Long-term Behavioral Abnormalities Research Articles

Minocycline mitigated enduring neurological consequences in the mice model of sepsis

AimSepsis-associated encephalopathy is a frequently observed consequence of sepsis, often resulting in chronic brain inflammation and injury, ultimately leading to a range of behavioral abnormalities. This study explores the potential preventive effects of minocycline on the long-lasting outcome of sepsis in a mice model of sepsis. MethodsAdult male C57 mice were subjected to experimental sepsis through a single intraperitoneal injection of 5 mg/kg lipopolysaccharide (LPS). Minocycline administration via oral gavage (12.5, 25, and 50 mg/kg) commenced three days before sepsis induction and continued on the day of induction. Mice underwent behavioral assessments one month post-sepsis, with subsequent brain tissue analysis to investigate oxidative stress markers and cholinergic function. Key findingsOne month following sepsis induction, mice exhibited significant anxiety- and depressive-like behaviors as determined by assessments in the elevated plus maze (EPM), open field, and tail suspension test (TST). Additionally, they displayed impaired recognition memory in the novel object recognition (NOR) test. Brain tissue analysis revealed a notable increase in oxidative stress markers and acetylcholinesterase (AChE) activity in septic mice. Notably, minocycline treatment effectively mitigated the long-term behavioral abnormalities resulting from sepsis, attenuated oxidative stress markers, and reduced AChE activity. SignificanceThese findings underscore the potential of minocycline as a therapeutic intervention during sepsis induction to prevent the enduring behavioral and neurological consequences of experimental sepsis.

The effects of maternal SSRI exposure on the serotonin system, prefrontal protein expression and behavioral development in male and female offspring rats

Fluoxetine (FLX), a commonly used selective serotonin reuptake inhibitor, is often used to treat depression during pregnancy. However, prenatal exposure to FLX has been associated with a series of neuropsychiatric illnesses. The use of a rodent model can provide a clear indication as to whether prenatal exposure to SSRIs, independent of maternal psychiatric disorders or genetic syndromes, can cause long-term behavioral abnormalities in offspring. Thus, the present study aimed to explore whether prenatal FLX exposure causes long-term neurobehavioral effects, and identify the underlying mechanism between FLX and abnormal behaviors. In our study, 12/mg/kg/day of FLX or equal normal saline (NS) was administered to pregnant Sprague-Dawley (SD) rats (FLX = 30, NS = 27) on gestation day 11 till birth. We assessed the physical development and behavior of offspring, and in vivo magnetic resonance spectroscopy (MRS) was conducted to quantify biochemical alterations in the prefrontal cortex (PFC). Ex vivo measurements of brain serotonin level and a proteomic analysis were also undertaken. Our results showed that the offspring (male offspring in particular) of fluoxetine exposed mothers showed delayed physical development, increased anxiety-like behavior, and impaired social interaction. Moreover, down-regulation of 5-HT and SERT expression were identified in the PFC. We also found that prenatal FLX exposure significantly decreased NAA/tCr with 1H-MRS in the PFC of offspring. Finally, a proteomic study revealed sex-dependent differential protein expression. These findings may have translational importance suggesting that using SSRI medication alone in pregnant mothers may result in developmental delay in their offspring. Our results also help guide the choice of outcome measures in identifying of molecular and developmental mechanisms.

Reelin deficiency contributes to long-term behavioral abnormalities induced by chronic adolescent exposure to Δ9-tetrahydrocannabinol in mice

Cannabis use is widespread among adolescents and has been associated with long-term negative outcomes on neurocognitive functions. However, the factors that contribute to the long-term detrimental effects of cannabis use remain poorly understood. Here, we studied how Reelin deficiency influences the behavior of mice exposed to cannabis during adolescence. Reelin is a gene implicated in the development of the brain and of psychiatric disorders. To this aim, heterozygous Reeler (HR) mice, that express reduced level of Reelin, were chronically injected during adolescence with high doses (10 mg/kg) of Δ9-tetrahydrocannabinol (THC), a major psychoactive component of cannabis. Two weeks after the last injection of THC, mice were tested with multiple behavioral assays, including working memory, social interaction, locomotor activity, anxiety-like responses, stress reactivity, and pre-pulse inhibition. Compared to wild-type (WT), HR mice treated with THC showed impaired social behaviors, elevated disinhibitory phenotypes and increased reactivity to aversive situations, in a sex-specific manner. Overall, these findings show that Reelin deficiency influences behavioral abnormalities caused by heavy consumption of THC during adolescence and suggest that elucidating Reelin signaling will improve our understanding of neurobiological mechanisms underlying behavioral traits relevant to the development of psychiatric conditions.

Intranasal Administration of Insulin Reduces Chronic Behavioral Abnormality and Neuronal Apoptosis Induced by General Anesthesia in Neonatal Mice.

Children, after multiple exposures to general anesthesia, appear to be at an increased risk of developing learning disabilities. Almost all general anesthetics—including sevoflurane, which is commonly used for children—are potentially neurotoxic to the developing brain. Anesthesia exposure during development might also be associated with behavioral deficiencies later in life. To date, there is no treatment to prevent anesthesia-induced neurotoxicity and behavioral changes. In this study, we anesthetized 7-day-old neonatal mice with sevoflurane for 3 h per day for three consecutive days and found that the anesthesia led to mild behavioral abnormalities later in life that were detectable by using the novel object recognition test, Morris water maze, and fear conditioning test. Biochemical and immunohistochemical studies indicate that anesthesia induced a decrease in brain levels of postsynaptic density 95 (PSD95), a postsynaptic marker, and marked activation of neuronal apoptosis in neonatal mice. Importantly, insulin administered through intranasal delivery prior to anesthesia was found to prevent the anesthesia-induced long-term behavioral abnormalities, reduction of PSD95, and activation of neuronal apoptosis. These findings suggest that intranasal insulin administration could be an effective approach to prevent the increased risk of neurotoxicity and chronic damage caused by anesthesia in the developing brain.

P1‐205: INTRANASAL ADMINISTRATION OF INSULIN PREVENTS CHRONIC COGNITIVE IMPAIRMENT AND NEURONAL APOPTOSIS INDUCED BY GENERAL ANESTHESIA IN NEONATAL MICE

General anesthesia increases the risk for cognitive impairment post operation, especially in children and elderly individuals. To date, there is no treatment to prevent the anesthesia-induced neurotoxicity and behavioral changes. We administered insulin through intranasal delivery to 7-day-old neonatal mice, followed by anesthesia with sevoflurane for 3 hrs per day for three consecutive days or once for 6 hrs. The mice were then assessed for cognitive function by using open field test, one-trial novel object recognition test, Morris water maze, and fear conditioning test when they reached to adult age. The neonatal mouse brains were also studied biochemically and immunohistochemically for changes in synaptic proteins, neuroinflammation, and neuronal apoptosis in the brain. We found that the anesthesia to neonatal mice led to mild behavioral abnormalities later in life that were detectable by using novel object recognition test, Morris water maze, and fear conditioning test. Biochemical and immunohistochemical studies indicate that anesthesia induced a decrease in brain level of postsynaptic density 95 (PSD95), a postsynaptic marker, and marked activation of neuronal apoptosis in neonatal mice. Importantly, insulin administered through intranasal delivery prior to anesthesia was found to prevent the anesthesia-induced long-term behavioral abnormalities, reduction of PSD95, and activation of neuronal apoptosis. Our results indicate that anesthesia exposure during development may be a risk factor for cognitive impairment later in life and that intranasal treatment with insulin can prevent this risk. These findings suggest that intranasal insulin administration could be an effective approach to prevent the increased risk of neurotoxicity and chronic damage caused by anesthesia to the developing brain.

Mouse Strain and Sex-Dependent Differences in Long-term Behavioral Abnormalities and Neuropathologies after Developmental Zika Infection.

Exposure of the developing fetus to Zika virus (ZIKV) results in a set of brain abnormalities described as the congenital Zika syndrome. Although microcephaly is the most obvious outcome, neuropathologies, such as intracranial calcifications and polymicrogyria, can occur in the absence of microcephaly. Moreover, the full impact of exposure on motor, social, and cognitive skills during development remains uncharacterized. We examined the long-term neurobehavioral consequences of neonatal ZIKV exposure in four genetically divergent inbred mouse strains (C57BL/6J, 129S1/SvImJ, FVB/NJ, and DBA/2J). Male and female mice were infected on postnatal day 1, considered comparable with exposure late in the second trimester of humans. We demonstrate strain differences in early susceptibility to the virus and the time course of glial reaction in the brain. These changes were associated with strain- and sex-dependent differences in long-term behavioral abnormalities that include hyperactivity, impulsiveness, and motor incoordination. In addition, the adult brains of susceptible mice exhibited widespread calcifications that may underlie the behavioral deficits observed. Characterization of the neuropathological sequelae of developmental exposure to the Zika virus in different immunocompetent mouse strains provides a foundation for identifying genetic and immune factors that contribute to long-term neurobehavioral consequences in susceptible individuals.SIGNIFICANCE STATEMENT Developmental Zika virus (ZIKV) infection is now known to cause brain abnormalities in infants that do not display microcephaly at birth, and the full impact of these more subtle neuropathologies has yet to be determined. We demonstrate in a mouse model that long-lasting behavioral aberrations occur after developmental ZIKV exposure. We compare four divergent mouse strains and find that the effects of Zika infection differ greatly between strains, in terms of behavioral changes, sex differences, and the intracranial calcifications that develop in the brains of susceptible mice. These findings provide a foundation for identifying susceptibility factors that lead to the development of abnormal behaviors secondary to ZIKV infection early in life.

Sevoflurane anesthesia during pregnancy in mice induces hearing impairment in the offspring.

IntroductionExposure to gamma-aminobutyric acid-mimetics and N-methyl-D-aspartate-receptor antagonists during pregnancy may lead to hearing loss and long-term behavioral abnormalities in the offspring. The purpose of this study was to explore the association between prenatal exposure to sevoflurane (SEV) anesthesia and hearing impairment in mice.Materials and methodsOn gestational day 15, pregnant Kunming mice were exposed for 2 hours to 2.5% SEV plus 100% oxygen (anesthesia group) or 100% oxygen alone (control group).ResultsDuring auditory brainstem response testing on P30, offspring of the anesthesia group mice exhibited higher hearing thresholds at 8, 16, 24, and 32 kHz; longer peak latency of wave II at all four frequencies; and longer interpeak latencies from waves II to V at 16, 24, and 32 kHz, compared to the control offspring. Caspase-3, iNOS, and COX-2 activation occurred in the fetal cochlea of the anesthesia group. Mitochondrial swelling was observed in the anesthesia group offspring at P1 and P15.ConclusionOur results suggest that SEV exposure during pregnancy may cause detrimental effects on the developing auditory system.

Chapter 48 - Neuroepigenetics and addiction

Drug addiction involves long-term behavioral abnormalities that arise in response to repeated exposure to drugs of abuse in vulnerable individuals. It is a multifactorial syndrome involving a complex interplay between genes and the environment. Evidence suggests that the underlying mechanisms regulating these persistent behavioral abnormalities involve changes in gene expression throughout the brain's reward circuitry, in particular, in the mesolimbic dopamine system. In the past decade, investigations have begun to reveal potential genes involved in the risk for addiction through genomewide association studies. Additionally, a crucial role for epigenetic mechanisms, which mediate the enduring effects of drugs of abuse on the brain in animal models of addiction, has been established. This chapter focuses on recent evidence that genetic and epigenetic regulatory events underlie the changes throughout the reward circuitry in humans, as well as animal models of addiction. While further investigations are necessary, a picture of genetic and epigenetic mechanisms involved in addiction is beginning to emerge and the insight gained from these studies will be key to the identification of novel targets for improved diagnosis and treatment of addiction syndromes in humans.

Effect of unilateral basal ganglia hemorrhage on long-term behavioral development of neonatal rats

Objective To investigate the effect of unilateral basal ganglia hemorrhage on long-term behavio-ral development of neonatal SD rats. Methods Forty-eight neonatal SD rats (10 days after birth) were equally divided into cerebral hemorrhage (CH) group, sham operation (SH) group and normal control (NC) group randomly, 16 cases for each group.Stereotaxic apparatus was used to inject autologous blood 25 μL into rats′ brain caudate nu-cleus to establish basal ganglia hemorrhage model, while SH group was injected with nothing, and NC group received no treatment.Berderson scoring method was used to test rats′ neurological functions on the first day, the third day, the seventh day, and the fourteenth day after operation, respectively.The open-field environment test and the Lat maze were used to assess behavior of the rats on the fourteenth day after operation, lasting for 3 days continuously. Results (1)After being modeled rats appeared with different degrees of neurological function damage.SH group rats′ neurological function damage was slight, which was completely restored in the 72 hours after surgery.CH group rats appeared hemiplegia and muscle tension change.CH rats neurological scores′ on the first and third day after modeling scores were separate (3.40±0.83) scores and (1.13±0.92) scores respectively, and the scores were statistically higher than SH group [(0.73±0.59) scores and (0.13±0.35) scores]and NC group (all P 0.05). (2)On the fourteenth day after modeling, in open-field test and the Lat maze, the number of passed panels, straightening(times) and grooming(times) in CH group were more than those in SH group and NC group respectively (all P 0.05). Conclusions Neonatal rats focal hemorrhage in unilateral basal ganglia is strongly repairable and compensatory, which leaves no severe neurological dysfunction.The neurological function damage which is caused by unilateral basal ganglia hemorrhage can lead to the increase of autonomic activities, the decrease of non-selective attention level, attention deficit and other long-term behavioral abnormalities. Key words: Cerebral hemorrhage; Basal ganglia; Behavioral development; Neonatal period; Rat

Lack of protracted behavioral abnormalities following intermittent or continuous chronic mild hypoxia in perinatal C57BL/6 mice

Several prospective studies indicated perinatal hypoxia as risk factor for psychiatric disorders like schizophrenia. It is thought that hypoxia prior to or during birth may contribute to alterations leading to the protracted clinical manifestation during young adulthood. However, only a small fraction of children with a history of perinatal hypoxia develop later psychotic symptoms, therefore it is not known if hypoxia alone is sufficient to trigger long-term behavioral changes. Here we exposed C57BL/6 mice from postnatal day 3–7 (P3–P7) to two established paradigms of chronic mild hypoxia (10% ambient O2), intermittent and continuous. Subsequently, mice were analysed during young adult stages using several basic behavioral tests. Previous studies demonstrated severe, but only transient, cortical damage in these paradigms; it is not clear, if these reversible morphological changes are accompanied by long-term behavioral effects. We found that neither intermittent nor continuous perinatal hypoxia induced long-term behavioral alterations. This may be due to the high regenerative capacity of the perinatal brain. Other possibilities include a potential resistance to perinatal hypoxia of the mouse strain used here or a level of hypoxia that was insufficient to trigger significant behavioral changes. Therefore, our data do not exclude a role of perinatal hypoxia as risk factor for psychiatric disorders. They rather suggest that either other, more severe hypoxic conditions like anoxia, or the presence of additional factors (as genetic risk factors) are necessary for generating long-term behavioral abnormalities.

Fluoxetine exposure during pregnancy and lactation: Effects on acute stress response and behavior in the novelty-suppressed feeding are age and gender-dependent in rats

Fluoxetine (FLX) is commonly used to treat anxiety and depressive disorders in pregnant women. Since FLX crosses the placenta and is excreted in milk, maternal treatment with this antidepressant may expose the fetus and neonate to increased levels of serotonin (5-HT). Long-term behavioral abnormalities have been reported in rodents exposed to higher levels of 5-HT during neurodevelopment. In this study we evaluated if maternal exposure to FLX during pregnancy and lactation would result in behavioral and/or stress response disruption in adolescent and adult rats. Our results indicate that exposure to FLX influenced restraint stress-induced Fos expression in the amygdala in a gender and age-specific manner. In male animals, a decreased expression was observed in the basolateral amygdala at adolescence and adulthood; whereas at adulthood, a decrease was also observed in the medial amygdala. A lack of FLX exposure effect was observed in females and also in the paraventricular nucleus of both genders. Regarding the behavioral evaluation, FLX exposure did not induce anhedonia in the sucrose preference test but decreased the latency to feed of both male and female adolescent rats evaluated in the novelty-suppressed feeding test. In conclusion, FLX exposure during pregnancy and lactation decreases acute amygdalar stress response to a psychological stressor in males (adolescents and adults) as well as influences the behavior of adolescents (males and females) in a model that evaluates anxiety and/or depressive-like behavior. Even though FLX seems to be a developmental neurotoxicant, the translation of these findings to human safe assessment remains to be determined since it is recognized that not treating a pregnant or lactating woman may also impact negatively the development of the descendants.

Ketamine reduces neuronal degeneration and anxiety levels when administered during early life-induced status epilepticus in rats

Status epilepticus (SE) when occurred during brain development can cause short- and long-term consequences, which are frequently associated with NMDA-mediated glutamatergic excitotoxicity. In the present work, we investigated the putative neuroprotective role of ketamine, an NMDA receptor antagonist, on early life SE-induced acute neuronal death and long-term behavioral abnormalities. Male Wistar rats (16 postnatal days) were induced to SE by LiCl–pilocarpine i.p. administration (3mEq/kg; 60mg/kg, respectively). Fifteen or 60min after pilocarpine injection, animals received a ketamine administration (22.5mg/kg i.p.). Neuronal degeneration was assessed 24h after SE induction. Another subset of animals was destined to behavioral tasks in adulthood (75–80 postnatal days). Fluoro-Jade C labeling revealed a marked neuronal death on CA1 hippocampal subfield, habenula, thalamus and amygdala in SE animals. Ketamine post-SE onset treatment prevented neuronal death in all regions assessed. In the elevated plus maze, SE induced an increase in anxiety-like behaviors whereas ketamine administration during seizures was able to prevent this alteration. Ketamine administration in non-SE animals resulted in high anxiety levels. There were no observed differences among groups in the open field task in all parameters analyzed. Our results suggest that ketamine post-SE onset treatment was effective in preventing acute and long-standing alterations caused by SE early in life, which indicates a putative role of glutamatergic system on SE-induced brain damage as well as long-lasting behavioral consequences.

Placental Source for 5-HT that Tunes Fetal Brain Development

Deciphering the influences of fetal programming on adult mental disorders causality depends on the identification of specific molecular pathways involved in their etiology. New insights will provide the means for reducing developmentally based disorder risk, and new therapeutic targets for treatments in adulthood. For example, our recent discovery of maternal–placental–fetal interactions that may influence brain development leads to new hypotheses regarding the mechanisms by which fetal programming of adult mental disorders may occur. A tryptophan (the precursor of serotonin—5-HT) metabolic pathway in the placenta (Bonnin et al, 2011) reflects the potential importance of extra-embryonically derived 5-HT in modulating developmental processes such as brain circuit wiring, thus affecting long-term brain function. This concept is consistent with classic genetic (5-HT1A knockout) and pharmacological (SSRI exposure) studies showing that disruption of 5-HT signaling transiently, during a restricted period of pre- or postnatal development, results in long-term behavioral abnormalities, such as increased anxiety in adulthood (Ansorge et al, 2008; Oberlander et al, 2009). Because many 5-HT receptors are expressed early and in complex temporal and spatial patterns during brain development (Bonnin et al, 2006), the full extent of the mechanisms through which disruption of 5-HT signaling leads to adult phenotypes is not yet understood. One possible route through which it could occur is the disruption of the modulatory activity of 5-HT signaling on fetal forebrain wiring. This was demonstrated in vitro via the modulation of netrin-1 axon guidance activity by 5-HT, and in vivo by simultaneous, targeted disruption of two 5-HT receptors (5-HT1B/1D) (Bonnin et al, 2007). Altered 5-HT signaling in the forebrain could preferentially influence wiring in this brain region in utero (Bonnin et al, 2007; Bonnin et al, 2011), ultimately leading to long-term dysfunction of circuits underlying mood and emotion. Control of 5-HT signaling, through the number and/or type of 5-HT receptors activated, may thus be critical for normal brain development.

Hippocampus specific iron deficiency alters competition and cooperation between developing memory systems

Iron deficiency (ID) is the most common gestational micronutrient deficiency in the world, targets the fetal hippocampus and striatum and results in long-term behavioral abnormalities. These structures primarily mediate spatial and procedural memory, respectively, in the rodent but have interconnections that result in competition or cooperation during cognitive tasks. We determined whether ID-induced impairment of one alters the function of the other by genetically inducing a 40% reduction of hippocampus iron content in late fetal life in mice and measuring dorsal striatal gene expression and metabolism and the behavioral balance between the two memory systems in adulthood. Slc11a2hipp/hipp mice had similar striatum iron content, but 18% lower glucose and 44% lower lactate levels, a 30% higher phosphocreatine:creatine ratio, and reduced iron transporter gene expression compared to wild type (WT) littermates, implying reduced striatal metabolic function. Slc11a2hipp/hipp mice had longer mean escape times on a cued task paradigm implying impaired procedural memory. Nevertheless, when hippocampal and striatal memory systems were placed in competition using a Morris Water Maze task that alternates spatial navigation and visual cued responses during training, and forces a choice between hippocampal and striatal strategies during probe trials, Slc11a2hipp/hipp mice used the hippocampus-dependent response less often (25%) and the visual cued response more often (75%) compared to WT littermates that used both strategies approximately equally. Hippocampal ID not only reduces spatial recognition memory performance but also affects systems that support procedural memory, suggesting an altered balance between memory systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s11689-010-9049-0) contains supplementary material, which is available to authorized users.

Anesthesia for Cesarean Delivery and Learning Disabilities in a Population-based Birth Cohort

Background Anesthetics administered to immature brains may cause histopathological changes and long-term behavioral abnormalities. The association between perinatal exposure to anesthetics during Cesarean delivery (CD) and development of learning disabilities (LD) was determined in a population-based birth cohort.

Anesthesia for Cesarean Delivery and Learning Disabilities in a Population-based Birth Cohort

Anesthetics administered to immature brains may cause histopathological changes and long-term behavioral abnormalities. The association between perinatal exposure to anesthetics during Cesarean delivery (CD) and development of learning disabilities (LD) was determined in a population-based birth cohort. The educational and medical records of all children born to mothers residing in five townships of Olmsted County, Minnesota from 1976-1982 and remaining in the community at age 5 were reviewed to identify those with LDs. Cox proportional hazards regression was used to compare rates of LD between children delivered vaginally and via CD (with general or regional anesthesia). Of the 5,320 children in this cohort, 497 were delivered via CD (under general anesthesia n = 193, and regional anesthesia n = 304). The incidence of LD depended on mode of delivery (P = 0.050, adjusted for sex, birth weight, gestational age, exposure to anesthesia before age 4 yr, and maternal education). LD risk was similar in children delivered by vagina or CD with general anesthesia, but was reduced in children receiving CD with regional anesthesia (hazard ratio = 0.64, 95% confidence interval 0.44 to 0.92; P = 0.017 for comparison of CD under regional anesthesia compared to vaginal delivery). Children exposed to general or regional anesthesia during CD are not more likely to develop LD compared to children delivered vaginally, suggesting that brief perinatal exposure to anesthetic drugs does not adversely affect long-term neurodevelopmental outcomes. The risk of LD may be lower in children delivered by CD whose mothers received regional anesthesia.

CON: The Toxic Effects of Anesthetics in the Developing Brain: The Clinical Perspective

Sulpicio G. Soriano, MD, FAAP‡ “All models are wrong, some models are useful” This quote by the statistician George E. P. Box seems to have relevance for the current preeminent controversy in pediatric anesthesiology, namely, developmental neuroapoptotic cell death after an anesthetic exposure in the immature brain. Worldwide, general anesthetics and sedatives are used in hundreds of thousands of neonates and infants every year during surgical operations, invasive procedures, and imaging studies. The possibility of anesthesiainduced neuronal cell loss, as suggested by animal models, during an otherwise uneventful procedure has sparked vigorous discussions among anesthesiologists about the safety of anesthesia in human newborns and infants. These concerns were recently addressed at the March 29, 2007, public hearing of the Anesthesia and Life Support Drugs Advisory Committee of the Food and Drug Administration (transcript available at http://www. fda.gov/ohrms/dockets/ac/07/transcripts/2007-4285t1.pdf). Although the exact mechanism of general anesthesia is not entirely understood, alterations of synaptic transmission involving -aminobutyric acid type A (GABAA) and N-methyl-d-aspartate (NMDA) glutamate receptors, to varying degrees, seem to play an important role. Because GABA and NMDA-mediated neuronal activity is essential for normal mammalian brain development, exposure to anesthetics could potentially interfere with brain maturation, learning, and neurocognitive function. Concerns about the effects of general anesthetics on neuronal structure and neurocognitive function were first raised more than two decades ago. In a series of studies, chronic subanesthetic exposure of pregnant rats to halothane led to delayed synaptogenesis and behavioral abnormalities in their pups. More recently, the potential for ketamine to cause increased neuronal cell death was documented in rat pups. However, although ketamine is rarely used for pediatric anesthesia, general anesthetics routinely used in pediatric practice have subsequently also been implicated not only in producing widespread neuronal cell death, but also in leading to long-term cognitive impairment in adult animals exposed to neonatal anesthesia. A 6-h exposure to a combination of isoflurane, nitrous oxide, and midazolam led to widespread apoptotic brain cell death in 7-day-old rats. When animals were examined in adulthood, many tests of behavior and attention remained normal. However, several tests of spatial learning and memory demonstrated impairment in adult animals that were exposed to the anesthetic cocktail as neonates, compared with their unanesthetized littermates. Several groups of investigators have now confirmed the neurotoxic effects of various anesthetics in a variety of in vivo and in vitro developing animal models. From the *Departments of Anesthesia and Pediatrics, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine; and †Institute of Pediatric Anesthesia, Cincinnati Children’s Research Foundation, Cincinnati, Ohio; and ‡Department of Anaesthesia, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts. Accepted for publication March 5, 2008. Address correspondence and reprint requests to Dr. Andreas Loepke, Department of Anesthesia, Cincinnati Children’s Hospital Medical Center, ML2001, 3333 Burnet Ave., Cincinnati, OH 45229. Address e-mail to Andreas.Loepke@cchmc.org. Copyright © 2008 International Anesthesia Research Society DOI: 10.1213/ane.0b013e3181733ef8