Shared Neural Markers of Decision Confidence and Error Detection

Empirical evidence indicates that people can provide accurate evaluations of their own thoughts and actions by means of both error detection and confidence judgments. This study investigates the foundations of these metacognitive abilities, specifically focusing on the relationship between confidence and error judgments in human perceptual decision making. Electroencephalography studies have identified the error positivity (Pe)—an event-related component observed following incorrect choices—as a robust neural index of participants' awareness of their errors in simple decision tasks. Here we assessed whether the Pe also varies in a graded way with participants' subjective ratings of decision confidence, as expressed on a 6-point scale after each trial of a dot count perceptual decision task. We observed clear, graded modulation of the Pe by confidence, with monotonic reduction in Pe amplitude associated with increasing confidence in the preceding choice. This effect was independent of objective accuracy. Multivariate decoding analyses indicated that neural markers of error detection were predictive of varying levels of confidence in correct decisions, including subtle shifts in high-confidence trials. These results suggest that shared mechanisms underlie two forms of metacognitive evaluation that are often treated separately, with consequent implications for current theories of their neurocognitive basis.

Parkinson's Disease Iron Deposition Caused by Nitric Oxide-Induced Loss of {beta}-Amyloid Precursor Protein

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP^–/– mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

Seasonal Plasticity of Precise Spike Timing in the Avian Auditory System

Vertebrate audition is a dynamic process, capable of exhibiting both short- and long-term adaptations to varying listening conditions. Precise spike timing has long been known to play an important role in auditory encoding, but its role in sensory plasticity remains largely unexplored. We addressed this issue in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii), a songbird that shows pronounced seasonal fluctuations in circulating levels of sex-steroid hormones, which are known to be potent neuromodulators of auditory function. We recorded extracellular single-unit activity in the auditory forebrain of males and females under different breeding conditions and used a computational approach to explore two potential strategies for the neural discrimination of sound level: one based on spike counts and one based on spike timing reliability. We report that breeding condition has robust sex-specific effects on spike timing. Specifically, in females, breeding condition increases the proportion of cells that rely solely on spike timing information and increases the temporal resolution required for optimal intensity encoding. Furthermore, in a functionally distinct subset of cells that are particularly well suited for amplitude encoding, female breeding condition enhances spike timing-based discrimination accuracy. No effects of breeding condition were observed in males. Our results suggest that high-resolution temporal discharge patterns may provide a plastic neural substrate for sensory coding.

Norepinephrine Activates Dopamine D4 Receptors in the Rat Lateral Habenula

The lateral habenula (LHb) is involved in reward and aversion and is reciprocally connected with dopamine (DA)-containing brain regions, including the ventral tegmental area (VTA). We used a multidisciplinary approach to examine the properties of DA afferents to the LHb in the rat. We find that >90% of VTA tyrosine hydroxylase (TH) neurons projecting to the LHb lack vesicular monoamine transporter 2 (VMAT2) mRNA, and there is little coexpression of TH and VMAT2 protein in this mesohabenular pathway. Consistent with this, electrical stimulation of LHb did not evoke DA-like signals, assessed with fast-scan cyclic voltammetry. However, electrophysiological currents that were inhibited by L741,742, a DA-D₄-receptor antagonist, were observed in LHb neurons when DA uptake or degradation was blocked. To prevent DA activation of D₄ receptors, we repeated this experiment in LHb slices from DA-depleted rats. However, this did not disrupt D₄ receptor activation initiated by the dopamine transporter inhibitor, GBR12935. As the LHb is also targeted by noradrenergic afferents, we examined whether GBR12935 activation of DA-D₄ receptors occurred in slices depleted of norepinephrine (NE). Unlike DA, NE depletion prevented the activation of DA-D₄ receptors. Moreover, direct application of NE elicited currents in LHb neurons that were blocked by L741,742, and GBR12935 was found to be a more effective blocker of NE uptake than the NE-selective transport inhibitor nisoxetine. These findings demonstrate that NE is released in the rat LHb under basal conditions and that it activates DA-D₄ receptors. Therefore, NE may be an important regulator of LHb function.

Active Avoidance Requires a Serial Basal Amygdala to Nucleus Accumbens Shell Circuit

Freezing is a species-typical defensive reaction to conditioned threats. While the neural circuitry of aversive Pavlovian behavior has been extensively studied, less is known about the circuitry underlying more active responses to danger. Here we show that the flow of information between the basal amygdala (BA) and the nucleus accumbens (NAcc) is necessary for signaled active avoidance behavior. Rats trained to avoid shock by shuttling during an auditory conditioned stimulus showed increased expression of the activity-dependent protein c-Fos in the NAcc, specifically the shell subregion (NAccSh). Silencing neural activity in the NAccSh, but not in the adjacent NAcc core, disrupted avoidance behavior. Disconnection of the BA and the NAccSh was just as effective at disrupting avoidance behavior as bilateral NAccSh inactivations, suggesting learned avoidance behavior requires an intact BA-NAccSh circuit. Together, these data highlight an essential role for the amygdalar projection to the ventral striatum in aversively motivated actions.

Optogenetic Stimulation of Accumbens Shell or Shell Projections to Lateral Hypothalamus Produce Differential Effects on the Motivation for Cocaine

Previous studies suggest that pharmacological or molecular activation of the nucleus accumbens shell (AcbSh) facilitates extinction of cocaine-seeking behavior. However, overexpression of CREB, which increases excitability of AcbSh neurons, enhances cocaine-seeking behavior while producing depression-like behavior in tests of mood. These discrepancies may reflect activity in differential AcbSh outputs, including those to the lateral hypothalamus (LH), a target region known to influence addictive behavior and mood. Presently, it is unknown whether there is a causal link between altered activity in the AcbSh-LH pathway and changes in the motivation for cocaine. In this study, we used an optogenetics approach to either globally stimulate AcbSh neurons or to selectively stimulate AcbSh terminal projections in the LH, in rats self-administering cocaine. We found that stimulation of the AcbSh-LH pathway enhanced the motivation to self-administer cocaine in progressive ratio testing, and led to long-lasting facilitation of cocaine-seeking behavior during extinction tests conducted after withdrawal from cocaine self-administration. In contrast, global AcbSh stimulation reduced extinction responding. We compared these opposing motivational effects with effects on mood using the forced swim test, where both global AcbSh neuron and selective AcbSh-LH terminal stimulation facilitated depression-like behavioral despair. Together, these findings suggest that the AcbSh neurons convey complex, pathway-specific modulation of addiction and depression-like behavior, and that these motivation and mood phenomenon are dissociable.

Integration of Canal and Otolith Inputs by Central Vestibular Neurons Is Subadditive for Both Active and Passive Self-Motion: Implication for Perception

Traditionally, the neural encoding of vestibular information is studied by applying either passive rotations or translations in isolation. However, natural vestibular stimuli are typically more complex. During everyday life, our self-motion is generally not restricted to one dimension, but rather comprises both rotational and translational motion that will simultaneously stimulate receptors in the semicircular canals and otoliths. In addition, natural self-motion is the result of self-generated and externally generated movements. However, to date, it remains unknown how information about rotational and translational components of self-motion is integrated by vestibular pathways during active and/or passive motion. Accordingly, here, we compared the responses of neurons at the first central stage of vestibular processing to rotation, translation, and combined motion. Recordings were made in alert macaques from neurons in the vestibular nuclei involved in postural control and self-motion perception. In response to passive stimulation, neurons did not combine canal and otolith afferent information linearly. Instead, inputs were subadditively integrated with a weighting that was frequency dependent. Although canal inputs were more heavily weighted at low frequencies, the weighting of otolith input increased with frequency. In response to active stimulation, neuronal modulation was significantly attenuated (~70%) relative to passive stimulation for rotations and translations and even more profoundly attenuated for combined motion due to subadditive input integration. Together, these findings provide insights into neural computations underlying the integration of semicircular canal and otolith inputs required for accurate posture and motor control, as well as perceptual stability, during everyday life.

Fast Gamma Oscillations Are Generated Intrinsically in CA1 without the Involvement of Fast-Spiking Basket Cells

Information processing in neuronal networks relies on the precise synchronization of ensembles of neurons, coordinated by the diverse family of inhibitory interneurons. Cortical interneurons can be usefully parsed by embryonic origin, with the vast majority arising from either the caudal or medial ganglionic eminences (CGE and MGE). Here, we examine the activity of hippocampal interneurons during gamma oscillations in mouse CA1, using an in vitro model where brief epochs of rhythmic activity were evoked by local application of kainate. We found that this CA1 KA-evoked gamma oscillation was faster than that in CA3 and, crucially, did not appear to require the involvement of fast-spiking basket cells. In contrast to CA3, we also found that optogenetic inhibition of pyramidal cells in CA1 did not significantly affect the power of the oscillation, suggesting that excitation may not be essential for gamma genesis in this region. We found that MGE-derived interneurons were generally more active than CGE interneurons during CA1 gamma, although a group of CGE-derived interneurons, putative trilaminar cells, were strongly phase-locked with gamma oscillations and, together with MGE-derived axo-axonic and bistratified cells, provide attractive candidates for being the driver of this locally generated, predominantly interneuron-driven model of gamma oscillations.

Melanin-Concentrating Hormone Neurons Release Glutamate for Feedforward Inhibition of the Lateral Septum

Melanin-concentrating hormone (MCH) regulates vital physiological functions, including energy balance and sleep. MCH cells are thought to be GABAergic, releasing GABA to inhibit downstream targets. However, there is little experimental support for this paradigm. To better understand the synaptic mechanisms of mouse MCH neurons, we performed neuroanatomical mapping and characterization followed by optogenetics to test their functional connectivity at downstream targets. Synaptophysin-mediated projection mapping showed that the lateral septal nucleus (LS) contained the densest accumulation of MCH nerve terminals. We then expressed channelrhodopsin-2 in MCH neurons and photostimulated MCH projections to determine their effect on LS activity. Photostimulation of MCH projections evoked a monosynaptic glutamate release in the LS. Interestingly, this led to a feedforward inhibition that depressed LS firing by a robust secondary GABA release. This study presents a circuit analysis between MCH and LS neurons and confirms their functional connection via monosynaptic and polysynaptic pathways. Our findings indicate that MCH neurons are not exclusively GABAergic and reveal a glutamate-mediated, feedforward mechanism that inhibits LS cells.

Absence of Plateau Potentials in dLGN Cells Leads to a Breakdown in Retinogeniculate Refinement

The link between neural activity and the refinement of projections from retina to the dorsal lateral geniculate nucleus (dLGN) of thalamus is based largely on studies that disrupt presynaptic retinogeniculate activity. Postsynaptic mechanisms responsible for implementing the activity-dependent remodeling in dLGN remain unknown. We tested whether L-type Ca²⁺ channel activity in the form of synaptically evoked plateau potentials in dLGN cells is needed for remodeling by using a mutant mouse that lacks the ancillary β₃ subunit and, as a consequence, has highly reduced L-type channel expression and attenuated L-type Ca²⁺ currents. In the dLGNs of β₃-null mice, glutamatergic postsynaptic activity evoked by optic tract stimulation was normal, but plateau potentials were rarely observed. The few plateaus that were evoked required high rates of retinal stimulation, but were still greatly attenuated compared with those recorded in age-matched wild-type mice. While β₃-null mice exhibit normal stage II and III retinal waves, their retinogeniculate projections fail to segregate properly and dLGN cells show a high degree of retinal convergence even at late postnatal ages. These structural and functional defects were also accompanied by a reduction in CREB phosphorylation, a signaling event that has been shown to be essential for retinogeniculate axon segregation. Thus, postsynaptic L-type Ca²⁺ activity plays an important role in mediating the refinement of the retinogeniculate pathway.

Divisive Gain Modulation of Motoneurons by Inhibition Optimizes Muscular Control

When using muscles, the precision with which force is delivered is as important as the delivery of force itself. Force is regulated by both the number of recruited motoneurons and their spike frequency. While it is known that the recruitment is ordered to reduce variability in force, it remains unclear whether the motoneuron gain, i.e., the slope of the transformation between synaptic input and spiking output, is also modulated to reduce variability in force. To address this issue, we use turtle hindlimb scratching as a model for fine motor control, since this behavior involves precise limb movement to rub the location of somatic nuisance touch. We recorded intracellularly from motoneurons in a reduced preparation where the limbs were removed to increase mechanical stability and the motor nerve activity served as a surrogate for muscle force. We found that not only is the gain of motoneurons regulated on a subsecond timescale, it is also adjusted to minimize variability. The modulation is likely achieved via an expansive nonlinearity between spike rate and membrane potential with inhibition having a divisive influence. These findings reveal a versatile mechanism of modulating neuronal sensitivity and suggest that such modulation is fundamentally linked to optimization.

Parkinson's Disease Iron Deposition Caused by Nitric Oxide-Induced Loss of {beta}-Amyloid Precursor Protein

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP^–/– mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

GABAergic Projections from Lateral Hypothalamus to Paraventricular Hypothalamic Nucleus Promote Feeding

Lesions of the lateral hypothalamus (LH) cause hypophagia. However, activation of glutamatergic neurons in LH inhibits feeding. These results suggest a potential importance for other LH neurons in stimulating feeding. Our current study in mice showed that disruption of GABA release from adult LH GABAergic neurons reduced feeding. LH GABAergic neurons project extensively to the paraventricular hypothalamic nucleus (PVH), and optogenetic stimulation of GABAergic LH -> PVH fibers induced monosynaptic IPSCs in PVH neurons, and potently increased feeding, which depended on GABA release. In addition, disruption of GABA-A receptors in the PVH reduced feeding. Thus, we have identified a new feeding pathway in which GABAergic projections from the LH to the PVH promote feeding.

Reactive Astrogliosis Causes the Development of Spontaneous Seizures

Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of β1-integrin (Itgβ1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted β1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl^– gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures.

Peripheral Nerve Injury Induces Persistent Vascular Dysfunction and Endoneurial Hypoxia, Contributing to the Genesis of Neuropathic Pain

Nerve injury is associated with microvascular disturbance; however, the role of the vascular system has not been well characterized in the context of neuropathic pain. Furthermore, ischemia is thought to play a role in a number of neuropathic pain conditions, and yet the role of hypoxia has also not been characterized in neuropathic pain conditions. In this study, we observed the presence of persistent endoneurial hypoxia in a mouse model of traumatic peripheral nerve injury, causing painful mononeuropathy. We attribute the ongoing hypoxia to microvascular dysfunction, endoneurial fibrosis, and increased metabolic requirements within the injured nerve. Increased lactate levels were observed in injured nerves, as well as increased oxygen consumption and extracellular acidification rates, suggesting that anaerobic glycolysis is required to maintain cellular ATP levels. Hypoxia causes a reduction in levels of the Na⁺/K⁺ ATPase ion transporter in both cultured primary dorsal root ganglion neurons and injured peripheral nerve. A reduction of Na⁺/K⁺ ATPase ion transporter levels likely contributes to the hyperexcitability of injured nerves. Physiological antagonism of hypoxia with hyperbaric oxygen alleviated mechanical allodynia in nerve-injured animals. These results suggest that hypoxia and the Na⁺/K⁺ ATPase ion transporter may be a novel mechanistic target for the treatment of neuropathic pain. In addition, the findings support the possibility of using hypoxia activated pro-drugs to localize treatments for neuropathic pain and nerve injury to injured nerves.

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Deficiency Attenuates Phagocytic Activities of Microglia and Exacerbates Ischemic Damage in Experimental Stroke

Clearing cellular debris after brain injury represents an important mechanism in regaining tissue homeostasis and promoting functional recovery. Triggering receptor expressed on myeloid cells-2 (TREM2) is a newly identified receptor expressed on microglia and is thought to phagocytose damaged brain cells. The precise role of TREM2 during ischemic stroke has not been fully understood. We explore TREM2 in both in vitro and in vivo stroke models and identify a potential endogenous TREM2 ligand. TREM2 knockdown in microglia reduced microglial activation to an amoeboid phenotype and decreased the phagocytosis of injured neurons. Phagocytosis and infarcted brain tissue resorption was reduced in TREM2 knock-out (KO) mice compared with wild-type (WT) mice. TREM2 KO mice also had worsened neurological recovery and decreased viable brain tissue in the ipsilateral hemisphere. The numbers of activated microglia and phagocytes in TREM2 KO mice were decreased compared with WT mice, and foamy macrophages were nearly absent in the TREM2 KO mice. Postischemia, TREM2 was highly expressed on microglia and TREM2-Fc fusion protein (used as a probe to identify potential TREM2 binding partners) bound to an unknown TREM2 ligand that colocalized to neurons. Oxygen glucose deprivation-exposed neuronal media, or cellular fractions containing nuclei or purified DNA, but not cytosolic fractions, stimulated signaling through TREM2. TREM2-Fc fusion protein pulled down nucleic acids from ischemic brain lysate. These findings establish the relevance of TREM2 in the phagocytosis of the infarcted brain and emphasize its role in influencing neurological outcomes following stroke. Further, nucleic acids may be one potential ligand of TREM2 in brain ischemia.

Migraine Mutations Impair Hippocampal Learning Despite Enhanced Long-Term Potentiation

To explain cognitive and memory difficulties observed in some familial hemiplegic migraine (FHM) patients, we examined hippocampal neurotransmission and plasticity in knock-in mice expressing the FHM type 1 (FHM1) R192Q gain-of function mutation in the CACNA1A gene that encodes the α_1A subunit of neuronal Ca_V2.1 channels. We determined stimulus intensity–response curves for anterior commissure-evoked hippocampal CA1 field potentials in strata pyramidale and radiatum and assessed neuroplasticity by inducing long-term potentiation (LTP) and long-term depression (LTD) in anesthetized mice in vivo. We also studied learning and memory using contextual fear-conditioning, Morris water maze, and novel object recognition tests. Hippocampal field potentials were significantly enhanced in R192Q mice compared with wild-type controls. Stimulus intensity–response curves were shifted to the left and displayed larger maxima in the mutants. LTP was augmented by twofold in R192Q mice, whereas LTD was unchanged compared with wild-type mice. R192Q mice showed significant spatial memory deficits in contextual fear-conditioning and Morris water maze tests compared with wild-type controls. Novel object recognition was not impaired in R192Q mice; however, mice carrying the more severe S218L CACNA1A mutation showed marked deficits in this test, suggesting a genotype–phenotype relationship. Thus, whereas FHM1 gain-of-function mutations enhance hippocampal excitatory transmission and LTP, learning and memory are paradoxically impaired, providing a possible explanation for cognitive changes detected in FHM. Data suggest that abnormally enhanced plasticity can be as detrimental to efficient learning as reduced plasticity and highlight how genetically enhanced neuronal excitability may impact cognitive function.

Mitochondrial ATP-Mg/Pi Carrier SCaMC-3/Slc25a23 Counteracts PARP-1-Dependent Fall in Mitochondrial ATP Caused by Excitotoxic Insults in Neurons

Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca²⁺ and Na⁺ influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca²⁺ deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca²⁺-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca²⁺ deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca²⁺ retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca²⁺ deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade.

Layer-Specific Refinement of Visual Cortex Function after Eye Opening in the Awake Mouse

The laminar structure and conserved cellular organization of mouse visual cortex provide a useful model to determine the mechanisms underlying the development of visual system function. However, the normal development of many receptive field properties has not yet been thoroughly quantified, particularly with respect to layer identity and in the absence of anesthesia. Here, we use multisite electrophysiological recording in the awake mouse across an extended period of development, starting at eye opening, to measure receptive field properties and behavioral-state modulation of responsiveness. We find selective responses for orientation, direction, and spatial frequency at eye opening, which are similar across cortical layers. After this initial similarity, we observe layer-specific maturation of orientation selectivity, direction selectivity, and linearity over the following week. Developmental increases in selectivity are most robust and similar between layers 2–4, whereas layers 5 and 6 undergo distinct refinement patterns. Finally, we studied layer-specific behavioral-state modulation of cortical activity and observed a striking reorganization in the effects of running on response gain. During week 1 after eye opening, running increases responsiveness in layers 4 and 5, whereas in adulthood, the effects of running are most pronounced in layer 2/3. Together, these data demonstrate that response selectivity is present in all layers of the primary visual cortex (V1) at eye opening in the awake mouse and identify the features of basic V1 function that are further shaped over this early developmental period in a layer-specific manner.

Anti-Muscarinic Adjunct Therapy Accelerates Functional Human Oligodendrocyte Repair

Therapeutic repair of myelin disorders may be limited by the relatively slow rate of human oligodendrocyte differentiation. To identify appropriate pharmacological targets with which to accelerate differentiation of human oligodendrocyte progenitors (hOPCs) directly, we used CD140a/O4-based FACS of human forebrain and microarray to hOPC-specific receptors. Among these, we identified CHRM3, a M₃R muscarinic acetylcholine receptor, as being restricted to oligodendrocyte-biased CD140a⁺O4⁺ cells. Muscarinic agonist treatment of hOPCs resulted in a specific and dose-dependent blockade of oligodendrocyte commitment. Conversely, when hOPCs were cocultured with human neurons, M₃R antagonist treatment stimulated oligodendrocytic differentiation. Systemic treatment with solifenacin, an FDA-approved muscarinic receptor antagonist, increased oligodendrocyte differentiation of transplanted hOPCs in hypomyelinated shiverer/rag2 brain. Importantly, solifenacin treatment of engrafted animals reduced auditory brainstem response interpeak latency, indicative of increased conduction velocity and thereby enhanced functional repair. Therefore, solifenacin and other selective muscarinic antagonists represent new adjunct approaches to accelerate repair by engrafted human progenitors.

Parkinson's Disease Iron Deposition Caused by Nitric Oxide-Induced Loss of {beta}-Amyloid Precursor Protein

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP^–/– mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

Divisive Gain Modulation of Motoneurons by Inhibition Optimizes Muscular Control

When using muscles, the precision with which force is delivered is as important as the delivery of force itself. Force is regulated by both the number of recruited motoneurons and their spike frequency. While it is known that the recruitment is ordered to reduce variability in force, it remains unclear whether the motoneuron gain, i.e., the slope of the transformation between synaptic input and spiking output, is also modulated to reduce variability in force. To address this issue, we use turtle hindlimb scratching as a model for fine motor control, since this behavior involves precise limb movement to rub the location of somatic nuisance touch. We recorded intracellularly from motoneurons in a reduced preparation where the limbs were removed to increase mechanical stability and the motor nerve activity served as a surrogate for muscle force. We found that not only is the gain of motoneurons regulated on a subsecond timescale, it is also adjusted to minimize variability. The modulation is likely achieved via an expansive nonlinearity between spike rate and membrane potential with inhibition having a divisive influence. These findings reveal a versatile mechanism of modulating neuronal sensitivity and suggest that such modulation is fundamentally linked to optimization.

{beta}-Secretase BACE1 Regulates Hippocampal and Reconstituted M-Currents in a {beta}-Subunit-Like Fashion

The β-secretase BACE1 is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease, but how its action on transmembrane proteins other than the amyloid precursor protein affects the nervous system is only beginning to be understood. We report here that BACE1 regulates neuronal excitability through an unorthodox, nonenzymatic interaction with members of the KCNQ (Kv7) family that give rise to the M-current, a noninactivating potassium current with slow kinetics. In hippocampal neurons from BACE1^–/– mice, loss of M-current enhanced neuronal excitability. We relate the diminished M-current to the previously reported epileptic phenotype of BACE1-deficient mice. In HEK293T cells, BACE1 amplified reconstituted M-currents, altered their voltage dependence, accelerated activation, and slowed deactivation. Biochemical evidence strongly suggested that BACE1 physically associates with channel proteins in a β-subunit-like fashion. Our results establish BACE1 as a physiologically essential constituent of regular M-current function and elucidate a striking new feature of how BACE1 impacts on neuronal activity in the intact and diseased brain.

Differential Role of the Proteasome in the Early and Late Phases of BDNF-Induced Facilitation of LTP

The neurotrophin brain-derived neurotrophic factor (BDNF) mediates activity-dependent long-term changes of synaptic strength in the CNS. The effects of BDNF are partly mediated by stimulation of local translation, with consequent alterations in the synaptic proteome. The ubiquitin-proteasome system (UPS) also plays an important role in protein homeostasis at the synapse by regulating synaptic activity. However, whether BDNF acts on the UPS to mediate the effects on long-term synaptic potentiation (LTP) has not been investigated. In the present study, we show similar and nonadditive effects of BDNF and proteasome inhibition on the early phase of synaptic potentiation (E-LTP) induced by theta-burst stimulation of rat hippocampal CA1 synapses. The effects of BDNF were blocked by the proteasome activator IU1, suggesting that the neurotrophin acts by decreasing proteasome activity. Accordingly, BDNF downregulated the proteasome activity in cultured hippocampal neurons and in hippocampal synaptoneurosomes. Furthermore, BDNF increased the activity of the deubiquitinating enzyme UchL1 in synaptoneurosomes and upregulated free ubiquitin. In contrast to the effects on posttetanic potentiation, proteasome activity was required for BDNF-mediated LTP. These results show a novel role for BDNF in UPS regulation at the synapse, which is likely to act together with the increased translation activity in the regulation of the synaptic proteome during E-LTP.

Ly6h Regulates Trafficking of Alpha7 Nicotinic Acetylcholine Receptors and Nicotine-Induced Potentiation of Glutamatergic Signaling

α7 nAChRs are expressed widely throughout the brain, where they are important for synaptic signaling, gene transcription, and plastic changes that regulate sensory processing, cognition, and neural responses to chronic nicotine exposure. However, the mechanisms by which α7 nAChRs are regulated are poorly understood. Here we show that trafficking of α7-subunits is controlled by endogenous membrane-associated prototoxins in the Ly6 family. In particular, we find that Ly6h reduces cell-surface expression and calcium signaling by α7 nAChRs. We detect Ly6h in several rat brain regions, including the hippocampus, where we find it is both necessary and sufficient to limit the magnitude of α7-mediated currents. Consistent with such a regulatory function, knockdown of Ly6h in rat hippocampal pyramidal neurons enhances nicotine-induced potentiation of glutamatergic mEPSC amplitude, which is known to be mediated by α7 signaling. Collectively our data suggest a novel cellular role for Ly6 proteins in regulating nAChRs, which may be relevant to plastic changes in the nervous system including rewiring of glutamatergic circuitry during nicotine addiction.

Use-Dependent Activation of Neuronal Kv1.2 Channel Complexes

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.

Dopamine Directly Modulates GABAA Receptors

Pharmacologically Distinct Nicotinic Acetylcholine Receptors Drive Efferent-Mediated Excitation in Calyx-Bearing Vestibular Afferents

Electrical stimulation of vestibular efferent neurons rapidly excites the resting discharge of calyx/dimorphic (CD) afferents. In turtle, this excitation arises when acetylcholine (ACh), released from efferent terminals, directly depolarizes calyceal endings by activating nicotinic ACh receptors (nAChRs). Although molecular biological data from the peripheral vestibular system implicate most of the known nAChR subunits, specific information about those contributing to efferent-mediated excitation of CD afferents is lacking. We sought to identify the nAChR subunits that underlie the rapid excitation of CD afferents and whether they differ from α9α10 nAChRs on type II hair cells that drive efferent-mediated inhibition in adjacent bouton afferents. We recorded from CD and bouton afferents innervating the turtle posterior crista during electrical stimulation of vestibular efferents while applying several subtype-selective nAChR agonists and antagonists. The α9α10 nAChR antagonists, α-bungarotoxin and α-conotoxin RgIA, blocked efferent-mediated inhibition in bouton afferents while leaving efferent-mediated excitation in CD units largely intact. Conversely, 5-iodo-A-85380, sazetidine-A, varenicline, α-conotoxin MII, and bPiDDB (N,N-dodecane-1,12-diyl-bis-3-picolinium dibromide) blocked efferent-mediated excitation in CD afferents without affecting efferent-mediated inhibition in bouton afferents. This pharmacological profile suggested that calyceal nAChRs contain α6 and β2, but not α9, nAChR subunits. Selective blockade of efferent-mediated excitation in CD afferents distinguished dimorphic from calyx afferents by revealing type II hair cell input. Dimorphic afferents differed in having higher mean discharge rates and a mean efferent-mediated excitation that was smaller in amplitude yet longer in duration. Molecular biological data demonstrated the expression of α9 in turtle hair cells and α4 and β2 in associated vestibular ganglia.

Monitoring of Vacuolar-Type H+ ATPase-Mediated Proton Influx into Synaptic Vesicles

Activity in Human Visual and Parietal Cortex Reveals Object-Based Attention in Working Memory

Visual attention enables observers to select behaviorally relevant information based on spatial locations, features, or objects. Attentional selection is not limited to physically present visual information, but can also operate on internal representations maintained in working memory (WM) in service of higher-order cognition. However, only little is known about whether attention to WM contents follows the same principles as attention to sensory stimuli. To address this question, we investigated in humans whether the typically observed effects of object-based attention in perception are also evident for object-based attentional selection of internal object representations in WM. In full accordance with effects in visual perception, the key behavioral and neuronal characteristics of object-based attention were observed in WM. Specifically, we found that reaction times were shorter when shifting attention to memory positions located on the currently attended object compared with equidistant positions on a different object. Furthermore, functional magnetic resonance imaging and multivariate pattern analysis of visuotopic activity in visual (areas V1–V4) and parietal cortex revealed that directing attention to one position of an object held in WM also enhanced brain activation for other positions on the same object, suggesting that attentional selection in WM activates the entire object. This study demonstrated that all characteristic features of object-based attention are present in WM and thus follows the same principles as in perception.

Microsaccade Control Signals in the Cerebellum

Microsaccades, the small saccades made when we try to keep the eyes still, were once believed to be inconsequential for vision, but recent studies suggest that they can precisely relocate gaze to tiny visual targets. Because the cerebellum is necessary for motor precision, we investigated whether microsaccades may exploit this neural machinery in monkeys. Almost all vermal Purkinje cells, which provide the eye-related output of the cerebellar cortex, were found to increase or decrease their simple spike firing rate during microsaccades. At both the single-cell and population level, microsaccade-related activity was highly similar to macrosaccade-related activity and we observed a continuous representation of saccade amplitude that spanned both the macrosaccade and microsaccade domains. Our results suggest that the cerebellum's role in fine-tuning eye movements extends even to the oculomotor system's smallest saccades and add to a growing list of observations that call into question the classical categorical distinction between microsaccades and macrosaccades.

Selective Increase of Intention-Based Economic Decisions by Noninvasive Brain Stimulation to the Dorsolateral Prefrontal Cortex

The intention behind another's action and the impact of the outcome are major determinants of human economic behavior. It is poorly understood, however, whether the two systems share a core neural computation. Here, we investigated whether the two systems are causally dissociable in the brain by integrating computational modeling, functional magnetic resonance imaging, and transcranial direct current stimulation experiments in a newly developed trust game task. We show not only that right dorsolateral prefrontal cortex (DLPFC) activity is correlated with intention-based economic decisions and that ventral striatum and amygdala activity are correlated with outcome-based decisions, but also that stimulation to the DLPFC selectively enhances intention-based decisions. These findings suggest that the right DLPFC is involved in the implementation of intention-based decisions in the processing of cooperative decisions. This causal dissociation of cortical and subcortical backgrounds may indicate evolutionary and developmental differences in the two decision systems.

Effector Specificity in Macaque Frontal and Parietal Cortex

Single neurons in the frontal eye fields (FEFs) and lateral intraparietal area (LIP) of macaques are preferentially activated by saccade- versus reach-related processes. fMRI studies focusing on saccade- and reach-specific activity in human cortex, however, provided conflicting evidence for effector specificity. To gain further insights into effector preferences throughout monkey cortex using the same technique as in humans, we performed a mixed block/event-related fMRI experiment in macaques. Within single fMRI runs, monkeys alternated between a visually guided saccade task, a visually guided arm movement task, and a fixation-only task requiring no saccades or arm movements. The detection of a peripheral pop-out go cue initiating the required operant behavior and the identification of a target among distractors was identical in the arm and saccade tasks. We found saccade-related activity in parietal areas V6, V6A, LIP, and caudal intraparietal area and frontal areas FEF, 45a, 45b, and 46. Areas 45 and FEF even showed markedly decreased fMRI activity during arm movements relative to fixation only. Conversely, medial and anterior intraparietal areas (MIP and AIP), and parietal area PEip; somatosensory areas S1 and S2; and (pre)motor areas F1, F3, F5, and F6 showed increased arm movement-related activity. F1, F5, PEip, and somatosensory cortex also showed deactivations during saccades relative to fixation only. Control experiments showed that such deactivations in both operant-specific functional networks did not depend on training history or rapid task switching requiring active suppression of the unpreferred operant behavior. Therefore, although both tasks required divided attention to detect a pop-out go cue and target, two largely segregated and mainly effector-driven cortical networks were activated.

Neural Mechanisms of Human Perceptual Choice Under Focused and Divided Attention

Perceptual decisions occur after the evaluation and integration of momentary sensory inputs, and dividing attention between spatially disparate sources of information impairs decision performance. However, it remains unknown whether dividing attention degrades the precision of sensory signals, precludes their conversion into decision signals, or dampens the integration of decision information toward an appropriate response. Here we recorded human electroencephalographic (EEG) activity while participants categorized one of two simultaneous and independent streams of visual gratings according to their average tilt. By analyzing trial-by-trial correlations between EEG activity and the information offered by each sample, we obtained converging behavioral and neural evidence that dividing attention between left and right visual fields does not dampen the encoding of sensory or decision information. Under divided attention, momentary decision information from both visual streams was encoded in slow parietal signals without interference but was lost downstream during their integration as reflected in motor mu- and beta-band (10–30 Hz) signals, resulting in a "leaky" accumulation process that conferred greater behavioral influence to more recent samples. By contrast, sensory inputs that were explicitly cued as irrelevant were not converted into decision signals. These findings reveal that a late cognitive bottleneck on information integration limits decision performance under divided attention, and places new capacity constraints on decision-theoretic models of information integration under cognitive load.

Distinct Neural Representation in the Dorsolateral, Dorsomedial, and Ventral Parts of the Striatum during Fixed- and Free-Choice Tasks

The striatum is a major input site of the basal ganglia, which play an essential role in decision making. Previous studies have suggested that subareas of the striatum have distinct roles: the dorsolateral striatum (DLS) functions in habitual action, the dorsomedial striatum (DMS) in goal-directed actions, and the ventral striatum (VS) in motivation. To elucidate distinctive functions of subregions of the striatum in decision making, we systematically investigated information represented by phasically active neurons in DLS, DMS, and VS. Rats performed two types of choice tasks: fixed- and free-choice tasks. In both tasks, rats were required to perform nose poking to either the left or right hole after cue-tone presentation. A food pellet was delivered probabilistically depending on the presented cue and the selected action. The reward probability was fixed in fixed-choice task and varied in a block-wise manner in free-choice task. We found the following: (1) when rats began the tasks, a majority of VS neurons increased their firing rates and information regarding task type and state value was most strongly represented in VS; (2) during action selection, information of action and action values was most strongly represented in DMS; (3) action-command information (action representation before action selection) was stronger in the fixed-choice task than in the free-choice task in both DLS and DMS; and (4) action-command information was strongest in DLS, particularly when the same choice was repeated. We propose a hypothesis of hierarchical reinforcement learning in the basal ganglia to coherently explain these results.

Recency, Repetition, and the Multidimensional Basis of Recognition Memory

Recency and repetition are two factors that have large effects on human memory performance. One way of viewing the beneficial impact of these variables on recognition memory is to assume that both factors modulate a unidimensional memory trace strength. Although previous functional neuroimaging studies have indicated that recency and repetition may modulate similar brain structures, particularly in the region of the inferior parietal cortex, there is extensive behavioral evidence that human subjects can make independent and accurate recognition memory judgments about both an item's recency and its frequency. In the present study, we used fMRI to examine patterns of brain activity during recognition memory for auditory–verbal stimuli that were parametrically and orthogonally manipulated in terms of recency and number of repetitions. We found in a continuous recognition paradigm that the lateral inferior parietal cortex, a region that has previously been associated with recollective forms of memory, is highly sensitive to recency but not repetition. In a multivariate analysis of whole-brain activation patterns, we found orthogonal components that dissociated recency and repetition variables, indicating largely independent neural bases underlying these two factors. The results demonstrate that although both recency and repetition dramatically improve recognition memory performance, the neural bases for this improvement are dissociable, and thus are difficult to explain in terms of access to a unitary memory trace.

Mouse Model of OPRM1 (A118G) Polymorphism Increases Sociability and Dominance and Confers Resilience to Social Defeat

A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in drug addiction, pain sensitivity, and, more recently, social behavior. The endogenous opioid system has been shown to regulate social distress and reward in a variety of animal models. However, mechanisms underlying the associations between the OPRM1 A118G SNP and these behaviors have not been clarified. We used a mouse model possessing the human equivalent nucleotide/amino acid substitution to study social affiliation and social defeat behaviors. In mice with the Oprm1 A112G SNP, we demonstrate that the G allele is associated with an increase in home-cage dominance and increased motivation for nonaggressive social interactions, similar to what is reported in human populations. When challenged by a resident aggressor, G-allele carriers expressed less submissive behavior and exhibited resilience to social defeat, demonstrated by a lack of subsequent social avoidance and reductions in anhedonia as measured by intracranial self-stimulation. Protection from social defeat in G-allele carriers was associated with a greater induction of c-fos in a resilience circuit comprising the nucleus accumbens and periaqueductal gray. These findings led us to test the role of endogenous opioids in the A112G mice. We demonstrate that the increase in social affiliation in G carriers is blocked by pretreatment with naloxone. Together, these data suggest a mechanism involving altered hedonic state and neural activation as well as altered endogenous opioid tone in the differential response to aversive and rewarding social stimuli in G-allele carriers.

A Causal Role for Posterior Medial Frontal Cortex in Choice-Induced Preference Change

After a person chooses between two items, preference for the chosen item will increase and preference for the unchosen item will decrease because of the choice made. In other words, we tend to justify or rationalize our past behavior by changing our attitude. This phenomenon of choice-induced preference change has been traditionally explained by cognitive dissonance theory. Choosing something that is disliked or not choosing something that is liked are both cognitively inconsistent and, to reduce this inconsistency, people tend to change their subsequently stated preference in accordance with their past choices. Previously, human neuroimaging studies identified posterior medial frontal cortex (pMFC) as a key brain region involved in cognitive dissonance. However, it remains unknown whether the pMFC plays a causal role in inducing preference change after cognitive dissonance. Here, we demonstrate that 25 min, 1 Hz repetitive transcranial magnetic stimulation applied over the pMFC significantly reduces choice-induced preference change compared with sham stimulation or control stimulation over a different brain region, demonstrating a causal role for the pMFC.

Revisiting the Role of Infralimbic Cortex in Fear Extinction with Optogenetics

Previous rodent studies have implicated the infralimbic (IL) subregion of the medial prefrontal cortex in extinction of auditory fear conditioning. However, these studies used pharmacological inactivation or electrical stimulation techniques, which lack temporal precision and neuronal specificity. Here, we used an optogenetic approach to either activate (with channelrhodopsin) or silence (with halorhodopsin) glutamatergic IL neurons during conditioned tones delivered in one of two phases: extinction training or extinction retrieval. Activating IL neurons during extinction training reduced fear expression and strengthened extinction memory the following day. Silencing IL neurons during extinction training had no effect on within-session extinction, but impaired the retrieval of extinction the following day, indicating that IL activity during extinction tones is necessary for the formation of extinction memory. Surprisingly, however, silencing IL neurons optogenetically or pharmacologically during the retrieval of extinction 1 day or 1 week following extinction training had no effect. Our findings suggest that IL activity during extinction training likely facilitates storage of extinction in target structures, but contrary to current models, IL activity does not appear to be necessary for retrieval of extinction memory.

Quantifying the Microvascular Origin of BOLD-fMRI from First Principles with Two-Photon Microscopy and an Oxygen-Sensitive Nanoprobe

The blood oxygenation level-dependent (BOLD) contrast is widely used in functional magnetic resonance imaging (fMRI) studies aimed at investigating neuronal activity. However, the BOLD signal reflects changes in blood volume and oxygenation rather than neuronal activity per se. Therefore, understanding the transformation of microscopic vascular behavior into macroscopic BOLD signals is at the foundation of physiologically informed noninvasive neuroimaging. Here, we use oxygen-sensitive two-photon microscopy to measure the BOLD-relevant microvascular physiology occurring within a typical rodent fMRI voxel and predict the BOLD signal from first principles using those measurements. The predictive power of the approach is illustrated by quantifying variations in the BOLD signal induced by the morphological folding of the human cortex. This framework is then used to quantify the contribution of individual vascular compartments and other factors to the BOLD signal for different magnet strengths and pulse sequences.

Persistent Pain Maintains Morphine-Seeking Behavior after Morphine Withdrawal through Reduced MeCP2 Repression of Glua1 in Rat Central Amygdala

As long-term opioids are increasingly used for control of chronic pain, how pain affects the rewarding effect of opioids and hence risk of prescription opioid misuse and abuse remains a healthcare concern and a challenging issue in current pain management. In this study, using a rat model of morphine self-administration, we investigated the molecular mechanisms underlying the impact of pain on operant behavior of morphine intake and morphine seeking before and after morphine withdrawal. We found that rats with persistent pain consumed a similar amount of daily morphine to that in control rats without pain, but maintained their level-pressing behavior of morphine seeking after abstinence of morphine at 0.2 mg/kg, whereas this behavior was gradually diminished in control rats. In the central nucleus of amygdala (CeA), a limbic structure critically involved in the affective dimension of pain, proteins of GluA1 subunits of glutamate AMPA receptors were upregulated during morphine withdrawal, and viral knockdown of CeA GluA1 eliminated the morphine-seeking behavior in withdrawn rats of the pain group. Chromatin immunoprecipitation analysis revealed that the methyl CpG-binding protein 2 (MeCP2) was enriched in the promoter region of Gria1 encoding GluA1 and this enrichment was significantly attenuated in withdrawn rats of the pain group. Furthermore, viral overexpression of CeA MeCP2 repressed the GluA1 level and eliminated the maintenance of morphine-seeking behavior after morphine withdrawal. These results suggest direct MeCp2 repression of GluA1 function as a likely mechanism for morphine-seeking behavior maintained by long-lasting affective pain after morphine withdrawal.

Life Extension Factor Klotho Prevents Mortality and Enhances Cognition in hAPP Transgenic Mice

Aging is the principal demographic risk factor for Alzheimer disease (AD), the most common neurodegenerative disorder. Klotho is a key modulator of the aging process and, when overexpressed, extends mammalian lifespan, increases synaptic plasticity, and enhances cognition. Whether klotho can counteract deficits related to neurodegenerative diseases, such as AD, is unknown. Here we show that elevating klotho expression decreases premature mortality and network dysfunction in human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Increasing klotho levels prevented depletion of NMDA receptor (NMDAR) subunits in the hippocampus and enhanced spatial learning and memory in hAPP mice. Klotho elevation in hAPP mice increased the abundance of the GluN2B subunit of NMDAR in postsynaptic densities and NMDAR-dependent long-term potentiation, which is critical for learning and memory. Thus, increasing wild-type klotho levels or activities improves synaptic and cognitive functions, and may be of therapeutic benefit in AD and other cognitive disorders.

Inspiration Is the Major Regulator of Human CSF Flow

The mechanisms behind CSF flow in humans are still not fully known. CSF circulates from its primary production sites at the choroid plexus through the brain ventricles to reach the outer surface of the brain in the subarachnoid spaces from where it drains into venous bloodstream and cervical lymphatics. According to a recent concept of brain fluid transport, established in rodents, CSF from the brain surface also enters the brain tissue along para-arterial routes and exits through paravenous spaces again into subarachnoid compartments. This unidirectional flow is mainly driven by arterial pulsation. To investigate how CSF flow is regulated in humans, we applied a novel real-time magnetic resonance imaging technique at high spatial (0.75 mm) and temporal (50 ms) resolution in healthy human subjects. We observed significant CSF flow exclusively with inspiration. In particular, during forced breathing, high CSF flow was elicited during every inspiration, whereas breath holding suppressed it. Only a minor flow component could be ascribed to cardiac pulsation. The present results unambiguously identify inspiration as the most important driving force for CSF flow in humans. Inspiratory thoracic pressure reduction is expected to directly modulate the hydrostatic pressure conditions for the low-resistance paravenous, venous, and lymphatic clearance routes of CSF. Furthermore, the experimental approach opens new clinical opportunities to study the pathophysiology of various forms of hydrocephalus and to design therapeutic strategies in relation to CSF flow alterations.

Anatomical Changes at the Level of the Primary Synapse in Neuropathic Pain: Evidence from the Spinal Trigeminal Nucleus

Accumulated evidence from experimental animal models suggests that neuronal loss within the dorsal horn is involved in the development and/or maintenance of peripheral neuropathic pain. However, to date, no study has specifically investigated whether such neuroanatomical changes also occur at this level in humans. Using brain imaging techniques, we sought to determine whether anatomical changes were present in the spinal trigeminal nucleus in subjects with chronic orofacial neuropathic pain. In 22 subjects with painful trigeminal neuropathy and 44 pain-free controls, voxel-based morphometry of T1-weighted anatomical images and diffusion tensor images were used to assess regional gray matter volume and microstructural changes within the brainstem. In addition, deterministic tractography was used to assess the integrity of ascending pain pathways. Orofacial neuropathic pain was associated with significant regional gray matter volume decreases, fractional anisotropy increases, and mean diffusivity decreases within the spinal trigeminal nucleus, specifically the subnucleus oralis. In addition, tractography revealed no significant differences in diffusivity properties in the root entry zone of the trigeminal nerve, the spinal trigeminal tract, or the ventral trigeminothalamic tracts in painful trigeminal neuropathy subjects compared with controls. These data reveal that chronic neuropathic pain in humans is associated with discrete alterations in the anatomy of the primary synapse. These neuroanatomical changes may be critical for the generation and/or maintenance of pathological pain.

Dysregulation of Glutamine Transporter SNAT1 in Rett Syndrome Microglia: A Mechanism for Mitochondrial Dysfunction and Neurotoxicity

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously, we and others reported a role of microglia in the pathophysiology of RTT. To understand the mechanism of microglia dysfunction in RTT, we identified a MeCP2 target gene, SLC38A1, which encodes a major glutamine transporter (SNAT1), and characterized its role in microglia. We found that MeCP2 acts as a microglia-specific transcriptional repressor of SNAT1. Because glutamine is mainly metabolized in the mitochondria, where it is used as an energy substrate and a precursor for glutamate production, we hypothesize that SNAT1 overexpression in MeCP2-deficient microglia would impair the glutamine homeostasis, resulting in mitochondrial dysfunction as well as microglial neurotoxicity because of glutamate overproduction. Supporting this hypothesis, we found that MeCP2 downregulation or SNAT1 overexpression in microglia resulted in (1) glutamine-dependent decrease in microglial viability, which was corroborated by reduced microglia counts in the brains of MECP2 knock-out mice; (2) proliferation of mitochondria and enhanced mitochondrial production of reactive oxygen species; (3) increased oxygen consumption but decreased ATP production (an energy-wasting state); and (4) overproduction of glutamate that caused NMDA receptor-dependent neurotoxicity. The abnormalities could be rectified by mitochondria-targeted expression of catalase and a mitochondria-targeted peptide antioxidant, Szeto-Schiller 31. Our results reveal a novel mechanism via which MeCP2 regulates bioenergetic pathways in microglia and suggest a therapeutic potential of mitochondria-targeted antioxidants for RTT.

Posttraumatic Stress Disorder-Like Induction Elevates {beta}-Amyloid Levels, Which Directly Activates Corticotropin-Releasing Factor Neurons to Exacerbate Stress Responses

Recent studies have found that those who suffer from posttraumatic stress disorder (PTSD) are more likely to experience dementia as they age, most often Alzheimer's disease (AD). These findings suggest that the symptoms of PTSD might have an exacerbating effect on AD progression. AD and PTSD might also share common susceptibility factors such that those who experience trauma-induced disease were already more likely to succumb to dementia with age. Here, we explored these two hypotheses using a mouse model of PTSD in wild-type and AD model animals. We found that expression of human familial AD mutations in amyloid precursor protein and presenilin 1 leads to sensitivity to trauma-induced PTSD-like changes in behavioral and endocrine stress responses. PTSD-like induction, in turn, chronically elevates levels of CSF β-amyloid (Aβ), exacerbating ongoing AD pathogenesis. We show that PTSD-like induction and Aβ elevation are dependent on corticotropin-releasing factor (CRF) receptor 1 signaling and an intact hypothalamic–pituitary–adrenal axis. Furthermore, we show that Aβ species can hyperexcite CRF neurons, providing a mechanism by which Aβ influences stress-related symptoms and PTSD-like phenotypes. Consistent with Aβ causing excitability of the stress circuitry, we attenuate PTSD-like phenotypes in vivo by lowering Aβ levels during PTSD-like trauma exposure. Together, these data demonstrate that exposure to PTSD-like trauma can drive AD pathogenesis, which directly perturbs CRF signaling, thereby enhancing chronic PTSD symptoms while increasing risk for AD-related dementia.

Modulation of Microglial Process Convergence Toward Neuronal Dendrites by Extracellular Calcium

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.

Characteristic Patterns of Dendritic Remodeling in Early-Stage Glaucoma: Evidence from Genetically Identified Retinal Ganglion Cell Types

Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1–2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

G-Protein-Coupled Estrogen Receptor 1 Is Anatomically Positioned to Modulate Synaptic Plasticity in the Mouse Hippocampus

Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and β, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions.

Responses to Conflicting Stimuli in a Simple Stimulus-Response Pathway

The "local bend response" of the medicinal leech (Hirudo verbana) is a stimulus–response pathway that enables the animal to bend away from a pressure stimulus applied anywhere along its body. The neuronal circuitry that supports this behavior has been well described, and its responses to individual stimuli are understood in quantitative detail. We probed the local bend system with pairs of electrical stimuli to sensory neurons that could not logically be interpreted as a single touch to the body wall and used multiple suction electrodes to record simultaneously the responses in large numbers of motor neurons. In all cases, responses lasted much longer than the stimuli that triggered them, implying the presence of some form of positive feedback loop to sustain the response. When stimuli were delivered simultaneously, the resulting motor neuron output could be described as an evenly weighted linear combination of the responses to the constituent stimuli. However, when stimuli were delivered sequentially, the second stimulus had greater impact on the motor neuron output, implying that the positive feedback in the system is not strong enough to render it immune to further input.

Photoreceptor Ablation Initiates the Immediate Loss of Glutamate Receptors in Postsynaptic Bipolar Cells in Retina

Structural changes underlying neurodegenerative diseases include dismantling of synapses, degradation of circuitry, and even massive rewiring. Our limited understanding of synapse dismantling stems from the inability to control the timing and extent of cell death. In this study, selective ablation of cone photoreceptors in live mouse retina and tracking of postsynaptic partners at the cone-to-ON cone bipolar cell synapse reveals that early reaction to cone loss involves rapid and local changes in postsynaptic glutamate receptor distribution. Glutamate receptors disappear with a time constant of 2 h. Furthermore, binding of glutamate receptors by agonists and antagonists is insufficient to rescue glutamate receptor loss, suggesting that receptor allocation depends on the physical presence of cones. These findings demonstrate that the initial step in synapse disassembly involves postsynaptic receptor loss rather than dendritic retraction, providing insight into the early stages of neurodegenerative disease.

The Nucleus Prepositus Hypoglossi Contributes to Head Direction Cell Stability in Rats

Head direction (HD) cells in the rat limbic system fire according to the animal's orientation independently of the animal's environmental location or behavior. These HD cells receive strong inputs from the vestibular system, among other areas, as evidenced by disruption of their directional firing after lesions or inactivation of vestibular inputs. Two brainstem nuclei, the supragenual nucleus (SGN) and nucleus prepositus hypoglossi (NPH), are known to project to the HD network and are thought to be possible relays of vestibular information. Previous work has shown that lesioning the SGN leads to a loss of spatial tuning in downstream HD cells, but the NPH has historically been defined as an oculomotor nuclei and therefore its role in contributing to the HD signal is less clear. Here, we investigated this role by recording HD cells in the anterior thalamus after either neurotoxic or electrolytic lesions of the NPH. There was a total loss of direction-specific firing in anterodorsal thalamus cells in animals with complete NPH lesions. However, many cells were identified that fired in bursts unrelated to the animals' directional heading and were similar to cells seen in previous studies that damaged vestibular-associated areas. Some animals with significant but incomplete lesions of the NPH had HD cells that were stable under normal conditions, but were unstable under conditions designed to minimize the use of external cues. These results support the hypothesis that the NPH, beyond its traditional oculomotor function, plays a critical role in conveying vestibular-related information to the HD circuit.

Behavioral Relevance Helps Untangle Natural Vocal Categories in a Specific Subset of Core Auditory Cortical Pyramidal Neurons

Sound categorization is essential for auditory behaviors like acoustic communication, but its genesis within the auditory pathway is not well understood—especially for learned natural categories like vocalizations, which often share overlapping acoustic features that must be distinguished (e.g., speech). We use electrophysiological mapping and single-unit recordings in mice to investigate how representations of natural vocal categories within core auditory cortex are modulated when one category acquires enhanced behavioral relevance. Taking advantage of a maternal mouse model of acoustic communication, we found no long-term auditory cortical map expansion to represent a behaviorally relevant pup vocalization category—contrary to expectations from the cortical plasticity literature on conditioning with pure tones. Instead, we observed plasticity that improved the separation between acoustically similar pup and adult vocalization categories among a physiologically defined subset of late-onset, putative pyramidal neurons, but not among putative interneurons. Additionally, a larger proportion of these putative pyramidal neurons in maternal animals compared with nonmaternal animals responded to the individual pup call exemplars having combinations of acoustic features most typical of that category. Together, these data suggest that higher-order representations of acoustic categories arise from a subset of core auditory cortical pyramidal neurons that become biased toward the combination of acoustic features statistically predictive of membership to a behaviorally relevant sound category.

Gain Modulation of Synaptic Inputs by Network State in Auditory Cortex In Vivo

The cortical network recurrent circuitry generates spontaneous activity organized into Up (active) and Down (quiescent) states during slow-wave sleep or anesthesia. These different states of cortical activation gain modulate synaptic transmission. However, the reported modulation that Up states impose on synaptic inputs is disparate in the literature, including both increases and decreases of responsiveness. Here, we tested the hypothesis that such disparate observations may depend on the intensity of the stimulation. By means of intracellular recordings, we studied synaptic transmission during Up and Down states in rat auditory cortex in vivo. Synaptic potentials were evoked either by auditory or electrical (thalamocortical, intracortical) stimulation while randomly varying the intensity of the stimulus. Synaptic potentials evoked by the same stimulus intensity were compared in Up/Down states. Up states had a scaling effect on the stimulus-evoked synaptic responses: the amplitude of weaker responses was potentiated whereas that of larger responses was maintained or decreased with respect to the amplitude during Down states. We used a computational model to explore the potential mechanisms explaining this nontrivial stimulus–response relationship. During Up/Down states, there is different excitability in the network and the neuronal conductance varies. We demonstrate that the competition between presynaptic recruitment and the changing conductance might be the central mechanism explaining the experimentally observed stimulus–response relationships. We conclude that the effect that cortical network activation has on synaptic transmission is not constant but contingent on the strength of the stimulation, with a larger modulation for stimuli involving both thalamic and cortical networks.

Prospective Coding of Dorsal Raphe Reward Signals by the Orbitofrontal Cortex

The orbitofrontal cortex (OFC) is important for the cognitive processes of learning and decision making. Previous recordings have revealed that OFC neurons encode predictions of reward outcomes. The OFC is interconnected with the dorsal raphe nucleus (DRN), which is a major serotonin (5-HT) center of the brain. Recent studies have provided increasing evidence that the DRN encodes reward signals. However, it remains unclear how the activity of DRN neurons affects the prospective reward coding of OFC neurons. By combining single-unit recordings from the OFC and optogenetic activation of the DRN in behaving mice, we found that DRN stimulation is sufficient to organize and modulate the anticipatory responses of OFC neurons. During pavlovian conditioning tasks for mice, odorant cues were associated with the delayed delivery of natural rewards of sucrose solution or DRN stimulation. After training, OFC neurons exhibited prospective responses to the sucrose solution. More importantly, the coupling of an odorant with delayed DRN stimulation resulted in tonic excitation or inhibition of OFC neurons during the delay period. The intensity of the prospective responses was affected by the frequency and duration of DRN stimulation. Additionally, DRN stimulation bidirectionally modulated the prospective responses to natural rewards. These experiments indicate that signals from the DRN are incorporated into the brain reward system to shape the cortical prospective coding of rewards.

A Functional Link between MT Neurons and Depth Perception Based on Motion Parallax

As an observer translates, objects lying at different distances from the observer have differential image motion on the retina (motion parallax). It is well established psychophysically that humans perceive depth rather precisely from motion parallax and that extraretinal signals may be used to correctly perceive the sign of depth (near vs far) when binocular and pictorial depth cues are absent or weak. However, the neural basis for this capacity remains poorly understood. We have shown previously that neurons in the macaque middle temporal (MT) area combine retinal image motion with smooth eye movement command signals to signal depth sign from motion parallax. However, those studies were performed in animals that were required simply to track a visual target, thus precluding direct comparisons between neural activity and behavior. Here, we examine the activity of MT neurons in rhesus monkeys that were trained to discriminate depth sign based on motion parallax, in the absence of binocular disparity and pictorial depth cues. We find that the most sensitive MT neurons approach behavioral sensitivity, whereas the average neuron is twofold to threefold less sensitive than the animal. We also find that MT responses are predictive of perceptual decisions (independent of the visual stimulus), consistent with a role for MT in providing sensory signals for this behavior. Our findings suggest that, in addition to its established roles in processing stereoscopic depth, area MT is well suited to contribute to perception of depth based on motion parallax.

Adult Cortical Plasticity Studied with Chronically Implanted Electrode Arrays

The functional architecture of adult cerebral cortex retains a capacity for experience-dependent change. This is seen after focal binocular lesions as rapid changes in receptive field (RF) of the lesion projection zone (LPZ) in the primary visual cortex (V1). To study the dynamics of the circuitry underlying these changes longitudinally, we implanted microelectrode arrays in macaque (Macaca mulatta) V1, eliminating the possibility of sampling bias, which was a concern in previous studies. With this method, we observed a rapid initial recovery in the LPZ and, during the following weeks, 63–89% of the sites in the LPZ showed recovery of visual responses with significant position tuning. The RFs shifted ~3° away from the scotoma. In the absence of a lesion, visual stimulation surrounding an artificial scotoma did not elicit visual responses, suggesting that the postlesion RF shifts resulted from cortical reorganization. Interestingly, although both spikes and LFPs gave consistent prelesion position tuning, only spikes reflected the postlesion remapping.

Aberrant Synaptic Integration in Adult Lamina I Projection Neurons Following Neonatal Tissue Damage

Mounting evidence suggests that neonatal tissue damage evokes alterations in spinal pain reflexes which persist into adulthood. However, less is known about potential concomitant effects on the transmission of nociceptive information to the brain, as the degree to which early injury modulates synaptic integration and membrane excitability in mature spinal projection neurons remains unclear. Here we demonstrate that neonatal surgical injury leads to a significant shift in the balance between synaptic excitation and inhibition onto identified lamina I projection neurons of the adult mouse spinal cord. The strength of direct primary afferent input to mature spino-parabrachial neurons was enhanced following neonatal tissue damage, whereas the efficacy of both GABAergic and glycinergic inhibition onto the same population was compromised. This was accompanied by reorganization in the pattern of sensory input to adult projection neurons, which included a greater prevalence of monosynaptic input from low-threshold A-fibers when preceded by early tissue damage. In addition, neonatal incision resulted in greater primary afferent-evoked action potential discharge in mature projection neurons. Overall, these results demonstrate that tissue damage during early life causes a long-term increase in the gain of spinal nociceptive circuits, and suggest that the prolonged consequences of neonatal trauma may not be restricted to the spinal cord but rather include excessive ascending signaling to supraspinal pain centers.

Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1

Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.

Functional Cooperation between the IP3 Receptor and Phospholipase C Secures the High Sensitivity to Light of Drosophila Photoreceptors In Vivo

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single G_qα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several G_qα molecules. We have recently shown that most of the spontaneous single G_qα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLCβ activity and cellular [Ca²⁺], which participates in light excitation via a still unclear mechanism. We show here that in IP₃ receptor (IP₃R)-deficient photoreceptors, both light-activated Ca²⁺ release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca²⁺ store depletion, linking Ca²⁺ release to light excitation. In IP₃R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca²⁺ release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca²⁺] required for excitation. Combination of IP₃R knockdown with reduced PLCβ catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca²⁺ elevation, showing a functional cooperation between IP₃R and PLCβ via released Ca²⁺. These findings suggest that in contrast to the current dogma that Ca²⁺ release via IP₃R does not participate in light excitation, we show that released Ca²⁺ plays a critical role in light excitation. The positive feedback between PLCβ and IP₃R found here may represent a common feature of the inositol-lipid signaling.

Trip6 Promotes Dendritic Morphogenesis through Dephosphorylated GRIP1-Dependent Myosin VI and F-Actin Organization

Thyroid receptor-interacting protein 6 (Trip6), a multifunctional protein belonging to the zyxin family of LIM proteins, is involved in various physiological and pathological processes, including cell migration and tumorigenesis. However, the role of Trip6 in neurons remains unknown. Here, we show that Trip6 is expressed in mouse hippocampal neurons and promotes dendritic morphogenesis. Through interaction with the glutamate receptor-interacting protein 1 (GRIP1) and myosin VI, Trip6 is crucial for the total dendritic length and the number of primary dendrites in cultured hippocampal neurons. Trip6 depletion reduces F-actin content and impairs dendritic morphology, and this phenocopies GRIP1 or myosin VI knockdown. Furthermore, phosphorylation of GRIP1^956T by AKT1 inhibits the interaction between GRIP1 and myosin VI, but facilitates GRIP1 binding to 14-3-3 protein, which is required for regulating F-actin organization and dendritic morphogenesis. Thus, the Trip6–GRIP1–myosin VI interaction and its regulation on F-actin network play a significant role in dendritic morphogenesis.

The Roles of Cdk5-Mediated Subcellular Localization of FOXO1 in Neuronal Death

Deficiency of cyclin-dependent kinase 5 (Cdk5) has been linked to the death of postmitotic cortical neurons during brain development. We now report that, in mouse cortical neurons, Cdk5 is capable of phosphorylating the transcription factor FOXO1 at Ser249 in vitro and in vivo. Cellular stresses resulting from extracellular stimulation by H₂O₂ or β-amyloid promote hyperactivation of Cdk5, FOXO1 nuclear export and inhibition of its downstream transcriptional activity. In contrast, a loss of Cdk5 leads to FOXO1 translocation into the nucleus: a shift due to decreased AKT activity but independent of S249 phosphorylation. Nuclear FOXO1 upregulates transcription of the proapoptotic gene, BIM, leading to neuronal death, which can be rescued when endogenous FOXO1 was replaced by the cytoplasmically localized form of FOXO1, FOXO1-S249D. Cytoplasmic, but not nuclear, Cdk5 attenuates neuronal death by inhibiting FOXO1 transcriptional activity and BIM expression. Together, our findings suggest that Cdk5 plays a novel and unexpected role in the degeneration of postmitotic neurons through modulation of the cellular location of FOXO1, which constitutes an alternative pathway through which Cdk5 deficiency leads to neuronal death.

Cooperative Roles of Hydrophilic Loop 1 and the C-Terminus of Presenilin 1 in the Substrate-Gating Mechanism of {gamma}-Secretase

-Secretase is a multisubunit protease complex that is responsible for generating amyloid-β peptides, which are associated with Alzheimer disease. The catalytic subunit of -secretase is presenilin 1 (PS1), which contains an initial substrate-binding site that is distinct from the catalytic site. Processive cleavage is suggested in the intramembrane-cleaving mechanism of -secretase. However, it largely remains unknown as to how -secretase recognizes its substrate during proteolysis. Here, we identified that the α-helical structural region of hydrophilic loop 1 (HL1) and the C-terminal region of human PS1 are distinct substrate-binding sites. Mutational analyses revealed that substrate binding to the HL1 region is critical for both - and -cleavage, whereas binding to the C-terminal region hampers -cleavage. Moreover, we propose that substrate binding triggers conformational changes in PS1, rendering it suitable for catalysis. Our data provide new insights into the complicated catalytic mechanism of PS1.

A Novel Size-Based Sorting Mechanism of Pinocytic Luminal Cargoes in Microglia

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4⁺ T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.

Speed and Sensitivity of Phototransduction in Drosophila Depend on Degree of Saturation of Membrane Phospholipids

Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP₂ hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ~5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca²⁺ influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ~10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ~2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.

PKC Enhances the Capacity for Secretion by Rapidly Recruiting Covert Voltage-Gated Ca2+ Channels to the Membrane

It is unknown whether neurons can dynamically control the capacity for secretion by promptly changing the number of plasma membrane voltage-gated Ca²⁺ channels. To address this, we studied peptide release from the bag cell neurons of Aplysia californica, which initiate reproduction by secreting hormone during an afterdischarge. This burst engages protein kinase C (PKC) to trigger the insertion of a covert Ca²⁺ channel, Apl Ca_v2, alongside a basal channel, Apl Ca_v1. The significance of Apl Ca_v2 recruitment to secretion remains undetermined; therefore, we used capacitance tracking to assay secretion, along with Ca²⁺ imaging and Ca²⁺ current measurements, from cultured bag cell neurons under whole-cell voltage-clamp. Activating PKC with the phorbol ester, PMA, enhanced Ca²⁺ entry, and potentiated stimulus-evoked secretion. This relied on channel insertion, as it was occluded by preventing Apl Ca_v2 engagement with prior whole-cell dialysis or the cytoskeletal toxin, latrunculin B. Channel insertion reduced the stimulus duration and/or frequency required to initiate secretion and strengthened excitation-secretion coupling, indicating that Apl Ca_v2 accesses peptide release more readily than Apl Ca_v1. The coupling of Apl Ca_v2 to secretion also changed with behavioral state, as Apl Ca_v2 failed to evoke secretion in silent neurons from reproductively inactive animals. Finally, PKC also acted secondarily to enhance prolonged exocytosis triggered by mitochondrial Ca²⁺ release. Collectively, our results suggest that bag cell neurons dynamically elevate Ca²⁺ channel abundance in the membrane to ensure adequate secretion during the afterdischarge.

Spillover Transmission Is Mediated by the Excitatory GABA Receptor LGC-35 in C. elegans

Under most circumstances, GABA activates chloride-selective channels and thereby inhibits neuronal activity. Here, we identify a GABA receptor in the nematode Caenorhabditis elegans that conducts cations and is therefore excitatory. Expression in Xenopus oocytes demonstrates that LGC-35 is a homopentameric cation-selective receptor of the cys-loop family exclusively activated by GABA. Phylogenetic analysis suggests that LGC-35 evolved from GABA-A receptors, but the pore-forming domain contains novel molecular determinants that confer cation selectivity. LGC-35 is expressed in muscles and directly mediates sphincter muscle contraction in the defecation cycle in hermaphrodites, and spicule eversion during mating in the male. In the locomotory circuit, GABA release directly activates chloride channels on the muscle to cause muscle relaxation. However, GABA spillover at these synapses activates LGC-35 on acetylcholine motor neurons, which in turn cause muscles to contract, presumably to drive wave propagation along the body. These studies demonstrate that both direct and indirect excitatory GABA signaling plays important roles in regulating neuronal circuit function and behavior in C. elegans.

Loss of Cyclin-Dependent Kinase 5 from Parvalbumin Interneurons Leads to Hyperinhibition, Decreased Anxiety, and Memory Impairment

Perturbations in fast-spiking parvalbumin (PV) interneurons are hypothesized to be a major component of various neuropsychiatric disorders; however, the mechanisms regulating PV interneurons remain mostly unknown. Recently, cyclin-dependent kinase 5 (Cdk5) has been shown to function as a major regulator of synaptic plasticity. Here, we demonstrate that genetic ablation of Cdk5 in PV interneurons in mouse brain leads to an increase in GABAergic neurotransmission and impaired synaptic plasticity. PVCre;fCdk5 mice display a range of behavioral abnormalities, including decreased anxiety and memory impairment. Our results reveal a central role of Cdk5 expressed in PV interneurons in gating inhibitory neurotransmission and underscore the importance of such regulation during behavioral tasks. Our findings suggest that Cdk5 can be considered a promising therapeutic target in a variety of conditions attributed to inhibitory interneuronal dysfunction, such as epilepsy, anxiety disorders, and schizophrenia.

Reversal Learning and Dopamine: A Bayesian Perspective

Memory Retrieval Requires Ongoing Protein Synthesis and NMDA Receptor Activity-Mediated AMPA Receptor Trafficking

Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA2_3Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA2_3Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

Revisiting the Evidence for Collapsing Boundaries and Urgency Signals in Perceptual Decision-Making

For nearly 50 years, the dominant account of decision-making holds that noisy information is accumulated until a fixed threshold is crossed. This account has been tested extensively against behavioral and neurophysiological data for decisions about consumer goods, perceptual stimuli, eyewitness testimony, memories, and dozens of other paradigms, with no systematic misfit between model and data. Recently, the standard model has been challenged by alternative accounts that assume that less evidence is required to trigger a decision as time passes. Such "collapsing boundaries" or "urgency signals" have gained popularity in some theoretical accounts of neurophysiology. Nevertheless, evidence in favor of these models is mixed, with support coming from only a narrow range of decision paradigms compared with a long history of support from dozens of paradigms for the standard theory. We conducted the first large-scale analysis of data from humans and nonhuman primates across three distinct paradigms using powerful model-selection methods to compare evidence for fixed versus collapsing bounds. Overall, we identified evidence in favor of the standard model with fixed decision boundaries. We further found that evidence for static or dynamic response boundaries may depend on specific paradigms or procedures, such as the extent of task practice. We conclude that the difficulty of selecting between collapsing and fixed bounds models has received insufficient attention in previous research, calling into question some previous results.