Annette S. Fincher

Sustained ethanol inhibition of native AMPA receptors on medial septum/diagonal band (MS/DB) neurons

The direct impact of ethanol on native, non‐NMDA glutamate receptors was examined in acutely isolated MS/DB neurons from rat. The impact of ethanol functional tolerance and physical dependence on non‐NMDA receptor function was also determined. Non‐NMDA receptors were defined pharmacologically as predominantly the AMPA subtype, because both AMPA‐ or kainate‐activated currents were blocked by GYKI 52466, a selective AMPA receptor antagonist. The relative magnitude of potentiation of AMPA‐activated currents by 10 or 100 μM cyclothiazide was consistent with recombinant AMPA flop‐subtype receptors. Finally, the selective kainate receptor agonist, SYM 8021, induced little current in MS/DB neurons. AMPA receptor currents when activated by kainate were sensitive to ethanol, showing inhibition of ∼5–50% when 10–300 mM ethanol and kainate were briefly co‐applied (3 s). Ethanol (100 mM) also inhibited both the initial transient peak and sustained currents activated by AMPA. Inhibition was sustained during continuous ethanol superfusions of 5 min, suggesting a lack of acute tolerance to ethanol‐induced AMPA receptor blockade. Rapid application of 3–3000 μM kainate activated concentration‐dependent currents in MS/DB neurons from Control and Ethanol Dependent animals that were not significantly different. Also, direct ethanol inhibition (300 mM) of kainate‐activated currents was not reduced by ethanol dependence, suggesting a lack of functional tolerance. These results suggest that native AMPA receptors on MS/DB neurons are inhibited by pharmacologically‐relevant concentrations of ethanol. However, these receptors, unlike NMDA receptors, do not undergo adaptation with sustained ethanol exposure sufficient to induce physical dependence.

Sensitivity of postsynaptic GABAB receptors on hippocampal CA1 and CA3 pyramidal neurons to ethanol

Baclofen-induced hyperpolarization of hippocampal CA1 and CA3 pyramidal neurons was examined to assess the impact of ethanol on postsynaptic GABAB receptors. These receptors activate outward K+ currents via a pertussis toxin-sensitive G protein cascade to reduce membrane potential during the slow inhibitory postsynaptic potential. This inhibitory action may play a role in ethanol intoxication and withdrawal excitability. In both types of pyramidal neurons, baclofen applied consecutively in increasing concentrations caused concentration dependent hyperpolarization. There were no significant differences in resting membrane potential, input resistance, maximum baclofen-induced hyperpolarization or EC50 between CA1 and CA3 neurons, although slope values were significantly smaller in the former neurons. These parameters were not significantly changed in the presence of ethanol 10-100 mM. Chronic ethanol treatment (12 days) sufficient to induce physical dependence also did not shift sensitivity or maximum response to baclofen in CA1 neurons. These results suggest that GABAB receptors in this model are essentially insensitive to ethanol and do not confirm our earlier preliminary observation of a possible down-regulation of postsynaptic GABAB receptor function by chronic ethanol treatment.

Effect of ethanol on γ-vinyl GABA-induced GABA accumulation in the substantia nigra and on synaptosomal GABA content in six rat brain regions

Abstract Two recently developed methods for estimating changes in presynaptic γ-aminobutyric acid (GABA) homeostasis were used for the first time to evaluate the effects of acute and chronic ethanol treatments on GABA utilization. GABA accumulation in the left substantia nigra zona reticulata (SNR) following unilateral microinjection of γ-vinyl GABA (GVG; 5 μg) was linear for at least 180 min while GABA concentrations in the uninjected right SNR did not change over this period. Net GABA accumulation (left minus right SNR) also increased linearly over this interval. Intraperitoneal (i.p.) admministration of ethanol (0.3, 1 or 3 g/kg) 15 min after GVG microinjection did not significantly change either the rate of GABA accumulation in left SNR, the net GABA accumulated or the concentration of GABA in the uninjected right SNR relative to saline injected controls over the 45-min test interval. Likewise, GABA accumulation in the left SNR or steady-state GABA concentrations in the right SNR of chronically intoxicated rats or physically dependent animals withdrawn from ethanol for 12 h did not change significantly from that of dextrose-fed controls. In a separate study, the effects of acute and chronic ethanol treatments on the concentration of GABA in synaptosomes isolated from the frontal cortex, hippocampus, tectum, striatum, cerebellum or brainstem were determined. Thirty min after acute treatment with ethanol (0.5, 1, 2 or 4 g/kg, i.p.) the concentration of GABA in synaptosomes from any of these brain regions was not significantly altered. Furthermore, chronic ethanol treatment sufficient to induced physical dependence and a severe ethanol withdrawal syndrome also did not significantly modify synaptosomal GABA concentrations. The present results do not support a role for changes in the availability of presynaptic GABA in either the acute or chronic central nervous system actions of ethanol.

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.

Lanthanum and zinc sensitivity of GABAA-activated currents in adult medial septum/diagonal band neurons from ethanol dependent rats

The impact of chronic ethanol treatment, sufficient to induce tolerance and physical dependence, on GABAA receptor function was studied in acutely isolated neurons from the medial septum/nucleus diagonal band (MS/nDB) of adult rats using whole cell, patch-clamp recordings. In ethanol-naive Controls, GABA (0.3-300 microM) induced concentration-dependent increases in Cl- current with a threshold of 0.3-1 microM, a mean maximal current of 7645 +/- 2148 pA at 100-300 microM, an EC50 of 11.3 +/- 1.3 microM and a slope of 1.53 +/- 0.07. GABA-activated currents in neurons from animals receiving two weeks of ethanol liquid diet treatment did not differ significantly on any of these measures. The rate of GABAA receptor desensitization (t1/2 = 6.49 +/- 1.19 s) estimated as the time required for loss of 50% of peak current during sustained application of 10 microM GABA, as well as the residual steady state current remaining following complete desensitization for controls was unchanged by chronic ethanol. The impact of chronic ethanol treatment on the GABAA receptor modulation by lanthanum and zinc which act as positive and negative allosteric modulators, respectively, was also evaluated. Test pulses of 3 microM GABA in control neurons showed maximal potentiation by 141 +/- 30% at approximately 1000 microM lanthanum with an EC50 of 107 +/- 34 microM and a slope of approximately 1. Lanthanum potentiation remained the same following chronic ethanol treatment. Initial estimates based on fitted concentration response curves suggested that maximal inhibition of 3 microM GABA responses by zinc at the level of 70.2 +/- 8.5% in control cells was significantly increased by chronic ethanol treatment to 95.3 +/- 2.5%, although the IC50 of 60.2 +/- 25 microM was not changed. However, this difference was not supported by direct tests of maximal 3-10 mM zinc concentrations. These results suggest that chronic ethanol treatment, sufficient to induce tolerance and physical dependence, probably does not lead to readily detectible changes in GABAA receptor function in MS/nDB neurons.

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 drugs 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 nicotines 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.

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.

Acute ethanol dependence or long-term ethanol treatment and abstinence do not reduce hippocampal responses to carbachol.

In the hippocampus of human alcoholics, prolonged ethanol treatment reduces the number of muscarinic ligand binding sites present at autopsy suggesting a decrease in functional muscarinic receptors. Whether these changes are due to alcohol-induced brain damage or ethanol dependence and represent a reduced level of cholinergic function is unknown. The present studies tested the impact of ethanol dependence or long-term ethanol treatment and subsequent withdrawal on the function of pre- and postsynaptic muscarinic receptors in the CA1 region of the rat hippocampus. Field excitatory postsynaptic potentials (EPSPs) were inhibited in a concentration-dependent manner by 0.1-100 microM carbachol. This presynaptic inhibitory action of carbachol involving muscarinic receptors was not significantly reduced either by ethanol treatment (12 days), causing physical dependence, or by long-term ethanol treatment (97-120 days) and abstinence (3-6 months). Postspike after hyperpolarizations (AHPs) were inhibited in a concentration-dependent manner by carbachol (6-2000 nM). This postsynaptic excitatory action of muscarinic receptors also was not significantly reduced either by 12-day ethanol treatment or by long-term ethanol treatment. Taken together, these results suggest that neither pre- nor postsynaptic muscarinic receptor function measured electrophysiologically is reduced by either ethanol dependence or long-term ethanol consumption and abstinence in the rat as suggested by reduced muscarinic ligand binding in the hippocampus of human alcoholics.

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 fluoxetines 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.