Fluoxetine disrupts motivation and GABAergic signaling in adolescent female hamsters.

Initial antidepressant treatment can paradoxically worsen symptoms in depressed adolescents by undetermined mechanisms. Interestingly, antidepressants modulate GABAA receptors, which mediate paradoxical effects of other therapeutic drugs, particularly in females. Although the neuroanatomic site of action for this paradox is unknown, elevated GABAA receptor signaling in the nucleus accumbens can disrupt motivation. We assessed fluoxetine's effects on motivated behaviors in pubescent female hamsters - anhedonia in the reward investigational preference (RIP) test as well as anxiety in the anxiety-related feeding/exploration conflict (AFEC) test. We also assessed accumbal signaling by RT-PCR and electrophysiology. Fluoxetine initially worsened motivated behaviors at puberty, relative to adulthood. It also failed to improve these behaviors as pubescent hamsters transitioned into adulthood. Low accumbal mRNA levels of multiple GABAA receptor subunits and GABA-synthesizing enzyme, GAD67, assessed by RT-PCR, suggested low GABAergic tone at puberty. Nonetheless, rapid fluoxetine-induced reductions of α5GABAA receptor and BDNF mRNA levels at puberty were consistent with age-related differences in GABAergic responses to fluoxetine and disruption of the motivational state. Whole-cell patch clamping of accumbal slices also suggested low GABAergic tone by the low amplitude of miniature inhibitory postsynaptic currents (mIPSCs) at puberty. It also confirmed age-related differences in GABAergic responses to fluoxetine. Specifically, fluoxetine potentiated mIPSC amplitude and frequency at puberty, but attenuated the amplitude during adulthood. These results implicate GABAergic tone and GABAA receptor plasticity in adverse motivational responses and resistance to fluoxetine during adolescence.

Long-lasting distortion of GABA signaling in MS/DB neurons after binge-like ethanol exposure during initial synaptogenesis.

Using a well-established model of binge-like ethanol treatment of rat pups on postnatal days (PD) 4-9, we found that maturation of GABAA receptor (GABAAR) miniature postsynaptic currents (mPSCs) was substantially blunted for medial septum/diagonal band (MS/DB) neurons in brain slices on PD 11-16. Ethanol reduced mPSC amplitude, frequency, and decay kinetics, while attenuating or exaggerating allosteric actions of zolpidem and allopregnanolone, respectively. The impact of ethanol in vivo was long lasting as most changes in MS/DB GABAAR mPSCs were still observed as late as PD 60-85. Maturing MS/DB neurons in naïve brain slices PD 4-16 showed increasing mPSC frequency, decay kinetics, and zolpidem sensitivity that were nearly identical to our earlier findings in cultured septal neurons (DuBois et al., 2004, 2006). These rapidly developing mPSC parameters continued to mature through the first month of life then stabilized throughout the remainder of the lifespan. Finally, equivalent ethanol-induced alterations in GABAAR mPSC signaling were present in MS/DB neurons from both male and female animals. Previously, we showed ethanol treatment of cultured embryonic day 20 septal neurons distorts the maturation of GABAAR mPSCs predicting that early stages of GABAergic transmission in MS/DB neurons are vulnerable to intoxication injury (DuBois et al., 2004, 2006). Since the overall character, timing, and magnitude of GABAergic mPSC developmental- and ethanol-induced changes in the in vivo model so closely mirror chronologically equivalent adaptations in cultured septal neurons, this suggests that such parallel models of ethanol impairment of GABAergic synaptic development in vivo and in vitro should be useful for translational studies exploring the efficacy and mechanism of action of potential therapeutic interventions from the cellular to whole animal level.

Varenicline and nicotine enhance GABAergic synaptic transmission in rat CA1 hippocampal and medial septum/diagonal band neurons.

AIMS: The FDA approved smoking cessation aid varenicline can effectively attenuate nicotine-stimulated dopamine release. Varenicline may also exert important actions on other transmitter systems that also influence nicotine reinforcement or contribute to the drug's cognitive and affective side effects. In this study, we determined if varenicline, like nicotine, can stimulate presynaptic GABA release. MAIN METHODS: Using whole-cell patch-clamp techniques, we measured GABA(A)R-mediated asynchronous, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in acute brain slices from two brain regions important for learning and memory, the hippocampus and basal forebrain. KEY FINDINGS: Both varenicline (10 µM) and nicotine (10 µM) applications alone resulted in small but significant increases in amplitude, as well as robustly enhanced frequency of mIPSCs in hippocampal CA1 pyramidal neurons and medial septum/diagonal band (MS/DB) neurons. A unique subpopulation of MS/DB neurons showed decreases in frequency. In the presence of nicotine, varenicline effectively attenuated the expected enhancement of hippocampal mIPSC frequency like a competitive antagonist. However, in the MS/DB, varenicline only partially attenuated nicotine's effects. Reversing the order of drug application by adding nicotine to varenicline-exposed slices had little effect. SIGNIFICANCE: Varenicline, like nicotine, stimulates presynaptic GABA release, and also exerts a partial agonist action by attenuating nicotine-stimulated release in both the hippocampus and basal forebrain. These effects could potentially affect cognitive functions.

Altered spatial learning and delay discounting in a rat model of human third trimester binge ethanol exposure.

Ethanol exposure during perinatal development can cause cognitive abnormalities including difficulties in learning, attention, and memory, as well as heightened impulsivity. The purpose of this study was to assess performance in spatial learning and impulsive choice tasks in rats subjected to an intragastric intubation model of binge ethanol exposure during human third trimester-equivalent brain development. Male and female Sprague-Dawley rat pups were intubated with ethanol (5.25 g/kg/day) on postnatal days 4-9. At adolescence (between postnatal days 35-38), these rats and sham intubated within-litter controls were trained in both spatial and cued versions of the Morris water maze. A subset of the male rats was subsequently tested on a delay-discounting task to assess impulsive choice. Ethanol-exposed rats were spatially impaired relative to controls, but performed comparably to controls on the cued version of the water maze. Ethanol-exposed rats also showed greater preference for large delayed rewards on the delay discounting task, but no evidence for altered reward sensitivity or perseverative behavior. These data demonstrate that early postnatal intermittent binge-like ethanol exposure has prolonged, detrimental, but selective effects on cognition, suggesting that even relatively brief ethanol exposure late in human pregnancy can be deleterious for cognitive function.

GABAergic miniature postsynaptic currents in septal neurons show differential allosteric sensitivity after binge-like ethanol exposure.

Binge-like ethanol treatment of septal neurons blunts GABAAR-mediated miniature postsynaptic currents (mPSCs), suggesting it arrests synaptic development. Ethanol may disrupt postsynaptic maturation by blunting feedback signaling through immature GABAARs. Here, the impact of ethanol on the sensitivity of mPSCs to zolpidem, zinc and 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha-OH-DHP) was tested. The decay phase of mPSCs showed concentration-dependent potentiation by zolpidem (0.03-100 microM), which was substantially blunted after ethanol exposure. Since zolpidem potentiation exhibited a substantial age-dependent increase in untreated neurons, this finding supported the idea that ethanol arrests synaptic development. GABAAR alpha1 subunit protein also increased with age in untreated neurons, paralleling enhanced sensitivity to zolpidem. Surprisingly, alpha1 levels were not reduced by binge ethanol even though mPSCs were relatively zolpidem-insensitive. Zinc (3-30 microM) decreased mPSC parameters in a concentration- and age-related manner with older untreated cells showing less inhibition. However, there was no increase in mPSC zinc sensitivity after binge ethanol as would be expected if a general arrest of synaptic maturation had occurred. 3alpha-OH-DHP (3-1000 nM) induced concentration-dependent potentiation of mPSC decay. Although potentiation was age-independent, binge ethanol treatment exaggerated sensitivity to this neurosteroid. Finally, chronic picrotoxin pretreatment (100 microM) intended to mimic GABAAR inhibition from ethanol pretreatment did not significantly change mPSC modulation by zolpidem, zinc or 3alpha-OH-DHP. These results suggest that binge ethanol treatment selectively arrests a subset of processes important for maturation of postsynaptic GABAA Rs. However, it is unlikely that ethanol causes a broad arrest of postsynaptic development through a direct inhibition of GABAAR signaling.

Chronic nicotine induces growth retardation in neonatal rat pups.

In the United State, 20% of pregnant women smoke. One of the most consistent adverse outcomes is reduced birth weight in the off-spring. Animal studies using chronic nicotine, the major psychoactive tobacco ingredient, have shown conflicting results, questioning the role of nicotine in growth retardation. To evaluate the direct effects of nicotine during a period equivalent to the human third trimester, we developed an oral gastric intubation model using neonatal rat pups. Nicotine (6 mg/kg/day) was dissolve in milk-formula and delivered during three feedings daily from postnatal day (P)1 to P7. Nicotine immediately and significantly [P<0.05] decreased weight gain per day (WGD) by 13.5% (+/-) 1 day after onset of treatment in both genders and throughout the treatment period. This resulted in significantly lower body weight at P4 and P5 in male and female pups, respectively. After nicotine withdrawal, WGD returned to control level within 1 day, whereas total body weight recovered by P18. There were no long-term consequences on body weight or growth pattern in either gender. The nicotinic acetylcholine receptor (nAChR) antagonist dihydro-beta-erythroidine (DHbetaE) reversed nicotine's effects on WGD suggesting an involvement of heteromeric alpha4beta2, whereas methyllycaconitine (MLA) an antagonist for the homomeric alpha7-type receptor was ineffective. The immediate decrease of growth in neonatal pups suggests that nicotine's effect on birth weight results from direct anorexic rather then indirect effects due to placental dysfunction or increased fetal hypoxia. The postnatal oral gastric intubation model seems to accurately reflect the direct effects of nicotine in neonates.

Binge ethanol exposure delays development of GABAergic miniature postsynaptic currents in septal neurons.

Whole cell GABA(A)R currents of septal neurons isolated from rat pups increase rapidly during the first weeks of life when inhibitory synapses are forming. Early postnatal binge ethanol intubation on days 4-9 delays this maturational up-regulation in septal neurons isolated several days later suggesting inhibitory synapse formation could be disrupted [S.-H. Hsiao, J.L. Acevedo, D.W. DuBois, K.R. Smith, J.R. West, G.D. Frye, Early postnatal ethanol intubation blunts GABA(A) receptor upregulation and modifies 3alpha-hydroxy-5alpha-pregnan-20-one sensitivity in rat MS/DB neurons, Brain Res. Dev. Brain Res. 130 (2001) 25-40]. Surprisingly, whole cell GABA(A)R function does not increase rapidly when septal neurons are grown for the same period in vitro and is not blunted by comparable ethanol exposure of the cultures [S.-H. Hsiao, D.W. DuBois, R.C. Miranda, G.D. Frye, Critically timed ethanol exposure reduces GABA(A)R function on septal neurons developing in vivo but not in vitro, Brain Res Dev. Brain Res. 1008 (2004) 69-80]. Because GABAergic miniature postsynaptic currents (mPSCs) show parallel patterns of maturation whether cortical neurons are growing in vivo or in vitro [D.D. Dunning, C.L. Hoover, I. Soltesz, M.A. Smith, D.K. ODowd, GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons, J. Neurophysiol. 82 (1999) 3286-3297], we examined the impact of binge ethanol exposure on synaptic receptors activated by these currents in septal cultures. Binge ethanol treatment of embryonic septal neurons over 6-11 days in vitro (DIV) slightly reduced GABA(A)R-mediated mPSC amplitude and frequency, but also substantially slowed decay kinetics when mPSCs were recorded later on DIV 13-18. Decreased frequency and slowed mPSC decay kinetics after ethanol were consistent with parameters measured in immature neurons. Untreated septal neurons exhibited decreased mPSC amplitude and frequency with acute 30-100 mM ethanol, without changing decay kinetics suggesting a direct inhibition of postsynaptic receptors. Sustained inhibition of GABA(A)Rs with 100 microM picrotoxin on DIV 6-11 decreased mPSC amplitude and frequency and slowed decay kinetics similar to binge ethanol exposure. These results suggest that binge ethanol exposure delays mPSC maturation by interfering with trophic postsynaptic GABA(A)R signaling during the early development of septal neurons.

Critically timed ethanol exposure reduces GABAAR function on septal neurons developing in vivo but not in vitro.

Six-day 'binge' ethanol intoxication postnatal days (PD) 4-9 delays up-regulation of gamma-aminobutyric acid type A receptors (GABAARs) in developing rat septal neurons [Dev. Brain Res. 130 (2001) 25]. This distortion occurs during synaptogenesis and could contribute to cognitive dysfunction in fetal alcohol syndrome (FAS). Here, we asked two questions concerning requirements for vulnerability to GABAAR blunting by ethanol. First, we asked whether receptor blunting required PD 4-9 ethanol exposure in rat pups and found that just a brief 2-day exposure (PD 8-9) was as effective as all 6 days. However, 2-day exposure on PD 4-5 was ineffective, showing that 'binge' timing was important. We also asked whether 'binge' exposure directly inhibited intrinsic processes of septal neurons and could blunt GABAARs on cells maturing outside the brain. Embryonic septal neurons grown in serum-free dispersed culture developed extensive dendritic arborizations, spontaneous synaptic activity and robust whole-cell GABAAR function, but surprisingly, did not show developmental up-regulation of GABAARs like septal neurons maturing in vivo [Brain Res. 810 (1998) 100]. Furthermore, age-matched 6-day 'binge' ethanol exposure did not blunt GABAAR function in septal neurons in vitro. These results suggest developmental mechanisms driving up-regulation of GABAAR function in septal neurons in vivo briefly becomes vulnerable to ethanol insult in early postnatal life. While septal neurons express comparable functional GABAARs whether maturing in vivo or in vitro, vulnerability to ethanol-induced receptor blunting requires elements of an intact brain environment not replicated in culture.

AMPA receptors on developing medial septum/diagonal band neurons are sensitive to early postnatal binge-like ethanol exposure.

The impact of binge-like, early postnatal ethanol treatment on AMPA or kainate whole cell currents was examined in acutely isolated medial septum/diagonal band (MS/DB) neurons. AMPA (10 or 100 microM) current was inhibited by GYKI 52466, a selective AMPA receptor (AMPAR) antagonist, in all neurons isolated on postnatal day (PD) 5-8, PD 12-15 or PD 32-35. Cyclothiazide, a selective inhibitor of AMPAR desensitization, also effectively potentiated AMPA currents. This suggests that non-NMDA, ionotropic glutamate receptors on immature MS/DB neuron are predominantly AMPARs. Concentration-dependent kainate (10-1000 microM) application evoked nondesensitizing currents that exhibited an increase in the maximum response by the end of first postnatal month, consistent with developmental regulation of AMPAR function. Acute 3 s ethanol application (100 mM) consistently blunted AMPA- and kainate currents approximately 20-30% across age groups. Inhibition was sustained during continuous ethanol superfusion lasting 10-12 min without evidence of acute tolerance. Repeated oral intubation of rat pups with ethanol (5.25 g/kg/day on PD 4-9), which models third trimester human binge drinking, resulted in peak blood ethanol levels of approximately 350 mg/dl (measured 90 min after PD 6 dosing). AMPA or kainate currents were upregulated in neurons isolated on PD 32-35 by earlier ethanol intubation suggesting that binge-like intoxication augments developing AMPAR function. Despite this augmentation of AMPAR function, no significant changes were found in the sensitivity of AMPA currents to GYKI 52466, cyclothiazide or acute ethanol (100 mM) sensitivity or in the levels of GluR1/GluR2 subunit proteins from MS/DB tissue. These results indicate that non-NMDA ionotrophic glutamate receptors on immature MS/DB neurons, which are largely of the AMPAR subtype, are moderately sensitive to immediate inhibition by ethanol. Repeating this inhibition during early postnatal binge-like intoxication can augment normal development of AMPAR function.

Effects of chronic ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons.

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor's response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanol's anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

Effects of early postnatal ethanol intubation on GABAergic synaptic proteins.

Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell-cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABA(A) receptor (GABA(A)R) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4-9 acutely inhibits whole cell GABA(A)R Cl(-) current and subsequently blunts GABA(A)R function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4-9 producing peak blood ethanol concentrations in the range of 302.5+/-6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13-14 when GABA(A)R currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. Surprisingly, ethanol did not decrease protein levels of GABA(A)R alpha1/beta2 subunits, GAD(67) or gephyrin in MS/DB at this time when whole cell recordings indicate GABA(A)R function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABA(A)R function remains normal on PDs 13-14 [Brain Res. 832 (1999) 124-135], reduced levels of several GABAergic synaptic proteins including: GAD(67), GABA(A)R alpha1 subunit, ClC-2 a voltage-gated Cl(-) channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABA(A)R function of individual neurons that survive this ethanol insult.