor in relation to behavior in both humans and animals (Yanai, 1984; Wallace, 1984; Smith et al, 1986). Experimental exposure to organochlorine compounds, like other neurotoxins with cholinotropic properties, damages cholinergic conducting pathways and leads to long-term alterations to the cholinergic system (Lauder, 1985; Dreyfus, 1998; Qiao et al., 2002; Slotkin et al., 2002; Slotkin, 2004). Dysfunction of cholinergic neurons plays a significant role in behavioral disorders seen in adult rats given organochlorine compounds

Behavioral abnormalities as long-term consequences of prenatal exposure to various factors are generally difficult to observe because of the large phenotypic variability of the developing organism (Nicholls, 2000). There is great value in studying sexual behavior in these situations, as sexual functions are the most sensitive and susceptible aspects of reproduction in males and, as has been demonstrated, are regulated by the activities of several neurotransmitter systems, including the cholinergic (Bitran & Hull, 1987; Mas et al.,

In addition, despite many studies of substances with cholinotropic properties and adverse influences on the developing brain during the prenatal period, the literature lacks reports of

Taking into consideration all these statements, the purpose of the present research is study of known selective blockers of M- and N-cholinergic systems, prescribed in various terms of a prenatal period, on development dynamics of neurotransmitter systems of the rats embryos brain, and track their dynamics in an adolescent period and in adulthood in comparison to the behavioural sexual status in the paste for a sexual behavior at rats males.




Investigations were performed on Wistar rats from the Rappolovo supplier, Russian Academy of Medical Sciences (Leningradskaya Oblast). Several series of experiments were

1997; Weissman et al., 1999; Slotkin et al., 2001).

during the prenatal period (Sherman et al., 1981).

1987; Hull et al., 1988; Retana et al., 1993; Gladkova, 2000).

Tasks of the present part of work were the following:

embryos brain, in various terms of a gestation.

effected by prenatal influence M- and N-cholinolytics.

pharmacological correction.

**2. Methods** 

the behavioral effects of prenatally administered selective cholinolytics.

performed. Female rats with a known date of pregnancy were obtained by mating females in proestrus-estrus with males. The day on which sperm were seen in vaginal smears was taken as the first day of pregnancy. Pregnant females, at different stages of gestation (9–11, 12–14, and 17–19 days of pregnancy), were given three i.m. injections (once daily) of the Ncholinoblocker ganglerone (10 mg/kg), while other groups received injections of the Mcholinoblocker methylbenactyzine (2 mg/kg) at the same time points. Doses were determined on the basis of the selectivities of cholinolytic actions and the absence of nonspecific actions. Control groups of females received injections of physiological saline. Experiments offspring groups (12–14 individuals per group) were formed in accord with the timing of prenatal administration of ganglerone (groups G10, G13, and G18, respectively) and methylbenactyzine (groups M10, M13, and M18). The offspring of intact rats served as the control group.

**Behavioral studies** were performed on rat offspring aged 3.5–4 months. Sexual experience was acquired in four sequential tests with receptive females. Sexual activity parameters were assessed using a standard sexual behavior test (3). Adult rats were kept in individual cages with food and water available *ad libitum*, in a room with controlled temperature and under an inverted 12 × 12 h light cycle (light off at 09:00 h). Tests for sexual behavior were done during the dark phase of the cycle and under dim red light illumination The test male was placed in the study chamber, of size 40 x 40 x 30 cm, for 5 min prior to presentation of a sexually susceptible female. Experiments were performed in dim red illumination. Receptivity in sterilized females was induced by sequential administration of estradiol dipropionate (25 mg, 48 h before the experiment) and progesterone (500 g, 4 h before the experiment). Components of sexual activity were recorded visually for 15 min in tests 1 and 4. The numbers of components of sexual behavior (mountings, intromissions, and ejaculations) and their latent periods were registered. During each behavioral test, the behavioral components recorded were mount latency (time from the introduction of a receptive female to the first mount), intromission latency (time from the introduction of a receptive female to the first intromission), ejaculation latency (interval between the first intromission and ejaculation), and postejaculatory interval (interval between the first ejaculation and the next intromission).

**Neurochemical studies** were performed using brains from 20-day embryos and brain structures (hypothalamus, hippocampus) from rat offspring two month age. The concentrations of the neurotransmitters dopamine (DA), noradrenaline (NA), and serotonin (5-HT) in brain tissues were measured by high-performance liquid chromatography using a Beckman System Gold with an LC-4C electrochemical detector. Brain structures were extracted on a cryostat at –20°C and were stored in liquid nitrogen until chromatographic analysis. Peaks were separated on a SphereClone 5 μ ODS 2 chromatography column (250 × 4.60 mm) with a Phenomenex precolumn. The mobile phase consisted of citratephosphate buffer pH 3.5, acetonitrile (88 ml/liter), and octanesulfonic acid (43 mg/liter). Chromatographic peaks were identified and assessed quantitatively in relation to peaks obtained from internal standards.

Serum hormone levels were assayed by immunoenzyme analysis using standard biochemical kits (Chema, Access) on a Uniplan immunoenzyme analyzer. Statistical analysis. Results were compared with control data and analyzed statistically by analysis of variance (ANOVA) on Origin 7.0.

Development of Male Sexual Function After Prenatal Modulation of Cholinergic System 97

administration of substances at different periods of pregnancy showed that sexual function in the offspring was most sensitive to injection of the N-cholinolytic ganglerone at 9–11 and 12–14 days of gestation and the M-cholinolytic methylbenactyzine at 9–11 days of gestation. The comparative analysis of the other parametres of males sexual behaviour from the groups G10-G18 and M10-M18 with the acquired sexual experience showed more appreciable sexual dysfunctions of offsprings G10-G18 in comparison with the offsprings, that were subjected to prenatal influence of methylbenactyzine (tab. 1). The structure of sexual function of males from group G10-G18 with the acquired sexual experience was characterized by very low value of copulatory components and their high latence. Level of ejaculatory activity of all groups G10-G18 was extremely low and authentically differed

Fig. 1. Dynamics of the acquisition of sexual activity in offspring; data based on ejaculatory and copulatory (mountings and intromissions) behavior in four sequential tests for sexual behavior (data from tests 1 and 4) (n = 14). Dark columns show numbers of inactive males in the first test; shaded columns show inactive males in the fourth test. **A)** Number of inactive males in terms of ejaculatory activity (EA) in offspring subjected to prenatal exposure to ganglerone at 9–11, 12–14, and 17–19 days of pregnancy (groups G10, G13, and G18, respectively) or methylbenactyzine (groups M10, M13, and M18, respectively) compared with control offspring; **B)** number of inactive males in terms of incomplete copulatory

activity (CA) (mountings and intromissions) in offspring of these groups.

from control group.
