Kay Jüngling

Neuropeptide S-Mediated Control of Fear Expression and Extinction: Role of Intercalated GABAergic Neurons in the Amygdala

A deficient extinction of memory is particularly important in the regime of fear, where it limits the beneficial outcomes of treatments of anxiety disorders. Fear extinction is thought to involve inhibitory influences of the prefrontal cortex on the amygdala, although the detailed synaptic mechanisms remain unknown. Here, we report that neuropeptide S (NPS), a recently discovered transmitter of ascending brainstem neurons, evokes anxiolytic effects and facilitates extinction of conditioned fear responses when administered into the amygdala in mice. An NPS receptor antagonist exerts functionally opposing responses, indicating that endogenous NPS is involved in anxiety behavior and extinction. Cellularly, NPS increases glutamatergic transmission to intercalated GABAergic neurons in the amygdala via presynaptic NPS receptors on connected principal neurons. These results identify mechanisms of NPS in the brain, a key role of intercalated neurons in the amygdala for fear extinction, and a potential pharmacological avenue for treating anxiety disorders.

Neuropeptide S: A transmitter system in the brain regulating fear and anxiety

The recently discovered Neuropeptide S (NPS) and its cognate receptor represent a highly interesting system of neuromodulation with unique physiological effects. On one hand, NPS increases wakefulness and arousal. On the other, NPS produces anxiolytic-like effects by acutely reducing fear responses as well as modulating long-term aspects of fear memory, such as attenuation of contextual fear or enhancement of fear extinction. The main sources of NPS in the brain are a few clusters of NPS-producing neurons in the brainstem. NPS binds to a G-protein-coupled receptor that is highly conserved among vertebrates and stimulates mobilization of intracellular Ca(2+) as well as activation of protein kinases. In synaptic circuits within the amygdala, which are important for processing of acute fear as well as formation and expression of fear memories, NPS causes increased release of the excitatory transmitter glutamate, especially in synaptic contacts to a subset of GABAergic interneurons. Polymorphisms in the human NPS receptor gene have been associated with altered sleep behavior and panic disorder. In conclusion, the NPS system displays a unique physiological profile with respect to the specificity and time course of its actions. These functions could provide interesting opportunities for both basic research and clinical applications.

N-Cadherin Transsynaptically Regulates Short-Term Plasticity at Glutamatergic Synapses in Embryonic Stem Cell-Derived Neurons

The cell adhesion molecule N-cadherin has been proposed to regulate synapse formation in mammalian central neurons. This is based on its synaptic localization enabling alignment of presynaptic and postsynaptic specializations by an adhesion mechanism. However, a potential role of N-cadherin in regulating synaptic transmission has remained elusive. In this paper, a functional analysis of N-cadherin knock-out synapses was enabled by in vitro neuronal differentiation of mouse embryonic stem cells circumventing the early embryonic lethality of mice genetically null for N-cadherin. In our in vitro system, initial synapse formation was not altered in the absence of N-cadherin, which might be attributable to compensatory mechanisms. Here, we demonstrate that N-cadherin is required for regulating presynaptic function at glutamatergic synapses. An impairment in the availability of vesicles for exocytosis became apparent selectively during high activity. Short-term plasticity was strongly altered with synaptic depression enhanced in the absence of N-cadherin. Most intriguingly, facilitation was converted to depression under specific stimulation conditions. This indicates an important role of N-cadherin in the control of short-term plasticity. To analyze, whether N-cadherin regulates presynaptic function by a transsynaptic mechanism, we studied chimeric cultures consisting of wild-type neocortical neurons and ES cell-derived neurons. With N-cadherin absent only postsynaptically, we observed a similar increase in short-term synaptic depression as found in its complete absence. This indicates a retrograde control of short-term plasticity by N-cadherin. In summary, our results revealed an unexpected involvement of a synaptic adhesion molecule in the regulation of short-term plasticity at glutamatergic synapses.

Neuropeptide S enhances memory during the consolidation phase and interacts with noradrenergic systems in the brain.

Neuropeptide S (NPS) has been shown to promote arousal and anxiolytic-like effects, as well as facilitation of fear extinction. In rodents, NPS receptors (NPSR) are prominently expressed in brain structures involved in learning and memory. Here, we investigate whether exogenous or endogenous NPS signaling can modulate acquisition, consolidation, or recall of emotional, spatial, and contextual memory traces, using two common behavioral paradigms, inhibitory avoidance (IA) and novel object recognition. In the IA paradigm, immediate and delayed post-training central NPS administration dose dependently enhanced memory retention in mice, indicating that NPS may act during the consolidation phase to enhance long-term memory. In contrast, pre-training or pre-test NPS injections were ineffective, suggesting that NPS had no effect on IA memory acquisition or recall. Peripheral administration of a synthetic NPSR antagonist attenuated NPS-induced IA memory enhancement, showing pharmacological specificity. NPS also enhanced hippocampal-dependent non-aversive memory in the novel object recognition task. In contrast, NPSR knockout mice displayed deficits in IA memory, novel object recognition, and novel place or context recognition, suggesting that activity of the endogenous NPS system is required for memory formation. Blockade of adrenergic signaling by propranolol attenuated NPS-induced memory enhancement in the IA task, indicating involvement of central noradrenergic systems. These results provide evidence for a facilitatory role of NPS in long-term memory, independent of memory content, possibly by acting as a salience signal or as an arousal-promoting factor.

Prevention of Stress-Impaired Fear Extinction Through Neuropeptide S Action in the Lateral Amygdala

Stressful and traumatic events can create aversive memories, which are a predisposing factor for anxiety disorders. The amygdala is critical for transforming such stressful events into anxiety, and the recently discovered neuropeptide S transmitter system represents a promising candidate apt to control these interactions. Here we test the hypothesis that neuropeptide S can regulate stress-induced hyperexcitability in the amygdala, and thereby can interact with stress-induced alterations of fear memory. Mice underwent acute immobilization stress (IS), and neuropeptide S and a receptor antagonist were locally injected into the lateral amygdala (LA) during stress exposure. Ten days later, anxiety-like behavior, fear acquisition, fear memory retrieval, and extinction were tested. Furthermore, patch-clamp recordings were performed in amygdala slices prepared ex vivo to identify synaptic substrates of stress-induced alterations in fear responsiveness. (1) IS increased anxiety-like behavior, and enhanced conditioned fear responses during extinction 10 days after stress, (2) neuropeptide S in the amygdala prevented, while an antagonist aggravated, these stress-induced changes of aversive behaviors, (3) excitatory synaptic activity in LA projection neurons was increased on fear conditioning and returned to pre-conditioning values on fear extinction, and (4) stress resulted in sustained high levels of excitatory synaptic activity during fear extinction, whereas neuropeptide S supported the return of synaptic activity during fear extinction to levels typical of non-stressed animals. Together these results suggest that the neuropeptide S system is capable of interfering with mechanisms in the amygdala that transform stressful events into anxiety and impaired fear extinction.

Purification of embryonic stem cell-derived neurons by immunoisolation

The pluripotency and high proliferative capacity of embryonic stem (ES) cells (1–3) makes them an attractive source of different cell types for biomedical research and cell replacement therapies. A major prerequisite for these applications is the availability of a homogeneous population of the desired cell type. However, ES cell‐derived material contains, for example, undifferentiated cells, which can cause tumor formation after transplantation into the brain (4). To avoid such unwanted side effects, effective purification of distinct types of cells needs to be developed. Here, we describe an immunoisolation procedure to purify neurons from in vitro differentiated mouse ES cells using an antibody against the neuronal cell adhesion molecule L1 (5, 6). Our procedure yields a pure population of differentiated neurons, which are electrically excitable and form excitatory, glutamatergic, and inhibitory GABAergic synapses. The ability to highly purify ES cell‐derived neurons will boost their molecular characterization and the further exploration of their therapeutic potential.

Heterosynaptic long-term potentiation at interneuron–principal neuron synapses in the amygdala requires nitric oxide signalling

Non‐technical summary  Long‐lasting changes in efficacy of cell–cell communication (long‐term potentiation; LTP) at specialized sites (synapses) between neurons in the brain are thought to underlie forms of learning and memory. These forms of LTP can occur at excitatory synapses and inhibitory synapses, thus in‐ or decreasing the activity of neurons. We provide evidence for a novel form of LTP at inhibitory synapses (LTPi) on a subset of neurons in the amygdala of mice, a brain region involved in fear and anxiety. This LTPi enhances the release of the inhibitory neurotransmitter GABA at synapses between inhibitory interneurons and excitatory principal neurons (PNs) in a sub‐region of the amygdala. The described LTPi is heavily dependent on the production and diffusion of the volatile gas nitric oxide (NO), produced by PNs during stages of increased activity. These findings indicate that NO‐mediated long‐term regulation of inhibitory transmission in the amygdala might contribute to the learning of fear.

Activation of neuropeptide S-expressing neurons in the locus coeruleus by corticotropin-releasing factor.

•  Neuropeptide S (NPS) and its cognate receptor represent a recently discovered transmitter system in the brain modulating anxiety‐ and stress‐related behaviour. •  Using a transgenic NPS‐EGFP‐expressing mouse line, the present study shows that NPS‐expressing neurons are situated in close proximity to corticotropin‐releasing factor (CRF)‐containing fibres at the locus coeruleus in the brain stem and express the CRF receptor 1 (CRF1). •  CRF depolarizes NPS neurons via activation of the CRF1 receptor through two different ionic mechanisms (a decrease in potassium and an increase in cation conductance) involving the cAMP signalling pathway. •  After acute immobilization stress, NPS neurons display an increased expression of c‐fos. •  This study identifies a mechanism by which stress‐related CRF release might activate NPS neurons in the brain stem, thereby triggering NPS release in target areas such as the amygdala, and functioning as a negative feedback control to buffer stress responsiveness.

Glutamic Acid Decarboxylase 65: A Link Between GABAergic Synaptic Plasticity in the Lateral Amygdala and Conditioned Fear Generalization

An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABAB receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.

Asymmetric N-Cadherin Expression Results in Synapse Dysfunction, Synapse Elimination, and Axon Retraction in Cultured Mouse Neurons

Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an in vivo asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.