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BackgroundThe authors tested the hypothesis that the effects of traumatic brain injury, surgery, and sevoflurane interact to induce neurobehavioral abnormalities in adult male rats and in their offspring (an animal model of intergenerational perioperative neurocognitive disorder).MethodsSprague-Dawley male rats (assigned generation F0) underwent a traumatic brain injury on postnatal day 60 that involved craniectomy (surgery) under 3% sevoflurane for 40 min followed by 2.1% sevoflurane for 3 h on postnatal days 62, 64, and 66 (injury group). The surgery group had craniectomy without traumatic brain injury, whereas the sevoflurane group had sevoflurane only. On postnatal day 90, F0 males and control females were mated to generate offspring (assigned generation F1).ResultsAcutely, F0 injury rats exhibited the greatest increases in serum corticosterone and interleukin-1β and -6, and activation of the hippocampal microglia. Long-term, compared to controls, F0 injury rats had the most exacerbated corticosterone levels at rest (mean ± SD, 2.21 ± 0.64 vs. 7.28 ± 1.95 ng/ml, n = 7 - 8; P < 0.001) and 10 min after restraint (133.12 ± 33.98 vs. 232.83 ± 40.71 ng/ml, n = 7 - 8; P < 0.001), increased interleukin-1β and -6, and reduced expression of hippocampal glucocorticoid receptor (Nr3c1; 0.53 ± 0.08 fold change relative to control, P < 0.001, n = 6) and brain-derived neurotrophic factor genes. They also exhibited greater behavioral deficiencies. Similar abnormalities were evident in their male offspring, whereas F1 females were not affected. The reduced Nr3c1 expression in F1 male, but not female, hippocampus was accompanied by corresponding Nr3c1 promoter hypermethylated CpG sites in F0 spermatozoa and F1 male, but not female, hippocampus.ConclusionsThese findings in rats suggest that young adult males with traumatic brain injury are at an increased risk of developing perioperative neurocognitive disorder, as are their unexposed male but not female offspring.Editor’s PerspectiveWhat We Already Know about This TopicWhat This Article Tells Us That Is New

Background Neonatal exposure to sevoflurane induces neurobehavioral and neuroendocrine abnormalities in exposed male rats (generation F0) and neurobehavioral, but not neuroendocrine, abnormalities in their male, but not female, offspring (generation F1). These effects of sevoflurane are accompanied by a hypermethylated neuron-specific K-2Cl (Kcc2) Cl exporter gene in the F0 spermatozoa and the F1 male hypothalamus, while the gene's expression is reduced in the F0 and F1 hypothalamus. We investigated whether inhibition of deoxyribonucleic acid methyltransferases (DNMTs) before paternal sevoflurane exposure could alleviate the anesthetic's F0 and F1 effects. Methods Sprague-Dawley male rats were anesthetized with 2.1% sevoflurane for 5 hours on postnatal day (P) 5 and mated with control females on P90 to generate offspring. The nonselective DNMT inhibitor decitabine (0.5 mg/kg, intraperitoneally) was administered 30 minutes before sevoflurane exposure. The F0 and F1 male rats were evaluated in in vivo and in vitro tests in adulthood. Results Paternal exposure to sevoflurane induced impaired prepulse inhibition of the acoustic startle response and exacerbated corticosterone responses to stress in F0 males and impaired prepulse inhibition of the startle responses in F1 males. These effects were accompanied in both generations by reduced and increased expressions of hypothalamic Kcc2 and Dnmt3a/b, respectively. Decitabine deterred the effects of paternal exposure to sevoflurane in F0 and F1 males. Conclusions These results suggest that similar decitabine-sensitive mechanisms regulating expression of multiple genes are involved in the mediation of neurobehavioral abnormalities in sires neonatally exposed to sevoflurane and in their future unexposed male offspring.

The progress of modern medicine would be impossible without the use of general anesthetics (GAs). Despite advancements in refining anesthesia approaches, the effects of GAs are not fully reversible upon GA withdrawal. Neurocognitive deficiencies attributed to GA exposure may persist in neonates or endure for weeks to years in the elderly. Human studies on the mechanisms of the long-term adverse effects of GAs are needed to improve the safety of general anesthesia but they are hampered not only by ethical limitations specific to human research, but also by a lack of specific biological markers that can be used in human studies to safely and objectively study such effects. The latter can primarily be attributed to an insufficient understanding of the full range of the biological effects induced by GAs and the molecular mechanisms mediating such effects even in rodents, which are far more extensively studied than any other species. Our most recent experimental findings in rodents suggest that GAs may adversely affect many more people than is currently anticipated. Specifically, we have shown that anesthesia with the commonly used GA sevoflurane induces in exposed animals not only neuroendocrine abnormalities (somatic effects), but also epigenetic reprogramming of germ cells (germ cell effects). The latter may pass the neurobehavioral effects of parental sevoflurane exposure to the offspring, who may be affected even at levels of anesthesia that are not harmful to the exposed parents. The large number of patients who require general anesthesia, the even larger number of their future unexposed offspring whose health may be affected, and a growing number of neurodevelopmental disorders of unknown etiology underscore the translational importance of investigating the intergenerational effects of GAs. In this mini review, we discuss emerging experimental findings on neuroendocrine, epigenetic, and intergenerational effects of GAs.

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Sevoflurane administered to neonatal rats induces neurobehavioral abnormalities and epigenetic reprogramming of their germ cells; the latter can pass adverse effects of sevoflurane to future offspring. As germ cells are susceptible to reprogramming by environmental factors across the lifespan, the authors hypothesized that sevoflurane administered to adult rats could induce neurobehavioral abnormalities in future offspring, but not in the exposed rats themselves. Methods Sprague-Dawley rats were anesthetized with 2.1% sevoflurane for 3 h every other day between postnatal days 56 and 60. Twenty-five days later, exposed rats and nonexposed controls were mated to produce offspring. Results Adult male but not female offspring of exposed parents of either sex exhibited deficiencies in elevated plus maze (mean ± SD, offspring of both exposed parents vs. offspring of control parents, 35 ± 12 vs. 15 ± 15 s, P < 0.001) and prepulse inhibition of acoustic startle (offspring of both exposed parents vs. offspring of control parents, 46.504 ± 13.448 vs. 25.838 ± 22.866%, P = 0.009), and increased methylation and reduced expression of the potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) in the hypothalamus. Kcc2 was also hypermethylated in sperm and ovary of the exposed rats. Surprisingly, exposed male rats also exhibited long-term abnormalities in functioning of the hypothalamic-pituitary-gonadal and -adrenal axes, reduced expression of hypothalamic and hippocampal Kcc2, and deficiencies in elevated plus maze (sevoflurane vs. control, 40 ± 24 vs. 25 ± 12 s, P = 0.038) and prepulse inhibition of startle (sevoflurane vs. control, 39.905 ± 21.507 vs. 29.193 ± 24.263%, P < 0.050). Conclusions Adult sevoflurane exposure affects brain development in male offspring by epigenetically reprogramming both parental germ cells, while it induces neuroendocrine and behavioral abnormalities only in exposed males. Sex steroids may be required for mediation of the adverse effects of adult sevoflurane in exposed males.

Background Deficiencies in neurocognitive function have been found in late childhood or adolescence in patients who had prolonged and/or repeated early-life general anesthesia. Animal studies suggest that anesthetic-induced impairment in the neuron-specific K+-2Cl− (Kcc2) Cl− exporter expression, which regulates developmental maturation of GABA type A receptor (GABAAR) signaling from excitatory to inhibitory, may play a mediating role. We tested whether the DNA methyltransferase (DNMT) inhibitor decitabine ameliorates the anesthetic’s adverse effects. Methods Sprague-Dawley male rats were injected with vehicle or decitabine 30 min before 2.1 % sevoflurane exposure for 5 h on postnatal day 5 (P5). On P19, P20, or P21, electroencephalography-detectable seizures were measured during 1 h of sevoflurane exposure, followed by collection of the trunk blood and brain tissue samples. Other rats were evaluated for changes in hippocampal CA1 dendrite morphology and gene expressions on ≥ P120. Results Rats in the vehicle plus sevoflurane group responded to sevoflurane exposure on P19, P20 or P21 with electroencephalography-detectable seizures and stress-like corticosterone secretion and had altered hippocampal dendrite morphology in adulthood. These rats had expressions of Kcc2 and Dnmt genes downregulated and upregulated, respectively, in the P19 - P21 cortex and hypothalamus and the ≥ P120 hippocampus. All measured parameters in the sevoflurane-exposed rats that were pretreated with decitabine were not different from those in the control group. Conclusions Neonatal exposure to sevoflurane sensitizes rats to adverse effects of repeated exposure to the anesthetic. The anesthetic-caused changes in the decitabine-sensitive mechanisms may play a mediating role in the developmental effects of early-life anesthesia.

Background The majority of studies evaluating neurocognition in humans who had procedures under anesthesia early in life found long-term deficits even though the typical anesthesia duration normalized to the human life span is much shorter than that shown to induce developmental abnormalities in rodents. Therefore, we studied whether subsequent environmental stressors contribute to deficiencies programmed by a brief neonatal etomidate exposure. Methods Postnatal days (P) 4, 5, or 6, Sprague-Dawley rats, pretreated with vehicle or the Na+-K+-2Cl− (NKCC1) inhibitor, bumetanide, received two injections of etomidate resulting in anesthesia for 2 h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3 h at P10. 3–7 days after exposure to etomidate the rats had increased hypothalamic NKCC1 mRNA and corticotropin releasing hormone (CRH) mRNA and decreased K+-2Cl− (KCC2) mRNA levels with greater changes in males. In rats neonatally exposed to both etomidate and maternal separation, these abnormalities persisted into adulthood. These animals also exhibited extended corticosterone responses to restraint stress with increases in total plasma corticosterone more robust in males, as well as behavioral abnormalities. Pretreatment with the NKCC1 inhibitor ameliorated most of these effects. Conclusions Post-anesthesia stressors may exacerbate/unmask neurodevelopmental abnormalities even after a relatively short anesthetic with etomidate, leading to dysregulated stress response systems and neurobehavioral deficiencies in adulthood. Amelioration by bumetanide suggests a mechanistic role for etomidate-enhanced gamma-aminobutyric acid type A receptor-mediated depolarization in initiating long-lasting alterations in gene expression that are further potentiated by subsequent maternal separation.

Human studies, and especially laboratory studies, provide evidence that early life exposure to general anesthesia may affect neurocognitive development via largely unknown mechanisms. We explored whether hippocampal histone acetylation had a role in neurodevelopmental effects of sevoflurane administered to neonatal rats. Male Sprague-Dawley rats were exposed to 3% sevoflurane or were subjected to maternal separation only for 2 h daily at postnatal days 6, 7, and 8. The histone deacetylase inhibitor, sodium butyrate (250 mg/kg, intraperitoneally), or saline was administered starting 2 h prior to anesthesia or maternal separation and continued daily until the end of behavioral tests, which were performed between postnatal days 33 and 50. Upon completion of the behavioral tests, the brain tissues were harvested for further analysis. Rats neonatally exposed to sevoflurane exhibited decreased freezing time in the fear conditioning contextual test and increased escape latency, decreased time in target quadrant, and number of platform crossings in the Morris water maze test. The sevoflurane-exposed rats had lower hippocampal density of dendritic spines, reduced levels of the brain-derived neurotrophic factor, c-fos protein, microtubule-associated protein 2, synapsin1, postsynaptic density protein 95, pCREB/CREB, CREB binding protein, and acetylated histones H3 and H4, and increased levels of histone deacetylases 3 and 8. These neurobehavioral abnormalities were normalized in the sevoflurane-exposed rats treated with sodium butyrate. Our findings provide evidence that neonatal exposure to sevoflurane induces neurobehavioral abnormalities and long-lasting alterations in histone acetylation; normalization of histone acetylation may alleviate the neurodevelopmental side effects of the anesthetic.

Animal studies provide strong evidence that general anesthetics (GAs), administered during the early postnatal period, induce long-term cognitive and neurological abnormalities. Because the brain growth spurt in rodents is delayed compared to that in humans, a fundamental question is whether the postnatal human brain is similarly vulnerable. Sevoflurane and propofol, GAs that share positive modulation of the gamma-aminobutyric acid type A receptor (GABAAR) function cause marked increase in corticosterone levels and induce long-term developmental alterations in synaptic activity in rodents. If synaptogenesis is affected, investigation of mechanisms of the synaptic effects of GAs is of high interest because synaptogenesis in humans continues for several years after birth. Here, we compared long-term synaptic effects of etomidate with those of propofol. Etomidate and propofol both positively modulate GABAAR activity, but in contrast to propofol, etomidate inhibits the adrenal synthesis of corticosterone. Postnatal day (P) 4, 5, or 6 rats received five injections of etomidate, propofol, or vehicle control during 5h of maternal separation. Endocrine effects of the anesthetics were evaluated by measuring serum levels of corticosterone immediately after anesthesia or maternal separation. The frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in hippocampal CA1 pyramidal neurons were measured at P24-40 and P≥80. Only propofol caused a significant increase in serum corticosterone levels (F(4.26)=17.739, P

Early life stressors, including general anesthesia, can have adverse effects on adult neural and behavioral outcomes, such as disruptions in inhibitory signaling, stress responsivity and increased risk of psychiatric disorders. Here we used a rat model to determine the effects of combined exposure to etomidate (ET) neonatal anesthesia and maternal separation on adult amygdala expression of genes for corticotropin-releasing hormone (Crh) and the chloride co-transporters Nkcc1 and Kcc2, as well as ethanol intake. Male and female Sprague-Dawley rats were subjected to 2 h of ET anesthesia on postnatal days (P) 4, 5, or 6 followed by maternal separation for 3 h on P10 (ET + SEP). During the P91-P120 period rats had daily 2 h access to three 0.05% saccharin solutions containing 0%, 5%, or 10% ethanol, followed by gene expression analyses. The ET + SEP group had increased Crh mRNA levels and Nkcc1/Kcc2 mRNA ratios in the amygdala, with greater increases in Nkcc1/Kcc2 mRNA ratios in males. A moderate increase in 5% ethanol intake was evident in the ET + SEP males, but not females, after calculation of the ratio of alcohol intake between the last week and first week of exposure. In contrast, control males tended to decrease alcohol consumption during the same period. A brief exposure to ET combined with a subsequent episode of stress early in life induced significant alterations in expression of amygdala Crh, Nkcc1 and Kcc2 with greater changes in the Cl− transporter expression in males. The possibility of increased alcohol intake in the exposed males requires further confirmation using different alcohol intake paradigms.

Background We tested the hypothesis that developmental effects of repeated neonatal exposure to sevoflurane in rats are exacerbated by stressful experiences received later in life. Methods Sprague-Dawley male rats received sequential exposures to 3% sevoflurane for two h on postnatal days (P) six, seven, and eight. After weaning at P21, rats were housed either in pairs in an enriched environment (EE) or singly in an enrichment-deprived environment (an adverse environment, AE). The hippocampal concentrations of brain-derived neurotrophic factor (BDNF), and synaptic markers were assessed at P8 and P53. The dentate gyrus neural progenitor proliferation was evaluated at P11 and P53 after administration of bromodeoyuridine (BrdU) at P8 to P10 and at P22 to P27, respectively. Neurobehavioural evaluations were performed at P49 to P53. Results Repeated sevoflurane exposure acutely reduced concentrations of BDNF, synaptic markers and neural progenitor proliferation. The sevoflurane group housed in the AE conditions (sevoflurane+AE) had decreased concentrations of BDNF and synaptic markers, and survival of new granule cells and impaired cognitive function compared with the control+AE, control+EE, and sevoflurane+EE groups. The neurobehavioural parameters in the sevoflurane+EE and control+EE groups were similar. Conclusions Neurocognitive abnormalities induced by repeated neonatal exposure to sevoflurane can be aggravated by stressful conditions such as social isolation and enrichment deprivation.

Background 1.5 million children under 12 months of age are exposed to general anesthesia annually in the United States alone. Human and especially animal studies provide evidence that exposure to general anesthesia during the early postnatal period may lead to long-term neurocognitive abnormalities via poorly understood mechanisms. We investigated whether an immature stress response system and γ-aminobutyric acid (GABA) type A receptor activities are involved in mediating these abnormalities. Methods Sprague–Dawley rats at postnatal days 4, 5 or 6 were anesthetized with 2.1% sevoflurane for 6 h; maternally separated and house reared rats served as controls. Results Sevoflurane anesthesia markedly increased corticosterone levels in rat pups of both genders. In adulthood, these rats responded to stress with heightened secretion of corticosterone and a greater increase in corticosterone levels in males versus females. Only male rats, previously exposed to neonatal sevoflurane, had a higher frequency of miniature inhibitory postsynaptic currents in CA1 neurons, spent a shorter time in open arms of the elevated plus maze (EPM) and exhibited impaired prepulse inhibition (PPI) of startle. Pretreatment of male rats prior to sevoflurane with the Na + -K + -2Cl − cotransporter inhibitor, bumetanide, or the mineralocorticoid receptor antagonist, RU28318, normalized endocrine responses to stress and the EPM behavior in adulthood, while only those pretreated with bumetanide exhibited normalized PPI of startle responses. Neither bumetanide nor RU28318 altered the effect of sevoflurane on synaptic activity. Conclusions Sevoflurane-enhanced neuronal excitation and elevated corticosteroid levels at the time of anesthesia contribute to the mechanisms initiating neonatal sevoflurane-induced long-term endocrine and neurobehavioral abnormalities.

Background Clinical studies report learning disabilities and attention-deficit/hyperactivity disorders in those exposed to general anaesthesia early in life. Rats, primarily males, exposed to GABAergic anaesthetics as neonates exhibit behavioural abnormalities, exacerbated responses to stress, and reduced expression of hypothalamic K+-2Cl− Cl− exporter (Kcc2). The latter is implicated in development of psychiatric disorders, including male predominant autism spectrum disorders. We tested whether parental early life exposure to sevoflurane, the most frequently used anaesthetic in paediatrics, affects the next generation of unexposed rats. Methods Offspring (F1) of unexposed or exposed to sevoflurane on postnatal day 5 Sprague-Dawley rats (F0) were subjected to behavioural and brain gene expression evaluations. Results Male, but not female, progeny of sevoflurane-exposed parents exhibited abnormalities in behavioural testing and Kcc2 expression. Male F1 rats of both exposed parents exhibited impaired spatial memory and expression of hippocampal and hypothalamic Kcc2. Offspring of only exposed sires had abnormalities in elevated plus maze and prepulse inhibition of startle, but normal spatial memory and impaired expression of hypothalamic, but not hippocampal, Kcc2. In contrast to exposed F0, their progeny exhibited normal corticosterone responses to stress. Bisulphite sequencing revealed increased CpG site methylation in the Kcc2 promoter in F0 sperm and F1 male hippocampus and hypothalamus that was in concordance with the changes in Kcc2 expression in specific F1 groups. Conclusions Neonatal exposure to sevoflurane can affect the next generation of males through epigenetic modification of Kcc2 expression, while F1 females are at diminished risk.

An imbalance between excitation and inhibition in the developing central nervous system may result in a pathophysiological outcome. We investigated the mechanistic roles of endocrine activity and γ-aminobutyric acid type A receptor (GABAAR)-mediated excitation in electroencephalographic seizures caused by the GABAAR-selective anesthetic propofol in neonatal rats.Postnatal day 4-6 Sprague Dawley rats underwent a minor surgical procedure to implant electrodes to measure electroencephalographic activity for 1 hour before and 1 hour after intraperitoneal administration of propofol (40 mg·kg). Various treatments were administered 15 minutes before administration of propofol.Episodes of electroencephalographic seizures and persistent low-amplitude spikes occurred during propofol anesthesia. Multifold increases in serum levels of corticosterone (t(10) = -5.062; P = 0.0005) and aldosterone (t(10) = -5.069; P = 0.0005) were detected 1 hour after propofol administration in animals that underwent experimental manipulations identical to those used to study electroencephalographic activity. Pretreatment with bumetanide, the Na-K-2Cl cotransporter inhibitor, which diminishes GABAAR-mediated excitation, eliminated both seizure and spike electroencephalographic activities caused by propofol. Mineralocorticoid and glucocorticoid receptor antagonists, RU 28318 and RU486, depressed electroencephalographic seizures but did not affect the spike electroencephalographic effects of propofol. Etomidate, at a dose sufficient to induce loss of righting reflex, was weak at increasing serum corticosteroid levels and eliciting electroencephalographic seizures. Etomidate given to corticosterone-pretreated rat pups further increased the total duration of electroencephalographic seizures caused by administration of exogenous corticosterone (t(21) = -2.512, P = 0.0203).Propofol increases systemic corticosteroid levels in neonatal rats, which along with GABAAR-mediated excitation appear to be required for propofol-induced neonatal electroencephalographic seizures. Enhancement of GABAAR activity alone may not be sufficient to elicit neonatal electroencephalographic seizures.

The effects of the halogenated aromatic amino acid 3,5-dibromo-D: -tyrosine (3,5-DBr-D: -Tyr) were studied in rat models of stroke and epileptic seizures caused by middle cerebral artery occlusion (MCAo) through respective intracerebral injection of endothelin-1 (ET-1) and intraperitoneal (i.p.) injection of pentylenetetrazole (PTZ). 3,5-DBr-D: -Tyr was administered as three bolus injections (30 or 90 mg/kg, i.p.) starting at 30, 90, and 180 min after ET-1 administration or as a single bolus (30 mg/kg, i.p.) 15 min prior to PTZ administration. Neurological deficits and infarct volume were estimated 3 days after ET-1 administration and seizure score was assessed during the first 20 min after PTZ administration. The safety of 3,5-DBr-D: -Tyr was evaluated in control animals using telemetry to measure cardiovascular parameters and immunostaining to assess the level of activated caspase-3. 3,5-DBr-D: -Tyr significantly improved neurological function and reduced infarct volume in the brain even when the treatment was initiated 3 h after the onset of MCAo. 3,5-DBr-D: -Tyr significantly depressed PTZ-induced seizures. 3,5-DBr-D: -Tyr did not cause significant changes in arterial blood pressure, heart rate and spontaneous locomotor activity, nor did it increase the number of activated caspase-3 positive cells in the brain. We conclude that 3,5-DBr-D: -Tyr, by alleviating the deleterious effects of MCAo and PTZ in rats with no obvious intrinsic effects on cardiovascular parameters and neurodegeneration, exhibits promising potential as a novel therapeutic direction for stroke and seizures.

Background We tested the hypothesis that in newborn rats, sevoflurane may cause seizures, neurotoxicity, and impairment in synaptic plasticity-effects that may be diminished by the Na-K-2Cl cotransporter 1 inhibitor, bumetanide. Methods Electroencephalography, activated caspase-3, and hippocampal long-term potentiation were measured in rats exposed to 2.1% sevoflurane for 0.5-6 h at postnatal days 4-17 (P4-P17). Results Arterial blood gas samples drawn at a sevoflurane concentration of 2.1% showed no evidence of either hypoxia or hypoventilation in spontaneously breathing rats. Higher doses of sevoflurane (e.g., 2.9%) caused respiratory depression. During anesthesia maintenance, the electroencephalography exhibited distinctive episodes of epileptic seizures in 40% of P4-P8 rats. Such seizure-like activity was not detected during anesthesia maintenance in P10-P17 rats. Emergence from 3 h of anesthesia with sevoflurane resulted in tonic/clonic seizures in some P10-P17 rats but not in P4-P8 rats. Bumetanide (5 micromol/kg, intraperitoneally) significantly decreased seizures in P4-P9 rats but did not affect the emergence seizures in P10-P17 rats. Anesthesia of P4 rats with sevoflurane for 6 h caused a significant increase in activated caspase-3 and impairment of long-term potentiation induction measured at 1 and 14-17 days after exposure to sevoflurane, respectively. Pretreatment of P4 rats with bumetanide nearly abolished the increase in activated caspase-3 but did not alleviate impairment of long-term potentiation. Conclusion These results support the possibility that excitatory output of sevoflurane-potentiated gamma-aminobutyric acid type A/glycine systems may contribute to epileptogenic and neurotoxic effects in early postnatal rats.

Phenylketonuria (PKU) is a metabolic disorder that results in significant brain dysfunction if untreated. Although phenylalanine restricted diets instituted at birth have clearly improved PKU outcomes, neuropsychological deficits and neurological changes still represent substantial problems. The specific mechanisms by which Phe affects the brains of individuals with PKU are yet fully determined. The use of animal models in PKU research significantly broadens the possibilities for investigating these mechanisms. This report presents an overview of findings from animal studies on the mechanisms of Phe action in the PKU brain, discussing the importance of changes in protein synthesis, transport of large neutral amino acids across the blood-brain barrier, synthesis of monoamine neurotransmitters, activity Of glutamate receptors, animal behavior, and translation of animal behavioral data to patients with PKU. This report shows that great progress has been made in past years and demonstrates the importance of further animal research to understand the neuropathological mechanisms underlying brain dysfunction in PKU. A better understanding of these mechanisms will guide the development of optimal treatment strategies for PKU. (C) 2009 Elsevier Inc. All rights reserved.

Background and purpose: Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5-dibromo-L-phenylalanine (3,5-DBr-L-Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit. Experimental approach: 3,5-DBr-L-Phe was administered intraperitoneally as three boluses after intracerebral injection of endothelin-1 (ET-1) adjacent to the middle cerebral artery to cause brain injury (a model of stroke). 3,5-DBr-L-Phe was also given as a single bolus prior to pentylenetetrazole (PTZ) injection to induce seizures or prior to the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) to cause disruption of prepulse inhibition (PPI) of startle (sensorimotor gating deficit). Key results: Brain damage caused by ET-1 was reduced by 52%, which is comparable with the effects of MK-801 in this model as reported by others. 3,5-DBr-L-Phe significantly reduced seizures induced by PTZ without the significant effects on arterial blood pressure and heart rate normally caused by NMDA antagonists. 3,5-DBr-L-Phe prevented the disruption of PPI measured 3 days after the administration of ET-1. 3,5-DBr-L-Phe also eliminated sensorimotor gating deficit caused by MK-801. Conclusion and implications: The pharmacological profile of 3,5-DBr-L-Phe might be beneficial not only for developing a therapy for the neurological and cognitive symptoms of stroke and seizures but also for some neuropsychiatric disorders.

In previous studies, we determined that macrophage migration inhibitory factor (MIF), acting intracellularly via its intrinsic thiol-protein oxidoreductase (TPOR) activity, stimulates basal neuronal delayed-rectifier K+ current ( IKv) and inhibits basal and angiotensin (ANG) II-induced increases in neuronal activity. These findings are the basis for our hypothesis that MIF is a negative regulator of ANG II actions in neurons. MIF has recently been recategorized as a member of the thioredoxin (Trx) superfamily of small proteins. In the present study we have examined whether Trx influences basal and ANG II-modulated IKv in an effort to determine whether the Trx superfamily can exert a general regulatory influence over neuronal activity and the actions of ANG II. Intracellular application of Trx (0.8–80 nM) into rat hypothalamic/brain stem neurons in culture increased neuronal IKv, as measured by voltage-clamp recordings. This effect of Trx was abolished in the presence of the TPOR inhibitor PMX 464 (800 nM). Furthermore, the mutant protein recombinant human C32S/C35S-Trx, which lacks TPOR activity, failed to alter neuronal IKv. Trx applied at a concentration (0.08 nM) that does not alter basal IKv abolished the inhibition of neuronal IKv produced by ANG II (100 nM). Given our observation that ANG II increases Trx levels in neuronal cultures, it is possible that Trx (like MIF) has a negative regulatory role over basal and ANG II-stimulated neuronal activity via modulation of IKv. Moreover, these data suggest that TPOR may be a general mechanism for negatively regulating neuronal activity.

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.

Recent studies have demonstrated that long-term developmental effects of neonatal anesthesia were more prominent in males. We tested whether steroids, in general, and sex steroids, in particular, are involved in the mediation of sevoflurane-caused paradoxical cortical seizures during the early postnatal period. Methods: Cortical electroencephalograms, hippocampal synaptic activity, serum levels of steroids and the loss of the righting reflex (LORR), a marker of anesthetic effect, were measured on postnatal days 4-6 in Sprague Dawley rats of both genders exposed to 2.1% sevoflurane. Results: Episodes of seizures, persistent spikes in electroencephalograms and increases in serum corticosterone were similar in both genders. In the order of increasing potency, the corticosteroid receptor antagonist RU 28318, the estradiol receptor antagonist ICI 182780 and the estradiol synthesis inhibitor formestane decreased sevoflurane-induced seizures. Exogenous estradiol increased sevoflurane-caused seizures, spikes and serum levels of corticosterone. These estradiol-enhanced seizures and spikes were depressed by ICI 182780 and the NKCC1 inhibitor, bumetanide, while RU 28318 decreased seizures only. In hippocampal CA1 neurons, estradiol increased the amplitude, rise time and area under the curve of gamma-aminobutyric acid type A receptor (GABAAR)-mediated miniature postsynaptic currents. Exogenous estradiol shortened, while ICI 182780 and formestane lengthened the time needed for sevoflurane to induce LORR. Conclusion: These findings provide evidence for gender-independent acute electroencephalographic effects of sevoflurane at this age. Corticosterone and estradiol are involved in the mediation of sevoflurane-induced seizures. Estradiol, but not corticosterone, also contributes to sevoflurane-caused spikes, by enhancing GABAAR-mediated excitation in the cortex. By increasing GABAAR-mediated inhibition in more mature caudal regions of the brain, estradiol contributes to sevoflurane-induced LORR.

Glutamate, the major excitatory neurotransmitter, is critical for normal brain development and function. Both extremes of glutamate receptor activity are detrimental for the brain. Glutamate's role in excitotoxicity has driven the search for receptor antagonists as neuroprotective agents, most of which have failed to achieve clinical, i.e. efficacious and safe, neuroprotection. High selectivity and potency provide potential explanations for this failure. For example, targeting individual glutamate receptor subtypes leaves other pathways of glutamatergic excitotoxicity intact. Furthermore, potent depression of glutamate receptor activity causes clinical side effects, such as the symptoms of schizophrenia produced by NMDA receptor antagonists. To produce efficacious neuroprotection devoid of significant side effects, it may be necessary to normalize the function of all components of the glutamatergic system, instead of blocking a single type of glutamate receptors. Halogenated derivatives of aromatic amino acids modulate glutamatergic activity via multiple pre- and postsynaptic actions with moderate efficacy. In addition, these compounds may trap hydroxyl radicals and facilitate hydroxyl radical-impaired glutamate uptake. Their balanced polyvalent action may overcome the limitations of previously tested glutamatergic agents and provide a basis for their use in the treatment of neurological and neuropsychiatric disorders. The properties of this class of compounds and relevant patents are reviewed in this article.

This paper reviews recent results of our investigation of the mechanisms whereby hyperphenylalaninemia may cause brain dysfunction in classical phenylketonuria (PKU). Acute applications of L-Phe in rat and mouse hippocampal and cerebrocortical cultured neurons, at a range of concentrations found in PKU brain, significantly and reversibly depressed glutamatergic synaptic transmission by a combination of pre- and postsynaptic actions: (1) competition for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptors; (2) attenuation of neurotransmitter release; (3) competition for the glutamate-binding site of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid and kainate (AMPA/kainate) receptors. Unlike L-Phe, its non-tyrosine metabolites, phenylacetic acid, phenylpyruvic acid, and phenyllactic acid, did not produce antiglutamatergic effects. L-Phe did not affect inhibitory gamma-aminobutyric (GABA)-ergic transmission. Consistent with this specific pattern of effects caused by L-Phe in neuronal cultures, the expression of NMDA receptor NR2A and AMPA receptor Glu1 and Glu2/3 subunits in brain of hyperphenylalaninemic PKU mice (Pah(enu2) strain) was significantly increased, whereas expression of the NMDA receptor NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Considering the important role of glutamatergic synaptic transmission in normal brain development and function, these L-Phe-induced changes in glutamatergic synaptic transmission in PKU brain may be a critical element of the neurological symptoms of PKU.

Background and Purpose— The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. Methods— We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). Results— 3,5-diiodo- l -tyrosine (DIT) and 3,5-dibromo- l -tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC 50 s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6±14.1 μmol/L and 127.5±13.3 μmol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7±14.1% and 57.1±12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. Conclusions— DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (I(NMDA)) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated I(NMDA) in a concentration-dependent manner (IC(50) = 1.71 +/- 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change I(NMDA). Although the L-Phe-I(NMDA) concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate I(NMDA) in the presence of D-alanine (10 microM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

We hypothesized that nitric oxide (NO) plays an important role in mediating the anti-adrenergic effect of adenosine on atrioventricular (AV) nodal conduction. In guinea-pig hearts instrumented for measurement of AV nodal conduction time (atrium-to-His bundle, A-H, interval), the NO synthase (NOS) inhibitor, l-NMMA (100 microm), reversibly inhibited 80% (P=0.009, n=6) of adenosine's anti-adrenergic action on the positive dromotropic effect of isoproterenol (0.01 microm). In parallel studies carried out in rabbit AV nodal myocytes, intracellular mechanisms whereby NO mediates the inhibitory effect of adenosine on isoproterenol-induced A-H interval shortening were studied. Adenosine (3 microm) inhibited isoproterenol-stimulated (0.1 microm) I(Ca,L)(beta -I(Ca,L)) by 46+/-6% (P0.001, n=17). Consistent with isolated heart data, the NOS inhibitors, l -NMMA (100 microm) and L-NNA (500 microm) attenuated the effect of adenosine on beta -I(Ca,L)by 69+/-8% (P0.001, n=16) and 69+/-7% (P0.001, n=10), respectively. An inhibitor of NO-stimulated guanylyl cyclase LY83538 (40 microm) reduced the inhibitory effect of adenosine on beta -I(Ca,L)by 97+/-6% (P=0.004, n=15). Similarly, the non-specific inhibitor of cAMP-phosphodiesterases IBMX (50 microm) decreased the anti-adrenergic effect of adenosine by 60% (P=0.02, n=6), whereas the extracellular application of the non-hydrolyzeable cAMP analog 8-Br-cAMP (500 microm) prevented this action of adenosine. Activation of cGMP-dependent protein kinase (PKG) by CPT-cGMP (300 microm) diminished beta -I(Ca,L), but to a significantly smaller degree (16+/-4%, P=0.025, n=12) than that caused by adenosine. NO mediates the anti-adrenergic effect of adenosine on AV nodal conduction by a mechanism predominately involving activation of cGMP-dependent cAMP-phosphodiesterase and to a lesser extent activation of PKG.

Background —The atrioventricular (AV) node is insensitive to changes in extracellular potassium concentration, [K + ] o , because of the absence of the inward rectifier potassium current (I K1 ). However, we propose that in the presence of adenosine, elevated [K + ] o should increase the adenosine-activated inward rectifier potassium current (I K,ADO ) in AV nodal myocytes and hence augment the negative dromotropic effect of the nucleoside. Methods and Results —The effects of normal (4.8 mmol/L) and high (8.0 mmol/L) [K + ] o on adenosine-induced changes in resting membrane potential (V m ), I K,ADO , and membrane resistance (R m ) in rabbit isolated AV nodal myocytes and in AV nodal conduction delay (atrium-to-His bundle, AH, interval) in guinea pig isolated hearts were determined with the use of whole-cell patch-clamp and His bundle electrogram techniques, respectively. High [K + ] o alone did not significantly affect membrane current, R m , or V m in AV nodal myocytes. However, high [K + ] o in the presence of adenosine (3 μmol/L) markedly increased I m (−0.249±0.038 to −0.571±0.111 nA, P m (151±21 to 77±8 MΩ, P m from −48±2 to −65±1 mV ( P + ] o alone did not significantly affect the AH interval in isolated hearts. However, high [K + ] o markedly lengthened the AH interval prolongation caused by adenosine (4 μmol/L, 7.9±0.8 vs 22.1±3.0 ms, P + ] o on adenosine-induced delay in AV nodal conduction was abolished by BaCl 2 (100 μmol/L). Conclusions —By increasing I K,ADO and decreasing R m of AV nodal myocytes, elevated [K + ] o , augments the depressant effect of adenosine on AV nodal conduction.

Background Dysrhythmias and death occur in patients with acquired long QT syndrome (LQTS). Little information exists regarding interactions between anesthetics and drugs that prolong ventricular repolarization. Therefore the effects of three commonly used intravenous anesthetics on ventricular repolarization were investigated in the setting of drug-induced, long QT syndrome. Methods The effects of increasing concentrations (0, 10, 25, and 50 microM) of propofol, ketamine, and thiopental on ventricular repolarization were evaluated by measuring the monophasic action potential duration at 90% repolarization (MAPD90) in guinea pig Langendorff-perfused hearts in the absence or presence of erythromycin (100 microM). If an anesthetic enhanced erythromycin-induced prolongation of MAPD90, its effects on the delayed rectifier (I[K]) and inward rectifier (I[Kl]) potassium currents were measured using the whole-cell patch-clamp technique. Results At clinically relevant concentrations, only thiopental significantly modulated erythromycin's effect on MAPD90. Thiopental at 10, 25, and 50 microM prolonged MAPD90 from a control of 163 +/- 6 ms by 18 +/- 4, 30 +/- 3, and 31 +/- 4 ms, respectively. In a separate group, erythromycin prolonged MAPD90 from 155 +/- 2 ms to 171 +/- 2 ms (n = 21, P < 0.001). In the presence of erythromycin, thiopental at 10, 25, and 50 microM caused significantly greater prolongation from a control of 171 +/- 2 ms by 39 +/- 2, 58 +/- 3, and 72 +/- 6 ms, respectively. Whole-cell patch-clamp experiments indicated that thiopental inhibited I(K) and I(Kl). Conclusions Intravenous anesthetics caused markedly different effects on ventricular repolarization. Thiopental, unlike propofol and ketamine, potentiated the effects of erythromycin on ventricular repolarization by inhibiting I(K) and I(Kl).

Objective: To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current ( I Ca) in atrioventricular (AV) nodal myocytes. Methods: The whole-cell patch-clamp technique was used to record I Ca from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Results: Isoprenaline, 0.1 μM, increased peak I Ca from 0.58±0.08 to 1.23±0.1 nA, and this increase was reversibly inhibited by adenosine, 10 μM (83±6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 μM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on I Ca. Methylene blue (10 μM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide ( · O2−), did not prevent, but increased, the inhibiting action of adenosine (49.5±6.6%, P

The aim of this study was to determine if adenosine exerts an anti-adrenergic effect on rabbit isolated atrioventricular (AV) nodal cells and, if so, the dependence of this effect on nitric oxide (NO) production. Inward Ca current, ICa, was measured in AV nodal cells, enzymatically isolated from rabbit hearts. Isoprenaline (0.1 microM) increased ICa from 676 +/-59 to 1102 +/-86 pA (n =25). This isoprenaline-induced increase in ICa(178 +/-15% of control) was abolished in the presence of 10 microM adenosine (ICa100 +/-2% of control, n =9, P

Objective: To study the actions of adenosine on the electrophysiology of spontaneously active, rod-shaped cells enzymatically isolated from rabbit atrioventricular (AV) node. Methods: Calcium-tolerant myocytes were isolated from the region of the AV node by enzymatic and mechanical dispersion. They were rod- or spindle-shaped, with spontaneous activity at 35–37 °C, and had higher membrane resistances (776 ± 283 MΩ, n = 13), compared to atrial cells (41 ± 18.2 MΩ, n = 7; P < 0.001). Membrane potential, spontaneous action potentials and transmembrane ionic currents were studied using the whole-cell patch-clamp technique, in current-clamp and voltage-clamp mode. Results: Adenosine (0.1–50 μM) slowed or abolished the spontaneous activity, with hyperpolarisation of the membrane potential. Voltage-clamp experiments showed that adenosine induced an inwardly rectifying time-independent current. The adenosine-induced current was shown to be carried by potassium ions by the effect of increasing external potassium, which altered the reversal potential in accordance with the calculated potassium equilibrium potential. The A1 adenosine receptor antagonist, CPDPX (8-cyclopentyl-1,3-dypropylxanthine), reversed the effects of adenosine and an A1 receptor agonist, R-PIA [ R (−) N 6-(2-phenylisopropyl)adenosine] had effects similar to adenosine. Adenosine also caused a small decrease in inward calcium current ( I Ca) in some AV nodal cells. Conclusions: These results indicate that adenosine acts at A1 adenosine receptors to suppress spontaneous activity, hyperpolarise membrane potential and induce a time-independent potassium current in AV nodal cells. These actions, combined with reduction in inward calcium current in some cells, may underlie the negative chronotropic and dromotropic actions of adenosine on rabbit AV nodal cells.

Calcium currents were recorded in cultured (5–7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of l -phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in l -phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at Vt = +20 mV became 40 ± 30% as contrasted to 416 ± 130% in neurones from control animals (the amplitude of the calcium currents at Vt = −10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of l -tyrosine to the cultivation medium (50 μM) restored the high-voltage calcium current, its relative amplitude reaching 280 ± 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.

Changes induced by internal administration of L-tyrosine and L-phenylalanine in high-threshold calcium currents have been studied on perfused PC12 pheochromocytoma cells using whole-cell voltage-clamp technique. A method for rapid changes of perfusing solutions has been used. L-Tyrosine (20 microM) slowed down the decline ('wash-out') of ICa occurring during intracellular perfusion and in most cells induced its temporary recovery. alpha-Methyl-D,L-tyrosine (a tyrosine hydroxylase blocker) exerted a similar effect. On the other hand, L-phenylalanine (20 microM) in most cells speeded-up the decline of ICa. Replacement of ATP in the perfusing solution by an equivalent amount of ADP (2 mM) did not alter the effects of amino acids. The possible mechanisms of the described changes are discussed in connection with the known role of L-tyrosine in posttranslational modifications of microtubular proteins.

Neurons from the hippocampus of one-day-old (controls) rats intraperitoneally injected with phenylalanine were isolated and cultured [3, 8]. The potential-dependent calcium input channel (ICa) in hippocampal neurons of controls after five-seven days of culture was made up of two components, low and high voltage, when the membrane potential was fixed under intracellular perfusion. The high-voltage channel was 69±13% of the total calcium channel at Vt = −10 mV. After rats were injected with phenylalanine, the high-voltage ICa significantly decreased to 32±14% of the total channel in the neurons at the same Vt. The low-voltage ICa did not change significantly.

Summary: Purpose: Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism that frequently results in epilepsy if a low Phe diet was not implemented at birth. The mechanisms by which Phe affects the brain are poorly understood. Methods: Audiogenic seizures (AGS) were studied in female homozygous Pahenu2 BTBR (PKU) mice. Results: Adult PKU mice, 18–20 weeks of age, in contrast to wild-type and heterozygous counterparts, exhibited a full range of AGS. Younger PKU mice, 5–7 weeks of age, had higher serum Phe levels (2.22 ± 0.20 mM) in comparison with the adult animals (1.72 ± 0.05 mM) and were not susceptible to AGS. Among adult mice, animals susceptible to AGS had significantly lower serum Phe levels (1.62 ± 0.06 mM) in comparison with those resistant to AGS (1.86 ± 0.07 mM). Susceptibility to AGS tended to increase in the afternoon when serum Phe concentration decreased in comparison to evening and morning. Normalization of serum Phe level by instituting a low Phe diet generally prevented susceptibility to AGS within 12 h. Although return to a standard diet raised Phe levels to hyperphenylalaninemic within 12 h in animals treated with a low Phe diet for 2 weeks, more than 7 weeks were needed for a complete resumption of AGS. Conclusions: Transient decrease in Phe levels within hyperphenylalaninemic range may be a necessary condition for PKU-related seizures to occur. A low Phe diet prevents susceptibility to seizures, which can resume with the significant delay after termination of dietary treatment.

Macrophage migration inhibitory factor (MIF) has widespread actions in the immune, endocrine, and nervous systems. Previously, we reported that increases in the intracellular levels of MIF depress the firing of hypothalamus/brain stem neurons in culture, including the chronotropic actions of angiotensin II. The objective of this study was to investigate the effects of MIF on delayed rectifier K+ current ( IKv), one of the component currents whose activity contributes to neuronal firing. Intracellular perfusion of MIF (80 nM) into Sprague–Dawley rat neuronal cultures caused a significant increase in IKv, as measured by patch-clamp recordings. This effect was apparent by 3 min, and was maximal after 20–30 min. IKv current density (pA/pF) increased from 31.58 ± 2.36 in controls to 41.88 ± 3.76 in MIF-treated neurons (mean ± SE; n = 9; P < 0.01). MIF that had been inactivated by boiling did not alter IKv, and MIF-neutralizing antibodies abolished the action of recombinant MIF (rMIF). The stimulatory effect of MIF on IKv current density was mimicked by intracellular application of either P1S-MIF (80 nM) or the peptide MIF-(50–65) (0.8–8 μM), both of which harbor the thiol-protein oxidoreductase (TPOR) activity of the MIF molecule. Conversely, neither C60S-MIF (80 nM) nor the MIF homologue D-dopachrome tautomerase (80 nM), both of which lack TPOR activity, altered IKv. Finally, the increase in IKv produced by rMIF was abolished by the superoxide scavenger Tiron (1 mM). These studies indicate that the neuronal action of MIF includes a stimulatory action on IKv that may be mediated by a TPOR/superoxide-scavenging mechanism.

An increasing body of evidence supports the hypothesis that diminished function of N-methyl-D-aspartate (NMDA) receptors and the associated increase in glutamate release and overstimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are critical elements of the pathophysiology of schizophrenia. Here, we describe a halogenated derivative of the aromatic amino acid L-phenylalanine that 1) activates NMDA receptors, 2) depresses presynaptic glutamate release, and 3) blocks AMPA/kainate receptors. The experiments were conducted in rat cerebrocortical cultured neurons by using the patch-clamp technique. 3,5-Dibromo-L-phenylalanine (3,5-DBr-L-Phe) augmented NMDA miniature excitatory postsynaptic currents (mEPSCs) and activated the steady-state current, effects that were eliminated by NMDA receptor antagonists DL-2-amino-5-phosphonopentanoic acid and MK-801 (dizocilpine maleate; 5H-dibenzo[a,d]cyclohepten-5,10-imine). 3,5-DBr-L-Phe was a partial agonist at the glutamate-binding site of NMDA receptors with an EC50 of 331.6 +/- 78.6 microM and with an efficacy of 30.5 +/- 4.7% compared with NMDA. 3,5-DBr-L-Phe depressed both amplitude and frequency of AMPA/kainate mEPSCs. The IC50 of 3,5-DBr-L-Phe to inhibit AMPA/kainate mEPSC frequency was 29.4 +/- 4.3 microM. 3,5-DBr-L-Phe significantly decreased paired pulse depression of AMPA/kainate EPSCs and attenuated current activated by AMPA with higher efficacy at lower concentration of AMPA. 3,5-DBr-L-Phe neither affected GABA miniature inhibitory postsynaptic currents nor elicited action potentials. By enhancing NMDA receptor function, reducing glutamate release and blocking AMPA/kainate receptors 3,5-DBr-L-Phe represents a new type of polyvalent modulator of glutamatergic synaptic transmission with potential therapeutic applications.

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.