*2.3. Seizure Threshold*

In all groups, females and males obtained similar results, with no gender distinction. The V2 group developed seizure behavior earlier in relation to the control group, requiring fewer booster doses (Figure 3).

In addition, in all experimental groups the animals showed more intense convulsions than in the control group (Figure 3).

**Figure 3.** Assessment of seizure threshold (*n* = 6 females and 6 males per group). (**a**) Number of doses needed to reach the seizure threshold. (**b**) Average intensity of seizure behavior. Data are expressed as means ± SEM. (\*) *p* < 0.01 and (\*\*) *p* < 0.05 compared to the control group (Kruskal Wallis test).

#### **3. Discussion**

Despite the increase in the number of scorpion accidents in the last few years, there is insufficient information on the effects of scorpion envenomation on pregnancy, lactation, and neonatal outcomes, and unfortunately the available data are controversial.

Experimental studies provide clues as to what might happen in an accident in humans. Thus, in the present study, we used lactating rats to evaluate the possible consequences for the offspring after a scorpion sting in the mother during breastfeeding.

A possible scorpion accident was simulated through a single subcutaneous injection of *T. serrulatus* venom, which is the most common method of inoculation in accidental bites. The dose of 4.0 mg/kg was determined in previous experiments in our laboratory (unpublished data) as causing symptoms observed in moderate to severe envenoming cases such as severe local pain, piloerection, respiratory perturbation and increased lacrimal and salivary secretions. The days of injection were chosen based on the different periods of brain maturation [27]. The development of the mammalian brain begins in embryogenesis, and its maturation continues in the postnatal period [27,28]. In rats, the neurogenesis in cortical regions starts on the ninth day of gestation and extends until the fifteenth postnatal day. The growth spurt of the brain corresponds to the period in which this organ increases in weight most rapidly. In rodents this event peaks around postnatal day 7, reviewed by [29].

Our results demonstrated a decrease in maternal care on the days of the venom injection, and offspring that were more susceptible to convulsion in adulthood. It is relevant to note that the earlier the envenoming, the lower the seizure threshold in the offspring.

In mammals, maternal care is the care that the mother provides to the offspring, and it is essential for the adequate development of the nervous system [15,17,30]. Maternal care modulates the expression of various genes and neurochemical content in one or more regions of the brain and changes in this process can cause variations in behavioral responses and the development of mood disorders [17,31].

Studies in animals and humans have shown that, during early childhood, the brain is particularly sensitive to stress [32], probably because in this period the system is still developing [19].

Maternal deprivation during the early postnatal period is one of the most potent stressors for pups [32] and it was experimentally demonstrated that it can affect behavior, ACTH and neurotrophin levels in rats, which persist into adulthood [33]. A stressful status can lead to permanent neurobehavioral alterations and an increased susceptibility to psychiatric disorders [34].

Epidemiological studies point to stress as a risk factor for epileptogenesis in adults and young people [20,35]. Experimentally, it was demonstrated that early-life stress in rats has long-lasting effects on brain excitability and may promote age-specific seizures and epilepsy [36]. On the other hand, increased maternal care makes mice genetically predisposed to epilepsy less susceptible to seizures [37].

In our experiments, we observed that mothers from groups V2, V10 and V16 remained away from the pups for a long period after venom injection. This is probably due to the fact that they were experiencing the symptoms of envenoming, mainly pain, as we could see from the vocalization that occurs with minimal contact by either the pups or the observers, with the mother, as well as frequent licking at the injection site. The symptoms of envenoming last for a few hours, creating a long period of separation between mothers and pups, which remained without maternal care.

In addition, mothers in group V2 did not collect the offspring during the observation period in the retrieval test. Retrieval is a common behavior displayed by rodents [38] and, usually, the assessment of maternal behavior is performed in the first week after delivery, as the offspring during this period are exclusively dependent on maternal care [15]. The time spent licking/grooming the pups decreases over the days, as does the time spent in contact with the pups. In the first 10 days after the delivery, the mothers stay longer in the nest and the pups grow, and maternal care tends to gradually decrease and the mother becomes less responsive towards her offspring [15,30]. Thus, the test was performed only in the V2 group, and was not performed in V10 and V16 groups.

Based on the above considerations, we believe that the stress of maternal deprivation could be responsible, at least in part, for the decrease in the convulsive threshold of the offspring.

However, we cannot disregard the possibility that, due to the physiological effects of the venom on the mother, some alteration occurs in the composition of the milk (milk components that could be in higher or lower concentrations), directly affecting the nervous system of the offspring, and studies are being developed in this regard in our laboratory.

It is also possible that some component of the venom or some cytokines produced by the mother are directly passed on, since scorpion accidents are capable of inducing an intense immune response in the injured individual. Several cytokines are increased in the plasma of envenomed patients, such as IL1-α, IL1-β, IL-6, IL-8, IL-10 TNF-α and IFN-γ [39–41]. In addition, it has been shown that scorpion venom can also alter some cytokines in pups of envenomed mothers [14].

These cytokines could affect the nervous system of the pups and be responsible for the decreased seizure threshold, as it is believed that inflammatory processes may be related to epileptogenesis [42]. Inflammation in neonates has already been shown to increase their susceptibility to seizures in adulthood and the induction of an inflammatory response was able to decrease the seizure threshold when performed between days 7 and 14 postnatally [43].

Evidence supports the hypothesis that cytokines not only act as inflammatory mediators, but also have neuromodulatory action. IL1-β is described as having excitatory effects in several brain regions [44]. In the hippocampus, TNF-α can increase the expression of AMPA receptors [45]. Concomitantly, this cytokine may be associated with decreased expression of GABAA receptors [46]. Epidemiological analyses have demonstrated that central nervous system infections are a major cause of acquired epilepsy revised by [47]. It is due to changes in the physiological properties of neurons within the hippocampus [48].

Thus, the inflammatory process resulting from the envenoming could also be responsible for the decrease in the convulsive threshold of the offspring.
