A descending dopamine pathway conserved from basal vertebrates to mammals

Dopamine neurons are classically known to modulate locomotion indirectly through ascending projections to the basal ganglia that project down to brainstem locomotor networks. Their loss in Parkinson’s disease is devastating. In lampreys, we recently showed that brainstem networks also receive direct descending dopaminergic inputs that potentiate locomotor output. Here, we provide evidence that this descending dopaminergic pathway is conserved to higher vertebrates, including mammals. In salamanders, dopamine neurons projecting to the striatum or brainstem locomotor networks were partly intermingled. Stimulation of the dopaminergic region evoked dopamine release in brainstem locomotor networks and concurrent reticulospinal activity. In rats, some dopamine neurons projecting to the striatum also innervated the pedunculopontine nucleus, a known locomotor center, and stimulation of the dopaminergic region evoked pedunculopontine dopamine release in vivo. Finally, we found dopaminergic fibers in the human pedunculopontine nucleus. The conservation of a descending dopaminergic pathway across vertebrates warrants re-evaluating dopamine’s role in locomotion.Dopaminergic neurons represent a vital neuromodulatory component essential for vertebrate motor control, and their loss in neurodegenerative disease is devastating. The meso-diencephalic dopamine (DA) neurons are known to provide ascending projections to the basal ganglia, which, in turn, provide input to cortical structure in mammals but also project caudally to the mesencephalic locomotor region (MLR), a highly conserved structure that controls locomotion in all vertebrates investigated to date (1–7; for review, see ref. 8). A growing body of evidence supports the view that basal ganglia connectivity is highly conserved among vertebrates, from lampreys to mammals (9–11; for review, see ref. 12), with some interspecies differences recently highlighted (13). As such, the homology between DA cell populations remains to be resolved in vertebrates. As a general rule, DA neurons from the meso-diencephalon send projections to the striatum in all vertebrates. In lampreys and teleosts, those neurons are located only in the diencephalon (posterior tuberculum), but in tetrapods and cartilaginous fishes (14) they are located in both the diencephalon and the mesencephalon. An increasing number of authors seem to agree with the hypothesis that at least some of the meso-diencephalic DA neurons located in the diencephalon are homologous in all vertebrates, and thus, homologous to at least a portion of the mammalian substantia nigra pars compacta (SNc)/ventral tegmental area (VTA) (13, 15–19; for review, see ref. 20). Alternatively, it was suggested that the posterior tuberculum DA neurons projecting to the striatum in zebrafish are homologs of the mammalian DA neurons of the A11 group (21). This will be discussed below in light of the results of the present study.In lampreys, only a few meso-diencephalic DA neurons send ascending projections to the striatum (9, 22); the majority of DA neurons send a direct descending projection to the MLR (22, 23), where DA is released and increases locomotor output through D1 receptors (22). These results demonstrate that the descending dopaminergic pathway to the MLR is an important modulator of locomotor output, but it remains to be determined whether this pathway is conserved in higher vertebrates.The existence of a descending dopaminergic pathway that powerfully increases locomotor output has important implications for Parkinson’s disease, which involves the meso-diencephalic DA neurons. A loss of descending dopaminergic projections could play a role in the locomotor deficits systematically observed in that disease. Because of the highly conserved nature of both the dopaminergic system and brainstem locomotor circuitry in vertebrates, we hypothesized that a direct descending dopaminergic pathway to the MLR also exists in higher vertebrates. Previous anatomical (24, 25) and electrophysiological (26) studies in rats support the idea of a descending connection from the SNc to the pedunculopontine nucleus [PPN, considered part of the MLR (2)]. Moreover, dopaminergic terminals were found in the PPN of monkeys (27), but the origin of this projection is still unknown in mammals.Here, we investigated whether the direct descending projection from meso-diencephalic DA neurons to the MLR is present in two tetrapods, the salamander and the rat. Moreover, we supplement our analyses with anatomical data from human brain tissue. Using traditional and virogenetic axonal tracing, immunofluorescence, in vivo voltammetry, and calcium imaging of reticulospinal neurons, we provide anatomical and functional evidence strongly supporting a conserved role for the descending projections of meso-diencephalic DA neurons in the regulation of brainstem locomotor networks across the vertebrate subphylum.     

Intrinsically organized resting state networks in the human spinal cord

Spontaneous fluctuations in functional magnetic resonance imaging (fMRI) signals of the brain have repeatedly been observed when no task or external stimulation is present. These fluctuations likely reflect baseline neuronal activity of the brain and correspond to functionally relevant resting-state networks (RSN). It is not known however, whether intrinsically organized and spatially circumscribed RSNs also exist in the spinal cord, the brain’s principal sensorimotor interface with the body. Here, we use recent advances in spinal fMRI methodology and independent component analysis to answer this question in healthy human volunteers. We identified spatially distinct RSNs in the human spinal cord that were clearly separated into dorsal and ventral components, mirroring the functional neuroanatomy of the spinal cord and likely reflecting sensory and motor processing. Interestingly, dorsal (sensory) RSNs were separated into right and left components, presumably related to ongoing hemibody processing of somatosensory information, whereas ventral (motor) RSNs were bilateral, possibly related to commissural interneuronal networks involved in central pattern generation. Importantly, all of these RSNs showed a restricted spatial extent along the spinal cord and likely conform to the spinal cord’s functionally relevant segmental organization. Although the spatial and temporal properties of the dorsal and ventral RSNs were found to be significantly different, these networks showed significant interactions with each other at the segmental level. Together, our data demonstrate that intrinsically highly organized resting-state fluctuations exist in the human spinal cord and are thus a hallmark of the entire central nervous system.Functional magnetic resonance imaging (fMRI) has been used to study the functional connectivity of the human brain, with spontaneous fluctuations in the resting-state fMRI signal (1–3) attracting much attention in the past few years (for review, see refs. 4–6). Brain regions showing temporally coherent spontaneous fluctuations constitute several anatomically consistent “resting state networks” (RSNs), such as visual, auditory, sensory-motor, executive control, and default mode networks (7–11). Consequently, analyses of RSNs are rapidly emerging as a powerful tool for in vivo mapping of neural circuitry in the human brain and one such approach for exploring RSNs is independent component analysis (ICA) (12–14). ICA decomposes the data into spatially independent and temporally coherent source signals/components. The advantage of ICA over more traditional seed-based approaches (15) is that it is a model-free, data-driven multiple-regression approach, i.e., within the ICA framework we can account for multiple underlying signal contributions (artifactual or neuronal in origin) simultaneously and thereby disentangle these different contributions to the measured observations (16). To date, ICA has been used not only to characterize brain connectivity in healthy adults (7, 10, 17), but also to assess the development of brain connectivity at various stages of (18, 19) as well as across the lifespan (20) and to investigate connectivity alterations in clinical populations (21–24).Here, we use this approach to investigate the intrinsic organization of RSNs in the human spinal cord. The spinal cord is the first part of the central nervous system (CNS) involved in the transmission of somatosensory information from the body periphery to the brain, as well as the last part of the CNS involved in relaying motor signals to the body periphery. This functional separation is also evident in the anatomical organization of the spinal cord, with the ventral part of gray matter involved in motor function and the dorsal part involved in somatosensory processing. The corresponding pairs of ventral and dorsal nerve roots convey information to and from the body periphery with a rostro-caudal topographical arrangement for both sensory (dermatomes) and motor innervation (myotomes).Although such a precise anatomical layout would suggest clear organizational principles for intrinsic spinal cord networks (similar to e.g., the visual and auditory RSNs in the brain), it is not known whether spatially consistent RSNs exist in the spinal cord. Distinct spatial maps due to cardiac and respiratory noise sources have been revealed by single subject ICA (25–27), and a seed-based approach demonstrated correlations between ventral horns and between dorsal horns (28), but no group patterns of circumscribed motor or sensory networks have yet been found; also only a few investigations of task-based functional connectivity have been performed (29–31). One reason for the apparent lack of relevant data is that fMRI is more challenging to perform in the spinal cord than in the brain (32, 33). The difficulties faced are mostly due to its small cross-sectional area (∼1 cm², necessitating the use of small voxel sizes, which leads to a low signal-to-noise ratio), magnetic susceptibility differences in tissues adjacent to the cord, e.g., vertebral bodies and spinous processes (causing signal loss and image distortion), as well as the influence of physiological noise (obscuring neuronally induced signal changes).Here, we used recent improvements in spinal fMRI [i.e., acquisition techniques that mitigate magnetic susceptibility differences (34), validated procedures for physiological noise reduction (35, 36) and techniques that allow voxel-wise group analyses (37, 38)] to overcome these difficulties and investigate the organizational principles of RSNs in the human spinal cord. We hypothesized that dorsal and ventral regions of the spinal cord would show different patterns of resting activity and furthermore investigated whether the segmental organization of the spinal cord would be evident in the rostro-caudal spatial layout of spinal RSNs.     

From the Cover: Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.Neurodegenerative diseases that affect motor neurons, such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA), have few treatment options due to the formidable challenges associated with drug delivery to the central nervous system (CNS) (1). The inability to cross the blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) after systemic delivery and insufficient penetration into the parenchyma from the cerebrospinal fluid (CSF) has hampered the use of promising biologic drugs such as recombinant neurotrophic factors and proteins (2, 3). Whereas there have been substantial recent advances in brain-targeted delivery of transcytosing peptides, antibodies, and exosomes to treat diseases like Alzheimer’s disease (4–6), few therapeutic options are available for degenerative diseases that affect motor neurons in the spinal cord. Furthermore, many potential drugs are biologics such as proteins, genes, and small interfering RNAs that are not readily transported into the nervous system (7, 8). Therefore, a significant need exists for the development of innovative technologies to delivery biologics into the spinal cord.Therapeutic molecules have been delivered to the spinal cord by systemic injection, direct injection, intrathecal transplantation of genetically modified cells secreting the molecules of interest, or remote delivery (9). Due to the BBB and BSCB, systemic delivery to the CNS by i.v. or intraarterial injection is limited to selected small molecule drugs. A comparison of adeno-associated virus (AAV) delivery by intraparenchymal versus intrathecal delivery revealed greater overall motor neuron transduction and distribution from intrathecal administration (10). More recently, intrathecal administration of AAV into the cerebral spinal fluid (CSF) has been shown to be effective in gene transfer to the CNS in nonhuman primates (11, 12) and has also been used in clinical trials to deliver engineered cells acting as protein expression depots (13). Nonetheless, the clinical application of these methods is limited because of the invasive nature of delivery and because delivery into the CNS parenchyma after intra-CSF injection is low due to limited diffusion and penetration (14).To develop minimally invasive technologies for biologics delivery into the spinal cord, we drew inspiration from viruses that transduce motor neurons. For decades, herpes simplex viruses (HSVs) have been known to enter the CNS by retrograde axonal transport, and HSV has been engineered for remote gene transfer in animal models of disease (15, 16). Engineered viruses and protein chimeras have also been used successfully in animal models to transfer therapeutic genes for conditions such as spinal cord injury, spinal muscular atrophy, chronic pain, and amyotrophic lateral sclerosis (17–21). However, virus-mediated gene therapy has been limited in clinical translation due to issues of immunogenicity, vector safety, and cost of production. Protein delivery methods have been likewise hindered by the high concentrations of protein required for cargo delivery and the lack of methods to dock and shuttle therapeutics into the CNS effectively (22). Still, the efficient retrograde transport of select viral vectors into the CNS led us to hypothesize that we could identify a peptide that functions similarly and is able to interact with and be internalized by motor axons for transport into the CNS.Armed with the precedent that HSV has evolved to deliver complex, biologically-active viral particles to the CNS from a peripheral skin lesion, we developed a strategy to screen bacteriophage display libraries in vivo to identify peptides that mediate M13 bacteriophage transport to the spinal cord. Here, we report the identification of a peptide that is trafficked into the spinal cord after intramuscular (IM) injection. We show that the peptide localizes with motor neurons after administration and can be used to carry proteins into the spinal cord via an intact motor axon that projects to the periphery. We further use this peptide, called targeted axonal import (TAxI) to deliver an active enzyme into spinal cord neurons after peripheral muscle injection in mice. This TAxI peptide shows potential for clinical relevance because it also binds to motor neurons in human spinal cord.     

Beneficial effects of IL-37 after spinal cord injury in mice

IL-37, a member of the IL-1 family, broadly reduces innate inflammation as well as acquired immunity. Whether the antiinflammatory properties of IL-37 extend to the central nervous system remains unknown, however. In the present study, we subjected mice transgenic for human IL-37 (hIL-37tg) and wild-type (WT) mice to spinal cord contusion injury and then treated them with recombinant human IL-37 (rIL-37). In the hIL-37tg mice, the expression of IL-37 was barely detectable in the uninjured cords, but was strongly induced at 24 h and 72 h after the spinal cord injury (SCI). Compared with WT mice, hIL-37tg mice exhibited increased myelin and neuronal sparing and protection against locomotor deficits, including 2.5-fold greater speed in a forced treadmill challenge. Reduced levels of cytokines (e.g., an 80% reduction in IL-6) were observed in the injured cords of hIL-37tg mice, along with lower numbers of blood-borne neutrophils, macrophages, and activated microglia. We treated WT mice with a single intraspinal injection of either full-length or processed rIL-37 after the injury and found that the IL-37–treated mice had significantly enhanced locomotor skills in an open field using the Basso Mouse Scale, as well as supported faster speed on a mechanical treadmill. Treatment with both forms of rIL-37 led to similar beneficial effects on locomotor recovery after SCI. This study presents novel data indicating that IL-37 suppresses inflammation in a clinically relevant model of SCI, and suggests that rIL-37 may have therapeutic potential for the treatment of acute SCI.The inflammatory response plays an essential role in tissue protection after injury or invasion by microorganisms (1, 2). Regardless of the tissue, unless regulated, inflammation can become chronic and result in tissue damage and loss of function (1, 2). This is particularly the case in spinal cord injury (SCI). After spinal cord contusion or compression injury, there is a rapid initiation of inflammation in rodents and in humans (2). This response is orchestrated by endogenous microglial cells and by circulating leukocytes, especially monocytes and neutrophils, which invade the lesion site during the first hours and days after injury (2–4). Although these cells are required for the clearance of cellular and myelin debris, they also release cytokines and cytotoxic factors, which are harmful to neurons, glia, axons, and myelin, resulting in secondary damage to adjacent regions of the spinal cord that had been previously unaffected by the insult (2, 5, 6). Indeed, it is currently well accepted that inflammation is a major contributor to secondary cell death after SCI. The damaging effects of inflammation are more pronounced in the central nervous system (CNS) than in other tissues, because of the limited capacity for axon regeneration and replenishment of damaged neurons and glial cells, which leads to irreversible functional disabilities (7, 8). Therefore, targeting inflammation is a valuable approach to promoting neuroprotection and limiting functional deficits in SCI.Cytokines are key players in the initiation, progression, and suppression of inflammation. Although several members of the IL-1 family are proinflammatory (9, 10), IL-37 has broad suppressive effects on innate inflammation and acquired immunity (11–14). Because a complete ORF for the mouse homolog of IL-37 has not yet been found, it was necessary to generate a strain of transgenic mice expressing human IL-37, designated hIL-37tg mice. These mice are protected against endotoxin shock, colitis, hepatitis, and myocardial infarction (9, 13, 15–18); however, a role for IL-37 after CNS trauma remains unexplored. In the present study, we subjected hIL-37tg mice to SCI and studied subsequent functional impairments in comparison with wild type (WT) mice. We also administered recombinant human IL-37 (rIL-37) to WT mice, to provide a rationale for clinical use of IL-37 as a therapeutic agent. We provide direct evidence for the first time, to our knowledge, that IL-37 exerts marked antiinflammatory properties on the contused spinal cord and confers protection from tissue damage and functional loss.     

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

Neuronal calcium (Ca²⁺)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca²⁺-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca²⁺-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca²⁺-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.Calcium (Ca²⁺) plays a crucial role in many and diverse cellular processes, including neurotransmission (1). Glutamate and neuropeptides are neurotransmitters released from the central terminals of dorsal root ganglion (DRG) neurons in the spinal dorsal horn, where signals for different sensory modalities, including pain, are conveyed to higher centers (2–12). Neurotransmitter release is tightly regulated by Ca²⁺-dependent SNARE proteins whose activity is regulated by Ca²⁺-binding proteins (CaBPs) (1, 7, 13).Parvalbumin (PV), calbindin D-28K (CB), calretinin (CR), and secretagogin (Scgn) are extensively studied EF-hand CaBPs, and they have also emerged as valuable anatomical markers for morphologically and functionally distinct neuronal subpopulations (14–17). The expression of CaBPs in DRG neurons has been thoroughly studied (18). Moreover, neuronal Ca²⁺ sensor 1 and downstream regulatory element-antagonist modulator (DREAM) are also EF-hand Ca²⁺-binding proteins in DRGs and the spinal cord (19, 20). Despite these advances, a CaBP has so far not been characterized in the majority of small- and medium-sized DRG neurons, many of which represent nociceptors.The subfamily of neuronal Ca²⁺-binding proteins (NECABs) consists of three members (NECAB1–NECAB3), probably as a result of gene duplication (21). NECABs are also EF-hand proteins, with one pair of EF-hand motifs in the N terminus and a putative antibiotic biosynthesis monooxygenase domain in the C terminus, which are linked by a NECAB homogeneous region (22). NECAB1/2 are restricted to the nervous system, whereas NECAB3 is also expressed in the heart and skeletal muscle (21).NECAB1 was first identified as the target protein of synaptotagmin I C2A-domain by affinity chromatography, with its expression restricted to layer 4 cortical pyramidal neurons, inhibitory interneurons, and hippocampal CA2 pyramidal cells in mouse brain (21, 23). The gene of the second member was cloned from mouse and initially named Necab. It encodes a 389-aa (NECAB2) (24). NECAB2 was identified as a downstream target of Pax6 in mouse retina, which is involved in retinal development (24, 25), as well as being a binding partner for the adenosine A_2A receptor (22). Furthermore, an interaction between NECAB2 and metabotropic glutamate receptor 5 (mGluR5) was demonstrated in rat hippocampal pyramidal cells, possibly regulating mGluR5’s coupling to its signaling machinery (26). Finally, NECAB3, also known as XB51, was isolated as an interacting target for the neuron-specific X11-like protein and is possibly involved in the pathogenesis of Alzheimer’s disease (27, 28).Very recently, NECAB1/2 were shown to have complementary expression patterns in mouse hippocampus at the mRNA and protein levels, whereas NECAB3 is broadly distributed in the hippocampus (29). NECAB1-expressing cells were seen throughout the cell-sparse layers of Ammon’s horn and the hilus of the dentate gyrus. In contrast, NECAB2 is enriched in pyramidal cells of the CA2 region. A minority of NECAB1⁺ neurons were GABAergic yet did not coexpress PV, CB, or CR (29).Here, we investigated the expression of NECAB1/2 in mouse DRGs and spinal cord using quantitative PCR (qPCR), immunohistochemistry (also combined with CLARITY) (30), and Western blotting. We compared the distribution of NECABs with that of the four CaBPs restricted to neurons, PV, CB, CR, or Scgn. NECAB⁺ neurons in the spinal dorsal horn were phenotyped using transgenic mice harboring genetic markers for excitatory [vesicular glutamate transporter 2 (VGLUT2)] (31) or inhibitory [glutamate decarboxylase 67 (GAD67)] (32) cell identities. Finally, the effect of peripheral nerve injury was analyzed.     

Long-term potentiation of glycinergic synapses triggered by interleukin 1β

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABA_A receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.Glycine mediates fast synaptic inhibition throughout the spinal cord, brainstem, and midbrain, controlling normal motor behavior and rhythm generation, somatosensory processing, auditory and retinal signaling, and coordination of reflex responses (1). Strychnine-sensitive glycine receptors (GlyRs) are pentameric ligand-gated chloride channels of the Cys-loop receptor family that together with GABA_A receptors (GABA_ARs) dynamically interact with the synaptic scaffold protein gephyrin to form inhibitory synapses (1, 2). In the dorsal horn of the spinal cord, glycinergic synapses are essential for nociceptive and tactile sensory processing both during adaptive and pathological pain states (3–7). However, compared with glutamatergic and GABAergic synapses, little is known about the regulation of their synaptic strength. Several studies have examined glycine receptor trafficking in cultured neurons and in heterologous expression systems (8, 9). Intracellular Ca²⁺ appears important in the stabilization of GlyRs at synapses in culture (10), and elevation of intracellular Ca²⁺ can also potently increase glycine receptor single channel openings in cultured cells and in heterologous systems (11). However, the modulation of glycinergic synaptic strength in native tissue remains relatively unexplored.Following peripheral injury or inflammation, changes in tactile perception develop, including hyperalgesia (exaggerated pain upon noxious stimulation), allodynia (pain in response to innocuous stimuli), and secondary hyperalgesia (pain spreading beyond the confines of the injured region). Inhibitory interneurons of the spinal dorsal horn have been proposed to gate the flow of innocuous and nociceptive sensory information from the periphery to higher brain centers (12), and supportive evidence for this idea is growing (13–17). Loss of GABAergic/glycinergic inhibition contributes to enhanced transmission of nociceptive signals through the dorsal horn circuit during pain states, resulting in hyperalgesia and allodynia (3, 18–20). For example, polysynaptic A-fiber inputs onto neurokinin 1 receptor (NK1R)-expressing projection neurons become apparent only when GABA_AR and GlyRs are pharmacologically blocked, indicating that under conditions of disinhibition, nonnoxious mechanical stimuli can drive nociceptive-specific projection pathways and elicit allodynia (21). The majority of neurons tested in the dorsal horn receive glycinergic synapses, including lamina I projection neurons, both excitatory and inhibitory interneurons of lamina II (22, 23), and inhibitory glycinergic neurons (24). Given the diversity of afferent targets, it is likely that glycinergic synapses are differentially modulated in a cell type- and subregion-specific manner. For example, during chronic inflammation, prostaglandin E₂ selectively depresses glycinergic synaptic inputs onto nonglycinergic neurons (24). Similarly, peripheral nerve injury suppresses glycinergic inhibition of a specific excitatory interneuron class [protein kinase C (PKC)γ⁺ neurons receiving Aβ fiber inputs], allowing excitatory afferents carrying nonnociceptive tactile information to activate ascending projections of nociceptive pathways that are normally under strong inhibitory control (23).Both hyperalgesia and allodynia occur within minutes of peripheral inflammation, but the mechanisms underlying these rapid perceptual alterations are poorly understood. The proinflammatory cytokine, IL-1β, is a potent hyperalgesic agent (25–27), contributing both to peripheral and central sensitization after tissue damage (28–31). Following tissue trauma, nerve injury, or inflammation, IL-1β levels are up-regulated in the spinal cord itself (29, 32, 33), and delivery of IL-1β intrathecally increases the activity of superficial dorsal horn neurons that transmit pain signals to the brain (34, 35). Intrathecal delivery of an IL-1 receptor antagonist blocks allodynia in rodent models of inflammatory pain (36, 37). A recent study also found that IL-1β application rapidly potentiated primary afferent (glutamatergic) synapses in dorsal horn slices, through unidentified signaling molecules released from glial cells (38). Here we report that IL-1β rapidly elicits a postsynaptic form of long-term potentiation (LTP) at glycinergic synapses on lamina II inhibitory neurons (GlyR LTP), and that the same glycinergic synapses are potentiated after peripheral inflammation.     

From the Cover: Degradation of mouse locomotor pattern in the absence of proprioceptive sensory feedback

Mammalian locomotor programs are thought to be directed by the actions of spinal interneuron circuits collectively referred to as “central pattern generators.” The contribution of proprioceptive sensory feedback to the coordination of locomotor activity remains less clear. We have analyzed changes in mouse locomotor pattern under conditions in which proprioceptive feedback is attenuated genetically and biomechanically. We find that locomotor pattern degrades upon elimination of proprioceptive feedback from muscle spindles and Golgi tendon organs. The degradation of locomotor pattern is manifest as the loss of interjoint coordination and alternation of flexor and extensor muscles. Group Ia/II sensory feedback from muscle spindles has a predominant influence in patterning the activity of flexor muscles, whereas the redundant activities of group Ia/II and group Ib afferents appear to determine the pattern of extensor muscle firing. These findings establish a role for proprioceptive feedback in the control of fundamental aspects of mammalian locomotor behavior.In mammals, walking and swimming represent favored terrestrial and aquatic solutions to the general challenge of locomotion. Both forms of movement depend on the temporal coordination of limb muscles at specific joints, driven by stereotypic and individualized patterns of flexor and extensor muscle activation (1–4). At a spinal level, locomotor programs are thought to emerge through the integrated actions of interneuronal circuits that function as central pattern generators (CPGs) and potentially through sensory feedback mediated by cutaneous and proprioceptive inputs (5–7). Advances in defining functional spinal motor circuitry in mammals (8) have nevertheless left unresolved the respective contributions of local interneuronal and sensory feedback systems to the coordination of locomotor activities in vivo. In part, this uncertainty stems from the inability to assess the impact of inactivating defined populations of sensory neurons with anatomical precision in vivo, under conditions in which locomotor output can be evaluated.Mammalian locomotion has traditionally been analyzed in cats by kinematic and electromyographic (EMG) evaluation of the walking step cycle, with a focus on the hindlimb (1, 2, 4, 9). These studies have shown that individual extensor and flexor muscles controlling the hip, knee, and ankle joints exhibit distinct and stereotypic onset and offset timing, as well as a pronounced alternation in flexor–extensor phasing that accompanies the transition from stance to swing, or swing to stance (1, 2, 4). To address the contribution of proprioceptive feedback to locomotor pattern generation, comparisons have been made between locomotor pattern in normal walking cats and fictive locomotion in the absence of phasic proprioceptive feedback (10–13). Under certain experimental conditions, normal and fictive motor output patterns are similar (10, 11), whereas other conditions reveal striking differences between the two motor programs (11, 13), often restricted to particular muscles (12). These observations suggested that the CPG may not be sufficient to reproduce normal locomotor output. Thus, the degree to which the spinal CPG directs functional locomotor patterns in the absence of proprioceptive sensory feedback remains uncertain.Prior studies in cat have, nevertheless, provided evidence that proprioceptive feedback modifies stance and swing phase transitions during walking to accommodate changes in task and terrain (14–18), but have not resolved the extent to which proprioceptive sensory feedback contributes to core elements of mammalian locomotor pattern. Nor have the individual contributions of the two main functional classes of proprioceptors—group Ia/II muscle spindle (MS) and group Ib Golgi tendon organ (GTO) afferents been examined. In this study we assessed the role of proprioceptive sensory feedback in mammalian locomotor pattern through an examination of mice in which a mutation in the Egr3 (early growth response 3) gene selectively impairs group Ia/II muscle spindle activation, eliminating one class of proprioceptive feedback. We assayed Egr3 mutants in two locomotor tasks—walking and swimming—which differ in the contribution of input from group Ib sensory afferents supplying GTOs (19, 20). Our studies, probe the role of sensory feedback in locomotor control under closed loop conditions and address the role of proprioceptive sensory feedback in assigning patterned motor output in vivo.Our analysis reveals that normal walking locomotor pattern in mice requires ongoing proprioceptive feedback to generate coordinated stepping movements. The absence of proprioceptive feedback from muscle spindles impairs locomotor pattern by perturbing the precise timing of ankle flexor muscle activity offset during swing phase. In addition, feedback from muscle spindles plays a more critical role in pattern generation when feedback from GTOs is absent—in the absence of feedback from muscle spindles and GTOs, coordinated stepping movements fail. These findings show that muscle spindle and GTO afferents provide essential and, in some instances, distinct functions in the patterning of locomotor output.     

Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis

Neural precursor cells (NPCs) offer a promising approach for treating demyelinating diseases. However, the cellular dynamics that underlie transplanted NPC-mediated remyelination have not been described. Using two-photon imaging of a newly developed ventral spinal cord preparation and a viral model of demyelination, we describe the motility and intercellular interactions of transplanted mouse NPCs expressing green fluorescent protein (GFP) with damaged axons expressing yellow fluorescent protein (YFP). Our findings reveal focal axonal degeneration that occurs in the ventral side of the spinal cord within 1 wk following intracranial instillation with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Axonal damage precedes extensive demyelination and is characterized by swelling along the length of the axon, loss of YFP signal, and transected appearance. NPCs engrafted into spinal cords of JHMV-infected mice exhibited diminished migration velocities and increased proliferation compared with transplanted cells in noninfected mice. NPCs preferentially accumulated within areas of axonal damage, initiated direct contact with axons, and subsequently expressed the myelin proteolipid protein gene, initiating remyelination. These findings indicate that NPCs transplanted into an inflammatory demyelinating microenvironment participate directly in therapeutic outcome through the wrapping of myelin around damaged neurons.Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function (1). The demyelination that defines MS pathology is progressive over time; however, spontaneous yet transient myelin repair can occur during the course of disease (2). Currently approved therapies for treating MS are designed to limit immune cell infiltration into the CNS to mute demyelination and impede the emergence of new lesions (3). Recent studies from our laboratory and others have shown that engraftment of neural precursor cells (NPCs) may provide an important unmet clinical need for MS patients by facilitating sustained remyelination that can restore motor function and ameliorate clinical symptoms associated with demyelinating disease. NPC engraftment is well tolerated in animal models and contributes to clinical recovery associated with remyelination, highlighting the feasibility of using NPCs for treating demyelinating diseases (4–8). Indeed, transplantation of human NPCs into the frontal lobes of children with Pelizaeus–Merzbacher disease (PMD), a rare genetic disorder that affects the growth of the myelin sheath, has revealed measurable gains in motor and/or cognition skills, emphasizing the translational relevance of NPCs for treatment of white matter diseases (9).Although NPCs have been shown to migrate and facilitate remyelination in preclinical animal models of demyelination, the motility characteristics and interactions of NPCs with damaged axons have not directly been visualized in real time within the intact cord. Instead, imaging has been limited to “snapshots” of NPCs transplanted into the spinal cord and visualized in fixed tissue by immunohistochemistry or with fluorescently labeled engrafted cells (8, 10, 11). In this study, we used a mouse model of viral-induced demyelination to establish an alternative imaging system enabling stable ex vivo imaging of transplanted GFP-labeled mouse NPCs within the ventral murine spinal cord through use of two-photon (2P) microscopy. Mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) develop MS-like symptoms ranging from partial to complete hind limb paralysis that are associated with immune cell accumulation within the CNS and white matter damage (12–16). We have previously demonstrated that engraftment of syngeneic mouse NPCs promotes clinical recovery that correlates with increased axonal integrity and remyelination (8), although neuroinflammation is not affected (17). Transplanted NPCs follow a CXCL12 chemokine gradient to preferentially colonize areas of white matter damage, where a majority of cells differentiate into an oligodendrocyte lineage (5, 8).Two-photon microscopy enables real-time visualization of cellular migration and intercellular interactions within intact organs (18). Several groups have used 2P microscopy to characterize axonal degradation and immune cell dynamics in the dorsal spinal cord during demyelinating disease progression (19–23). However, because engrafted NPCs preferentially migrate to regions deep within the ventral spinal cord (24), standard dorsal-side 2P in vivo imaging techniques are not suitable for visualization deep in the ventral side. Using a ventral-side imaging preparation, we now demonstrate that NPCs transplanted into the spinal cords of JHMV-infected mice under pathologic conditions migrate directionally, take up residence in regions of axonal degradation, colocalize with damaged axons, and facilitate remyelination through direct interactions with axons.     

From the Cover: PNAS Plus: Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.Induced pluripotent stem cell (iPSC) technology is currently being used to model human diseases in vitro and may contribute to the discovery and validation of new pharmacological treatments (1–3). In particular, neuroscientists have seized the opportunity to culture neurons from patients with neurological and psychiatric disorders and have demonstrated that phenotypes associated with particular disorders can be recapitulated in the dish (4–7). However, the basic culture conditions for growing neurons in vitro have not been updated to reflect fundamental principles of brain physiology. Currently, most human neuronal cultures are grown in media based on DMEM/F12 (4, 5, 7–24), Neurobasal (25–30), or a mixture of DMEM and Neurobasal (DN) (31–34). To promote neuronal differentiation and survival, a variety of supplements, such as serum, growth factors, hormones, proteins, and antioxidants, are typically added to these basal media. Although these media were designed and optimized to promote neuronal survival in vitro, they were not tested for their ability to support fundamental neuronal functions. Using several electrophysiological techniques such as patch clamping, calcium imaging, and multielectrode arrays, we found that widely used tissue culture media (e.g., DMEM basal, Neurobasal, serum) actually impaired neurophysiological functions.We identified several neuroactive components in these media that acutely interfered with neuronal function. To solve these issues, we designed a chemically defined basal medium: BrainPhys basal. We used human neurons in vitro to demonstrate in a series of experiments that this new medium, combined with the appropriate supplements, better supports important neuronal functions while sustaining cell survival in vitro. Notably, BrainPhys-based medium better mimics the environment present in healthy living brains, unlike previous media based on DMEM, Neurobasal or serum. Although BrainPhys basal was specifically designed for the culturing of mature human neurons, our studies also showed that BrainPhys provided a functional environment for ex vivo brain slices and for culturing rodent primary neurons.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

Optogenetic activation of cholinergic neurons in the PPT or LDT induces REM sleep

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.Rapid eye movement (REM) sleep is tightly regulated, yet the mechanisms that control REM sleep remain incompletely understood. Early pharmacological and unit recording studies suggested that ACh was important for REM sleep regulation (1, 2). For example, injection of cholinergic drugs into the dorsal mesopontine tegmentum reliably induced a state very similar to natural REM sleep in cats (3–6). Unit recordings from the cholinergic areas of the mesopontine tegmentum revealed cells that were active during wakefulness and REM sleep, as well as neurons active only during REM sleep (7–13). Electrical stimulation of the laterodorsal tegmentum (LDT) in cats increased the percentage of time spent in REM sleep (14), and activation of the pedunculopontine tegmentum (PPT) in rats induced wakefulness and REM sleep (15). If cholinergic PPT and LDT neurons are necessary for REM sleep to occur, as the early studies suggest, then lesioning the PPT or LDT should decrease REM sleep. In cats, lesions of the PPT and LDT do disrupt REM sleep (16, 17), but lesions in rodents have had little effect on REM sleep or increased REM sleep (18–22). Additionally, c-fos studies have found very few cholinergic cells activated under high-REM sleep conditions. When c-fos–positive cholinergic neurons in the PPT and LDT are found to correlate with the percentage of REM sleep, they still account for only a few of the total cholinergic cells in the area (23). Juxtacellular recordings of identified cholinergic neurons in the LDT found these cells had wake and REM active firing profiles, with the majority firing the highest during REM sleep (13). These discrepancies have led to alternative theories of REM sleep regulation, where cholinergic neurons do not play a key role (18, 19, 23, 24 and reviewed in 25, 26).The PPT and LDT are made up of heterogeneous populations of cells, including distinct populations of cholinergic, GABAergic, and glutamatergic neurons (27–29). Many GABAergic neurons are active during REM sleep, as indicated by c-fos (23), and both GABAergic and glutamatergic neurons have been found with maximal firing rates during REM sleep in the LDT and medial PPT (13). To distinguish the differential roles of each cell type in REM sleep regulation, a method that can modulate specific cell types in the behaving animal is needed. Optogenetics now provides this ability to target specific subpopulations of neurons and control them with millisecond temporal resolution (30). Therefore, we aimed to determine the role of cholinergic neurons in the PPT and LDT in REM sleep regulation using optogenetics.     

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43^Q331K and TDP-43^M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43^Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43^Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are progressive, adult-onset neurodegenerative diseases with overlapping clinical and pathological features (1–3). ALS is characterized by the selective loss of upper and lower motor neurons, leading to progressive fatal paralysis and muscle atrophy. A large majority (∼90%) of ALS and FTLD-U cases are without a known genetic cause. Importantly, in these sporadic cases, the appearance of ubiquitinated inclusions within the affected neurons of the nervous system characterizes both ALS and FTLD-U patients, suggesting an overlapping mechanism underlying both diseases. Biochemical characterization of brains and spinal cords from ALS and FTLD-U patients identified transactivating response region (TAR) DNA binding protein (TDP-43) as the major protein component of these ubiquitinated inclusions (4, 5). The discovery of ALS-linked mutations in the glycine-rich C-terminal domain of TDP-43 (6-8) demonstrated a pathological role of TDP-43 in both diseases. The subsequent identification of mutations in a structurally and functionally related nucleic acid binding protein, FUS/TLS (fused in sarcoma/translocated in liposarcoma) (9, 10), further implicated defects in RNA processing in ALS pathogenesis.TDP-43 is a multifunctional nucleic acid binding protein. Within the nervous system, TDP-43 binds to >6,000 pre-mRNAs and affects the levels of ∼600 mRNAs and the splicing patterns of another 950 (11). Structurally, the 414-aa protein consists of two RNA recognition motifs (RRM1 and RRM2) (12, 13), nuclear import and export signal (14), and a glycine-rich region implicated in protein–protein interactions (15, 16) that include components of the RNA splicing machinery (17, 18).Disruption in mice of the highly conserved Tardbp gene is embryonically lethal (19–22). Similarly, postnatal inactivation of Tardbp (by Cre recombinase-mediated gene excision encoded by a ubiquitously-expressed CAG-Cre transgene) results in rapid postnatal death accompanied by defects in fat metabolism (22). TDP-43 autoregulates its own RNA level (11, 23) at least in part by stimulating excision of an intron in its 3′ untranslated region, thereby making the spliced RNA a substrate for nonsense-mediated RNA degradation (11). Furthermore, transgenic rodent models have been used to demonstrate that overriding the autoregulatory mechanism by overexpression of unregulated wild-type (24–28) or disease-linked mutant (26, 28–35) TDP-43 transgenes can produce neurodegeneration in mice.ALS and FTLD-U patient brain and spinal cord samples are characterized by the accumulation of cytoplasmic TDP-43 aggregates accompanied by a distinct clearing of nuclear TDP-43 within affected neurons and glia (36, 37), implicating possible loss of nuclear TDP-43 function in disease pathogenesis. In human disease, TDP-43 has been reported to be abnormally phosphorylated, ubiquitinated, and cleaved to produce C-terminal fragments (4, 5, 38, 39). Ectopic expression of these C-terminal fragments in cell-culture models (40–42) has shown that they are aggregation-prone and confer an intrinsic toxicity. However, the extent of the contribution of these C-terminal fragments to disease pathogenesis is undetermined. Indeed, double-immunofluorescent labeling of ALS patient spinal cords using N-terminal–specific and C-terminal–specific antibodies suggests that inclusions in spinal cord motor neurons are comprised primarily of full-length TDP-43 (37). Importantly, retention of ability to bind RNA by full-length TDP-43 has been demonstrated to be required for toxicity in yeast, fly, and Caenorhabditis elegans models (43–46).Nevertheless, it remains unresolved whether toxicity to motor neurons from mutations in TDP-43 is mediated through a gain of toxic property, loss-of-function, or a combination of both. By generation of transgenic mice encoding levels of wild-type or mutant human TDP-43 comparable to endogenous TDP-43, we demonstrate mutant-dependent, age-dependent motor neuron disease from ALS-linked TDP-43 mutants in the absence of overexpression, cytoplasmic accumulation of a 35 kDa TDP-43 fragment, or insoluble TDP-43 aggregates. Accompanying autoregulation-mediated reduction of endogenous wild-type TDP-43 are splicing alterations previously identified to be TDP-43–dependent (11). Additional splicing alterations are identified by systematic genome-wide analyses of alternative splicing that are indicative of both enhancement and loss-of-function by the TDP-43 mutants for individual RNA substrates, from which we conclude that ALS-linked mutations confer both loss- and gain-of-function properties to TDP-43, and that these act intranuclearly to induce splicing alterations that may underlie age-dependent motor neuron disease.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.