Stabilization of V1 interneuron-motor neuron connectivity ameliorates motor phenotype in a mouse model of ALS.

Loss of connectivity between spinal V1 inhibitory interneurons and motor neurons is found early in disease in the SOD1G93A mice. Such changes in premotor inputs can contribute to homeostatic imbalance of motor neurons. Here, we show that the Extended Synaptotagmin 1 (Esyt1) presynaptic organizer is downregulated in V1 interneurons. V1 restricted overexpression of Esyt1 rescues inhibitory synapses, increases motor neuron survival, and ameliorates motor phenotypes. Two gene therapy approaches overexpressing ESYT1 were investigated; one for local intraspinal delivery, and the other for systemic administration using an AAV-PHP.eB vector delivered intravenously. Improvement of motor functions is observed in both approaches, however systemic administration appears to significantly reduce onset of motor impairment in the SOD1G93A mice in absence of side effects. Altogether, we show that stabilization of V1 synapses by ESYT1 overexpression has the potential to improve motor functions in ALS, demonstrating that interneurons can be a target to attenuate ALS symptoms.

Role of dopamine neurons in familiarity.

Dopamine neurons signal the salience of environmental stimuli and influence learning, although it is less clear if these neurons also determine the salience of memories. Ventral tegmental area (VTA) dopamine neurons increase their firing in the presence of new objects and reduce it upon repeated, inconsequential exposures, marking the shift from novelty to familiarity. This study investigates how dopamine neuron activity during repeated familiar object exposure affects an animal's preference for new objects in a subsequent novel object recognition (NOR) test. We hypothesize that a single familiarization session will not sufficiently lower dopamine activity, such that the memory of a familiar object remains salient, leading to equal exploration of familiar and novel objects and weaker NOR discrimination. In contrast, multiple familiarization sessions likely suppress dopamine activity more effectively, reducing the salience of the familiar object and enhancing subsequent novelty discrimination. Our experiments in mice indicated that multiple familiarization sessions reduce VTA dopamine neuron activation, as measured by c-Fos expression, and enhance novelty discrimination compared with a single familiarization session. Dopamine neurons that show responsiveness to novelty were primarily located in the paranigral nucleus of the VTA and expressed vesicular glutamate transporter 2 transcripts, marking them as dopamine-glutamate neurons. Chemogenetic inhibition of dopamine neurons during a single session paralleled the effects of multiple sessions, improving NOR. These findings suggest that a critical role of dopamine neurons during the transition from novelty to familiarity is to modulate the salience of an object's memory.

Role of Dopamine Neurons in Familiarity.

Dopamine neurons signal the salience of environmental stimuli, influencing learning and motivation. However, research has not yet identified whether dopamine neurons also modulate the salience of memory content. Dopamine neuron activity in the ventral tegmental area (VTA) increases in response to novel objects and diminishes as objects become familiar through repeated presentations. We proposed that the declined rate of dopamine neuron activity during familiarization affects the salience of a familiar object's memory. This, in turn, influences the degree to which an animal distinguishes between familiar and novel objects in a subsequent novel object recognition (NOR) test. As such, a single familiarization session may not sufficiently reduce dopamine activity, allowing the memory of a familiar object to maintain its salience and potentially attenuating NOR. In contrast, multiple familiarization sessions could lead to more pronounced dopamine activity suppression, strengthening NOR. Our data in mice reveals that, compared to a single session, multiple sessions result in decreased VTA dopamine neuron activation, as indicated by c-Fos measurements, and enhanced novelty discrimination. Critically, when VTA dopamine neurons are chemogenetically inhibited during a single familiarization session, NOR improves, mirroring the effects of multiple familiarization sessions. In summary, our findings highlight the pivotal function of dopamine neurons in familiarity and suggest a role in modulating the salience of memory content.

Detection of endogenous NPY release determined by novel GRAB sensor in cultured cortical neurons.

Neuropeptide Y (NPY) is an abundantly expressed peptide in the nervous system. Its widespread distribution along with its receptors, both centrally and peripherally, indicates its broad functions in numerous biological processes. However, the low endogenous concentration and diffuse distribution of NPY make it challenging to study its actions and dynamics directly and comprehensively. Studies on the role of NPY have primarily been limited to exogenous application, transgene expression, or knock-out in biological systems, which are often combined with pharmacological probes to delineate the involvement of specific NPY receptors. Therefore, to better understand the function of NPY in time and space, direct visualization of the real-time dynamics of endogenous NPY is a valuable and desired tool. Using the first-generation and newly developed intensiometric green fluorescent G-protein-coupled NPY sensor (GRAB NPY1.0), we, for the first time, demonstrate and characterize the direct detection of endogenously released NPY in cultured cortical neurons. A dose-dependent fluorescent signal was observed upon exogenous NPY application in nearly all recorded neurons. Pharmacologically evoked neuronal activity induced a significant increase in fluorescent signal in 32% of neurons, reflecting the release of NPY, despite only 3% of all neurons containing NPY. The remaining pool of neurons expressing the sensor were either non-responsive or displayed a notable decline in the fluorescent signal. Such decline in fluorescent signal was not rescued in cortical cultures transduced with an NPY overexpression vector, where 88% of the neurons were NPY-positive. Overexpression of NPY did, however, result in sensor signals that were more readily distinguishable. This may suggest that biological factors, such as subtle changes in intracellular pH, could interfere with the fluorescent signal, and thereby underestimate the release of endogenous NPY when using this new sensor in its present configuration. However, the development of next-generation NPY GRAB sensor technology is expected soon, and will eventually enable much-wanted studies on endogenous NPY release dynamics in both cultured and intact biological systems.

Neuronal activity dynamics in the dentate gyrus during early epileptogenesis.

Epileptogenesis is a complex process involving a multitude of changes at the molecular, cellular and network level. Previous studies have identified several key alterations contributing to epileptogenesis and the development of hyper-excitability in different animal models, but only a few have focused on the early stages of this process. For post status epilepticus (SE) temporal lobe epilepsy in particular, understanding network dynamics during the early phases might be crucial for developing accurate preventive treatments to block the development of chronic spontaneous seizures. In this study, we used a viral vector mediated approach to examine activity of neurons in the dentate gyrus of the hippocampus during early epileptogenesis. We find that while granule cells are active 8 h after SE and then gradually decrease their activity, Calretinin-positive mossy cells and Neuropeptide Y-positive GABAergic interneurons in the hilus show a delayed activation pattern starting at 24 and peaking at 48 h after SE. These data suggest that indirect inhibition of granule cells by mossy cells through recruitment of local GABAergic interneurons could be an important mechanisms of excitability control during early epileptogenesis, and contribute to our understanding of the complex role of these cells in normal and pathological conditions.

The Scaffold Protein PICK1 as a Target in Chronic Pain.

Well-tolerated and effective drugs for treating chronic pain conditions are urgently needed. Most chronic pain patients are not effectively relieved from their pain and suffer from debilitating drug side effects. This has not only drastic negative consequences for the patients' quality of life, but also constitute an enormous burden on society. It is therefore of great interest to explore new potent targets for effective pain treatment with fewer side effects and without addiction liability. A critical component of chronic pain conditions is central sensitization, which involves the reorganization and strengthening of synaptic transmission within nociceptive pathways. Such changes are considered as maladaptive and depend on changes in the surface expression and signaling of AMPA-type glutamate receptors (AMPARs). The PDZ-domain scaffold protein PICK1 binds the AMPARs and has been suggested to play a key role in these maladaptive changes. In the present paper, we review the regulation of AMPARs by PICK1 and its relation to pain pathology. Moreover, we highlight other pain-relevant PICK1 interactions, and we evaluate various compounds that target PICK1 and have been successfully tested in pain models. Finally, we evaluate the potential on-target side effects of interfering with the action of PICK1 action in CNS and beyond. We conclude that PICK1 constitutes a valid drug target for the treatment of inflammatory and neuropathic pain conditions without the side effects and abuse liability associated with current pain medication.

Bidirectional protein-protein interactions control liquid-liquid phase separation of PSD-95 and its interaction partners.

The organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein-protein interactions (PPIs) which constitute a functional postsynapse. The PSD protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the Stargazin (TARP-γ2)-mediated liquid-liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate the formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions, while in other cases short, highly charged peptides are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems.

A Novel Peripheral Action of PICK1 Inhibition in Inflammatory Pain.

Chronic pain is a major healthcare problem that impacts one in five adults across the globe. Current treatment is compromised by dose-limiting side effects including drowsiness, apathy, fatigue, loss of ability to function socially and professionally as well as a high abuse liability. Most of these side effects result from broad suppression of excitatory neurotransmission. Chronic pain states are associated with specific changes in the efficacy of synaptic transmission in the pain pathways leading to amplification of non-noxious stimuli and spontaneous pain. Consequently, a reversal of these specific changes may pave the way for the development of efficacious pain treatment with fewer side effects. We have recently described a high-affinity, bivalent peptide TAT-P4-(C5)2, enabling efficient targeting of the neuronal scaffold protein, PICK1, a key protein in mediating chronic pain sensitization. In the present study, we demonstrate that in an inflammatory pain model, the peptide does not only relieve mechanical allodynia by targeting PICK1 involved in central sensitization, but also by peripheral actions in the inflamed paw. Further, we assess the effects of the peptide on novelty-induced locomotor activity, abuse liability, and memory performance without identifying significant side effects.

Modulating Dopamine Signaling and Behavior with Chemogenetics: Concepts, Progress, and Challenges.

For more than 60 years, dopamine (DA) has been known as a critical modulatory neurotransmitter regulating locomotion, reward-based motivation, and endocrine functions. Disturbances in DA signaling have been linked to an array of different neurologic and psychiatric disorders, including Parkinson's disease, schizophrenia, and addiction, but the underlying pathologic mechanisms have never been fully elucidated. One major obstacle limiting interpretation of standard pharmacological and transgenic interventions is the complexity of the DA system, which only appears to widen as research progresses. Nonetheless, development of new genetic tools, such as chemogenetics, has led to an entirely new era for functional studies of neuronal signaling. By exploiting receptors that are engineered to respond selectively to an otherwise inert ligand, so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), chemogenetics enables pharmacological remote control of neuronal activity. Here we review the recent, extensive application of this technique to the DA field and how its use has advanced the study of the DA system and contributed to our general understanding of DA signaling and related behaviors. Moreover, we discuss the challenges and pitfalls associated with the chemogenetic technology, such as the metabolism of the DREADD ligand clozapine N-oxide (CNO) to the D2 receptor antagonist clozapine. We conclude that despite the recent concerns regarding CNO, the chemogenetic toolbox provides an exceptional approach to study neuronal function. The huge potential should promote continued investigations and additional refinements to further expound key mechanisms of DA signaling and circuitries in normal as well as maladaptive behaviors.

The C-terminal flanking peptide of neuropeptide Y (NPY) is not essential for seizure-suppressant actions of prepro-NPY overexpression in male rats.

The full coding sequence of neuropeptide Y (NPY), prepro-NPY, is sequentially metabolized into three peptides; an N-terminus 28-amino acid signaling peptide, the NPY peptide itself (NPY1-36), and a 30-amino acid C-terminus peptide, known as the C-terminal flanking peptide of neuropeptide-Y (CPON). While the signaling peptide directs intracellular trafficking and NPY1-36 is well characterized, the biological function of CPON is unknown. This is noteworthy because CPON is co-stored and co-released along with NPY1-36 and could thus potentially serve important functions. To assess the role of CPON, we adapted a viral genetic approach using two different vector designs encoding NPY, but where the CPON coding sequence was excluded from one of the vectors. Thus, the effect of CPON was indirectly assessed. Male rats received intrahippocampal injections of either a vector encoding NPY1-39 whose metabolism yields NPY1-36 and not CPON, or a prepro-NPY vector encoding both NPY1-36 and CPON. A third vector encoding EGFP served as control. We subsequently studied to what extent CPON might affect seizure susceptibility and memory performance, respectively, to address two important questions to evaluate the potential of NPY gene therapy in epilepsy. Both NPY vectors, as compared to EGFP control, were found to be equally effective at suppressing acute kainate-induced seizures, and both did not influence learning and memory performance in the Morris water maze. Thus CPON itself does not appear to aid actions governed by vector-mediated overexpression of NPY1-36 within the hippocampus. Whether CPON serves other important functions remains to be determined.

Npas4 Is a Critical Regulator of Learning-Induced Plasticity at Mossy Fiber-CA3 Synapses during Contextual Memory Formation.

Synaptic connections between hippocampal mossy fibers (MFs) and CA3 pyramidal neurons are essential for contextual memory encoding, but the molecular mechanisms regulating MF-CA3 synapses during memory formation and the exact nature of this regulation are poorly understood. Here we report that the activity-dependent transcription factor Npas4 selectively regulates the structure and strength of MF-CA3 synapses by restricting the number of their functional synaptic contacts without affecting the other synaptic inputs onto CA3 pyramidal neurons. Using an activity-dependent reporter, we identified CA3 pyramidal cells that were activated by contextual learning and found that MF inputs on these cells were selectively strengthened. Deletion of Npas4 prevented both contextual memory formation and this learning-induced synaptic modification. We further show that Npas4 regulates MF-CA3 synapses by controlling the expression of the polo-like kinase Plk2. Thus, Npas4 is a critical regulator of experience-dependent, structural, and functional plasticity at MF-CA3 synapses during contextual memory formation.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Optogenetic inhibition of chemically induced hypersynchronized bursting in mice.

Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.

Activity-dependent long-term plasticity of afferent synapses on grafted stem/progenitor cell-derived neurons.

Stem cell-based cell replacement therapies aiming at restoring injured or diseased brain function ultimately rely on the capability of transplanted cells to promote functional recovery. The mechanisms by which stem cell-based therapies for neurological conditions can lead to functional recovery are uncertain, but structural and functional repair appears to depend on integration of transplanted cell-derived neurons into neuronal circuitries. The nature by which stem/progenitor cell-derived neurons synaptically integrate into neuronal circuitries is largely unexplored. Here we show that transplanted GFP-labeled neuronal progenitor cells into the rat hippocampus exhibit mature neuronal morphology following 4-10 weeks. GFP-positive cells were preferentially integrated into the principal cell layers of hippocampus, particularly CA3. Patch-clamp recordings from GFP-expressing cells revealed that they generated fast action potentials, and their intrinsic membrane properties were overall similar to endogenous host neurons recorded in same areas. As judged by occurrence of spontaneous excitatory postsynaptic currents (EPSCs), transplanted GFP-positive cells were synaptically integrated into the host circuitry. Comparable to host neurons, both paired-pulse depression and facilitation of afferent fiber stimulation-evoked EPSCs were observed in GFP-positive cells. Upon high-frequency stimulation, GFP-positive cells displayed post-tetanic potentiation of EPSCs, in some cases followed by long-term potentiation (LTP) lasting for more than 30 min. Our data show for the first time that transplanted neuronal progenitor cells can become functional neurons and their afferent synapses are capable of expressing activity-dependent short and long-term plasticity. These synaptic properties may facilitate host-to-graft interactions and regulate activity of the grafted cells promoting functional recovery of the diseased brain.

Tuning afferent synapses of hippocampal interneurons by neuropeptide Y.

Cholecystokinin (CCK)-expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory-forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65-GFP transgenic mice for prospective identification of CCK basket cells and whole-cell patch-clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation-evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation-evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities.

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice.

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Functional properties and synaptic integration of genetically labelled dopaminergic neurons in intrastriatal grafts.

Intrastriatal grafts of fetal ventral mesencephalic tissue, rich in dopaminergic neurons, can reverse symptoms in Parkinson's disease. For development of effective cell replacement therapy, other sources of dopaminergic neurons, e.g. derived from stem cells, are needed. However, the electrophysiological properties grafted cells need to have in order to induce substantial functional recovery are poorly defined. It has not been possible to prospectively identify and record from dopaminergic neurons in fetal transplants. Here we used transgenic mice expressing green fluorescent protein under control of the rat tyrosine hydroxylase promoter for whole-cell patch-clamp recordings of endogenous and grafted dopaminergic neurons. We transplanted ventral mesencephalic tissue from E12.5 transgenic mice into striatum of neonatal rats with or without lesions of the nigrostriatal dopamine system. The transplanted cells exhibited intrinsic electrophysiological properties typical of substantia nigra dopaminergic neurons, i.e. broad action potentials, inward rectifying currents with characteristic 'sag', and spontaneous action potentials. The grafted dopaminergic neurons also received functional excitatory and inhibitory synaptic inputs from the host brain, as shown by the presence of both spontaneous and stimulation-evoked excitatory and inhibitory postsynaptic currents. Occurrence of spontaneous excitatory and inhibitory currents was lower, and of spontaneous action potentials was higher, in neurons placed in the dopamine-depleted striatum than of those in the intact striatum. Our findings define specific electrophysiological characteristics of transplanted fetal dopaminergic neurons, and we provide the first direct evidence of functional synaptic integration of these neurons into host neural circuitries.