Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

Evolution of connectivity architecture in the Drosophila mushroom body.

Brain evolution has primarily been studied at the macroscopic level by comparing the relative size of homologous brain centers between species. How neuronal circuits change at the cellular level over evolutionary time remains largely unanswered. Here, using a phylogenetically informed framework, we compare the olfactory circuits of three closely related Drosophila species that differ in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans and Drosophila sechellia that specializes on ripe noni fruit. We examine a central part of the olfactory circuit that, to our knowledge, has not been investigated in these species-the connections between projection neurons and the Kenyon cells of the mushroom body-and identify species-specific connectivity patterns. We found that neurons encoding food odors connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific connectivity differences reflect two distinct neuronal phenotypes: in the number of projection neurons or in the number of presynaptic boutons formed by individual projection neurons. Finally, behavioral analyses suggest that such increased connectivity enhances learning performance in an associative task. Our study shows how fine-grained aspects of connectivity architecture in an associative brain center can change during evolution to reflect the chemical ecology of a species.

Cortical nitric oxide required for presynaptic long-term potentiation in the insular cortex.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse retrogradely into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is required for kainate receptor (KAR)-dependent presynaptic form of LTP (pre-LTP) in the adult insular cortex (IC). In the IC, we found that inhibition of NO synthase erased the maintenance of pre-LTP, while the induction of pre-LTP required the activation of KAR. Furthermore, NO is essential for pre-LTP induced between two pyramidal cells in the IC using the double patch-clamp recording. These results suggest that NO is required for homosynaptic pre-LTP in the IC. Our results present strong evidence for the critical roles of NO in pre-LTP in the IC. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Presynaptic regulators in memory formation.

The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

The Lack of Synapsin Alters Presynaptic Plasticity at Hippocampal Mossy Fibers in Male Mice.

Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons, it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity, i.e., decreased facilitation and post-tetanic potentiation, but increased long-term potentiation in male synapsin triple knock-out (KO) mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from KO animals. Our results indicate that all synapsin isoforms are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse.

Effects of Time-Dependent Adenosine Triphosphate Consumption Caused by Neuron Firing on Adenosine Triphosphate Concentrations in Synaptic Boutons Containing and Lacking a Stationary Mitochondrion.

The precise mechanism behind the supply of adenosine triphosphate (ATP) to approximately half of the presynaptic release sites in axons that lack a stationary mitochondrion is not fully understood. This paper presents a mathematical model designed to simulate the transient ATP concentration in presynaptic en passant boutons. The model is utilized to investigate how the ATP concentration responds to increased ATP demand during neuronal firing in boutons with a stationary mitochondrion and those without one. The analysis suggests that neuron firing may cause oscillations in the ATP concentrations, with peak-to-peak amplitudes ranging from 0.06% to 5% of their average values. However, this does not deplete boutons lacking a mitochondrion of ATP; for physiologically relevant values of model parameters, their concentration remains approximately 3.75 times higher than the minimum concentration required for synaptic activity. The variance in average ATP concentrations between boutons containing a stationary mitochondrion and those lacking one ranges from 0.3% to 0.8%, contingent on the distance between the boutons. The model indicates that diffusion-driven ATP transport is rapid enough to adequately supply ATP molecules to boutons lacking a stationary mitochondrion.

Human iPSC-Derived Neurons with Reliable Synapses and Large Presynaptic Action Potentials.

Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9 weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100 Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9 weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to ∼25 mV and short durations of ∼0.5 ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.

Location analysis of presynaptically active and silent synapses in single-cultured hippocampal neurons.

A morphologically present but non-functioning synapse is termed a silent synapse. Silent synapses are categorized into "postsynaptically silent synapses," where AMPA receptors are either absent or non-functional, and "presynaptically silent synapses," where neurotransmitters cannot be released from nerve terminals. The presence of presynaptically silent synapses remains enigmatic, and their physiological significance is highly intriguing. In this study, we examined the distribution and developmental changes of presynaptically active and silent synapses in individual neurons. Our findings show a gradual increase in the number of excitatory synapses, along with a corresponding decrease in the percentage of presynaptically silent synapses during neuronal development. To pinpoint the distribution of presynaptically active and silent synapses, i.e., their positional information, we employed Sholl analysis. Our results indicate that the distribution of presynaptically silent synapses within a single neuron does not exhibit a distinct pattern during synapse development in different distance from the cell body. However, irrespective of neuronal development, the proportion of presynaptically silent synapses tends to rise as the projection site moves farther from the cell body, suggesting that synapses near the cell body may exhibit higher synaptic transmission efficiency. This study represents the first observation of changes in the distribution of presynaptically active and silent synapses within a single neuron.

Anomalous diffusion of synaptic vesicles and its influences on spontaneous and evoked neurotransmission.

Neurons in the central nervous system communicate with each other by activating billions of tiny synaptic boutons distributed along their fine axons. These presynaptic varicosities are very crowded environments, comprising hundreds of synaptic vesicles. Only a fraction of these vesicles can be recruited in a single release episode, either spontaneous or evoked by action potentials. Since the seminal work by Fatt and Katz, spontaneous release has been modelled as a memoryless process. Nevertheless, at central synapses, experimental evidence indicates more complex features, including non-exponential distributions of release intervals and power-law behaviour in their rate. To describe these features, we developed a probabilistic model of spontaneous release based on Brownian motion of synaptic vesicles in the presynaptic environment. To account for different diffusion regimes, we based our simulations on fractional Brownian motion. We show that this model can predict both deviation from the Poisson hypothesis and power-law features in experimental quantal release series, thus suggesting that the vesicular motion by diffusion could per se explain the emergence of these properties. We demonstrate the efficacy of our modelling approach using electrophysiological recordings at single synaptic boutons and ultrastructural data. When this approach was used to simulate evoked responses, we found that the replenishment of the readily releasable pool driven by Brownian motion of vesicles can reproduce the characteristic binomial release distributions seen experimentally. We believe that our modelling approach supports the idea that vesicle diffusion and readily releasable pool dynamics are crucial factors for the physiological functioning of neuronal communication. KEY POINTS: We developed a new probabilistic model of spontaneous and evoked vesicle fusion based on simple biophysical assumptions, including the motion of vesicles before they dock to the release site. We provide closed-form equations for the interval distribution of spontaneous releases in the special case of Brownian diffusion of vesicles, showing that a power-law heavy tail is generated. Fractional Brownian motion (fBm) was exploited to simulate anomalous vesicle diffusion, including directed and non-directed motion, by varying the Hurst exponent. We show that our model predicts non-linear features observed in experimental spontaneous quantal release series as well as ultrastructural data of synaptic vesicles spatial distribution. Evoked exocytosis based on a diffusion-replenished readily releasable pool might explain the emergence of power-law behaviour in neuronal activity.

Parallel processing of quickly and slowly mobilized reserve vesicles in hippocampal synapses.

Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.

Distributed feature representations of natural stimuli across parallel retinal pathways.

How sensory systems extract salient features from natural environments and organize them across neural pathways is unclear. Combining single-cell and population two-photon calcium imaging in mice, we discover that retinal ON bipolar cells (second-order neurons of the visual system) are divided into two blocks of four types. The two blocks distribute temporal and spatial information encoding, respectively. ON bipolar cell axons co-stratify within each block, but separate laminarly between them (upper block: diverse temporal, uniform spatial tuning; lower block: diverse spatial, uniform temporal tuning). ON bipolar cells extract temporal and spatial features similarly from artificial and naturalistic stimuli. In addition, they differ in sensitivity to coherent motion in naturalistic movies. Motion information is distributed across ON bipolar cells in the upper and the lower blocks, multiplexed with temporal and spatial contrast, independent features of natural scenes. Comparing the responses of different boutons within the same arbor, we find that axons of all ON bipolar cell types function as computational units. Thus, our results provide insights into the visual feature extraction from naturalistic stimuli and reveal how structural and functional organization cooperate to generate parallel ON pathways for temporal and spatial information in the mammalian retina.

Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal.

The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and "flash-and-freeze" electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations.

PTPN11/Corkscrew Activates Local Presynaptic Mapk Signaling to Regulate Synapsin, Synaptic Vesicle Pools, and Neurotransmission Strength, with a Dual Requirement in Neurons and Glia.

Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.

Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery.

Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

Frequency of Spontaneous Neurotransmission at Individual Boutons Corresponds to the Size of the Readily Releasable Pool of Vesicles.

Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential (AP) across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle (SV) fusing (Pv) and in the number of vesicles available for immediate release, known as the readily releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of APs. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses. Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release.

Ca2+ imaging of synaptic compartments using subcellularly targeted GCaMP8f in Drosophila.

GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail "quantal" imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4.

CaV 2.1 α1 subunit motifs that control presynaptic CaV 2.1 subtype abundance are distinct from CaV 2.1 preference.

Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2 subtype levels are largely unsolved. Because the CaV 2 α1  subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1  subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1  subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1  subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1  subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1  subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2 subtype abundance and preference remain enigmatic. The CaV α1  subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1  subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1  subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2 subtype abundance but not preference, whereas the CaV 2.2 α1  subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2 subtype abundance or preference. The CaV 2 α1  subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophila and mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

Dopamine-Dependent Plasticity Is Heterogeneously Expressed by Presynaptic Calcium Activity across Individual Boutons of the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium influx and dopaminergic input in the presynaptic terminals of Kenyon cells (KCs), the principal neurons of the MB, triggers long-term depression (LTD), which plays a critical role in olfactory learning. However, it is controversial whether such synaptic plasticity is accompanied by a corresponding decrease in odor-evoked calcium activity in the KC presynaptic terminals. Here, we address this question by inducing LTD by pairing odor presentation with optogenetic activation of dopaminergic neurons (DANs). This allows us to rigorously compare the changes at the presynaptic and postsynaptic sites in the same conditions. By imaging presynaptic acetylcholine release in the condition where LTD is reliably observed in the postsynaptic calcium signals, we show that neurotransmitter release from KCs is depressed selectively in the MB compartments innervated by activated DANs, demonstrating the presynaptic nature of LTD. However, total odor-evoked calcium activity of the KC axon bundles does not show concurrent depression. We further conduct calcium imaging in individual presynaptic boutons and uncover the highly heterogeneous nature of calcium plasticity. Namely, only a subset of boutons, which are strongly activated by associated odors, undergo calcium activity depression, while weakly responding boutons show potentiation. Thus, our results suggest an unexpected nonlinear relationship between presynaptic calcium influx and the results of plasticity, challenging the simple view of cooperative actions of presynaptic calcium and dopaminergic input.

Localization and neurochemical identity of alpha1-adrenergic receptor-containing elements in the mouse locus coeruleus.

The locus coeruleus (LC) is the major source for norepinephrine (NE) in the brain and projects to areas involved in learning and memory, reward, arousal, attention, and autonomic functions related to stress. There are three types of adrenergic receptors that respond to NE: alpha1-, alpha2-, and beta-adrenergic receptors. Previous behavioral studies have shown the alpha1-adrenergic receptor (α1AR) to be present in the LC, however, with conflicting results. For example, it was shown that α1ARs in the LC are involved in some of the motivational effects of stimulation of the medial forebrain bundle, which was reduced by α1AR antagonist terazosin. Another study showed that during novelty-induced behavioral activation, the α1AR antagonist prazosin reduced c-fos expression in brain regions known to contain motoric α1ARs, except for the LC, where c-fos expression was enhanced. Despite new research delineating more specific connectivity of the neurons in the LC, and some roles of the adrenergic receptors, the α1ARs have not been localized at the subcellular level. Therefore, in order to gain a greater understanding of the aforementioned studies, we used immunohistochemistry at the electron microscopic (EM) level to determine which neuronal or glial elements in the LC express the α1AR. We hypothesized, based on previous work in the ventral periaqueductal gray area, that the α1AR would be found mainly presynaptically in axon terminals, and possibly in glial elements. Single labeling immunohistochemistry at the EM revealed that about 40% of labeled elements that contained the α1AR were glial elements, while approximately 50% of the labeled neuronal elements were axon terminals or small unmyelinated axons in the LC. Double labeling immunohistochemistry found the α1AR expressed in GFAP-labeled astrocytes, in both GABAergic and glutamatergic axon terminals, and in a portion of the α1AR dendrites, colocalized with tyrosine hydroxylase (TH, a marker for noradrenergic neurons). This study sheds light on the neuroanatomical framework underlying the effects of NE and pharmaceuticals acting directly or indirectly on α1ARs in the LC.