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Background Identifying effective strategies to prevent memory loss in AD has eluded researchers to date, and likely reflects insufficient understanding of early pathogenic mechanisms directly affecting memory encoding. As synaptic loss best correlates with memory loss in AD, refocusing efforts to identify factors driving synaptic impairments may provide the critical insight needed to advance the field. In this study, we reveal a previously undescribed cascade of events underlying pre and postsynaptic hippocampal signaling deficits linked to cognitive decline in AD. These profound alterations in synaptic plasticity, intracellular Ca2+ signaling, and network propagation are observed in 3–4 month old 3xTg-AD mice, an age which does not yet show overt histopathology or major behavioral deficits. Methods In this study, we examined hippocampal synaptic structure and function from the ultrastructural level to the network level using a range of techniques including electron microscopy (EM), patch clamp and field potential electrophysiology, synaptic immunolabeling, spine morphology analyses, 2-photon Ca2+ imaging, and voltage-sensitive dye-based imaging of hippocampal network function in 3–4 month old 3xTg-AD and age/background strain control mice. Results In 3xTg-AD mice, short-term plasticity at the CA1-CA3 Schaffer collateral synapse is profoundly impaired; this has broader implications for setting long-term plasticity thresholds. Alterations in spontaneous vesicle release and paired-pulse facilitation implicated presynaptic signaling abnormalities, and EM analysis revealed a reduction in the ready-releasable and reserve pools of presynaptic vesicles in CA3 terminals; this is an entirely new finding in the field. Concurrently, increased synaptically-evoked Ca2+ in CA1 spines triggered by LTP-inducing tetani is further enhanced during PTP and E-LTP epochs, and is accompanied by impaired synaptic structure and spine morphology. Notably, vesicle stores, synaptic structure and short-term plasticity are restored by normalizing intracellular Ca2+ signaling in the AD mice. Conclusions These findings suggest the Ca2+ dyshomeostasis within synaptic compartments has an early and fundamental role in driving synaptic pathophysiology in early stages of AD, and may thus reflect a foundational disease feature driving later cognitive impairment. The overall significance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic vesicle stores, synaptic plasticity, and network propagation, which directly impact memory encoding. Electronic supplementary material The online version of this article (10.1186/s13024-019-0307-7) contains supplementary material, which is available to authorized users.

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is extensive, long-lasting, and develops well after the obvious physical symptoms of acute withdrawal. The neurobiological mechanisms underlying this prolonged withdrawal-induced anxiety are not well understood. The basolateral amygdala is a major emotional center in the brain and regulates the expression of anxiety. New evidence suggests that increased glutamatergic function in the basolateral amygdala may contribute to withdrawal-related anxiety following chronic ethanol exposure. Recent evidence also suggests that kainate-type ionotropic glutamate receptors are inhibited by intoxicating concentrations of acute ethanol. This acute sensitivity suggests potential contributions by these receptors to the increased glutamatergic function seen during chronic exposure. Therefore we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on kainate receptor (KAR) mediated synaptic transmission in the basolateral amygdala (BLA) of Sprague Dawley rats. Our study showed that CIE, but not withdrawal, increased synaptic responses mediated by KARs. Interestingly, both CIE and WD occluded KAR-mediated synaptic plasticity. Finally, we found that BLA fEPSP responses were increased during CIE and WD via a mechanism that is independent from glutamate release from presynaptic terminals. Taken together, these data suggest that KARs might also contribute to postsynaptic increases in glutamatergic synaptic transmission during CIE and that the mechanisms responsible for the expression of KAR-dependent synaptic plasticity might be engaged by chronic ethanol exposure and withdrawal.

The lateral/basolateral amygdala (BLA) forms an integral part of the neural circuitry controlling innate anxiety and learned fear. More recently, BLA dependent modulation of self-administration behaviors suggests a much broader role in the regulation of reward evaluation. To test this, we employed a self-administration paradigm that procedurally segregates ‘seeking’ (exemplified as lever-press behaviors) from consumption (drinking) directed at a sweetened ethanol solution. Microinjection of the nonselective serotonin type-2 receptor agonist, alpha-methyl-5-hydroxytryptamine (-m5HT) into the BLA reduced lever pressing behaviors in a dose-dependent fashion. This was associated with a significant reduction in the number of response-bouts expressed during non-reinforced sessions without altering the size of a bout or the rate of responding. Conversely, intra-BLA -m5HT only modestly effected consumption-related behaviors; the highest dose reduced the total time spent consuming a sweetened ethanol solution but did not inhibit the total number of licks, number of lick bouts, or amount of solution consumed during a session. In vitro neurophysiological characterization of BLA synaptic responses showed that -m5HT significantly reduced extracellular field potentials. This was blocked by the 5-HT2A/C antagonist ketanserin suggesting that 5-HT2-like receptors mediate the behavioral effect of -m5HT. During whole-cell patch current-clamp recordings, we subsequently found that -m5HT increased action potential threshold and hyperpolarized the resting membrane potential of BLA pyramidal neurons. Together, our findings show that the activation of BLA 5-HT2A/C receptors inhibits behaviors related to reward-seeking by suppressing BLA principal neuron activity. These data are consistent with the hypothesis that the BLA modulates reward-related behaviors and provides specific insight into BLA contributions during operant self-administration of a sweetened ethanol solution.

Amygdala glutamatergic neurotransmission regulates withdrawal induced anxiety-like behaviors following chronic ethanol exposure. The lateral/basolateral amygdala receives multiple glutamatergic projections that contribute to overall amygdala function. Our lab has previously shown that rat cortical (external capsule) afferents express postsynaptic alterations during chronic intermittent ethanol exposure and withdrawal. However, thalamic (internal capsule) afferents also provide crucial glutamatergic input during behavioral conditioning, and they have not been studied in the context of chronic drug exposure. We report here that these thalamic inputs express altered presynaptic function during withdrawal from chronic ethanol exposure. This is characterized by enhanced release probability, as exemplified by altered paired-pulse ratios and decreased failure rates of unitary events, and increased concentrations of synaptic glutamate. Quantal analysis further implicates a withdrawal-dependent enhancement of the readily releasable pool of vesicles as a probable mechanism. These functional alterations are accompanied by increased expression of vesicle associated protein markers. These data demonstrate that chronic ethanol modulation of glutamate neurotransmission in the rat lateral/basolateral amygdala is afferent-specific. Further, presynaptic regulation of lateral/basolateral amygdala thalamic inputs by chronic ethanol may be a novel neurobiological mechanism contributing to the increased anxiety-like behaviors that characterize withdrawal.

The amygdala plays a critical role in the generation and expression of anxiety-like behaviors including those expressed following withdrawal (WD) from chronic intermittent ethanol (CIE) exposure. In particular, the BLA glutamatergic system controls the expression of both innate and pathological anxiety. Recent data suggests that CIE and WD may functionally alter this system in a manner that closely parallels memory-related phenomena like long-term potentiation (LTP). We therefore specifically dissected CIE/WD-induced changes in glutamatergic signaling using electrophysiological and biochemical approaches with a particular focus on the plasticity-related components of this neurotransmitter system. Our results indicate that cortical glutamatergic inputs arriving at BLA principal via the external capsule undergo predominantly post-synaptic alterations in AMPA receptor function following CIE and WD. Biochemical analysis revealed treatment-dependent changes in AMPA receptor surface expression and subunit phosphorylation that are complemented by changes in total protein levels and/or phosphorylation status of several key, plasticity-associated protein kinases such as calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). Together, these data show that CIE- and WD-induced changes in BLA glutamatergic function both functionally and biochemically mimic plasticity-related states. These mechanisms likely contribute to long-term increases in anxiety-like behavior following chronic ethanol exposure.

Withdrawal-related anxiety is cited as a major contributor to relapse in recovering alcoholics. Changes in lateral/basolateral amygdala (BLA) neurotransmission could directly influence anxiety-like behaviors after chronic ethanol exposure and withdrawal. We have shown that these treatments enhance BLA glutamatergic function and neurotransmission. However, the BLA GABAergic system tightly controls the expression of anxiety-like behavior, and additional neuroadaptations in this system are potentially important as well. The intrinsic BLA GABAergic system consists of at least two populations of interneurons: local feed-back interneurons scattered throughout the region and feed-forward interneurons concentrated within groups found in the lateral/paracapsular region of the BLA. In the present study, we found that withdrawal from chronic ethanol robustly decreased presynaptic function at feed-forward GABA synapses but did not alter neurotransmitter release from local interneurons. Differential presynaptic changes at these synapses were complemented by decreased zolpidem sensitivity at feed-forward synapses and decreased midazolam sensitivity at local synapses. Consistent with this, chronic ethanol/withdrawal decreased expression of GABA α1-subunit total protein and increased surface expression of α4-subunit protein. We also found transient increases in GABA-receptor-associated protein levels and persistent increases in γ2-subunit and gephyrin proteins that would suggest alterations in GABA(A) receptor trafficking that might help regulate changes in α4-subunit localization. These data together suggest that chronic ethanol and withdrawal differentially modulate local and lateral paracapsular cell GABAergic synapses via distinct presynaptic and postsynaptic mechanisms. These findings extend our understanding of the neurobiological mechanisms governing changes in anxiety-like behavior after chronic ethanol exposure and withdrawal.

Central among the brain regions that regulate fear/anxiety behaviors is the lateral/basolateral amygdala (BLA). BLA output is tightly controlled by the relative activity of two populations of inhibitory GABAergic interneurons, local feedback cells distributed throughout the nucleus, and feedforward cells found along the lateral paracapsular border of this subdivision. Recent studies suggest that dopamine (DA) can modulate the BLA GABAergic system, thus linking fear/anxiety states with mesolimbic reward/attentional processes. However, the precise dopaminergic mechanisms regulating the activity of the two BLA GABAergic neuron populations have not been fully explored. We therefore examined the effects of DA D3-like receptors on BLA-dependent anxiety-like behavior and neurophysiology. After confirming the presence of D3-like receptors within the BLA, we found that microinjection of a D3-selective antagonist into the BLA decreased anxiety-like behavior expressed in both the light/dark transition test and the elevated plus maze. Consistent with this, we found that in vitro D3-like receptor activation selectively inhibits synaptic transmission at both BLA feedback and feedforward GABAergic interneuron populations, with no effect on glutamatergic transmission. This inhibition of GABAergic transmission is a result of a D3-like receptor-mediated, dynamin-dependent process that presumably reflects endocytosis of postsynaptic GABA(A) receptors found on principal BLA neurons. Because environmental cues alter both DA release and relative activity states of the BLA, our data strongly suggest that DA, potentially acting through D3-like receptors, may suppress the relative contribution by inhibitory processes in the BLA and modify the expression of BLA-related behaviors.

Plasticity at glutamatergic synapses is believed to be the cellular correlate of learning and memory. Classic fear conditioning, for example, is dependent upon NMDA-type glutamate receptor activation in the lateral/basolateral amygdala followed by increased synaptic expression of AMPA-type glutamate receptors. This review provides an extensive comparison between the initiation and expression of glutamatergic plasticity during learning/memory and glutamatergic alterations associated with chronic ethanol exposure and withdrawal. The parallels between these neuro-adaptive processes suggest that long-term ethanol exposure might "chemically condition" amygdala-dependent fear/anxiety via the increased function of pre- and post-synaptic glutamate signaling.

This article summarizes the proceedings of a symposium that was presented at a conference entitled "Alcoholism and Stress: A Framework for Future Treatment Strategies." The conference was held in Volterra, Italy on May 6-9, 2008 and this symposium was chaired by Jeff L. Weiner. The overall goal of this session was to review recent findings that may shed new light on the neurobiological mechanisms that underlie the complex relationships between stress, anxiety, and alcoholism. Dr. Danny Winder described a novel interaction between D1 receptor activation and the corticotrophin-releasing factor (CRF) system that leads to an increase in glutamatergic synaptic transmission in the bed nucleus of the stria terminalis. Dr. Marisa Roberto presented recent data describing how protein kinase C epsilon, ethanol, and CRF interact to alter GABAergic inhibition in the central nucleus of the amygdala. Dr. Jeff Weiner presented recent advances in our understanding of inhibitory circuitry within the basolateral amygdala (BLA) and how acute ethanol exposure enhances GABAergic inhibition in these pathways. Finally, Dr. Brian McCool discussed recent findings on complementary glutamatergic and GABAergic adaptations to chronic ethanol exposure and withdrawal in the BLA. Collectively, these investigators have identified novel mechanisms through which neurotransmitter and neuropeptide systems interact to modulate synaptic activity in stress and anxiety circuits. Their studies have also begun to describe how acute and chronic ethanol exposure influence excitatory and inhibitory synaptic communication in these pathways. These findings point toward a number of novel neurobiological targets that may prove useful for the development of more effective treatment strategies for alcohol use disorders.