Activation of a Locus Coeruleus to Dorsal Hippocampus Noradrenergic Circuit Facilitates Associative Learning.

Processing of contextual information during a new episodic event is crucial for learning and memory. Neuromodulation in the hippocampus and prefrontal cortex plays an important role in the formation of associations between environmental cues and an aversive experience. Noradrenergic neurons in the locus coeruleus send dense projections to both regions, but their contribution to contextual associative learning has not been established. Here, we utilize selective optogenetic and pharmacological manipulations to control noradrenergic transmission in the hippocampus during the encoding of a contextual fear memory. We find that boosting noradrenergic terminal release in the dorsal CA1 enhances the acquisition of contextual associative learning and that this effect requires local activation of ß-adrenenergic receptors. Moreover, we show that increasing norepinephrine release can ameliorate contextual fear learning impairments caused by dopaminergic dysregulation in the hippocampus. Our data suggest that increasing of hippocampal noradrenergic activity can have important implications in the treatment of cognitive disorders that involve problems in contextual processing.

Dopaminergic regulation of hippocampal plasticity, learning, and memory.

The hippocampus is responsible for encoding behavioral episodes into short-term and long-term memory. The circuits that mediate these processes are subject to neuromodulation, which involves regulation of synaptic plasticity and local neuronal excitability. In this review, we present evidence to demonstrate the influence of dopaminergic neuromodulation on hippocampus-dependent memory, and we address the controversy surrounding the source of dopamine innervation. First, we summarize historical and recent retrograde and anterograde anatomical tracing studies of direct dopaminergic projections from the ventral tegmental area and discuss dopamine release from the adrenergic locus coeruleus. Then, we present evidence of dopaminergic modulation of synaptic plasticity in the hippocampus. Plasticity mechanisms are examined in brain slices and in recordings from in vivo neuronal populations in freely moving rodents. Finally, we review pharmacological, genetic, and circuitry research that demonstrates the importance of dopamine release for learning and memory tasks while dissociating anatomically distinct populations of direct dopaminergic inputs.

Midbrain dopaminergic innervation of the hippocampus is sufficient to modulate formation of aversive memories.

Aversive memories are important for survival, and dopaminergic signaling in the hippocampus has been implicated in aversive learning. However, the source and mode of action of hippocampal dopamine remain controversial. Here, we utilize anterograde and retrograde viral tracing methods to label midbrain dopaminergic projections to the dorsal hippocampus. We identify a population of midbrain dopaminergic neurons near the border of the substantia nigra pars compacta and the lateral ventral tegmental area that sends direct projections to the dorsal hippocampus. Using optogenetic manipulations and mutant mice to control dopamine transmission in the hippocampus, we show that midbrain dopamine potently modulates aversive memory formation during encoding of contextual fear. Moreover, we demonstrate that dopaminergic transmission in the dorsal CA1 is required for the acquisition of contextual fear memories, and that this acquisition is sustained in the absence of catecholamine release from noradrenergic terminals. Our findings identify a cluster of midbrain dopamine neurons that innervate the hippocampus and show that the midbrain dopamine neuromodulation in the dorsal hippocampus is sufficient to maintain aversive memory formation.

Fear conditioning potentiates the hippocampal CA1 commissural pathway in vivo and increases awake phase sleep.

The hippocampus is essential for spatial learning and memory. To assess learning we used contextual fear conditioning (cFC), where animals learn to associate a place with aversive events like foot-shocks. Candidate memory mechanisms for cFC are long-term potentiation (LTP) and long-term depression (LTD), but there is little direct evidence of them operating in the hippocampus in vivo following cFC. Also, little is known about the behavioral state changes induced by cFC. To address these issues, we recorded local field potentials in freely behaving mice by stimulating in the left dorsal CA1 region and recording in the right dorsal CA1 region. Synaptic strength in the commissural pathway was monitored by measuring field excitatory postsynaptic potentials (fEPSPs) before and after cFC. After cFC, the commissural pathway's synaptic strength was potentiated. Although recordings occurred during the wake phase of the light/dark cycle, the mice slept more in the post-conditioning period than in the pre-conditioning period. Relative to awake periods, in non-rapid eye movement (NREM) sleep the fEPSPs were larger in both pre- and post-conditioning periods. We also found a significant negative correlation between the animal's speed and fEPSP size. Therefore, to avoid confounds in the fEFSP potentiation estimates, we controlled for speed-related and sleep-related fEPSP changes and still found that cFC induced long-term potentiation, but no significant long-term depression. Synaptic strength changes were not found in the control group that simply explored the fear-conditioning chamber, indicating that exploration of the novel place did not produce the measurable effects caused by cFC. These results show that following cFC, the CA1 commissural pathway is potentiated, likely contributing to the functional integration of the left and right hippocampi in fear memory consolidation. In addition, the cFC paradigm produces significant changes in an animal's behavioral state, which are observable as proximal changes in sleep patterns.

Synaptic neurexin-1 assembles into dynamically regulated active zone nanoclusters.

Neurexins are well-characterized presynaptic cell adhesion molecules that engage multifarious postsynaptic ligands and organize diverse synapse properties. However, the precise synaptic localization of neurexins remains enigmatic. Using super-resolution microscopy, we demonstrate that neurexin-1 forms discrete nanoclusters at excitatory synapses, revealing a novel organizational feature of synaptic architecture. Synapses generally contain a single nanocluster that comprises more than four neurexin-1 molecules and that also includes neurexin-2 and/or neurexin-3 isoforms. Moreover, we find that neurexin-1 is physiologically cleaved by ADAM10 similar to its ligand neuroligin-1, with ∼4-6% of neurexin-1 and ∼2-3% of neuroligin-1 present in the adult brain as soluble ectodomain proteins. Blocking ADAM10-mediated neurexin-1 cleavage dramatically increased the synaptic neurexin-1 content, thereby elevating the percentage of Homer1(+) excitatory synapses containing neurexin-1 nanoclusters from 40-50% to ∼80%, and doubling the number of neurexin-1 molecules per nanocluster. Taken together, our results reveal an unexpected nanodomain organization of synapses in which neurexin-1 is assembled into discrete presynaptic nanoclusters that are dynamically regulated via ectodomain cleavage.

Postsynaptic adhesion GPCR latrophilin-2 mediates target recognition in entorhinal-hippocampal synapse assembly.

Synapse assembly likely requires postsynaptic target recognition by incoming presynaptic afferents. Using newly generated conditional knock-in and knockout mice, we show in this study that latrophilin-2 (Lphn2), a cell-adhesion G protein-coupled receptor and presumptive α-latrotoxin receptor, controls the numbers of a specific subset of synapses in CA1-region hippocampal neurons, suggesting that Lphn2 acts as a synaptic target-recognition molecule. In cultured hippocampal neurons, Lphn2 maintained synapse numbers via a postsynaptic instead of a presynaptic mechanism, which was surprising given its presumptive role as an α-latrotoxin receptor. In CA1-region neurons in vivo, Lphn2 was specifically targeted to dendritic spines in the stratum lacunosum-moleculare, which form synapses with presynaptic entorhinal cortex afferents. In this study, postsynaptic deletion of Lphn2 selectively decreased spine numbers and impaired synaptic inputs from entorhinal but not Schaffer-collateral afferents. Behaviorally, loss of Lphn2 from the CA1 region increased spatial memory retention but decreased learning of sequential spatial memory tasks. Thus, Lphn2 appears to control synapse numbers in the entorhinal cortex/CA1 region circuit by acting as a domain-specific postsynaptic target-recognition molecule.

Monitoring Synapses Via Trans-Synaptic GFP Complementation.

Over the last years, the analysis of synaptic connectivity in the mammalian brain has been accelerated by the use of techniques combining electrophysiology, light microscopy, viral tracing, and genetic manipulations in animal models. Of particular interest are methods that aim to label synapses by tethering complementary split GFP fragments in opposing sites of the synaptic cleft. Here, I describe SynView, a method for monitoring synapse formation based on GFP complementation, and provide a detailed protocol for use in neuronal cultures from mouse hippocampus.

Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories.

Dopamine Regulates Aversive Contextual Learning and Associated In Vivo Synaptic Plasticity in the Hippocampus.

Dopamine release during reward-driven behaviors influences synaptic plasticity. However, dopamine innervation and release in the hippocampus and its role during aversive behaviors are controversial. Here, we show that in vivo hippocampal synaptic plasticity in the CA3-CA1 circuit underlies contextual learning during inhibitory avoidance (IA) training. Immunohistochemistry and molecular techniques verified sparse dopaminergic innervation of the hippocampus from the midbrain. The long-term synaptic potentiation (LTP) underlying the learning of IA was assessed with a D1-like dopamine receptor agonist or antagonist in ex vivo hippocampal slices and in vivo in freely moving mice. Inhibition of D1-like dopamine receptors impaired memory of the IA task and prevented the training-induced enhancement of both ex vivo and in vivo LTP induction. The results indicate that dopamine-receptor signaling during an aversive contextual task regulates aversive memory retention and regulates associated synaptic mechanisms in the hippocampus that likely underlie learning.

Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation.

Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1ß and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1ß to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1ß and neuroligin-1 if and after neurexin-1ß bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1ß and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1ß forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

Rab3B protein is required for long-term depression of hippocampal inhibitory synapses and for normal reversal learning.

Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1α and RIM2α as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1α are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1α is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.

A neural switch for active and passive fear.

The central nucleus of the amygdala (CeA) serves as a major output of this structure and plays a critical role in the expression of conditioned fear. By combining cell- and tissue-specific pharmacogenetic inhibition with functional magnetic resonance imaging (fMRI), we identified circuits downstream of CeA that control fear expression in mice. Selective inhibition of a subset of neurons in CeA led to decreased conditioned freezing behavior and increased cortical arousal as visualized by fMRI. Correlation analysis of fMRI signals identified functional connectivity between CeA, cholinergic forebrain nuclei, and activated cortical structures, and cortical arousal was blocked by cholinergic antagonists. Importantly, inhibition of these neurons switched behavioral responses to the fear stimulus from passive to active responses. Our findings identify a neural circuit in CeA that biases fear responses toward either passive or active coping strategies.

Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro.

Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we show that maintenance of continuous presynaptic SNARE-complex assembly required a nonclassical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) and promoted SNARE-complex assembly. Moreover, triple-knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and died prematurely. Thus, synucleins may function to sustain normal SNARE-complex assembly in a presynaptic terminal during aging.

GABA homeostasis contributes to the developmental programming of anxiety-related behavior.

During development, when inhibitory and excitatory synapses are formed and refined, homeostatic mechanisms act to adjust inhibitory input in order to maintain neural activity within a normal range. As the brain matures, synaptogenesis slows and a relatively stable level of inhibition is achieved. Deficits in inhibitory neurotransmission are associated with increased anxiety-related behavior and drugs that potentiate GABA function, the major inhibitory neurotransmitter in the brain, are effective anxiolytics. These observations raise the possibility that transient perturbations in the activity of neural circuits during development might induce compensatory changes in inhibition that could persist into adulthood and contribute to changes in anxiety-related behavior. To test this hypothesis, we treated mice continuously during the major period of forebrain synaptogenesis (P14-28) with the GABA-A receptor positive modulator diazepam and assessed anxiety-related behavior in adulthood. Control experiments confirmed anxiolytic effects of the drug following one day of treatment and the development of tolerance following two weeks of treatment. When tested in adulthood, one month after the end of treatment, diazepam-treated mice exhibited significantly increased behavioral inhibition in the open-field, elevated-plus maze, and novel object behavioral paradigms. Levels of benzodiazepine binding sites in amygdala and frontal cortex were specifically decreased in diazepam-treated mice demonstrating that homeostatic adjustments in GABA function persist into adulthood. Our results show that increased GABAergic activity can affect the developmental programming of anxiety-related behavior.

Suppression of conditioning to ambiguous cues by pharmacogenetic inhibition of the dentate gyrus.

Serotonin receptor 1A knockout (Htr1a(KO)) mice show increased anxiety-related behavior in tests measuring innate avoidance. Here we demonstrate that Htr1a(KO) mice show enhanced fear conditioning to ambiguous conditioned stimuli, a hallmark of human anxiety. To examine the involvement of specific forebrain circuits in this phenotype, we developed a pharmacogenetic technique for the rapid tissue- and cell type-specific silencing of neural activity in vivo. Inhibition of neurons in the central nucleus of the amygdala suppressed conditioned responses to both ambiguous and nonambiguous cues. In contrast, inhibition of hippocampal dentate gyrus granule cells selectively suppressed conditioned responses to ambiguous cues and reversed the knockout phenotype. These data demonstrate that Htr1a(KO) mice have a bias in the processing of threatening cues that is moderated by hippocampal mossy-fiber circuits, and suggest that the hippocampus is important in the response to ambiguous aversive stimuli.

Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation.

Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS. The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244). Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6. A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production. Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism. Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.

Emerging interest in the kallikrein gene family for understanding and diagnosing cancer.

Kallikreins are proteolytic enzymes that constitute a subfamily of serine proteases. Novel kallikrein genes were cloned recently, and it was shown that the human kallikrein family contains 15 genes tandemly aligned on chromosomal locus 19q13.3-q13.4. Based on their altered expression in tumor cells, kallikreins may be involved in the pathogenesis and/or progression of cancer. Evidence is presented that certain kallikreins may be exploited as diagnostic cancer biomarkers. Although the function(s) of novel kallikreins is currently unknown, increasing evidence suggests that kallikreins may participate in regulatory enzymatic cascade(s). Elucidation of the function of novel kallikreins largely depends on the availability of active recombinant proteins. Here, the zymogen for kallikrein 13 was overexpressed in Pichia pastoris and biochemically characterized. It was shown that the kallikrein 13 zymogen displays intrinsic catalytic activity leading to autoactivation. A clipped form of kallikrein 13 was identified, indicating autocatalytic cleavage at the internal bond R114-S115. Mature kallikrein 13 displays trypsin-like activity with restricted specificity on synthetic and protein substrates. Combinatorial P1-Lys libraries of tetrapeptide fluorogenic substrates were synthesized and used for the profiling of the P2 specificity of selected kallikreins. Interestingly, it was shown that human kallikrein 13, similarly to PSA, could specifically cleave human plasminogen to generate angiostatin-like fragments, suggesting that specific kallikreins may have antiangiogenic actions. An understanding of the physiology of human kallikreins is emerging with potential clinical applications.

The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family.

Zyme/protease M/neurosin/human kallikrein 6 (hK6) is a member of the human kallikrein family of trypsin-like serine proteinases and was originally identified as being down-regulated in metastatic breast and ovarian tumors when compared with corresponding primary tumors. Recent evidence suggests that hK6 may serve as a circulating tumor marker in ovarian cancers. In addition, it was described in the brain of Parkinson's disease and Alzheimer's disease patients, where it is implicated in amyloid precursor protein processing. It is thus a biomarker for these diseases. To examine the mechanism of activation of hK6, we have solved the structure of its proform, the first of a human kallikrein family member. The proenzyme displays a fold that exhibits chimeric features between those of trypsinogen and other family members. It lacks the characteristic "kallikrein loop" and forms the six disulfide bridges of trypsin. Pro-hK6 displays a completely closed specificity pocket and a unique conformation of the regions involved in structural rearrangements upon proteolytic cleavage activation. This points to a novel activation mechanism, which could be extrapolated to other human kallikreins.