BDNF signaling in context: From synaptic regulation to psychiatric disorders.

Brain-derived neurotrophic factor (BDNF) is a neuropeptide that plays numerous important roles in synaptic development and plasticity. While its importance in fundamental physiology is well established, studies of BDNF often produce conflicting and unclear results, and the scope of existing research makes the prospect of setting future directions daunting. In this review, we examine the importance of spatial and temporal factors on BDNF activity, particularly in processes such as synaptogenesis, Hebbian plasticity, homeostatic plasticity, and the treatment of psychiatric disorders. Understanding the fundamental physiology of when, where, and how BDNF acts and new approaches to control BDNF signaling in time and space can contribute to improved therapeutics and patient outcomes.

Brain-derived neurotrophic factor scales presynaptic calcium transients to modulate excitatory neurotransmission.

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF's function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.

Ketamine: Mechanisms and Relevance to Treatment of Depression.

Major depressive disorder (MDD) is a leading cause of suicide in the world. Monoamine-based antidepressant drugs are a primary line of treatment for this mental disorder, although the delayed response and incomplete efficacy in some patients highlight the need for improved therapeutic approaches. Over the past two decades, ketamine has shown rapid onset with sustained (up to several days) antidepressant effects in patients whose MDD has not responded to conventional antidepressant drugs. Recent preclinical studies have started to elucidate the underlying mechanisms of ketamine's antidepressant properties. Herein, we describe and compare recent clinical and preclinical findings to provide a broad perspective of the relevant mechanisms for the antidepressant action of ketamine.

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action.

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine's antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine's antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor-mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine's antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine's antidepressant action.

MeCP2 loss-of-function dysregulates microRNAs regionally and disrupts excitatory/inhibitory synaptic transmission balance.

Rett syndrome is a leading cause of intellectual disability in females primarily caused by loss of function mutations in the transcriptional regulator MeCP2. Loss of MeCP2 leads to a host of synaptic phenotypes that are believed to underlie Rett syndrome pathophysiology. Synaptic deficits vary by brain region upon MeCP2 loss, suggesting distinct molecular alterations leading to disparate synaptic outcomes. In this study, we examined the contribution of MeCP2's newly described role in miRNA regulation to regional molecular and synaptic impairments. Two miRNAs, miR-101a and miR-203, were identified and confirmed as upregulated in MeCP2 KO mice in the hippocampus and cortex, respectively. miR-101a overexpression in hippocampal cultures led to opposing effects at excitatory and inhibitory synapses and in spontaneous and evoked neurotransmission, revealing the potential for a single miRNA to broadly regulate synapse function in the hippocampus. These results highlight the importance of regional alterations in miRNA expression and the specific impact on synaptic function with potential implications for Rett syndrome.

VAMP4 Maintains a Ca2+-Sensitive Pool of Spontaneously Recycling Synaptic Vesicles.

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca2+ signaling pathways. Here, we probed the mechanisms that maintain Ca2+ sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca2+-dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augmentsCa2+-sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release.SIGNIFICANCE STATEMENT Spontaneous neurotransmitter release that occurs independent of presynaptic action potentials (APs) shows significant sensitivity to intracellular Ca2+ levels. In this study, we identify the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecule vesicle-associated membrane protein 4 (VAMP4) as a key component of the machinery that maintains these Ca2+-sensitive fraction of spontaneous release events. Following brief intense activity, VAMP4-dependent synaptic vesicle retrieval supports a pool of vesicles that fuse spontaneously in the long term. We propose that this vesicle trafficking pathway acts to shape spontaneous release and associated signaling based on previous activity history of synapses.

Inactivation of NMDA Receptors in the Ventral Tegmental Area during Cocaine Self-Administration Prevents GluA1 Upregulation but with Paradoxical Increases in Cocaine-Seeking Behavior.

Cocaine self-administration increases expression of GluA1 subunits in ventral tegmental area (VTA) dopamine neurons, which subsequently enhance the motivation for cocaine. This increase in GluA1 may be dependent on concomitant NMDA receptor (NMDAR) activation during self-administration, similar to cocaine-induced long-term potentiation in the VTA. In this study, we used viral-mediated expression of a dominant-negative GluN1 subunit (HSV-dnGluN1) in VTA neurons to study the effect of transient NMDAR inactivation on the GluA1 increases induced by chronic cocaine self-administration in male rats. We found that dnGluN1 expression in the VTA limited to the 3 weeks of cocaine self-administration prevents the subsequent increase in tissue GluA1 levels when compared with control infusions of HSV-LacZ. Surprisingly, dnGluN1 expression led to an enhancement in the motivation to self-administer cocaine as measured using a progressive ratio reinforcement schedule and to enhanced cocaine seeking measured in extinction/reinstatement tests following an extended 3 week withdrawal period. Despite blocking tissue GluA1 increases in cocaine self-administering animals, the HSV-dnGluN1 treatment resulted in increased membrane levels of GluA1 and GluN2B, along with markedly higher locomotor responses to intra-VTA infusions of AMPA, suggesting a paradoxical increase in VTA AMPA receptor responsiveness. Together, these data suggest that NMDARs mediate cocaine-induced increases in VTA GluA1 expression, but such transient NMDAR inactivation also leads to compensatory scaling of synaptic AMPA receptors that enhance the motivational for cocaine.SIGNIFICANCE STATEMENT Dopamine neurons in the ventral tegmental area (VTA) are critical substrates of drug rewards. Animal models indicate that chronic cocaine use enhances excitatory glutamatergic input to these neurons, making them more susceptible to environmental stimuli that trigger drug craving and relapse. We previously found that self-administration of cocaine increases AMPA glutamate receptors in the VTA, and this effect enhances motivation for cocaine. Here we report that the mechanism for this upregulation involves NMDA receptor activity during cocaine use. While interference with NMDA receptor function blocks AMPA receptor upregulation, it also produces a paradoxical enhancement in membrane AMPA receptor subunits, AMPA responsiveness, and the motivation for cocaine. Thus, pharmacotherapy targeting NMDA receptors may inadvertently produce substantial adverse consequences for cocaine addiction.

Loss of Doc2-Dependent Spontaneous Neurotransmission Augments Glutamatergic Synaptic Strength.

Action potential-evoked vesicle fusion comprises the majority of neurotransmission within chemical synapses, but action potential-independent spontaneous neurotransmission also contributes to the collection of signals sent to the postsynaptic cell. Previous work has implicated spontaneous neurotransmission in homeostatic synaptic scaling, but few studies have selectively manipulated spontaneous neurotransmission without substantial changes in evoked neurotransmission to study this function in detail. Here we used a quadruple knockdown strategy to reduce levels of proteins within the soluble calcium-binding double C2 domain (Doc2)-like protein family to selectively reduce spontaneous neurotransmission in cultured mouse and rat neurons. Activity-evoked responses appear normal while both excitatory and inhibitory spontaneous events exhibit reduced frequency. Excitatory miniature postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), increase in amplitude after quadruple knockdown. This increase in synaptic efficacy correlates with reduced phosphorylation levels of eukaryotic elongation factor 2 and also requires the presence of elongation factor 2 kinase. Together, these data suggest that spontaneous neurotransmission independently contributes to the regulation of synaptic efficacy, and action potential-evoked and spontaneous neurotransmission can be segregated at least partially on a molecular level.SIGNIFICANCE STATEMENT Action potential-evoked and spontaneous neurotransmission have been observed in nervous system circuits as long as methods have existed to measure them. Despite being well studied, controversy still remains about whether these forms of neurotransmission are regulated independently on a molecular level or whether they are simply a continuum of neurotransmission modes. In this study, members of the Doc2 family of presynaptic proteins were eliminated, which caused a reduction in spontaneous neurotransmission, whereas action potential-evoked neurotransmission remained relatively normal. This protein loss also caused an increase in synaptic strength, suggesting that spontaneous neurotransmission is able to communicate independently with the postsynaptic neuron and trigger downstream signaling cascades that regulate the synaptic state.

NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses.

Clinical studies consistently demonstrate that a single sub-psychomimetic dose of ketamine, an ionotropic glutamatergic NMDAR (N-methyl-D-aspartate receptor) antagonist, produces fast-acting antidepressant responses in patients suffering from major depressive disorder, although the underlying mechanism is unclear. Depressed patients report the alleviation of major depressive disorder symptoms within two hours of a single, low-dose intravenous infusion of ketamine, with effects lasting up to two weeks, unlike traditional antidepressants (serotonin re-uptake inhibitors), which take weeks to reach efficacy. This delay is a major drawback to current therapies for major depressive disorder and faster-acting antidepressants are needed, particularly for suicide-risk patients. The ability of ketamine to produce rapidly acting, long-lasting antidepressant responses in depressed patients provides a unique opportunity to investigate underlying cellular mechanisms. Here we show that ketamine and other NMDAR antagonists produce fast-acting behavioural antidepressant-like effects in mouse models, and that these effects depend on the rapid synthesis of brain-derived neurotrophic factor. We find that the ketamine-mediated blockade of NMDAR at rest deactivates eukaryotic elongation factor 2 (eEF2) kinase (also called CaMKIII), resulting in reduced eEF2 phosphorylation and de-suppression of translation of brain-derived neurotrophic factor. Furthermore, we find that inhibitors of eEF2 kinase induce fast-acting behavioural antidepressant-like effects. Our findings indicate that the regulation of protein synthesis by spontaneous neurotransmission may serve as a viable therapeutic target for the development of fast-acting antidepressants.

Mechanisms underlying differential effectiveness of memantine and ketamine in rapid antidepressant responses.

Ketamine is an NMDA receptor (NMDAR) antagonist that elicits rapid antidepressant responses in patients with treatment-resistant depression. However, ketamine can also produce psychotomimetic effects that limit its utility as an antidepressant, raising the question of whether the clinically tolerated NMDAR antagonist memantine possesses antidepressant properties. Despite its similar potency to ketamine as an NMDAR antagonist, clinical data suggest that memantine does not exert rapid antidepressant actions for reasons that are poorly understood. In this study, we recapitulate the ketamine and memantine clinical findings in mice, showing that ketamine, but not memantine, has antidepressant-like effects in behavioral models. Using electrophysiology in cultured hippocampal neurons, we show that ketamine and memantine effectively block NMDAR-mediated miniature excitatory postsynaptic currents in the absence of Mg(2+). However, in physiological levels of extracellular Mg(2+), we identified key functional differences between ketamine and memantine in their ability to block NMDAR function at rest. This differential effect of ketamine and memantine extends to intracellular signaling coupled to NMDAR at rest, in that memantine does not inhibit the phosphorylation of eukaryotic elongation factor 2 or augment subsequent expression of BDNF, which are critical determinants of ketamine-mediated antidepressant efficacy. These results demonstrate significant differences between the efficacies of ketamine and memantine on NMDAR-mediated neurotransmission that have impacts on downstream intracellular signaling, which we hypothesize is the trigger for rapid antidepressant responses. These data provide a novel framework on the necessary functional requirements of NMDAR-mediated neurotransmission as a critical determinant necessary to elicit rapid antidepressant responses.

Impact of DNMT1 and DNMT3a forebrain knockout on depressive- and anxiety like behavior in mice.

DNA methylation has been shown to impact certain forms of synaptic and behavioral plasticity that have been implicated in the development in psychiatric disorders. DNA methylation is catalyzed by DNA methyltransferase (DNMT) enzymes that continue to be expressed in postmitotic neurons in the forebrain. Using a conditional forebrain knockout of DNMT1 or DNMT3a we assessed the role of these DNMTs in anxiety and depressive-like behavior in mice using an array of behavioral testing paradigms. Forebrain deletion of DNMT1 had anxiolytic and antidepressant-like properties as assessed by elevated plus maze, novelty suppressed feeding, forced swim, and social interaction tests. DNMT3a knockout mice, by contrast, did not exhibit significant behavioral alterations in these tests. Given the putative role of altered DNA methylation patterns in the development of schizophrenia, we also assessed DNMT1 and DNMT3a knockout mice in a prepulse inhibition task and found an enhanced prepulse inhibition of startle in DNMT1 knockouts relative to wild type mice, with no change evident in DNMT3a knockout mice. Our data suggest that DNMT1 and DNMT3a are distinctly involved in affective behavior and that DNMT1 may ultimately represent a potential target for treatment of certain affective behavioral disorders.

Brain-derived neurotrophic factor and neuropsychiatric disorders.

Brain derived neurotrophic factor (BDNF) is the most prevalent growth factor in the central nervous system (CNS). It is essential for the development of the CNS and for neuronal plasticity. Because BDNF plays a crucial role in development and plasticity of the brain, it is widely implicated in psychiatric diseases. This review provides a summary of clinical and preclinical evidence for the involvement of this ubiquitous growth factor in major depressive disorder, schizophrenia, addiction, Rett syndrome, as well as other psychiatric and neurodevelopmental diseases. In addition, the review includes a discussion of the role of BDNF in the mechanism of action of pharmacological therapies currently used to treat these diseases, such antidepressants and antipsychotics. The review also covers a critique of experimental therapies such as BDNF mimetics and discusses the value of BDNF as a target for future drug development.

A role for histone deacetylases in the cellular and behavioral mechanisms underlying learning and memory.

Histone deacetylases (HDACs) are a family of chromatin remodeling enzymes that restrict access of transcription factors to the DNA, thereby repressing gene expression. In contrast, histone acetyltransferases (HATs) relax the chromatin structure allowing for an active chromatin state and promoting gene transcription. Accumulating data have demonstrated a crucial function for histone acetylation and histone deacetylation in regulating the cellular and behavioral mechanisms underlying synaptic plasticity and learning and memory. In trying to delineate the roles of individual HDACs, genetic tools have been used to manipulate HDAC expression in rodents, uncovering distinct contributions of individual HDACs in regulating the processes of memory formation. Moreover, recent findings have suggested an important role for HDAC inhibitors in enhancing learning and memory processes as well as ameliorating symptoms related to neurodegenerative diseases. In this review, we focus on the role of HDACs in learning and memory, as well as significant data emerging from the field in support of HDAC inhibitors as potential therapeutic targets for the treatment of cognitive disorders.

Loss of histone deacetylase 2 improves working memory and accelerates extinction learning.

Histone acetylation and deacetylation can be dynamically regulated in response to environmental stimuli and play important roles in learning and memory. Pharmacological inhibition of histone deacetylases (HDACs) improves performance in learning tasks; however, many of these classical agents are "pan-HDAC" inhibitors, and their use makes it difficult to determine the roles of specific HDACs in cognitive function. We took a genetic approach using mice lacking the class I HDACs, HDAC1 or HDAC2, in postmitotic forebrain neurons to investigate the specificity or functional redundancy of these HDACs in learning and synaptic plasticity. We show that selective knock-out of Hdac2 led to a robust acceleration of the extinction rate of conditioned fear responses and a conditioned taste aversion as well as enhanced performance in an attentional set-shifting task. Hdac2 knock-out had no impact on episodic memory or motor learning, suggesting that the effects are task-dependent, with the predominant impact of HDAC2 inhibition being an enhancement in an animal's ability to rapidly adapt its behavioral strategy as a result of changes in associative contingencies. Our results demonstrate that the loss of HDAC2 improves associative learning, with no effect in nonassociative learning tasks, suggesting a specific role for HDAC2 in particular types of learning. HDAC2 may be an intriguing target for cognitive and psychiatric disorders that are characterized by an inability to inhibit behavioral responsiveness to maladaptive or no longer relevant associations.

Acute suppression of spontaneous neurotransmission drives synaptic potentiation.

The impact of spontaneous neurotransmission on neuronal plasticity remains poorly understood. Here, we show that acute suppression of spontaneous NMDA receptor-mediated (NMDAR-mediated) neurotransmission potentiates synaptic responses in the CA1 regions of rat and mouse hippocampus. This potentiation requires protein synthesis, brain-derived neurotrophic factor expression, eukaryotic elongation factor-2 kinase function, and increased surface expression of AMPA receptors. Our behavioral studies link this same synaptic signaling pathway to the fast-acting antidepressant responses elicited by ketamine. We also show that selective neurotransmitter depletion from spontaneously recycling vesicles triggers synaptic potentiation via the same pathway as NMDAR blockade, demonstrating that presynaptic impairment of spontaneous release, without manipulation of evoked neurotransmission, is sufficient to elicit postsynaptic plasticity. These findings uncover an unexpectedly dynamic impact of spontaneous glutamate release on synaptic efficacy and provide new insight into a key synaptic substrate for rapid antidepressant action.

Selective role for DNMT3a in learning and memory.

Methylation of cytosine nucleotides is governed by DNA methyltransferases (DNMTs) that establish de novo DNA methylation patterns in early embryonic development (e.g., DNMT3a and DNMT3b) or maintain those patterns on hemimethylated DNA in dividing cells (e.g., DNMT1). DNMTs continue to be expressed at high levels in mature neurons, however their impact on neuronal function and behavior are unclear. To address this issue we examined DNMT1 and DNMT3a expression following associative learning. We also generated forebrain specific conditional Dnmt1 or Dnmt3a knockout mice and characterized them in learning and memory paradigms as well as for alterations in long-term potentiation (LTP) and synaptic plasticity. Here, we report that experience in an associative learning task impacts expression of Dnmt3a, but not Dnmt1, in brain areas that mediate learning of this task. We also found that Dnmt3a knockout mice, and not Dnmt1 knockouts have synaptic alterations as well as learning deficits on several associative and episodic memory tasks. These findings indicate that the de novo DNA methylating enzyme DNMT3a in postmitotic neurons is necessary for normal memory formation and its function cannot be substituted by the maintenance DNA methylating enzyme DNMT1.

Ketamine induced synaptic plasticity operates independently of long-term potentiation.

Synaptic plasticity occurs via multiple mechanisms to regulate synaptic efficacy. Homeostatic and Hebbian plasticity are two such mechanisms by which neuronal synapses can be altered. Although these two processes are mechanistically distinct, they converge on downstream regulation of AMPA receptor activity to modify glutamatergic neurotransmission. However, much remains to be explored regarding how these two prominent forms of plasticity interact. Ketamine, a rapidly acting antidepressant, increases glutamatergic transmission via pharmacologically-induced homeostatic plasticity. Here, we demonstrate that Hebbian plasticity mechanisms are still intact in synapses that have undergone homeostatic scaling by ketamine after either systemic injection or perfusion onto hippocampal brain slices. We also investigated this relationship in the context of stress induced by chronic exposure to corticosterone (CORT) to better model the circumstances under which ketamine may be used as an antidepressant. We found that CORT induced an anhedonia-like behavioral phenotype in mice but did not impair long-term potentiation (LTP) induction. Furthermore, corticosterone exposure does not impact the intersection of homeostatic and Hebbian plasticity mechanisms, as synapses from CORT-exposed mice also demonstrated intact ketamine-induced plasticity and LTP in succession. These results provide a mechanistic explanation for how ketamine used for the treatment of depression does not impair the integrity of learning and memory processes encoded by mechanisms such as LTP.

Ketamine and rapid antidepressant action: new treatments and novel synaptic signaling mechanisms.

Ketamine is an open channel blocker of ionotropic glutamatergic N-Methyl-D-Aspartate (NMDA) receptors. The discovery of its rapid antidepressant effects in patients with depression and treatment-resistant depression fostered novel effective treatments for mood disorders. This discovery not only provided new insight into the neurobiology of mood disorders but also uncovered fundamental synaptic plasticity mechanisms that underlie its treatment. In this review, we discuss key clinical aspects of ketamine's effect as a rapidly acting antidepressant, synaptic and circuit mechanisms underlying its action, as well as how these novel perspectives in clinical practice and synapse biology form a road map for future studies aimed at more effective treatments for neuropsychiatric disorders.