Carlos Fernando Valenzuela

Low Concentrations of Alcohol Inhibit BDNF-Dependent GABAergic Plasticity via L-type Ca2+ Channel Inhibition in Developing CA3 Hippocampal Pyramidal Neurons

Fetal alcohol spectrum disorder (FASD) is associated with learning and memory alterations that could be, in part, a consequence of hippocampal damage. The CA3 hippocampal subfield is one of the regions affected by ethanol (EtOH), including exposure during the third trimester-equivalent (i.e., neonatal period in rats). However, the mechanism of action of EtOH is poorly understood. In CA3 pyramidal neurons from neonatal rats, dendritic BDNF release causes long-term potentiation of the frequency of GABAA receptor-mediated spontaneous postsynaptic currents (LTP-GABAA) and this mechanism is thought to play a role in GABAergic synapse maturation. Here, we show that short- and long-term exposure of neonatal male rats to low EtOH concentrations abolishes LTP-GABAA by inhibiting L-type voltage-gated Ca2+ channels. These findings support the recommendation that even light drinking should be avoided during pregnancy.

Ethanol Decreases Purkinje Neuron Excitability by Increasing GABA Release in Rat Cerebellar Slices

Cerebellar Purkinje neurons (PNs) receive inhibitory GABAergic input from stellate and basket cells, which are located in the outer and inner portions of the molecular layer, respectively. Ethanol (EtOH) was recently shown to increase GABAergic transmission at PNs via a mechanism that involves enhanced calcium release from presynaptic internal stores (J Pharmacol Exp Ther 323:356–364, 2007). Here, we further characterized the effect of EtOH on GABA release and assessed its impact on PN excitability. Using whole-cell patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that EtOH acutely increases the frequency but not the amplitude or half-width of miniature and spontaneous inhibitory postsynaptic currents (IPSCs). EtOH significantly increased the amplitude and decreased the paired pulse ratio of IPSCs evoked by stimulation in the outer but not inner molecular layer. In current clamp, EtOH decreased both the amplitude of excitatory postsynaptic potentials evoked in PNs by granule cell axon stimulation and the number of action potentials triggered by these events; these effects depended on GABAA receptor activation because they were not observed in presence of bicuculline. Loose-patch cell-attached PN recordings revealed that neither the spontaneous action potential firing frequency nor the coefficient of variation of the interspike interval was altered by acute EtOH exposure. These findings suggest that EtOH differentially affects GABAergic transmission at stellate cell- and basket cell-to-PN synapses and that it modulates PN firing triggered by granule cell axonal input. These effects could be in part responsible for the cerebellar impairments associated with acute EtOH intoxication.

Platelet-derived Growth Factor Receptor-induced Feed-forward Inhibition of Excitatory Transmission between Hippocampal Pyramidal Neurons

Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity ofN-methyl-d-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-γ, and by blocking the intracellular release of Ca2+. Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca2+- and PKA-dependent inhibition of their modulation by c-Src.

Moderate prenatal alcohol exposure reduces plasticity and alters NMDA receptor subunit composition in the dentate gyrus

Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semiquantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2′-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.

Prenatal ethanol exposure persistently impairs NMDA receptor‐dependent activation of extracellular signal‐regulated kinase in the mouse dentate gyrus

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal‐dependent learning deficits and a decreased ability to elicit long‐term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal‐regulated kinase 1/2 (ERK1/2) signaling cascade by NMDA receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol‐exposed adult animals would have deficits in hippocampal NMDA receptor‐dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA‐stimulated ERK1/2 activation in acute hippocampal slices prepared from adult fetal alcohol‐exposed mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor‐dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life‐long cognitive deficits, associated with prenatal alcohol exposure.

Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs.

Moderate Alcohol Exposure during the Rat Equivalent to the Third Trimester of Human Pregnancy Alters Regulation of GABAA Receptor-Mediated Synaptic Transmission by Dopamine in the Basolateral Amygdala

Fetal ethanol (EtOH) exposure leads to a range of neurobehavioral alterations, including deficits in emotional processing. The basolateral amygdala (BLA) plays a critical role in modulating emotional processing, in part, via dopamine (DA) regulation of GABA transmission. This BLA modulatory system is acquired during the first 2 weeks of postnatal life in rodents (equivalent to the third trimester of human pregnancy) and we hypothesized that it could be altered by EtOH exposure during this period. We found that exposure of rats to moderate levels of EtOH vapor during the third trimester-equivalent [postnatal days (P) 2–12] alters DA modulation of GABAergic transmission in BLA pyramidal neurons during periadolescence. Specifically, D1R-mediated potentiation of spontaneous inhibitory postsynaptic currents (IPSCs) was significantly attenuated in EtOH-exposed animals. However, this was associated with a compensatory decrease in D3R-mediated suppression of miniature IPSCs. Western blot analysis revealed that these effects were not a result of altered D1R or D3R levels. BLA samples from EtOH-exposed animals also had significantly lower levels of the DA precursor (L-3,4-dihydroxyphenylalanine) but DA levels were not affected. This is likely a consequence of reduced catabolism of DA, as indicated by reduced levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the BLA samples. Anxiety-like behavior was not altered in EtOH-exposed animals. This is the first study to demonstrate that the modulatory actions of DA in the BLA are altered by developmental EtOH exposure. Although compensatory adaptations were engaged in our moderate EtOH exposure paradigm, it is possible that these are not able to restore homeostasis and correct anxiety-like behaviors under conditions of heavier EtOH exposure. Therefore, future studies should investigate the potential role of alterations in the modulatory actions of DA in the pathophysiology of fetal alcohol spectrum disorders.

Opposite effects of acute ethanol exposure on GAP-43 and BDNF expression in the hippocampus versus the cerebellum of juvenile rats.

The adolescent brain is particularly vulnerable to the effects of alcohol, with intoxications at this developmental age often producing long-lasting effects. The present study addresses the effects of a single acute ethanol exposure on growth-associated protein-43 (GAP-43) and brain-derived neurotrophic factor (BDNF) gene expression in neurons in the cerebellum and hippocampus of adolescent rats. Male postnatal day 23 (P23) Sprague-Dawley rats were exposed to ethanol vapors for 2h and after a recovery period of 2h, the cerebellum and hippocampus were harvested and samples were taken for blood alcohol concentration (BAC) determinations. We found that this exposure resulted in a mean BAC of 174 mg/dL, which resembles levels in human adolescents after binge drinking. Analyses of total RNA and protein by quantitative reverse transcription PCR and western blotting, respectively, revealed that this single ethanol exposure significantly decreased the levels of GAP-43 mRNA and protein in the cerebellum but increased the levels of mRNA and protein in the hippocampus. BDNF mRNA and protein levels were also increased in the hippocampus but not in the cerebellum of these animals. In situ hybridizations revealed that GAP-43 and BDNF mRNA levels were primarily increased by alcohol exposure in hippocampal dentate granule cells and CA3 neurons. Overall, the reported alterations in the expression of the plasticity-associated genes GAP-43 and BDNF in juvenile rats are consistent with the known deleterious effects of binge drinking on motor coordination and cognitive function.

Acute ethanol exposure inhibits silencing of cerebellar Golgi cell firing induced by granule cell axon input

Golgi cells (GoCs) are specialized interneurons that provide inhibitory input to granule cells in the cerebellar cortex. GoCs are pacemaker neurons that spontaneously fire action potentials, triggering spontaneous inhibitory postsynaptic currents in granule cells and also contributing to the generation tonic GABAA receptor-mediated currents in granule cells. In turn, granule cell axons provide feedback glutamatergic input to GoCs. It has been shown that high frequency stimulation of granule cell axons induces a transient pause in GoC firing in a type 2-metabotropic glutamate receptor (mGluR2)-dependent manner. Here, we investigated the effect ethanol on the pause of GoC firing induced by high frequency stimulation of granule cell axons. GoC electrophysiological recordings were performed in parasagittal cerebellar vermis slices from postnatal day 23 to 26 rats. Loose-patch cell-attached recordings revealed that ethanol (40 mM) reversibly decreases the pause duration. An antagonist of mGluR2 reduced the pause duration but did not affect the effect of ethanol. Whole-cell voltage-clamp recordings showed that currents evoked by an mGluR2 agonist were not significantly affected by ethanol. Perforated-patch experiments in which hyperpolarizing and depolarizing currents were injected into GoCs demonstrated that there is an inverse relationship between spontaneous firing and pause duration. Slight inhibition of the Na+/K+ pump mimicked the effect of ethanol on pause duration. In conclusion, ethanol reduces the granule cell axon-mediated feedback mechanism by reducing the input responsiveness of GoCs. This would result in a transient increase of GABAA receptor-mediated inhibition of granule cells, limiting information flow at the input stage of the cerebellar cortex.

AMPAR-mediated synaptic transmission in the CA1 hippocampal region of neonatal rats: unexpected resistance to repeated ethanol exposure

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate glutamatergic receptors (AMPAR) mediate most of the fast excitatory synaptic transmission in mature neurons. In contrast, a number of developing synapses do not express AMPARs; these are gradually acquired in an activity-driven manner during the first week of life in rats, which is equivalent to the third trimester of human pregnancy. Neuronal stimulation has been shown to drive high conductance Ca(2+)-permeable AMPARs into the synapse, strengthening glutamatergic synaptic transmission. Alterations in this process could induce premature stabilization or inappropriate elimination of newly formed synapses and contribute to the hippocampal abnormalities associated with fetal alcohol spectrum disorder. Previous studies from our laboratory performed with hippocampal slices from neonatal rats showed that acute ethanol exposure exerts potent stimulant effects on CA1 and CA3 neuronal networks. However, the impact of these in vitro actions of acute ethanol exposure is unknown. Here, we tested the hypothesis that repeated in vivo exposure to ethanol strengthens AMPAR-mediated neurotransmission in the CA1 region by means of an increase in synaptic expression of Ca(2+)-permeable AMPARs. We exposed rats to ethanol vapor (serum ethanol concentration approximately 40 mM) or air for 4h/day from postnatal day (P) 2-6. In brain slices prepared at P4-6, we found no significant effect of ethanol exposure on input-output curves for AMPAR-mediated field excitatory postsynaptic potentials (fEPSPs), the contribution of Ca(2+)-permeable AMPARs to these fEPSPs, or the acute effect of ethanol on fEPSP amplitude. These results suggest that homeostatic plasticity mechanisms act to maintain glutamatergic synaptic strength and ethanol sensitivity in response to repeated developmental ethanol exposure.