Optogenetic stimulation of corticostriatal circuits improves behavioral flexibility in mice with prenatal alcohol exposure.

Fetal alcohol spectrum disorder (FASD) is the most common preventable form of developmental and neurobehavioral disability. Animal models have demonstrated that even low to moderate prenatal alcohol exposure (PAE) is sufficient to impair behavioral flexibility in multiple domains. Previously, utilizing a moderate limited access drinking in the dark paradigm, we have shown that PAE 1) impairs touchscreen pairwise visual reversal in male adult offspring 2) leads to small but significant decreases in orbitofrontal (OFC) firing rates 3) significantly increases dorsal striatum (dS) activity and 4) aberrantly sustains OFC-dS synchrony across early reversal. In the current study, we examined whether optogenetic stimulation of OFC-dS projection neurons would be sufficient to rescue the behavioral inflexibility induced by PAE in male C57BL/6J mice. Following discrimination learning, we targeted OFC-dS projections using a retrograde adeno-associated virus (AAV) delivered to the dS which expressed channel rhodopsin (ChR2). During the first four sessions of reversal learning, we delivered high frequency optogenetic stimulation to the OFC via optic fibers immediately following correct choice responses. Our results show that optogenetic stimulation significantly reduced the number of sessions, incorrect responses, and correction errors required to move past the early perseverative phase for both PAE and control mice. In addition, OFC-dS stimulation during early reversal learning reduced the increased sessions, correct and incorrect responding seen in PAE mice during the later learning phase of reversal but did not significantly alter later performance in control ChR2 mice. Taken together these results suggest that stimulation of OFC-dS projections can improve early reversal learning in PAE and control mice, and these improvements can persist even into later stages of the task days later. These studies provide an important foundation for future clinical approaches to improve executive control in those with FASD. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Prenatal alcohol exposure promotes NLRP3 inflammasome-dependent immune actions following morphine treatment and paradoxically prolongs nerve injury-induced pathological pain in female mice.

BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.

Recent Breakthroughs in Understanding the Cerebellum's Role in Fetal Alcohol Spectrum Disorder: A Systematic Review.

Exposure to alcohol during fetal development can lead to structural and functional abnormalities in the cerebellum, a brain region responsible for motor coordination, balance, and specific cognitive functions. In this systematic review, we comprehensively analyze a vast body of research conducted on vertebrate animals and humans over the past 13 years. We identified studies through PubMed and screened them following PRISMA guidelines. Data extraction and quality analysis were conducted using Covidence systematic review software. A total of 108 studies met our inclusion criteria, with the majority (79 studies) involving vertebrate animal models and 29 studies focusing on human subjects. Animal models included zebrafish, mice, rats, sheep, and non-human primates, investigating the impact of ethanol on cerebellar structure, gene/protein expression, physiology, and cerebellar-dependent behaviors. Additionally, some animal studies explored potential therapeutic interventions against ethanol-induced cerebellar damage. The human studies predominantly adopted cohort designs, exploring the effects of prenatal alcohol exposure on cerebellar structure and function. Certain human studies delved into innovative cerebellar-based diagnostic approaches for fetal alcohol spectrum disorder (FASD). The collective findings from these studies clearly indicate that the cerebellum is involved in various neurophysiological deficits associated with FASD, emphasizing the importance of evaluating both cerebellar structure and function in the diagnostic process for this condition. Moreover, this review sheds light into potential therapeutic strategies that can mitigate prenatal alcohol exposure-induced cerebellar damage.

Long-term effects of low prenatal alcohol exposure on GABAergic interneurons of the murine posterior parietal cortex.

BACKGROUND: Fetal alcohol spectrum disorders (FASDs) are characterized by a wide range of physical, cognitive, and behavioral impairments that occur throughout the lifespan. Prenatal alcohol exposure (PAE) can lead to adult impairments in cognitive control behaviors mediated by the posterior parietal cortex (PPC). The PPC plays a fundamental role in the performance of response tasks in both primates and rodents, specifically when choices between similar target and nontarget stimuli are required. Furthermore, the PPC is reciprocally connected with other cortical areas. Despite the extensive literature investigating the molecular mechanisms underlying PAE impairments in cognitive functions mediated by cortical areas, little is known regarding the long-term effects of PAE on PPC development and function. Here, we examined changes in the cellular organization of GABAergic interneurons and their function in PPC using behaviorally naïve control and PAE mice. METHODS: We used a limited access model of PAE in which C57BL/6J females were exposed to a solution of 10% (w/v) ethanol and 0.066% (w/V) saccharin for 4 h/day throughout gestation. Using high-throughput fluorescent microscopy, we quantified the levels of GABAergic interneurons in the PPC of adult PAE and control offspring. In a separate cohort, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) using whole-cell patch clamp recordings from PPC layer 5 pyramidal neurons. RESULTS: PAE led to a significant overall reduction of parvalbumin-expressing GABAergic interneurons in PAE mice regardless of sex. Somatostatin- and calretinin-expressing GABAergic interneurons were not affected. Interestingly, PAE did not modulate sIPSC amplitude or frequency. CONCLUSIONS: These results suggest that impairments in cognitive control observed in FASD may be due to the significant reduction of parvalbumin-expressing GABAergic interneurons in the PPC. PAE animals may show compensatory changes in GABAergic function following developmental reduction of these interneurons.

Binge-like ethanol exposure during the brain growth spurt disrupts the function of retrosplenial cortex-projecting anterior thalamic neurons in adolescent mice.

Ethanol (EtOH) exposure during late pregnancy leads to enduring impairments in learning and memory that may stem from damage to components of the posterior limbic memory system, including the retrosplenial cortex (RSC) and anterior thalamic nuclei (ATN). In rodents, binge-like EtOH exposure during the first week of life (equivalent to the third trimester of human pregnancy) triggers apoptosis in these brain regions. We hypothesized that this effect induces long-lasting alterations in the function of RSC-projecting ATN neurons. To test this hypothesis, vesicular GABA transporter-Venus mice (expressing fluorescently tagged GABAergic interneurons) were subjected to binge-like EtOH vapor exposure on postnatal day (P) 7. This paradigm activated caspase 3 in the anterodorsal (AD), anteroventral (AV), and reticular thalamic nuclei at P7 but did not reduce neuronal density in these areas at P60-70. At P40-60, we injected red retrobeads into the RSC and performed patch-clamp slice electrophysiological recordings from retrogradely labeled neurons in the AD and AV nuclei 3-4 days later. We found significant effects of treatment on instantaneous action potential (AP) frequency and AP overshoot, as well as sex × treatment interactions for AP threshold and overshoot in AD neurons. A sex × treatment interaction was detected for AP number in AV neurons. EtOH exposure also reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents and increased the charge transfer of spontaneous inhibitory postsynaptic currents. These results highlight a novel cellular mechanism that could contribute to the lasting learning and memory deficits associated with developmental EtOH exposure.

Prenatal alcohol exposure alters mRNA expression for stress peptides, glucocorticoid receptor function and immune factors in acutely stressed neonatal brain.

Background: The amygdala, hippocampus and hypothalamus are critical stress regulatory areas that undergo functional maturation for stress responding initially established during gestational and early postnatal brain development. Fetal alcohol spectrum disorder (FASD), a consequence of prenatal alcohol exposure (PAE), results in cognitive, mood and behavioral disorders. Prenatal alcohol exposure negatively impacts components of the brain stress response system, including stress-associated brain neuropeptides and glucocorticoid receptors in the amygdala, hippocampus and hypothalamus. While PAE generates a unique brain cytokine expression pattern, little is known about the role of Toll-like receptor 4 (TLR4) and related proinflammatory signaling factors, as well as anti-inflammatory cytokines in PAE brain stress-responsive regions. We hypothesized that PAE sensitizes the early brain stress response system resulting in dysregulated neuroendocrine and neuroimmune activation. Methods: A single, 4-h exposure of maternal separation stress in male and female postnatal day 10 (PND10) C57Bl/6 offspring was utilized. Offspring were from either prenatal control exposure (saccharin) or a limited access (4 h) drinking-in-the-dark model of PAE. Immediately after stress on PND10, the hippocampus, amygdala and hypothalamus were collected, and mRNA expression was analyzed for stress-associated factors (CRH and AVP), glucocorticoid receptor signaling regulators (GAS5, FKBP51 and FKBP52), astrocyte and microglial activation, and factors associated with TLR4 activation including proinflammatory interleukin-1ß (IL-1ß), along with additional pro- and anti-inflammatory cytokines. Select protein expression analysis of CRH, FKBP and factors associated with the TLR4 signaling cascade from male and female amygdala was conducted. Results: The female amygdala revealed increased mRNA expression in stress-associated factors, glucocorticoid receptor signaling regulators and all of the factors critical in the TLR4 activation cascade, while the hypothalamus revealed blunted mRNA expression of all of these factors in PAE following stress. Conversely, far fewer mRNA changes were observed in males, notably in the hippocampus and hypothalamus, but not the amygdala. Statistically significant increases in CRH protein, and a strong trend in increased IL-1ß were observed in male offspring with PAE independent of stressor exposure. Conclusion: Prenatal alcohol exposure creates stress-related factors and TLR-4 neuroimmune pathway sensitization observed predominantly in females, that is unmasked in early postnatal life by a stress challenge.

Experimental data on developmental elimination of retrosplenial cortex GABAergic interneurons in a mouse model of ethanol exposure during the last trimester of human pregnancy.

It has been previously shown that 40% of murine cortical interneurons are eliminated via apoptosis during the first two weeks of postnatal development [1], [2], [3]. Here, we report data on the effect of ethanol exposure on this process in a mouse model of binge-like alcohol exposure during last trimester of human pregnancy (equivalent to the first postnatal week in mice). We used transgenic mice that express the Venus fluorescent protein in GABAergic interneurons under the control of the vesicular GABA transporter promoter (VGAT-Venus mice) [4]. Mice were exposed to air (controls) or ethanol for 4 hr/day on postnatal days 4 to 9 using vapor inhalation chambers [5]. This exposure paradigm produces peak blood ethanol concentrations between 300 and 400 mg/dl. Transcardial perfusions were performed under anesthesia at postnatal days 5, 7, 10 and 30. Cryostat-prepared floating sections were stained with the fluorescent DNA dye, 4'6-diamidino-2-phenylindole (DAPI). We then quantified the density of Venus-positive GABAergic interneurons in layers I, II-IV and V of the retrosplenial cortex, which is part of the limbic memory system [6], and is sensitive to ethanol-induced apoptosis during the first postnatal week in mice [7], [8], [9], [10], [11]. The data show that density of interneurons decreases in the retrosplenial cortex layers during the first week of life and that ethanol exposure does not significantly alter this process. These data may be of interest to investigators who are studying the effect of ethanol and other teratogenic agents on developing interneurons in the cerebral cortex.

Sex-specific effect of prenatal alcohol exposure on N-methyl-D-aspartate receptor function in orbitofrontal cortex pyramidal neurons of mice.

BACKGROUND: Alcohol consumption during pregnancy can produce behavioral and cognitive deficits that persist into adulthood. These include impairments in executive functions, learning, planning, and cognitive flexibility. We have previously shown that moderate prenatal alcohol exposure (PAE) significantly impairs reversal learning, a measure of flexibility mediated across species by different brain areas that include the orbital frontal cortex (OFC). Reversal learning is likewise impaired by genetic or pharmacological inactivation of GluN2B subunit-containing N-methyl-D-aspartate receptors (NMDARs). In the current study, we tested the hypothesis that moderate PAE persistently alters the number and function of GluN2B subunit-containing NMDARs in OFC pyramidal neurons of adult mice. METHODS: We used a rodent model of fetal alcohol spectrum disorders and left offspring undisturbed until adulthood. Using whole-cell, patch-clamp recordings, we assessed NMDAR function in slices from 90- to 100-day-old male and female PAE and control mice. Pharmacologically isolated NMDA receptor-mediated evoked excitatory postsynaptic currents (NMDA-eEPSCs) were recorded in the absence and presence of the GluN2B antagonist, Ro25-6981(1 µM). In a subset of littermates, we evaluated the level of GluN2B protein expression in the synaptic fraction using Western blotting technique. RESULTS: Our results indicate that PAE females show significantly larger (~23%) NMDA-eEPSC amplitudes than controls, while PAE induced a significant decrease (~17%) in NMDA-eEPSC current density of pyramidal neurons recorded in slices from male mice. NMDA-eEPSC decay time was not affected in PAE-exposed mice from either sex. The contribution of GluN2B subunit-containing NMDARs to the eEPSCs was not significantly altered by PAE. Moreover, there were no significant changes in protein expression in the synaptic fraction of either PAE males or females. CONCLUSIONS: These findings suggest that low-to-moderate PAE modulates NMDAR function in pyramidal neurons in a sex-specific manner, although we did not find evidence that the effect is mediated by dysfunction of synaptic GluN2B subunit-containing NMDARs.

Enhancement of parvalbumin interneuron-mediated neurotransmission in the retrosplenial cortex of adolescent mice following third trimester-equivalent ethanol exposure.

Prenatal ethanol exposure causes a variety of cognitive deficits that have a persistent impact on quality of life, some of which may be explained by ethanol-induced alterations in interneuron function. Studies from several laboratories, including our own, have demonstrated that a single binge-like ethanol exposure during the equivalent to the third trimester of human pregnancy leads to acute apoptosis and long-term loss of interneurons in the rodent retrosplenial cortex (RSC). The RSC is interconnected with the hippocampus, thalamus, and other neocortical regions and plays distinct roles in visuospatial processing and storage, as well as retrieval of hippocampal-dependent episodic memories. Here we used slice electrophysiology to characterize the acute effects of ethanol on GABAergic neurotransmission in the RSC of neonatal mice, as well as the long-term effects of neonatal ethanol exposure on parvalbumin-interneuron mediated neurotransmission in adolescent mice. Mice were exposed to ethanol using vapor inhalation chambers. In postnatal day (P) 7 mouse pups, ethanol unexpectedly failed to potentiate GABAA receptor-mediated synaptic transmission. Binge-like ethanol exposure of P7 mice expressing channel rhodopsin in parvalbumin-positive interneurons enhanced the peak amplitudes, asynchronous activity and total charge, while decreasing the rise-times of optically-evoked GABAA receptor-mediated inhibitory postsynaptic currents in adolescent animals. These effects could partially explain the learning and memory deficits that have been documented in adolescent and young adult mice exposed to ethanol during the third trimester-equivalent developmental period.

Characterization of motor function in mice developmentally exposed to ethanol using the Catwalk system: Comparison with the triple horizontal bar and rotarod tests.

Studies with human subjects indicate that ethanol exposure during fetal development causes long-lasting alterations in motor coordination that are, in part, a consequence of cerebellar damage. Studies with rats exposed to ethanol during the neonatal brain growth spurt have consistently recapitulated these deficits. However, studies with mice have yielded mixed results. We hypothesized that the use of highly sensitive motor function tests, such as the Catwalk test, would reliably detect motor function deficits in mice developmentally exposed to ethanol. Venus-vesicular GABA transporter transgenic mice were ethanol exposed during postnatal days 4-9 using vapor inhalation chambers and then subjected to the Catwalk test during adolescence. Catwalk data were rigorously analyzed using an innovative multistep statistical approach. For comparison, motor coordination and strength were assessed with the triple horizontal bar and rotarod tests. Unexpectedly, we found that out of 186 parameters analyzed in the Catwalk test, only one was affected by ethanol exposure (i.e., reduced coupling between left front paw and the right hind paw). In the triple horizontal bar test, ethanol-exposed mice were able to hold to the bars for less time than controls. Surprisingly, ethanol-exposed mice performed better in the rotarod test than controls. These data indicate that neonatal ethanol exposure of mice causes mixed effects on motor function during adolescence. The Catwalk test suggests that gait is generally preserved in these mice, whereas the triple horizontal bar test revealed deficits on motor strength and the rotarod test an increase in motor coordination.

Moderate prenatal alcohol exposure alters the number and function of GABAergic interneurons in the murine orbitofrontal cortex.

Exposure to alcohol during development produces Fetal Alcohol Spectrum Disorders (FASD), characterized by a wide range of effects that include deficits in multiple cognitive domains. Early identification and treatment of individuals with FASD remain a challenge because neurobehavioral alterations do not become a significant problem until late childhood and early adolescence. Understanding the mechanisms underlying low and moderate prenatal alcohol exposure (PAE) effects on behavior and cognition is essential for improved diagnosis and treatment. Here, we examined the functional and morphological changes in an area known to be involved in executive control, the orbitofrontal cortex (OFC). We found that a moderate PAE model, previously shown to impair behavioral flexibility and to alter OFC activity in vivo, produced moderate functional and morphological changes within the OFC of mice in vitro. Specifically, slice electrophysiological recordings of spontaneous inhibitory post-synaptic currents in OFC pyramidal neurons revealed a significant increase in the amplitude and area in PAE mice relative to controls. Immunohistochemistry uncovered an increase in calretinin-, but not somatostatin- or parvalbumin-expressing cortical interneurons in the OFC of PAE mice. Together, these data suggest that moderate prenatal alcohol exposure alters the disinhibitory function in the OFC, which may contribute to the executive function deficits associated with FASD.

The mouse-equivalent of the human BDNF VAL66MET polymorphism increases dorsal hippocampal volume and does not interact with developmental ethanol exposure.

A relatively common polymorphism in the human brain-derived neurotrophic factor (BDNF) gene (Val66Met, which corresponds to Val68Met in mice) has been shown to modulate cognitive function and vulnerability to mental health disorders. This substitution impairs trafficking and activity-dependent release of BDNF. A number of studies with both humans and transgenic mice suggest that carriers of the Met allele have deficits in the structure and/or function of the hippocampal formation. Using a relatively new transgenic mouse model of this polymorphism, we recently demonstrated that it modulates the effects of developmental ethanol exposure in the hippocampus. Here, we further characterized the effect of this polymorphism on hippocampal morphology and its interaction with ethanol vapor exposure during the 2nd and 3rd trimester equivalents of human pregnancy. We found that BDNFmet/met mice have slightly larger hippocampal volumes than BDNFval/val mice. Ethanol vapor exposure during the 2nd and 3rd trimester equivalents of human pregnancy increased hippocampal volume in a single hippocampal subregion, the CA1 stratum radiatum. Ethanol exposure did not interact with BDNF genotype to affect volume in any hippocampal subregion. These results suggest that the Val66Met polymorphism does not reduce hippocampal size (i.e., it rather increases it slightly) or increase susceptibility to prenatal ethanol exposure-induced structural hippocampal damage during adulthood.

Neonatal ethanol exposure triggers apoptosis in the murine retrosplenial cortex: Role of inhibition of NMDA receptor-driven action potential firing.

Exposure to ethanol during the last trimester equivalent of human pregnancy causes apoptotic neurodegeneration in the developing brain, an effect that is thought to be mediated, in part, by inhibition of NMDA receptors. However, NMDA receptors can rapidly adapt to the acute effects of ethanol and are ethanol resistant in some populations of developing neurons. Here, we characterized the effect of ethanol on NMDA and non-NMDA receptor-mediated synaptic transmission in the retrosplenial cortex (RSC), a brain region involved in the integration of different modalities of spatial information that is among the most sensitive regions to ethanol-induced neurodegeneration. A single 4-h exposure to ethanol vapor of 7-day-old transgenic mice that express the Venus fluorescent protein in interneurons triggered extensive apoptosis in the RSC. Slice electrophysiological recordings showed that bath-applied ethanol inhibits NMDA and non-NMDA receptor excitatory postsynaptic currents (EPSCs) in pyramidal neurons and interneurons; however, we found no evidence of acute tolerance development to this effect after the 4-h in-vivo ethanol vapor exposure. Acute bath application of ethanol reduced action potential firing evoked by synaptic stimulation to a greater extent in pyramidal neurons than interneurons. Submaximal inhibition of NMDA EPSCs, but not non-NMDA EPSCs, mimicked the acute effect of ethanol on synaptically-evoked action potential firing. These findings indicate that partial inhibition of NMDA receptors by ethanol has sizable effects on the excitability of glutamatergic and GABAergic neurons in the developing RSC, and suggest that positive allosteric modulators of these receptors could ameliorate ethanol intoxication-induced neurodegeneration during late stages of fetal development.

Long-term Reductions in the Population of GABAergic Interneurons in the Mouse Hippocampus following Developmental Ethanol Exposure.

Developmental exposure to ethanol leads to a constellation of cognitive and behavioral abnormalities known as Fetal Alcohol Spectrum Disorders (FASDs). Many cell types throughout the central nervous system are negatively impacted by gestational alcohol exposure, including inhibitory, GABAergic interneurons. Little evidence exists, however, describing the long-term impact of fetal alcohol exposure on survival of interneurons within the hippocampal formation, which is critical for learning and memory processes that are impaired in individuals with FASDs. Mice expressing Venus yellow fluorescent protein in inhibitory interneurons were exposed to vaporized ethanol during the third trimester equivalent of human gestation (postnatal days 2-9), and the long-term effects on interneuron numbers were measured using unbiased stereology at P90. In adulthood, interneuron populations were reduced in every hippocampal region examined. Moreover, we found that a single exposure to ethanol at P7 caused robust activation of apoptotic neurodegeneration of interneurons in the hilus, granule cell layer, CA1 and CA3 regions of the hippocampus. These studies demonstrate that developmental ethanol exposure has a long-term impact on hippocampal interneuron survivability, and may provide a mechanism partially explaining deficits in hippocampal function and hippocampus-dependent behaviors in those afflicted with FASDs.

Prenatal Alcohol Exposure Increases Histamine H3 Receptor-Mediated Inhibition of Glutamatergic Neurotransmission in Rat Dentate Gyrus.

BACKGROUND: We have reported that prenatal alcohol exposure (PAE)-induced deficits in dentate gyrus, long-term potentiation (LTP), and memory are ameliorated by the histamine H3 receptor inverse agonist ABT-239. Curiously, ABT-239 did not enhance LTP or memory in control offspring. Here, we initiated an investigation of how PAE alters histaminergic neurotransmission in the dentate gyrus and other brain regions employing combined radiohistochemical and electrophysiological approaches in vitro to examine histamine H3 receptor number and function. METHODS: Long-Evans rat dams voluntarily consumed either a 0% or 5% ethanol solution 4 hours each day throughout gestation. This pattern of drinking, which produces a mean peak maternal serum ethanol concentration of 60.8 ± 5.8 mg/dl, did not affect maternal weight gain, litter size, or offspring birthweight. RESULTS: Radiohistochemical studies in adult offspring revealed that specific [3 H]-A349821 binding to histamine H3 receptors was not different in PAE rats compared to controls. However, H3 receptor-mediated Gi /Go protein-effector coupling, as measured by methimepip-stimulated [35 S]-GTPγS binding, was significantly increased in cerebral cortex, cerebellum, and dentate gyrus of PAE rats compared to control. A LIGAND analysis of detailed methimepip concentration-response curves in dentate gyrus indicated that PAE significantly elevates receptor-effector coupling by a lower affinity H3 receptor population without significantly altering the affinities of H3 receptor subpopulations. In agreement with the [35 S]-GTPγS studies, a similar range of methimepip concentrations also inhibited electrically evoked field excitatory postsynaptic potential responses and increased paired-pulse ratio, a measure of decreased glutamate release, to a significantly greater extent in dentate gyrus slices from PAE rats than in controls. CONCLUSIONS: These results suggest that a PAE-induced elevation in H3 receptor-mediated inhibition of glutamate release from perforant path terminals as 1 mechanism contributing the LTP deficits previously observed in the dentate gyrus of PAE rats, as well as providing a mechanistic basis for the efficacy of H3 receptor inverse agonists for ameliorating these deficits.

Prenatal Alcohol Exposure Leads to Enhanced Serine 9 Phosphorylation of Glycogen Synthase Kinase-3ß (GSK-3ß) in the Hippocampal Dentate Gyrus of Adult Mouse.

BACKGROUND: The goal of this study was to evaluate the expression and serine 9 phosphorylation of glycogen synthase kinase (GSK-3ß) within the adult hippocampal dentate gyrus (DG) in a preclinical mouse model of fetal alcohol spectrum disorders. GSK-3ß is a multifunctional kinase that modulates many hippocampal processes affected by gestational alcohol, including synaptic plasticity and adult neurogenesis. GSK-3ß is a constitutively active kinase that is negatively regulated by phosphorylation at the serine 9 residue. METHODS: We utilized a well-characterized limited access "drinking-in-the-dark" paradigm of prenatal alcohol exposure (PAE) and measured p(Ser9)GSK-3ß and total GSK-3ß within adult DG by Western blot analysis. In addition, we evaluated the expression pattern of both p(Ser9)GSK-3ß and total GSK-3ß within the adult hippocampal dentate of PAE and control mice using high-resolution confocal microscopy. RESULTS: Our findings demonstrate a marked 2.0-fold elevation of p(Ser9)GSK-3ß in PAE mice, concomitant with a more moderate 36% increase in total GSK-3ß. This resulted in an approximate 63% increase in the p(Ser9)GSK-3ß/GSK-3ß ratio. Immunostaining revealed robust GSK-3ß expression within Cornu Ammonis (CA) pyramidal neurons, hilar mossy cells, and a subset of GABAergic interneurons, with low levels of expression within hippocampal progenitors and dentate granule cells. CONCLUSIONS: These findings suggest that PAE may lead to a long-term disruption of GSK-3ß signaling within the DG, and implicate mossy cells, GABAergic interneurons, and CA primary neurons as major targets of this dysregulation.

Ethanol exposure during development reduces GABAergic/glycinergic neuron numbers and lobule volumes in the mouse cerebellar vermis.

Cerebellar alterations are a hallmark of Fetal Alcohol Spectrum Disorders and are thought to be responsible for deficits in fine motor control, motor learning, balance, and higher cognitive functions. These deficits are, in part, a consequence of dysfunction of cerebellar circuits. Although the effect of developmental ethanol exposure on Purkinje and granule cells has been previously characterized, its actions on other cerebellar neuronal populations are not fully understood. Here, we assessed the impact of repeated ethanol exposure on the number of inhibitory neurons in the cerebellar vermis. We exposed pregnant mice to ethanol in vapor inhalation chambers during gestational days 12-19 and offspring during postnatal days 2-9. We used transgenic mice expressing the fluorescent protein, Venus, in GABAergic/glycinergic neurons. Using unbiased stereology techniques, we detected a reduction in Venus positive neurons in the molecular and granule cell layers of lobule II in the ethanol exposed group at postnatal day 16. In contrast, ethanol produced a more widespread reduction in Purkinje cell numbers that involved lobules II, IV-V and IX. We also found a reduction in the volume of lobules II, IV-V, VI-VII, IX and X in ethanol-exposed pups. These findings indicate that second and third trimester-equivalent ethanol exposure has a greater impact on Purkinje cells than interneurons in the developing cerebellar vermis. The decrease in the volume of most lobules could be a consequence of a reduction in cell numbers, dendritic arborizations, or axonal projections.

Third Trimester Equivalent Alcohol Exposure Reduces Modulation of Glutamatergic Synaptic Transmission by 5-HT1A Receptors in the Rat Hippocampal CA3 Region.

Fetal alcohol exposure has been associated with many neuropsychiatric disorders that have been linked to altered serotonin (5-hydroxytryptamine; 5-HT) signaling, including depression and anxiety. During the first 2 weeks of postnatal life in rodents (equivalent to the third trimester of human pregnancy) 5-HT neurons undergo significant functional maturation and their axons reach target regions in the forebrain (e.g., cortex and hippocampus). The objective of this study was to identify the effects of third trimester ethanol (EtOH) exposure on hippocampal 5-HT signaling. Using EtOH vapor inhalation chambers, we exposed rat pups to EtOH for 4 h/day from postnatal day (P) 2 to P12. The average serum EtOH concentration in the pups was 0.13 ± 0.04 g/dl (legal intoxication limit in humans = 0.08 g/dl). We used brain slices to assess the modulatory actions of 5-HT on field excitatory postsynaptic potentials in the hippocampal CA3 region at P13-P15. Application of the GABAA/glycine receptor antagonist, picrotoxin, caused broadening of field excitatory postsynaptic potentials (fEPSPs), an effect that was reversed by application of 5-HT in slices from air exposed rats. However, this effect of 5-HT was absent in EtOH exposed animals. In slices from naïve animals, application of a 5-HT1A receptor antagonist blocked the effect of 5-HT on the fEPSPs recorded in presence of picrotoxin, suggesting that third trimester ethanol exposure acts by inhibiting the function of these receptors. Studies indicate that 5-HT1A receptors play a critical role in the development of hippocampal circuits. Therefore, inhibition of these receptors by third trimester ethanol exposure could contribute to the pathophysiology of fetal alcohol spectrum disorders.

Prenatal alcohol exposure alters synaptic activity of adult hippocampal dentate granule cells under conditions of enriched environment.

Prenatal alcohol exposure (PAE) results in fetal alcohol spectrum disorder (FASD), which is characterized by a wide range of cognitive and behavioral deficits that may be linked to impaired hippocampal function and adult neurogenesis. Preclinical studies in mouse models of FASD indicate that PAE markedly attenuates enrichment-mediated increases in the number of adult-generated hippocampal dentate granule cells (aDGCs), but whether synaptic activity is also affected has not been studied. Here, we utilized retroviral birth-dating coupled with whole cell patch electrophysiological recordings to assess the effects of PAE on enrichment-mediated changes in excitatory and inhibitory synaptic activity as a function of DGC age. We found that exposure to an enriched environment (EE) had no effect on baseline synaptic activity of 4- or 8-week-old aDGCs from control mice, but significantly enhanced the excitatory/inhibitory ratio of synaptic activity in 8-week-old aDGCs from PAE mice. In contrast, exposure to EE significantly enhanced the excitatory/inhibitory ratio of synaptic activity in older pre-existing DGCs situated in the outer dentate granule cell layer (i.e., those generated during embryonic development; dDGCs) in control mice, an effect that was blunted in PAE mice. These findings indicate distinct electrophysiological responses of hippocampal DGCs to behavioral challenge based on cellular ontogenetic age, and suggest that PAE disrupts EE-mediated changes in overall hippocampal network activity. These findings may have implications for future therapeutic targeting of hippocampal dentate circuitry in clinical FASD. © 2016 Wiley Periodicals, Inc.