Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.

Cortical circuit activity underlying sleep slow oscillations and spindles.

Slow oscillations and sleep spindles are hallmarks of the EEG during slow-wave sleep (SWS). Both oscillatory events, especially when co-occurring in the constellation of spindles nesting in the slow oscillation upstate, are considered to support memory formation and underlying synaptic plasticity. The regulatory mechanisms of this function at the circuit level are poorly understood. Here, using two-photon imaging in mice, we relate EEG-recorded slow oscillations and spindles to calcium signals recorded from the soma of cortical putative pyramidal-like (Pyr) cells and neighboring parvalbumin-positive interneurons (PV-Ins) or somatostatin-positive interneurons (SOM-Ins). Pyr calcium activity was increased more than threefold when spindles co-occurred with slow oscillation upstates compared with slow oscillations or spindles occurring in isolation. Independent of whether or not a spindle was nested in the slow oscillation upstate, the slow oscillation downstate was preceded by enhanced calcium signal in SOM-Ins that vanished during the upstate, whereas spindles were associated with strongly increased PV-In calcium activity. Additional wide-field calcium imaging of Pyr cells confirmed the enhanced calcium activity and its widespread topography associated with spindles nested in slow oscillation upstates. In conclusion, when spindles are nested in slow oscillation upstates, maximum Pyr activity appears to concur with strong perisomatic inhibition of Pyr cells via PV-Ins and low dendritic inhibition via SOM-Ins (i.e., conditions that might optimize synaptic plasticity within local cortical circuits).

Amygdala interneuron subtypes control fear learning through disinhibition.

Learning is mediated by experience-dependent plasticity in neuronal circuits. Activity in neuronal circuits is tightly regulated by different subtypes of inhibitory interneurons, yet their role in learning is poorly understood. Using a combination of in vivo single-unit recordings and optogenetic manipulations, we show that in the mouse basolateral amygdala, interneurons expressing parvalbumin (PV) and somatostatin (SOM) bidirectionally control the acquisition of fear conditioning--a simple form of associative learning--through two distinct disinhibitory mechanisms. During an auditory cue, PV(+) interneurons are excited and indirectly disinhibit the dendrites of basolateral amygdala principal neurons via SOM(+) interneurons, thereby enhancing auditory responses and promoting cue-shock associations. During an aversive footshock, however, both PV(+) and SOM(+) interneurons are inhibited, which boosts postsynaptic footshock responses and gates learning. These results demonstrate that associative learning is dynamically regulated by the stimulus-specific activation of distinct disinhibitory microcircuits through precise interactions between different subtypes of local interneurons.

Sleep supports cued fear extinction memory consolidation independent of circadian phase.

Sleep promotes memory, particularly for declarative learning. However, its role in non-declarative, emotional memories is less well understood. Some studies suggest that sleep may influence fear-related memories, and thus may be an important factor determining the outcome of treatments for emotional disorders such as post-traumatic stress disorder. Here, we investigated the effect of sleep deprivation and time of day on fear extinction memory consolidation. Mice were subjected to a cued Pavlovian fear and extinction paradigm at the beginning of their resting or active phase. Immediate post-extinction learning sleep deprivation for 5h compromised extinction memory when tested 24h after learning. Context-dependent extinction memory recall was completely prevented by sleep-manipulation during the resting phase, while impairment was milder during the active phase and extinction memory retained its context-specificity. Importantly, control experiments excluded confounding factors such as differences in baseline locomotion, fear generalization and stress hormone levels. Together, our findings indicate that post-learning sleep supports cued fear extinction memory consolidation in both circadian phases. The lack of correlation between memory efficacy and sleep time suggests that extinction memory may be influenced by specific sleep events in the early consolidation period.

Encoding of conditioned fear in central amygdala inhibitory circuits.

The central amygdala (CEA), a nucleus predominantly composed of GABAergic inhibitory neurons, is essential for fear conditioning. How the acquisition and expression of conditioned fear are encoded within CEA inhibitory circuits is not understood. Using in vivo electrophysiological, optogenetic and pharmacological approaches in mice, we show that neuronal activity in the lateral subdivision of the central amygdala (CEl) is required for fear acquisition, whereas conditioned fear responses are driven by output neurons in the medial subdivision (CEm). Functional circuit analysis revealed that inhibitory CEA microcircuits are highly organized and that cell-type-specific plasticity of phasic and tonic activity in the CEl to CEm pathway may gate fear expression and regulate fear generalization. Our results define the functional architecture of CEA microcircuits and their role in the acquisition and regulation of conditioned fear behaviour.

Postnatal maturation of GABAergic modulation of sensory inputs onto lateral amygdala principal neurons.

KEY POINTS: Throughout life, fear learning is indispensable for survival and neural plasticity in the lateral amygdala underlies this learning and storage of fear memories. During development, properties of fear learning continue to change into adulthood, but currently little is known about changes in amygdala circuits that enable these behavioural transitions. In recordings from neurons in lateral amygdala brain slices from infant up to adult mice, we show that spontaneous and evoked excitatory and inhibitory synaptic transmissions mature into adolescence. At this time, increased inhibitory activity and signalling has the ability to restrict the function of excitation by presynaptic modulation, and may thus enable precise stimulus associations to limit fear generalization from adolescence onward. Our results provide a basis for addressing plasticity mechanisms that underlie altered fear behaviour in young animals. ABSTRACT: Convergent evidence suggests that plasticity in the lateral amygdala (LA) participates in acquisition and storage of fear memory. Sensory inputs from thalamic and cortical areas activate principal neurons and local GABAergic interneurons, which provide feed-forward inhibition that tightly controls LA activity and plasticity via pre- and postsynaptic GABAA and GABAB receptors. GABAergic control is also critical during fear expression, generalization and extinction in adult animals. During rodent development, properties of fear and extinction learning continue to change into early adulthood. Currently, few studies have assessed physiological changes in amygdala circuits that may enable these behavioural transitions. To obtain first insights, we investigated changes in spontaneous and sensory input-evoked inhibition onto LA principal neurons and then focused on GABAB receptor-mediated modulation of excitatory sensory inputs in infant, juvenile, adolescent and young adult mice. We found that spontaneous and sensory-evoked inhibition increased during development. Physiological changes were accompanied by changes in dendritic morphology. While GABAB heteroreceptors were functionally expressed on sensory afferents already early in development, they could only be physiologically recruited by sensory-evoked GABA release to mediate heterosynaptic inhibition from adolescence onward. Furthermore, we found GABAB -mediated tonic inhibition of sensory inputs by ambient GABA that also emerged in adolescence. The observed increase in GABAergic drive may be a substrate for providing modulatory GABA. Our data suggest that GABAB -mediated tonic and evoked presynaptic inhibition can suppress sensory input-driven excitation in the LA to enable precise stimulus associations and limit generalization of conditioned fear from adolescence onward.

Limits of the detection of microplastics in fish tissue using stimulated Raman scattering microscopy.

We demonstrate the detection sensitivity of microplastic beads within fish tissue using stimulated Raman scattering (SRS) microscopy. The intrinsically provided chemical contrast distinguishes different types of plastic compounds within fish tissue. We study the size-dependent signal-to-noise ratio of the microplastic beads and determine a lower boundary for the detectable size. Our findings demonstrate how SRS microscopy can serve as a complementary modality to conventional Raman scattering imaging in order to detect and identify microplastic particles in fish tissue.

Disrupting 5-HT(2A) receptor/PDZ protein interactions reduces hyperalgesia and enhances SSRI efficacy in neuropathic pain.

Antidepressants are one of the first-line treatments for neuropathic pain. Despite the influence of serotonin (5-hydroxytryptamine, 5-HT) in pain modulation, selective serotonin reuptake inhibitors (SSRIs) are less effective than tricyclic antidepressants. Here, we show, in diabetic neuropathic rats, an alteration of the antihyperalgesic effect induced by stimulation of 5-HT(2A) receptors, which are known to mediate SSRI-induced analgesia. 5-HT(2A) receptor density was not changed in the spinal cord of diabetic rats, whereas postsynaptic density protein-95 (PSD-95), one of the PSD-95/disc large suppressor/zonula occludens-1 (PDZ) domain containing proteins interacting with these receptors, was upregulated. Intrathecal injection of a cell-penetrating peptidyl mimetic of the 5-HT(2A) receptor C-terminus, which disrupts 5-HT(2A) receptor-PDZ protein interactions, induced an antihyperalgesic effect in diabetic rats, which results from activation of 5-HT(2A) receptors by endogenous 5-HT. The peptide also enhanced antihyperalgesia induced by the SSRI fluoxetine. Its effects likely resulted from an increase in receptor responsiveness, because it revealed functional 5-HT(2A) receptor-operated Ca(2+) responses in neurons, an effect mimicked by knockdown of PSD-95. Hence, 5-HT(2A) receptor/PDZ protein interactions might contribute to the resistance to SSRI-induced analgesia in painful diabetic neuropathy. Disruption of these interactions might be a valuable strategy to design novel treatments for neuropathic pain and to increase the effectiveness of SSRIs.

Midbrain dopaminergic inputs gate amygdala intercalated cell clusters by distinct and cooperative mechanisms in male mice.

Dopaminergic signaling plays an important role in associative learning, including fear and extinction learning. Dopaminergic midbrain neurons encode prediction error-like signals when threats differ from expectations. Within the amygdala, GABAergic intercalated cell (ITC) clusters receive one of the densest dopaminergic projections, but their physiological consequences are incompletely understood. ITCs are important for fear extinction, a function thought to be supported by activation of ventromedial ITCs that inhibit central amygdala fear output. In mice, we reveal two distinct novel mechanisms by which mesencephalic dopaminergic afferents control ITCs. Firstly, they co-release GABA to mediate rapid, direct inhibition. Secondly, dopamine suppresses inhibitory interactions between distinct ITC clusters via presynaptic D1 receptors. Early extinction training augments both GABA co-release onto dorsomedial ITCs and dopamine-mediated suppression of dorso- to ventromedial inhibition between ITC clusters. These findings provide novel insights into dopaminergic mechanisms shaping the activity balance between distinct ITC clusters that could support their opposing roles in fear behavior.

Fear Memory Retrieval Is Associated With a Reduction in AMPA Receptor Density at Thalamic to Amygdala Intercalated Cell Synapses.

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cell masses (ITC) are tight clusters of GABAergic neurons, which are distributed around the basolateral amygdala complex. Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsal medio-paracapsular cluster. Here, we investigate the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses and, in particular, whether specific changes in the synaptic density of AMPA receptors underlie the observed change. To this aim, we used a detergent-digested freeze-fracture replica immunolabeling technique (FRIL) approach that enables to visualize the spatial distribution of intrasynaptic AMPA receptors at high resolution. AMPA receptors were detected using an antibody raised against an epitope common to all AMPA subunits. To visualize thalamic inputs, we virally transduced the posterior thalamic complex with Channelrhodopsin 2-YFP, which is anterogradely transported along axons. Using face-matched replica, we confirmed that the postsynaptic elements were ITC neurons due to their prominent expression of µ-opioid receptors. With this approach, we show that, following auditory fear conditioning in mice, the formation and retrieval of fear memory is linked to a significant reduction in the density of AMPA receptors, particularly at spine synapses formed by inputs of the posterior intralaminar thalamic and medial geniculate nuclei onto identified ITC neurons. Our study is one of the few that has directly linked the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks, by showing that fear-memory induced reduction in AMPA/NMDA ratio at thalamic-ITC synapses is associated with a reduced postsynaptic AMPA receptor density.

Short-term high-fat feeding induces a reversible net decrease in synaptic AMPA receptors in the hypothalamus.

Dietary obesity compromises brain function, but the effects of high-fat food on synaptic transmission in hypothalamic networks, as well as their potential reversibility, are yet to be fully characterized. We investigated the impact of high-fat feeding on a hallmark of synaptic plasticity, i.e., the expression of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) that contain the subunits GluA1 and GluA2, in hypothalamic and cortical synaptoneurosomes of male rats. In the main experiment (experiment 1), three days, but not one day of high-fat diet (HFD) decreased the levels of AMPAR GluA1 and GluA2 subunits, as well as GluA1 phosphorylation at Ser845, in hypothalamus but not cortex. In experiment 2, we compared the effects of the three-day HFD with those a three-day HFD followed by four recovery days of normal chow. This experiment corroborated the suppressive effect of high-fat feeding on hypothalamic but not cortical AMPAR GluA1, GluA2, and GluA1 phosphorylation at Ser845, and indicated that the effects are reversed by normal-chow feeding. High-fat feeding generally increased energy intake, body weight, and serum concentrations of insulin, leptin, free fatty acids, and corticosterone; only the three-day HFD increased wakefulness assessed via video analysis. Results indicate a reversible down-regulation of hypothalamic glutamatergic synaptic strength in response to short-term high-fat feeding. Preceding the manifestation of obesity, this rapid change in glutamatergic neurotransmission may underlie counter-regulatory efforts to prevent excess body weight gain, and therefore, represent a new target of interventions to improve metabolic control.

Neuronal circuits of fear extinction.

Fear extinction is a form of inhibitory learning that allows for the adaptive control of conditioned fear responses. Although fear extinction is an active learning process that eventually leads to the formation of a consolidated extinction memory, it is a fragile behavioural state. Fear responses can recover spontaneously or subsequent to environmental influences, such as context changes or stress. Understanding the neuronal substrates of fear extinction is of tremendous clinical relevance, as extinction is the cornerstone of psychological therapy of several anxiety disorders and because the relapse of maladaptative fear and anxiety is a major clinical problem. Recent research has begun to shed light on the molecular and cellular processes underlying fear extinction. In particular, the acquisition, consolidation and expression of extinction memories are thought to be mediated by highly specific neuronal circuits embedded in a large-scale brain network including the amygdala, prefrontal cortex, hippocampus and brain stem. Moreover, recent findings indicate that the neuronal circuitry of extinction is developmentally regulated. Here, we review emerging concepts of the neuronal circuitry of fear extinction, and highlight novel findings suggesting that the fragile phenomenon of extinction can be converted into a permanent erasure of fear memories. Finally, we discuss how research on genetic animal models of impaired extinction can further our understanding of the molecular and genetic bases of human anxiety disorders.

Studying Neuronal Function Ex Vivo Using Optogenetic Stimulation and Patch Clamp.

Optogenetics has become a key method to interrogate the function of neural populations and circuits in the brain. This technique combines the targeted expression of light-activated proteins with subsequent manipulation of neural activity by light. Opsins such as Channelrhodopsin-2 (ChR2), which is a light-gated cation-channel, can be fused to or coexpressed with fluorescent proteins to allow for visualization and concurrent activation of neurons and their axonal projections. Via stereotaxic delivery of viral vectors, ChR2 can be constitutively or conditionally expressed in specific neurons in defined brain regions. Subsequently, identified axonal projections can be studied functionally ex vivo in combination with patch-clamp recordings in brain slices. This optogenetic mapping of neural circuitry has enabled the identification and characterization of novel synaptic connections and the detailed investigation of known anatomical connections previously not amenable with electrical stimulation techniques. Here, we describe a protocol for investigating functional properties of local and long-range connectivity in the brain using blue-light activated ChR2 variants and whole-cell patch-clamp recordings in acute brain slices.

Mistletoe-Based Drugs Work in Synergy with Radio-Chemotherapy in the Treatment of Glioma In Vitro and In Vivo in Glioblastoma Bearing Mice.

BACKGROUND: Extracts from Viscum album L. (VE) are used in the complementary cancer therapy in Europe for decades. VE contain several compounds like the mistletoe lectins (MLs) 1-3 and viscotoxins and also several minor ingredients. Since mistletoe lectin 1 (ML-1) has been described as the main component of VE harboring antitumor activity, purified native or recombinant ML-1 has been recently used in clinical trials. MLs stimulate the immune system, induce cytotoxicity, are able to modify the expression of cancer-associated genes, and influence the proliferation and motility of tumor cells. OBJECTIVE: In this study our goal was to determine anticancer effects of the VE ISCADOR Qu, of recombinant ML-1 (Aviscumine), and of native ML-1 in the treatment of glioblastoma (GBM), the most common and highly malignant brain tumor in adults. Additionally we were interested whether these drugs, used in combination with a temozolomide-(TMZ)-based radio-chemotherapy, provide synergistic effects. METHODS: Cell culture assays, ex vivo murine hippocampal brain slice cultures, human GBM cryosections, and a xenograft orthotopic glioblastoma mouse model were used. RESULTS: In cells, the expression of the ML receptor CD75s, which is also expressed in GBM specimen, but not in normal brain, correlates with the drug-induced cytotoxicity. In GBM cells, the drugs induce cell death in a concentration-dependent manner and reduce cell growth by inducing cell cycle arrest in the G2/M phase. The cell cycle arrest was paralleled by modifications in the expression of cell cycle regulating genes. ML containing drugs, if combined with glioma standard therapy, provide synergistic and additive anticancer effects. Despite not reaching statistical significance, a single intratumoral application of Aviscumine prolonged the median survival of GBM mice longer than tumor irradiation. Moreover, intratumorally applied Aviscumine prolonged the survival of GBM-bearing mice if used in combination with irradiation and TMZ for further 6.5 days compared to the radio-chemotherapy. CONCLUSION: Our results suggest that an adjuvant treatment of glioma patients with ML-containing drugs might be beneficial.

In Vivo Regulation of Oligodendrocyte Precursor Cell Proliferation and Differentiation by the AMPA-Receptor Subunit GluA2.

The functional role of AMPA receptor (AMPAR)-mediated synaptic signaling between neurons and oligodendrocyte precursor cells (OPCs) remains enigmatic. We modified the properties of AMPARs at axon-OPC synapses in the mouse corpus callosum in vivo during the peak of myelination by targeting the GluA2 subunit. Expression of the unedited (Ca2+ permeable) or the pore-dead GluA2 subunit of AMPARs triggered proliferation of OPCs and reduced their differentiation into oligodendrocytes. Expression of the cytoplasmic C-terminal (GluA2(813-862)) of the GluA2 subunit (C-tail), a modification designed to affect the interaction between GluA2 and AMPAR-binding proteins and to perturb trafficking of GluA2-containing AMPARs, decreased the differentiation of OPCs without affecting their proliferation. These findings suggest that ionotropic and non-ionotropic properties of AMPARs in OPCs, as well as specific aspects of AMPAR-mediated signaling at axon-OPC synapses in the mouse corpus callosum, are important for balancing the response of OPCs to proliferation and differentiation cues.

Environmental Enrichment Prevents Transcriptional Disturbances Induced by Alpha-Synuclein Overexpression.

Onset and progression of neurodegenerative disorders, including synucleinopathies such as Parkinson's disease, have been associated with various environmental factors. A highly compelling association from a therapeutic point of view has been found between a physically active lifestyle and a significantly reduced risk for Parkinson's disease. Mimicking such conditions in animal models by promoting physical activity, social interactions, and novel surroundings yields in a so-called enriched environment known to enhance adult neurogenesis, increase synaptic plasticity, and decelerate neuronal loss. Yet, the genes that connect beneficial environmental cues to the genome and delay disease-related symptoms have remained largely unclear. To identify such mediator genes, we used a 2 × 2 factorial design opposing genotype and environment. Specifically, we compared wildtype to transgenic mice overexpressing human SNCA, a key gene in synucleinopathies encoding alpha-synuclein, and housed them in a standard and enriched environment from weaning to 12 months of age before profiling their hippocampal transcriptome using RNA-sequencing. Under standard environmental conditions, differentially expressed genes were overrepresented for calcium ion binding, membrane, synapse, and other Gene Ontology terms previously linked to alpha-synuclein biology. Upregulated genes were significantly enriched for genes attributed to astrocytes, microglia, and oligodendrocytes. These disturbances in gene activity were accompanied by reduced levels of several presynaptic proteins and the immediate early genes EGR1 and NURR1. Intriguingly, housing transgenic animals in the enriched environment prevented most of these perturbations in gene activity. In addition, a sustained activation specifically in transgenic animals housed in enriched conditions was observed for several immediate early genes including Egr1, Nr4a2/Nurr1, Arc, and Homer1a. These findings suggest a compensatory mechanism through an enriched environment-activated immediate early gene network that prevented most disturbances induced by alpha-synuclein overexpression. This regulatory framework might harbor attractive targets for novel therapeutic approaches that mimic beneficial environmental stimuli.

GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Projection Neurons Modulate Fear Extinction.

Inhibitory synaptic transmission in the amygdala has a pivotal role in fear learning and its extinction. However, the local circuits formed by GABAergic inhibitory interneurons within the amygdala and their detailed function in shaping these behaviors are not well understood. Here we used lentiviral-mediated knockdown of the cell adhesion molecule neurofascin in the basolateral amygdala (BLA) to specifically remove inhibitory synapses at the axon initial segment (AIS) of BLA projection neurons. Quantitative analysis of GABAergic synapse markers and measurement of miniature inhibitory postsynaptic currents in BLA projection neurons after neurofascin knockdown ex vivo confirmed the loss of GABAergic input. We then studied the impact of this manipulation on anxiety-like behavior and auditory cued fear conditioning and its extinction as BLA related behavioral paradigms, as well as on long-term potentiation (LTP) in the ventral subiculum-BLA pathway in vivo. BLA knockdown of neurofascin impaired ventral subiculum-BLA-LTP. While this manipulation did not affect anxiety-like behavior and fear memory acquisition and consolidation, it specifically impaired extinction. Our findings indicate that modification of inhibitory synapses at the AIS of BLA projection neurons is sufficient to selectively impair extinction behavior. A better understanding of the role of distinct GABAergic synapses may provide novel and more specific targets for therapeutic interventions in extinction-based therapies.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL). The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain. Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity.

The regulated delivery of AMPA-type glutamate receptors (AMPARs) to synapses is an important mechanism underlying synaptic plasticity. Here, we ask whether the synaptic scaffolding protein PSD-95 (postsynaptic density 95) participates in AMPAR incorporation during two forms of synaptic plasticity. In hippocampal slice cultures, the expression of PSD-95-green fluorescent protein (PSD-95-GFP) increases AMPAR currents by selectively delivering glutamate receptor 1 (GluR1)-containing receptors to synapses, thus mimicking long-term potentiation (LTP). Mutational analysis shows that the N terminal of PSD-95 including the first two PDZ [PSD-95/Discs large (Dlg)/zona occludens-1 (ZO-1)] domains is necessary and sufficient to mediate this effect. Further supporting a role in synaptic plasticity, wild-type PSD-95 occludes LTP and dominant negative forms block LTP. Moreover, we demonstrate that PSD-95 also participates in AMPAR delivery during experience-driven plasticity in vivo. In the barrel cortex from experience-deprived animals, the expression of PSD-95-GFP selectively increases AMPAR currents, mimicking experience-driven plasticity. In nondeprived animals, PSD-95-GFP produces no additional potentiation, indicating common mechanisms between PSD-95-mediated potentiation and experience-driven synaptic strengthening. A dominant negative form of PSD-95 blocks experience-driven potentiation of synapses. Pharmacological analysis in slice cultures reveals that PSD-95 acts downstream of other signaling pathways involved in LTP. We conclude that PSD-95 controls activity-dependent AMPAR incorporation at synapses via PDZ interactions not only during LTP in vitro but also during experience-driven synaptic strengthening by natural stimuli in vivo.