Spinal neuropeptide Y Y1 receptor-expressing neurons are a pharmacotherapeutic target for the alleviation of neuropathic pain.

Peripheral nerve injury sensitizes a complex network of spinal cord dorsal horn (DH) neurons to produce allodynia and neuropathic pain. The identification of a druggable target within this network has remained elusive, but a promising candidate is the neuropeptide Y (NPY) Y1 receptor-expressing interneuron (Y1-IN) population. We report that spared nerve injury (SNI) enhanced the excitability of Y1-INs and elicited allodynia (mechanical and cold hypersensitivity) and affective pain. Similarly, chemogenetic or optogenetic activation of Y1-INs in uninjured mice elicited behavioral signs of spontaneous, allodynic, and affective pain. SNI-induced allodynia was reduced by chemogenetic inhibition of Y1-INs, or intrathecal administration of a Y1-selective agonist. Conditional deletion of Npy1r in DH neurons, but not peripheral afferent neurons prevented the anti-hyperalgesic effects of the intrathecal Y1 agonist. We conclude that spinal Y1-INs are necessary and sufficient for the behavioral symptoms of neuropathic pain and represent a promising target for future pharmacotherapeutic development of Y1 agonists.

Proliferating NG2-Cell-Dependent Angiogenesis and Scar Formation Alter Axon Growth and Functional Recovery After Spinal Cord Injury in Mice.

Spinal cord injury (SCI) induces a centralized fibrotic scar surrounded by a reactive glial scar at the lesion site. The origin of these scars is thought to be perivascular cells entering lesions on ingrowing blood vessels and reactive astrocytes, respectively. However, two NG2-expressing cell populations, pericytes and glia, may also influence scar formation. In the periphery, new blood vessel growth requires proliferating NG2+ pericytes; if this were also true in the CNS, then the fibrotic scar would depend on dividing NG2+ pericytes. NG2+ glial cells (also called oligodendrocyte progenitors or polydendrocytes) also proliferate after SCI and accumulate in large numbers among astrocytes in the glial scar. Their effect there, if any, is unknown. We show that proliferating NG2+ pericytes and glia largely segregate into the fibrotic and glial scars, respectively; therefore, we used a thymidine kinase/ganciclovir paradigm to ablate both dividing NG2+ cell populations to determine whether either scar was altered. Results reveal that loss of proliferating NG2+ pericytes in the lesion prevented intralesion angiogenesis and completely abolished the fibrotic scar. The glial scar was also altered in the absence of acutely dividing NG2+ cells, displaying discontinuous borders and significantly reduced GFAP density. Collectively, these changes enhanced edema, prolonged hemorrhage, and impaired forelimb functional recovery. Interestingly, after halting GCV at 14 d postinjury, scar elements and vessels entered the lesions over the next 7 d, as did large numbers of axons that were not present in controls. Collectively, these data reveal that acutely dividing NG2+ pericytes and glia play fundamental roles in post-SCI tissue remodeling.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) is characterized by formation of astrocytic and fibrotic scars, both of which are necessary for lesion repair. NG2+ cells may influence both scar-forming processes. This study used a novel transgenic mouse paradigm to ablate proliferating NG2+ cells after SCI to better understand their role in repair. For the first time, our data show that dividing NG2+ pericytes are required for post-SCI angiogenesis, which in turn is needed for fibrotic scar formation. Moreover, loss of cycling NG2+ glia and pericytes caused significant multicellular tissue changes, including altered astrocyte responses and impaired functional recovery. This work reveals previously unknown ways in which proliferating NG2+ cells contribute to endogenous repair after SCI.

Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels.

The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Secretin deficiency causes impairment in survival of neural progenitor cells in mice.

Hippocampal neurogenesis is the lifelong production of new neurons in the central nervous system (CNS), and affects many physiological and pathophysiological conditions, including neurobehavioral disorders. The early postnatal stage is the most prominent neurogenesis period; however, the functional role of neurogenesis in this developing stage has not been well characterized. To understand the role of hippocampal neurogenesis in the postnatal developing period, we analyzed secretin, a neuropeptide, which is expressed significantly higher in the development stage. Secretin is a pleiotropic neuropeptide hormone that belongs to the secretin/VIP/glucagon peptide family. Although secretin was originally isolated in the gastrointestinal system, it has been found that secretin itself acts as a neuropeptide in the CNS. Here, we report a new function of secretin as a survival factor for neural progenitor cells in the hippocampus. We found that secretin-deficient mice exhibit decreased numbers of BrdU-labeled new neurons and dramatically increased apoptosis of doublecortin-positive neural progenitor cells in the subgranular zone of the dentate gyrus (DG) during the early postnatal period. Furthermore, we found that reduced survival of neural progenitor cells leads to decreased volume of DG, reduced long-term potentiation and impaired spatial learning ability in adults. Our studies demonstrate that secretin has important implications for neurogenesis in postnatal development, and affects neurobehavioral function in the adult mouse.

K+ channel alterations in the progression of experimental autoimmune encephalomyelitis.

Voltage-gated K(+) (Kv) channels play critical roles not only in regulating synaptic transmission and intrinsic excitability of neurons, but also in controlling the function and proliferation of other cells in the central nervous system (CNS). The non-specific Kv channel blocker, 4-AminoPyridine (4-AP) (Dalfampridine, Ampyra®), is currently used to treat multiple sclerosis (MS), an inflammatory demyelinating disease. However, little is known how various types of Kv channels are altered in any inflammatory demyelinating diseases. By using established animal models for MS, experimental autoimmune encephalomyelitis (EAE), we report that expression and distribution patterns of Kv channels are altered in the CNS correlating with EAE severity. The juxtaparanodal (JXP) targeting of Kv1.2/Kvß2 along myelinated axons is disrupted within demyelinated lesions in the white matter of spinal cord in EAE. Moreover, somatodendritic Kv2.1 channels in the motor neurons of lower spinal cord significantly decrease correlating with EAE severity. Interestingly, Kv1.4 expression surrounding lesions is markedly up-regulated in the initial acute phase of both EAE models. Its expression in glial fibrillary acidic protein (GFAP)-positive astrocytes further increases in the remitting phase of remitting-relapsing EAE (rrEAE), but decreases in late chronic EAE (chEAE) and the relapse of rrEAE, suggesting that Kv1.4-positive astrocytes may be neuroprotective. Taken together, our studies reveal myelin-dependent and -independent alterations of Kv channels in the progression of EAE and lay a solid foundation for future study in search of a better treatment for MS.

Innate and adaptive immune activation in the brain of MPS IIIB mouse model.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

Copper chelation and autoimmunity differentially impact myelin in the hippocampal-prefrontal circuit.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. About 50% of MS patients develop deficits in learning, memory and executive function, which are accompanied by demyelinating lesions in the hippocampus and/or prefrontal cortex (PFC). Why demyelination in these regions occurs in some patients but not in others and what is the underlying mechanism remain unclear. Here we report that myelin density in the hippocampus and PFC is markedly reduced in the cuprizone model, but not in the chronic experimental autoimmune encephalomyelitis. These two models can be used for studying different neuropathophysiological aspects of demyelinating diseases.

Expression of GABA A receptor alpha1 subunit mRNA and protein in rat neocortex following photothrombotic infarction.

Photothrombotic infarcts of the neocortex result in structural and functional alterations of cortical networks, including decreased GABAergic inhibition, and can generate epileptic seizures within 1 month of lesioning. In our study, we assessed the involvement and potential changes of cortical GABA A receptor (GABA AR) alpha1 subunits at 1, 3, 7, and 30 days after photothrombosis. Quantitative competitive reverse transcription-polymerase chain reaction (cRT-PCR) and semi-quantitative Western blot analysis were used to investigate GABA AR alpha1 subunit mRNA and protein levels in proximal and distal regions of perilesional cortex and in homotopic areas of young adult Sprague-Dawley rats. GABA AR alpha1 subunit mRNA levels were decreased ipsilateral and contralateral to the infarct at 7 days, but were increased bilaterally at 30 days. GABA AR alpha1 subunit protein levels revealed no significant change in neocortical areas of both hemispheres of lesioned animals compared with protein levels of sham-operated controls at 1, 3, 7, and 30 days. At 30 days, GABA AR alpha1 subunit protein expression was significantly increased in lesioned animals within proximal and distal regions of perilesional cortex compared with distal neocortical areas contralaterally (Student's t-test, p<0.05). Short- and long-term alterations of mRNA and protein levels of the GABA AR alpha1 subunit ipsilateral and contralateral to the lesion may influence alterations in cell surface receptor subtype expression and GABA AR function following ischemic infarction and may be associated with formative mechanisms of poststroke epileptogenesis.

Poststroke epilepsy following transient unilateral middle cerebral and common carotid artery occlusion in young adult and aged F344 rats.

The mechanisms of injured brain that establish poststroke seizures and epilepsy are not well understood, largely because animal modeling has had limited development. The main objective of this study was to determine whether an arterial occlusion model of cortical stroke in young adult and aged rats was capable of generating either focal or generalized epileptic seizures within 2 months of lesioning. Four- and 20-month-old male Fischer 344 (F344) sham-operated controls and those lesioned by transient (3 h) unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion (MCA/CCAo) were studied by video-EEG recordings up to 2 months post-procedure. The main findings were: 1) seizures (grade 3 and above) were recorded within 2 months in both young (4-month; 0.23/h) and aged (20-month; 1.93/h) MCA/CCAo rat groups; both MCA/CCAo rat groups had more seizures recorded than the respective control groups, i.e., no seizures in young controls and 0.52/h in old controls; 2) both age and infarction independently had effects on seizure frequency; however, there was no demonstrated interaction between the two factors; and 3) there was no difference in infarct volumes comparing 4- to 20-month-old MCA/CCAo animals. In addition, all lesioned and sham-operated animals demonstrated intermittent solitary myoclonic convulsions arising out of sleep. Morbidity and mortality of animals limited the extent to which the animals could be evaluated, especially 20-month-old animals. These results suggest that transient unilateral MCA/CCAo can result in poststroke epileptic seizures in both young adult and aged F344 rats within a relatively brief period of time following lesioning.

Heterogeneity of astrocyte and NG2 cell insertion at the node of ranvier.

The node of Ranvier is a functionally important site on the myelinated axon where sodium channels are clustered and regeneration of action potentials occurs, allowing fast saltatory conduction of action potentials. Early ultrastructural studies have revealed the presence of "glia" or "astrocytes" at the nodes. NG2 cells, also known as oligodendrocyte precursor cells or polydendrocytes, which are a resident glial cell population in the mature mammalian central nervous system that is distinct from astrocytes, have also been shown to extend processes that contact the nodes. However, the prevalence of the two types of glia at the node has remained unknown. We have used specific cell surface markers to examine the association of NG2 cells and astrocytes with the nodes of Ranvier in the optic nerve, corpus callosum, and spinal cord of young adult mice or rats. We show that more than 95% of the nodes in all three regions contained astrocyte processes, while 33-49% of nodes contained NG2 cell processes. NG2 cell processes were associated more frequently with larger nodes. A few nodes were devoid of glial apposition. Electron microscopy and stimulated emission depletion (STED) super-resolution microscopy confirmed the presence of dual glial insertion at some nodes and further revealed that NG2 cell processes contacted the nodal membrane at discrete points, while astrocytes had broader processes that surrounded the nodes. The study provides the first systematic quantitative analysis of glial cell insertions at central nodes of Ranvier. J. Comp. Neurol. 525:535-552, 2017. © 2016 Wiley Periodicals, Inc.

Suppression of Inflammatory Demyelinaton and Axon Degeneration through Inhibiting Kv3 Channels.

The development of neuroprotective and repair strategies for treating progressive multiple sclerosis (MS) requires new insights into axonal injury. 4-aminopyridine (4-AP), a blocker of voltage-gated K+ (Kv) channels, is used in symptomatic treatment of progressive MS, but the underlying mechanism remains unclear. Here we report that deleting Kv3.1-the channel with the highest 4-AP sensitivity-reduces clinical signs in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. In Kv3.1 knockout (KO) mice, EAE lesions in sensory and motor tracts of spinal cord were markedly reduced, and radial astroglia were activated with increased expression of brain derived neurotrophic factor (BDNF). Kv3.3/Kv3.1 and activated BDNF receptors were upregulated in demyelinating axons in EAE and MS lesions. In spinal cord myelin coculture, BDNF treatment promoted myelination, and neuronal firing via altering channel expression. Therefore, suppressing Kv3.1 alters neural circuit activity, which may enhance BNDF signaling and hence protect axons from inflammatory insults.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.

Regulation of neurovascular coupling in autoimmunity to water and ion channels.

Much progress has been made in understanding autoimmune channelopathies, but the underlying pathogenic mechanisms are not always clear due to broad expression of some channel proteins. Recent studies show that autoimmune conditions that interfere with neurovascular coupling in the central nervous system (CNS) can lead to neurodegeneration. Cerebral blood flow that meets neuronal activity and metabolic demand is tightly regulated by local neural activity. This process of reciprocal regulation involves coordinated actions of a number of cell types, including neurons, glia, and vascular cells. In particular, astrocytic endfeet cover more than 90% of brain capillaries to assist blood-brain barrier (BBB) function, and wrap around synapses and nodes of Ranvier to communicate with neuronal activity. In this review, we highlight four types of channel proteins that are expressed in astrocytes, regarding their structures, biophysical properties, expression and distribution patterns, and related diseases including autoimmune disorders. Water channel aquaporin 4 (AQP4) and inwardly rectifying potassium (Kir4.1) channels are concentrated in astrocytic endfeet, whereas some voltage-gated Ca(2+) and two-pore domain K(+) channels are expressed throughout the cell body of reactive astrocytes. More channel proteins are found in astrocytes under normal and abnormal conditions. This research field will contribute to a better understanding of pathogenic mechanisms underlying autoimmune disorders.

Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons.

Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav, but not Kv1.2, channels in mouse cerebellum. These results indicate that AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport before anchoring them to the AIS and nodes of Ranvier.

Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity.

BACKGROUND: Neuronal activity intimately communicates with blood flow through the blood-brain barrier (BBB) in the central nervous system (CNS). Astrocyte endfeet cover more than 90% of brain capillaries and interact with synapses and nodes of Ranvier. The roles of astrocytes in neurovascular coupling in the CNS remain poorly understood. RESULTS: Here we show that astrocytes that are intrinsically different are activated by inflammatory autoimmune insults and alterations of neuronal activity. In the progression of experimental autoimmune encephalomyelitis (EAE), both fibrous and protoplasmic astrocytes were broadly and reversibly activated in the brain and spinal cord, indicated by marked upregulation of glial fibrillary acidic protein (GFAP) and other astrocytic proteins. In early and remitting EAE, upregulated GFAP and astrocytic endfoot water channel aquaporin 4 (AQP4) enclosed white matter lesions in spinal cord, whereas they markedly increased and formed bundles in exacerbated lesions in late EAE. In cerebellar cortex, upregulation of astrocytic proteins correlated with EAE severity. On the other hand, protoplasmic astrocytes were also markedly activated in the brains of ankyrin-G (AnkG) and Kv3.1 KO mice, where neuronal activities are altered. Massive astrocytes replaced degenerated Purkinje neurons in AnkG KO mice. In Kv3.1 KO mice, GFAP staining significantly increased in cerebellar cortex, where Kv3.1 is normally highly expressed, but displayed in a patchy pattern in parts of the hippocampus. CONCLUSIONS: Thus, astrocytes can detect changes in both blood and neurons, which supports their central role in neurovascular coupling. These studies contribute to the development of new strategies of neuroprotection and repair for various diseases, through activity-dependent regulation of neurovascular coupling.

Myelination of rodent hippocampal neurons in culture.

Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer's disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.

Effects of age and cortical infarction on EEG dynamic changes associated with spike wave discharges in F344 rats.

Rodent models of absence seizures are used to investigate the network properties and regulatory mechanisms of the seizure's generalized spike and wave discharge (SWD). As rats age, SWDs occur more frequently, suggesting aging-related changes in the regulation of the corticothalamic mechanisms generating the SWD. We hypothesized that brain resetting mechanisms - how the brain "resets" itself to a more normal functional state following a transient period of abnormal function, e.g., a SWD - are impaired in aged animals and that brain infarction would further affect these resetting mechanisms. The main objective of this study was to determine the effects of aging, infarction, and their potential interaction on the resetting of EEG dynamics assessed by quantitative EEG (qEEG) measures of linear (signal energy measured by amplitude variation; signal frequency measured by mean zero-crossings) and nonlinear (signal complexity measured by the pattern match regularity statistic and the short-term maximum Lyapunov exponent) brain EEG dynamics in 4- and 20-month-old F344 rats with and without brain infarction. The main findings of the study were: 1) dynamic resetting of both linear and nonlinear EEG characteristics occurred following SWDs; 2) animal age significantly affected the degree of dynamic resetting in all four qEEG measures: SWDs in older rats exhibited a lower degree of dynamic resetting; 3) infarction significantly affected the degree of dynamic resetting only in terms of EEG signal complexity: SWDs in infarcted rats exhibited a lower degree of dynamic resetting; and 4) in all four qEEG measures, there was no significant interaction effect between age and infarction on dynamic resetting. We conclude that recovery of the brain to its interictal state following SWDs was better in young adult animals compared with aged animals, and to a lesser degree, in age-matched controls compared with infarction-injured animal groups, suggesting possible effects of brain resetting mechanisms and/or the disruption of the epileptogenic network that triggers SWDs.

Long-term video-EEG recordings following transient unilateral middle cerebral and common carotid artery occlusion in Long-Evans rats.

The mechanisms of injured brain that establish poststroke seizures and epilepsy are not well understood, largely because animal modeling of these phenomena has had limited development. We studied the electrobehavioral properties of 2.5-month-old male Long-Evans rats by video-electroencephalogram (EEG) recordings during the 6 months following sham operation or lesioning by transient unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion (MCA/CCAO). The main findings of this study were: (1) control animals demonstrated interictal focal or restricted bilateral 7-8 Hz spike-wave discharges (SWDs) lasting 1-2 s without behavioral change and ictal generalized 7-8 Hz SWDs (absence seizures), which were prolonged, frequent, and associated with motor arrest of the animal; (2) lesioned animals demonstrated cortical infarction associated with interictal SWDs similar to controls, except that focal discharges were more numerous relative to bilateral discharges, and ictal SWDs, which were of shorter duration and less frequent than those of controls; (3) lesioned animals demonstrated decreased hemispheric and regional spectral power at approximately 7 and 15 Hz compared with controls, directly related to the reduced occurrence of ictal SWDs; and (4) lesioning did not independently generate either focal or generalized epileptic seizures. These studies demonstrate distinct electrobehavioral properties of Long-Evans rats lesioned by MCA/CCAO as juveniles and monitored by video-EEG recordings during young adulthood but fail to provide evidence of poststroke seizures or epilepsy.