Maintenance of optogenetic channel rhodopsin (ChR2) function in aging mice: Implications for pharmacological studies of inhibitory synaptic transmission, quantal content, and calcium homeostasis.

Disruption of synaptic function is believed to represent a common pathway contributing to cognitive decline during aging. Optogenetics is a prodigious tool for studying relationships between function and synaptic circuitry but models utilizing viral vectors present limitations. Careful characterization of the functionality of channel rhodopsin in transgenic models is crucial for determining whether they can be used across aging. This includes verifying the light sensitivity of the protein and confirming its ability to generate action potentials in response to light stimulation. We combined in vitro optogenetic methodology and a reduced synaptic preparation of acutely isolated neurons to determine if the ChR2(H134R)-eYFP vGAT mouse model is well-suited for aging studies. We used neurons from young (2-6 mo), middle-aged (10-14 mo) and aged (17-25 mo) bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R in GABAergic cell populations. Cellular physiology and calcium dynamics were assessed in basal forebrain (BF) neurons using patch-clamp recording and fura-2 microfluorimetry, alongside 470 nm light stimulation of the transgenic ChR2 channel to characterize a wide array of physiological functions known to decline with age. We found ChR2 expression is functionally maintained across aging, while spontaneous and optically evoked inhibitory postsynaptic currents, and quantal content were decreased. Aged mice also showed an increase in intracellular calcium buffering. These results, which are on par with previous observations, demonstrate that the optogenetic vGAT BAC mouse model is well-suited for investigating age-related changes in calcium signaling and synaptic transmission.

Effects of ethanol and varenicline on female Sprague-Dawley rats in a third trimester model of fetal alcohol syndrome.

Perinatal ethanol exposure disrupts a variety of developmental processes in neurons important for establishing a healthy brain. These ethanol-induced impairments known as fetal alcohol spectrum disorder (FASD) are not fully understood, and currently, there is no effective treatment. Further, growing evidence suggests that adult females are more susceptible to ethanol, with the effects of perinatal ethanol exposure also being sexually divergent. Female models have been historically underutilized in neurophysiological investigations, but here, we used a third-trimester binge-ethanol model of FASD to examine changes to basal forebrain (BF) physiology and behavior in female Sprague-Dawley rats. We also tested varenicline as a potential cholinomimetic therapeutic. Rat pups were gavage-treated with binge-like ethanol, varenicline and ethanol, and varenicline alone. Using patch-clamp electrophysiology in BF slices, we observed that binge-ethanol exposure increased spontaneous post-synaptic current (sPSC) frequency. Varenicline exposure alone also enhanced sPSC frequency. Varenicline plus ethanol co-treatment prevented the sPSC frequency increase. Changes in BF synaptic transmission persisted into adolescence after binge-ethanol treatment. Behaviorally, binge-ethanol treated females displayed increased anxiety (thigmotaxis) and demonstrated learning deficits in the water maze. Varenicline/ethanol co-treatment was effective at reducing these behavioral deficits. In the open field, ethanol-treated rats displayed longer distances traveled and spent less time in the center of the open field box. Co-treated rats displayed less anxiety, demonstrating a possible effect of varenicline on this measure. In conclusion, ethanol-induced changes in both BF synaptic transmission and behavior were reduced by varenicline in female rats, supporting a role for cholinergic therapeutics in FASD treatment.

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.

Characterization of age-related changes in synaptic transmission onto F344 rat basal forebrain cholinergic neurons using a reduced synaptic preparation.

Basal forebrain (BF) cholinergic neurons participate in a number of cognitive processes that become impaired during aging. We previously found that age-related enhancement of Ca(2+) buffering in rat cholinergic BF neurons was associated with impaired performance in the water maze spatial learning task (Murchison D, McDermott AN, Lasarge CL, Peebles KA, Bizon JL, and Griffith WH. J Neurophysiol 102: 2194-2207, 2009). One way that altered Ca(2+) buffering could contribute to cognitive impairment involves synaptic function. In this report we show that synaptic transmission in the BF is altered with age and cognitive status. We have examined the properties of spontaneous postsynaptic currents (sPSCs) in cholinergic BF neurons that have been mechanically dissociated without enzymes from behaviorally characterized F344 rats. These isolated neurons retain functional presynaptic terminals on their somata and proximal dendrites. Using whole cell patch-clamp recording, we show that sPSCs and miniature PSCs are predominately GABAergic (bicuculline sensitive) and in all ways closely resemble PSCs recorded in a BF in vitro slice preparation. Adult (4-7 mo) and aged (22-24 mo) male rats were cognitively assessed using the water maze. Neuronal phenotype was identified post hoc using single-cell RT-PCR. The frequency of sPSCs was reduced during aging, and this was most pronounced in cognitively impaired subjects. This is the same population that demonstrated increased intracellular Ca(2+) buffering. We also show that increasing Ca(2+) buffering in the synaptic terminals of young BF neurons can mimic the reduced frequency of sPSCs observed in aged BF neurons.

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.

Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala.

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is long-lasting, can manifest well after the overt physical symptoms of withdrawal, and is frequently associated with relapse in recovering alcoholics. The neurobiological mechanisms governing these withdrawal-associated increases in anxiety are currently unknown. The basolateral amygdala (BLA) is a major emotional center in the brain and regulates the expression of both learned fear and anxiety. Neurotransmitter system alterations within this brain region may therefore contribute to withdrawal-associated anxiety. Because evidence suggests that glutamate-gated neurotransmitter receptors are sensitive to acute ethanol exposure, we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on glutamatergic synaptic transmission in the BLA. We found that slices prepared from CIE and WD animals had significantly increased contributions by synaptic NMDA receptors. In addition, CIE increased the amplitude of AMPA-receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs), whereas only WD altered the amplitude and kinetics of tetrodotoxin-resistant spontaneous events (mEPSCs). Similarly, the frequency of sEPSCs was increased in both CIE and WD neurons, although only WD increased the frequency of mEPSCs. These data suggest that CIE and WD differentially alter both pre- and postsynaptic properties of BLA glutamatergic synapses. Finally, we show that microinjection of the AMPA-receptor antagonist, DNQX, can attenuate withdrawal-related anxiety-like behavior. Together, our results suggest that increased glutamatergic function may contribute to anxiety expressed during withdrawal from chronic ethanol.

Distinct functional characteristics of the lateral/basolateral amygdala GABAergic system in C57BL/6J and DBA/2J mice.

It is generally understood that genetic mechanisms contribute to pathological anxiety and that C57BL/6 (B6) and DBA/2J (D2) mice, inbred strains differing markedly in their anxiety-like behaviors, may represent a model system to study these contributions. Because lateral/basolateral amygdala (BLA) GABA(A) receptors help regulate anxiety-like behaviors, we have tested the hypothesis that differences in receptor function/expression may be related to strain-specific differences in experimentally measured anxiety. First, we demonstrated that anxiety-like behaviors in two separate assays were more substantial in D2 mice. Then, using whole-cell electrophysiology of isolated neurons, we found that D2 BLA neurons expressed significantly greater GABA-gated responses than B6 BLA neurons. This was specific for GABA(A) receptors, because N-methyl-d-aspartate-gated responses were similar between strains. At the molecular level, this increased GABA(A) function was associated with higher levels of alpha 2 subunit mRNA expression in D2 BLA. Finally, to understand the ramifications of these functional and molecular biological differences, we examined both electrically evoked GABAergic responses and spontaneous synaptic currents using whole-cell recordings with in vitro slice preparations. Presynaptic GABAergic function was more robust in D2 compared with B6 slices. Together, our findings suggest that genetic mechanisms differentially represented in these two inbred mouse strains lead to robust differences in pre- and postsynaptic aspects of amygdala GABAergic 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.

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.