Reflections on the past two decades of neuroscience.

The first issue of Nature Reviews Neuroscience was published 20 years ago, in 2000. To mark this anniversary, in this Viewpoint article we asked a selection of researchers from across the field who have authored pieces published in the journal in recent years for their thoughts on notable and interesting developments in neuroscience, and particularly in their areas of the field, over the past two decades. They also provide some thoughts on current lines of research and questions that excite them.

Differential role of pre- and postsynaptic neurons in the activity-dependent control of synaptic strengths across dendrites.

Neurons receive a large number of active synaptic inputs from their many presynaptic partners across their dendritic tree. However, little is known about how the strengths of individual synapses are controlled in balance with other synapses to effectively encode information while maintaining network homeostasis. This is in part due to the difficulty in assessing the activity of individual synapses with identified afferent and efferent connections for a synapse population in the brain. Here, to gain insights into the basic cellular rules that drive the activity-dependent spatial distribution of pre- and postsynaptic strengths across incoming axons and dendrites, we combine patch-clamp recordings with live-cell imaging of hippocampal pyramidal neurons in dissociated cultures and organotypic slices. Under basal conditions, both pre- and postsynaptic strengths cluster on single dendritic branches according to the identity of the presynaptic neurons, thus highlighting the ability of single dendritic branches to exhibit input specificity. Stimulating a single presynaptic neuron induces input-specific and dendritic branchwise spatial clustering of presynaptic strengths, which accompanies a widespread multiplicative scaling of postsynaptic strengths in dissociated cultures and heterosynaptic plasticity at distant synapses in organotypic slices. Our study provides evidence for a potential homeostatic mechanism by which the rapid changes in global or distant postsynaptic strengths compensate for input-specific presynaptic plasticity.

Integrins in synapse regulation.

Integrins are a large family of extracellular matrix (ECM) receptors. In the developing and adult brain, many integrins are present at high levels at synapses. The tetrapartite structure of synapses - which comprises presynaptic and postsynaptic neurons, the ECM and glial processes - places synaptic integrins in an excellent position to sense dynamic changes in the synaptic environment and use this information to coordinate further changes in synapse structure and function that will shape neural circuit properties. Recent developments in our understanding of the cellular and physiological roles of integrins, which range from control of neural process outgrowth and synapse formation to regulation of synaptic plasticity and memory, enable us to attempt a synthesis of synaptic integrin function.

Developmental excitation-inhibition imbalance underlying psychoses revealed by single-cell analyses of discordant twins-derived cerebral organoids.

Despite extensive genetic and neuroimaging studies, detailed cellular mechanisms underlying schizophrenia and bipolar disorder remain poorly understood. Recent progress in single-cell RNA sequencing (scRNA-seq) technologies enables identification of cell-type-specific pathophysiology. However, its application to psychiatric disorders is challenging because of methodological difficulties in analyzing human brains and the confounds due to a lifetime of illness. Brain organoids derived from induced pluripotent stem cells (iPSCs) of the patients are a powerful avenue to investigate the pathophysiological processes. Here, we generated iPSC-derived cerebral organoids from monozygotic twins discordant for psychosis. scRNA-seq analysis of the organoids revealed enhanced GABAergic specification and reduced cell proliferation following diminished Wnt signaling in the patient, which was confirmed in iPSC-derived forebrain neuronal cells. Two additional monozygotic twin pairs discordant for schizophrenia also confirmed the excess GABAergic specification of the patients' neural progenitor cells. With a well-controlled genetic background, our data suggest that unbalanced specification of excitatory and inhibitory neurons during cortical development underlies psychoses.

Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks.

Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca(2+) signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca(2+) channels. Intracellular infusion of NMDARs or Ca(2+)-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites.

ß3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons.

The integrins are transmembrane receptors for ECM proteins, and they regulate various cellular functions in the central nervous system. In hippocampal neurons, the ß3 integrin subtype is required for homeostatic synaptic scaling of AMPA receptors (AMPARs) induced by chronic activity deprivation. The surface level of ß3 integrin in postsynaptic neurons directly correlates with synaptic strength and the abundance of synaptic GluA2 AMPAR subunit. Although these observations suggest a functional link between ß3 integrin and AMPAR, little is known about the mechanistic basis for the connection. Here we investigate the nature of ß3 integrin and AMPAR interaction underlying the ß3 integrin-dependent control of synaptic AMPAR expression and thus synaptic strength. We show that ß3 integrin and GluA2 subunit form a complex in mouse brain that involves the direct binding between their cytoplasmic domains. In contrast, ß3 integrin associates with GluA1 AMPAR subunit only weakly, and, in a heterologous expression system, the interaction requires the coexpression of GluA2. Surprisingly, in hippocampal pyramidal neurons, expressing ß3 integrin mutants with either increased or decreased affinity for extracellular ligands has no differential effects in elevating excitatory synaptic currents and surface GluA2 levels compared with WT ß3 integrin. Our findings identify an integrin family member, ß3, as a direct interactor of an AMPAR subunit and provide molecular insights into how this cell-adhesion protein regulates the composition of cell-surface AMPARs.

[Switching from oral oxycodone to oxycodone injection - an investigation into the dosage amount ratio].

In switching from oraloxycodone to oxycodone injection, clinical guidelines recommend a conversion dose ratio of 0.75. However, in clinical sites, a higher dosage may be needed due to characteristics of cancer pain. In the present study, we investigated changing the dosage amount of oxycodone before and after switching from oraloxycodone administration to oxycodone injection in patients (n=14) who reported suffering from cancer pain. As a result, we found the ratio of the amount used after switching to be 0.91 ± 0.25 (mean ± SD) on the first day, increasing to 1.46 ± 0.48 on the fifth day. Our findings suggest that the dosage amount was the correct one for each patient's condition and also that adequate injection dosage to manage cancer pain was greater than that of calculated by the conversion ratio.

Subanalgesic ketamine enhances morphine-induced antinociceptive activity without cortical dysfunction in rats.

PURPOSE: Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, has been used for the treatment of cancer pain as an analgesic adjuvant to opioids. However, ketamine is known to produce psychotomimetic side effects including cognitive impairments under a high-dose situation, presumably as the result of cortical dysfunction. Here, we investigated whether low-dose ketamine was useful as an analgesic adjuvant to morphine for pain control, focusing on frontocortical function. METHODS: To assess the analgesic effects of ketamine with or without morphine, we performed behavioral and histochemical experiments, using the hot plate test and c-Fos expression analysis in rats. The effect on cortical function was also determined by prepulse inhibition (PPI) of the acoustic startle and evoked potentials in the hippocampal CA1-medial prefrontal cortex (mPFC) synapses as measures of synaptic efficacy. RESULTS: Coadministration of ketamine as a subanalgesic dose significantly enhanced intraperitoneal morphine-induced antinociceptive response, which was measured as the increased reaction latency in the hot plate test. In addition, the noxious thermal stimulus-induced c-Fos expression in the ventrolateral periaqueductal gray matter was significantly suppressed by concomitant ketamine and morphine. In contrast, the subanalgesic dose of ketamine did not impair PPI and synaptic efficacy in the mPFC. CONCLUSION: The present results indicate that the morphine-induced analgesic effect is enhanced by a concomitant subanalgesic dose of ketamine without affecting cortical function. Our findings possibly support the clinical notion that low-dose ketamine as an analgesic adjuvant has therapeutic potential to reduce opioid dosage, thereby improving the quality of life in cancer pain patients.

Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy.

Synapse regulation exploits the capacity of actin to function as a stable structural component or as a dynamic filament. Beyond its well-appreciated role in eliciting visible morphological changes at the synapse, the emerging picture points to an active contribution of actin to the modulation of the efficacy of pre- and postsynaptic terminals. Moreover, by engaging distinct pools of actin and divergent signalling pathways, actin-dependent morphological plasticity could be uncoupled from modulation of synaptic strength. The aim of this Review is to highlight some of the recent progress in elucidating the role of the actin cytoskeleton in synaptic function.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity.

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

Astrocyte-synapse interactions and cell adhesion molecules.

Astrocytes are increasingly gaining attention as a major player in regulating brain functions. Not only are astrocytes important for their supporting roles in maintaining optimal neuronal activity, they also dynamically interact with synapses through their highly ramified morphology to directly influence information processing by the neural circuits and the behaviours that depend on it. Here, we take a close look at astrocyte-synapse interactions involved in the coordination of synaptogenesis and astrocyte maturation in the developing brain through to the contribution of astrocytes in synaptic plasticity in the adult brain, and end with a perspective on astrocyte function in behaviours and diseases. In particular, we focus on the roles of synapse adhesion proteins. While cell adhesion proteins that form a bridge between the presynaptic and the postsynaptic compartments have been extensively studied, recent reports highlighting the striking participation of astrocytic cell adhesion proteins in synapse formation and function underscores the importance of reconsidering the conventional neurocentric view of synaptic adhesive interactions and the underlying logic.

Linking spontaneous and stimulated spine dynamics.

Our brains continuously acquire and store memories through synaptic plasticity. However, spontaneous synaptic changes can also occur and pose a challenge for maintaining stable memories. Despite fluctuations in synapse size, recent studies have shown that key population-level synaptic properties remain stable over time. This raises the question of how local synaptic plasticity affects the global population-level synaptic size distribution and whether individual synapses undergoing plasticity escape the stable distribution to encode specific memories. To address this question, we (i) studied spontaneously evolving spines and (ii) induced synaptic potentiation at selected sites while observing the spine distribution pre- and post-stimulation. We designed a stochastic model to describe how the current size of a synapse affects its future size under baseline and stimulation conditions and how these local effects give rise to population-level synaptic shifts. Our study offers insights into how seemingly spontaneous synaptic fluctuations and local plasticity both contribute to population-level synaptic dynamics.

The Shaping of AMPA Receptor Surface Distribution by Neuronal Activity.

Neurotransmission is critically dependent on the number, position, and composition of receptor proteins on the postsynaptic neuron. Of these, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are responsible for the majority of postsynaptic depolarization at excitatory mammalian synapses following glutamate release. AMPARs are continually trafficked to and from the cell surface, and once at the surface, AMPARs laterally diffuse in and out of synaptic domains. Moreover, the subcellular distribution of AMPARs is shaped by patterns of activity, as classically demonstrated by the synaptic insertion or removal of AMPARs following the induction of long-term potentiation (LTP) and long-term depression (LTD), respectively. Crucially, there are many subtleties in the regulation of AMPARs, and exactly how local and global synaptic activity drives the trafficking and retention of synaptic AMPARs of different subtypes continues to attract attention. Here we will review how activity can have differential effects on AMPAR distribution and trafficking along with its subunit composition and phosphorylation state, and we highlight some of the controversies and remaining questions. As the AMPAR field is extensive, to say the least, this review will focus primarily on cellular and molecular studies in the hippocampus. We apologise to authors whose work could not be cited directly owing to space limitations.

How Staying Negative Is Good for the (Adult) Brain: Maintaining Chloride Homeostasis and the GABA-Shift in Neurological Disorders.

Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the "dematuration" of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities.

Activity-dependent coordination of presynaptic release probability and postsynaptic GluR2 abundance at single synapses.

The strength of an excitatory synapse depends on both the presynaptic release probability (p(r)) and the abundance of functional postsynaptic AMPA receptors. How these parameters are related or balanced at a single synapse remains unknown. By taking advantage of live fluorescence imaging in cultured hippocampal neurons where individual synapses are readily resolved, we estimate p(r) by labeling presynaptic vesicles with a styryl dye, FM1-43, while concurrently measuring postsynaptic AMPA receptor abundance at the same synapse by immunolabeling surface GluR2. We find no appreciable correlation between p(r) and the level of surface synaptic GluR2 under basal condition, and blocking basal neural activity has no effect on the observed lack of correlation. However, elevating network activity drives their correlation, which accompanies a decrease in mean GluR2 level. These findings provide the direct evidence that the coordination of pre- and postsynaptic parameters of synaptic strength is not intrinsically fixed but that the balance is tuned by synaptic use at individual synapses.

My Neighbour Hetero-deconstructing the mechanisms underlying heterosynaptic plasticity.

Synapses change in strength following patterns of activity, but in many cases seemingly inactive neighbouring synapses also undergo changes in strength. These heterosynaptic changes occur across developmental time-points in various brain circuits in different species, but their precise molecular mechanisms are not well understood. Additionally, heterosynaptic changes can mirror homosynaptic plasticity or occur in opposition to homosynaptic changes. In this review we consider what useful functionality heterosynaptic dynamics could potentially endow the circuit with, and the underlying signalling events that implement heterosynaptic changes. We discuss what unanswered questions remain, and what the future looks like for understanding the logic of synaptic plasticity.

Heterosynaptic cross-talk of pre- and postsynaptic strengths along segments of dendrites.

Dendrites are crucial for integrating incoming synaptic information. Individual dendritic branches are thought to constitute a signal processing unit, yet how neighboring synapses shape the boundaries of functional dendritic units is not well understood. Here, we address the cellular basis underlying the organization of the strengths of neighboring Schaffer collateral-CA1 synapses by optical quantal analysis and spine size measurements. Inducing potentiation at clusters of spines produces NMDA-receptor-dependent heterosynaptic plasticity. The direction of postsynaptic strength change shows distance dependency to the stimulated synapses where proximal synapses predominantly depress, whereas distal synapses potentiate; potentiation and depression are regulated by CaMKII and calcineurin, respectively. In contrast, heterosynaptic presynaptic plasticity is confined to weakening of presynaptic strength of nearby synapses, which requires CaMKII and the retrograde messenger nitric oxide. Our findings highlight the parallel engagement of multiple signaling pathways, each with characteristic spatial dynamics in shaping the local pattern of synaptic strengths.

Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum.

Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs, although their exact function has remained controversial. Here, we identify in mouse hippocampus, a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron.

Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.

The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye-labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.