Citation: Mujtaba, S.; Patro, I.K.; Patro, N. Multiple Early Life Stressors as Risk Factors for Neurodevelopmental Abnormalities in the F1 Wistar Rats. Brain Sci. 2023, 13, 1360. https://doi.org/10.3390/ brainsci13101360 Academic Editor: Nelida M. Conejo Received: 7 July 2023 Revised: 7 September 2023 Accepted: 20 September 2023 Published: 22 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). brain sciences Article Multiple Early Life Stressors as Risk Factors for Neurodevelopmental Abnormalities in the F1 Wistar Rats Syed Mujtaba 1,2 , Ishan Kumar Patro 1,2 and Nisha Patro 1, * 1 School of Studies in Neuroscience, Jiwaji University, Gwalior 474011, India; mujtabasyed.ju@gmail.com (S.M.); ikpatro@jiwaji.edu (I.K.P.) 2 School of Studies in Zoology, Jiwaji University, Gwalior 474011, India * Correspondence: nishapatro@gmail.com Abstract: Cumulative exposure to multiple early life stressors is expected to affect behavioral de- velopment, causing increased susceptibility to neuropsychiatric disorders. The present study was designed to mimic such conditions in a rat model to study behavioral impairments during adoles- cence and adulthood. Female Wistar rats (n = 32; 140–150 gm) were switched to a low protein (LP; 8% protein) or control (20% protein) diet 15 days prior to conception, and then the diet regime was maintained throughout the experimental period. Pups born to control and LP dams were intraperi- toneally injected with deltamethrin (DLT—pyrethroid insecticide; 0.7 mg/kg body weight; PND 1 to 7), lipopolysaccharide (LPS—bacterial endotoxin; 0.3 mg/kg body weight; PND 3 and 5), or DLT+LPS, on designated days forming eight experimental groups (Control, LP, Control+LPS, LP+LPS, Control+DLT, LP+DLT, Control+DLT+LPS and LP+DLT+LPS). Neurobehavioral assessments were performed in F1 rats (1, 3, 6 months) by open field, elevated plus maze, light and dark box, and rotarod tests. LP rats were found to be highly susceptible to either singular or cumulative exposure as compared to their age-matched control counterparts, showing significantly severe behavioral abnormalities, such as hyperactivity, attention deficits and low anxiety, the hallmark symptoms of neuropsychiatric disorders like schizophrenia and ADHD, suggesting thereby that early life multi-hit exposure may predispose individuals to developmental disorders. Keywords: multi-hit stress; protein malnourishment; low anxiety; hyperactivity; schizophrenia; ADHD 1. Introduction The developing brain is highly susceptible to adverse environmental conditions, lead- ing to many potential and permanent structural alterations and adverse long-term effects on the behavioral and cognitive abilities of an individual. The early stages of life are marked by rapid brain growth and a dynamic process of synapse sculpting and pruning, making this time particularly prone to harmful disruptions [1]. Perinatal stressors like protein malnutrition, infections and neurotoxicant exposure have been implicated as risk factors for developing brain, leading to cognitive, behavioral and emotional impairments in both animals and humans, raising susceptibility to neuropsychiatric and neurodegen- erative illnesses later in life [2–4]. Clinical investigations have revealed how exposure to environmental stressors early in life and throughout the developmental years of childhood and adolescence results in resilient or maladaptive behavior with a significant impact on cognition later in life [5]. Multiple types of stressors (multi-hit), such as exposure to prenatal and postnatal malnutrition [6,7], viral and bacterial infections, trauma, and social maltreatment and neurotoxins [8,9] act in variable combinations and profoundly disrupt brain development, thus may considerably increase the pathophysiology of neuropsychi- atric disorders in affected individuals. Moreover, maternal inflammatory reactions during pregnancy [10], antenatal infection in preterm newborns [11] and neonatal infection [12] have also been linked to a lower intelligence quotient in offspring and increased chances Brain Sci. 2023, 13, 1360. https://doi.org/10.3390/brainsci13101360 https://www.mdpi.com/journal/brainsci