Excitatory Interneurons Research Articles

Expression of Neuropeptide FF Defines a Population of Excitatory Interneurons in the Superficial Dorsal Horn of the Mouse Spinal Cord that Respond to Noxious and Pruritic Stimuli

The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch.

The Role of Intracellular Calcium in Changing of ElectricalCharacteristics of Premotor Interneurons in Intact Snails and Snails During Various Forms of Plasticity

It was previously shown that both associative learning and the formation of long-term sensitization led to the increase in excitability of premotor interneurons of the defensive behavior of terrestrial snail Helix lucorum. In the present study, we analyzed the role of intracellular calcium ions in the maintenance of increased excitability in premotor interneurons of terrestrial snail after the formation of a conditioned defensive reflex. It was shown that the increase of the intracellular Ca2+ concentration after adding caffeine to the solution washing the nervous system of the mollusk led to a decrease of the threshold of action potential and to an increase of the critical level of depolarization without a change of the membrane potential of premotor interneurons in both intact and trained snails. The decrease of the intracellular Ca2+ concentration in premotor interneurons by the intracellular injection of (ethylene glycol-bis (2-aminoethylether)-N, N, N, N-tetraacetic acid) (EGTA) resulted in a significant increase of the threshold of generation of the action potential in intact snails. But the values of threshold of generation of the action potential in trained snails after injection of EGTA did not significantly differ from the values of studied parameters before injection. After application of the membrane-penetrating chelator, BAPTA-AM, the changes in the membrane and threshold potentials of premotor interneurons of intact and trained snails were not observed. Our results demonstrated that both the increase and decrease of intracellular Ca2+ concentration were not involved in maintaining the changes of membrane characteristics of premotor interneurons observed after associative learning.

Recruitment of Spinoparabrachial Neurons by Dorsal Horn Calretinin Neurons

The dorsal horn of the spinal cord is the first integration site of somatosensory inputs from the periphery. In the superficial layers of the dorsal horn, nociceptive inputs are processed by a complex network of excitatory and inhibitory interneurons whose function and connectivity remain poorly understood. We examined the role of calretinin-expressing interneurons (CR neurons) in such processing and show that they receive direct inputs from nociceptive fibers and polysynaptic inputs from touch-sensitive Aβ fibers. Their activation by chemogenetic or optogenetic stimulation produces mechanical allodynia and nocifensive responses. Furthermore, they monosynaptically engage spinoparabrachial (SPb) neurons in lamina I, suggesting CR neurons modulate one of the major ascending pain pathways of the dorsal horn. In conclusion, we propose a neuronal pathway in which CR neurons are positioned at the junction between nociceptive and innocuous circuits and directly control SPb neurons in lamina I.

Diverse Mechanisms Lead to Common Dysfunction of Striatal Cholinergic Interneurons in Distinct Genetic Mouse Models of Dystonia.

Clinical and experimental data indicate striatal cholinergic dysfunction in dystonia, a movement disorder typically resulting in twisted postures via abnormal muscle contraction. Three forms of isolated human dystonia result from mutations in the TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) genes. Experimental models carrying these mutations facilitate identification of possible shared cellular mechanisms. Recently, we reported elevated extracellular striatal acetylcholine by in vivo microdialysis and paradoxical excitation of cholinergic interneurons (ChIs) by dopamine D2 receptor (D2R) agonism using ex vivo slice electrophysiology in Dyt1ΔGAG/+ mice. The paradoxical excitation was caused by overactive muscarinic receptors (mAChRs), leading to a switch in D2R coupling from canonical Gi/o to noncanonical β-arrestin signaling. We sought to determine whether these mechanisms in Dyt1ΔGAG/+ mice are shared with Thap1C54Y/+ knock-in and Gnal+/- knock-out dystonia models and to determine the impact of sex. We found Thap1C54Y/+ mice of both sexes have elevated extracellular striatal acetylcholine and D2R-induced paradoxical ChI excitation, which was reversed by mAChR inhibition. Elevated extracellular acetylcholine was absent in male and female Gnal+/- mice, but the paradoxical D2R-mediated ChI excitation was retained and only reversed by inhibition of adenosine A2ARs. The Gi/o-preferring D2R agonist failed to increase ChI excitability, suggesting a possible switch in coupling of D2Rs to β-arrestin, as seen previously in a DYT1 model. These data show that, whereas elevated extracellular acetylcholine levels are not always detected across these genetic models of human dystonia, the D2R-mediated paradoxical excitation of ChIs is shared and is caused by altered function of distinct G-protein-coupled receptors.SIGNIFICANCE STATEMENT Dystonia is a common and often disabling movement disorder. The usual medical treatment of dystonia is pharmacotherapy with nonselective antagonists of muscarinic acetylcholine receptors, which have many undesirable side effects. Development of new therapeutics is a top priority for dystonia research. The current findings, considered in context with our previous investigations, establish a role for cholinergic dysfunction across three mouse models of human genetic dystonia: DYT1, DYT6, and DYT25. The commonality of cholinergic dysfunction in these models arising from diverse molecular etiologies points the way to new approaches for cholinergic modulation that may be broadly applicable in dystonia.

Human cerebrospinal fluid promotes spontaneous gamma oscillations in the hippocampus in vitro

Gamma oscillations (30-80 Hz) are fast network activity patterns frequently linked to cognition. They are commonly studied in hippocampal brain slices in vitro, where they can be evoked via pharmacological activation of various receptor families. One limitation of this approach is that neuronal activity is studied in a highly artificial extracellular fluid environment, as provided by artificial cerebrospinal fluid (aCSF). Here, we examine the influence of human cerebrospinal fluid (hCSF) on kainate-evoked and spontaneous gamma oscillations in mouse hippocampus. We show that hCSF, as compared to aCSF of matched electrolyte and glucose composition, increases the power of kainate-evoked gamma oscillations and induces spontaneous gamma activity in areas CA3 and CA1 that is reversed by washout. Bath application of atropine entirely abolished hCSF-induced gamma oscillations, indicating critical contribution from muscarinic acetylcholine receptor-mediated signaling. In separate whole-cell patch clamp recordings from rat hippocampus, hCSF increased theta resonance frequency and strength in pyramidal cells along with enhancement of h-current (Ih ) amplitude. We found no evidence of intrinsic gamma frequency resonance at baseline (aCSF) among fast-spiking interneurons, and this was not altered by hCSF. However, hCSF increased the excitability of fast-spiking interneurons, which likely contributed to gamma rhythmogenesis. Our findings show that hCSF promotes network gamma oscillations in the hippocampus in vitro and suggest that neuromodulators distributed in CSF could have significant influence on neuronal network activity in vivo.

Identification of a Spinal Circuit for Mechanical and Persistent Spontaneous Itch

Lightly stroking the lips or gently poking some skin regions can evoke mechanical itch in healthy human subjects. Sensitization of mechanical itch and persistent spontaneous itch are intractable symptoms in chronic itch patients. However, the underlying neural circuits are not well defined. We identified a subpopulation of excitatory interneurons expressing Urocortin 3::Cre (Ucn3+) in the dorsal spinal cord as a central node in the pathway that transmits acute mechanical itch and mechanical itch sensitization as well as persistent spontaneous itch under chronic itch conditions. This population receives peripheral inputs from Toll-like receptor 5-positive (TLR5+) Aβ low-threshold mechanoreceptors and is directly innervated by inhibitory interneurons expressing neuropeptide Y::Cre (NPY+) in the dorsal spinal cord. Reduced synaptic inhibition and increased intrinsic excitability of Ucn3+ neurons lead to chronic itch sensitization. Our study sheds new light on the neural basis of chronic itch and unveils novel avenues for developing mechanism-specific therapeutic advancements.

Modeling the daily rhythm of human pain processing in the dorsal horn.

Experimental studies show that human pain sensitivity varies across the 24-hour day, with the lowest sensitivity usually occurring during the afternoon. Patients suffering from neuropathic pain, or nerve damage, experience an inversion in the daily modulation of pain sensitivity, with the highest sensitivity usually occurring during the early afternoon. Processing of painful stimulation occurs in the dorsal horn (DH), an area of the spinal cord that receives input from peripheral tissues via several types of primary afferent nerve fibers. The DH circuit is composed of different populations of neurons, including excitatory and inhibitory interneurons, and projection neurons, which constitute the majority of the output from the DH to the brain. In this work, we develop a mathematical model of the dorsal horn neural circuit to investigate mechanisms for the daily modulation of pain sensitivity. The model describes average firing rates of excitatory and inhibitory interneuron populations and projection neurons, whose activity is directly correlated with experienced pain. Response in afferent fibers to peripheral stimulation is simulated by a Poisson process generating nerve fiber spike trains at variable firing rates. Model parameters for fiber response to stimulation and the excitability properties of neuronal populations are constrained by experimental results found in the literature, leading to qualitative agreement between modeled responses to pain and experimental observations. We validate our model by reproducing the wind-up of pain response to repeated stimulation. We apply the model to investigate daily modulatory effects on pain inhibition, in which response to painful stimuli is reduced by subsequent non-painful stimuli. Finally, we use the model to propose a mechanism for the observed inversion of the daily rhythmicity of pain sensation under neuropathic pain conditions. Underlying mechanisms for the shift in rhythmicity have not been identified experimentally, but our model results predict that experimentally-observed dysregulation of inhibition within the DH neural circuit may be responsible. The model provides an accessible, biophysical framework that will be valuable for experimental and clinical investigations of diverse physiological processes modulating pain processing in humans.

Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia

SummaryChronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I–IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.

A more efficient conditional mouse model of Dravet syndrome: Implications for epigenetic selection and sex-dependent behaviors

BackgroundDravet Syndrome (DS) is an epileptic disorder characterized by spontaneous and thermally-induced seizures, hyperactivity, cognitive deficits, autistic-like behaviors, and Sudden Unexpected Death in Epilepsy (SUDEP). DS is caused by de novo loss-of-function mutations in the SCN1A gene. Selective loss of GABAergic interneuron excitability is the primary cause of the disease. Up to 60% of Scn1a+/− mice die from SUDEP before sexual maturity. New methodWe used Cre-Lox technology to conditionally delete Scn1a in all epiblast-derived somatic cells by crossing a floxed Scn1a mouse with a mouse expressing Cre under the Meox2 promoter. ResultsParental Scn1a flox (F) mice, parental Meox2 Cre+ mice, and their F/+:Meox2-Cre− offspring were phenotypically normal and did not prematurely die. In contrast, F/+:Meox2-Cre+ offspring recapitulated DS seizure and behavioral phenotypes. Unexpectedly, male F/+:Meox2-Cre+ mice demonstrated impaired social interaction, while females did not. Comparison with existing methodIn the previous models, colony maintenance required breeding SUDEP survivors, which greatly increased colony size required to sustain experimental animal production, and raised the concern that surviving breeders have epigenetic traits that impart new phenotypes to their offspring. Our method greatly facilitates breeding, recapitulates DS phenotypes, eliminates concerns about parents that are survivors, and provides initial evidence for unexpected sex-dependent social interaction impairment. ConclusionsWe introduce a more efficient mouse model of human DS that demonstrates an efficient breeding strategy free from potential inherited epigenetic changes and reveals an unexpected sex-specific impairment of social interaction in DS. This new model should have great value to investigators of DS.

NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10 [corrected

Unprocessed pro-neuregulin 2 (pro-NRG2) accumulates on neuronal cell bodies at junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). NMDA receptors (NMDARs) trigger NRG2 ectodomain shedding from these sites followed by activation of ErbB4 receptor tyrosine kinases, and ErbB4 signaling cell-autonomously downregulates intrinsic excitability of GABAergic interneurons by reducing voltage-gated sodium channel currents. NMDARs also promote dispersal of Kv2.1 clusters from ER-PM junctions and cause a hyperpolarizing shift in its voltage-dependent channel activation, suggesting that NRG2/ErbB4 and Kv2.1 work together to regulate intrinsic interneuron excitability in an activity-dependent manner. Here we explored the cellular processes underlying NMDAR-dependent NRG2 shedding in cultured rat hippocampal neurons. We report that NMDARs control shedding by two separate but converging mechanisms. First, NMDA treatment disrupts binding of pro-NRG2 to ER-PM junctions by post-translationally modifying conserved Ser/Thr residues in its intracellular domain. Second, using a mutant NRG2 protein that cannot be modified at these residues and that fails to accumulate at ER-PM junctions, we demonstrate that NMDARs also directly promote NRG2 shedding by ADAM-type metalloproteinases. Using pharmacological and shRNA-mediated knockdown, and metalloproteinase overexpression, we unexpectedly find that ADAM10, but not ADAM17/TACE, is the major NRG2 sheddase acting downstream of NMDAR activation. Together, these findings reveal how NMDARs exert tight control over the NRG2/ErbB4 signaling pathway, and suggest that NRG2 and Kv2.1 are co-regulated components of a shared pathway that responds to elevated extracellular glutamate levels.

Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system.

Acetylcholine Release Inhibits Distinct Excitatory Inputs Onto Hippocampal CA1 Pyramidal Neurons via Different Cellular and Network Mechanisms.

In hippocampal CA1, muscarinic acetylcholine (ACh) receptor (mAChR) activation via exogenous application of cholinergic agonists has been shown to presynaptically inhibit Schaffer collateral (SC) glutamatergic inputs in stratum radiatum (SR), and temporoammonic (TA) and thalamic nucleus reuniens (RE) glutamatergic inputs in stratum lacunosum-moleculare (SLM). However, steady-state uniform mAChR activation may not mimic the effect of ACh release in an intact hippocampal network. To more accurately examine the effect of ACh release on glutamatergic synaptic efficacy, we measured electrically evoked synaptic responses in CA1 pyramidal cells (PCs) following the optogenetic release of ACh in genetically modified mouse brain slices. The ratio of synaptic amplitudes in response to paired-pulse SR stimulation (stimulus 2/stimulus 1) was significantly reduced by the optogenetic release of ACh, consistent with a postsynaptic decrease in synaptic efficacy. The effect of ACh release was blocked by the M3 receptor antagonist 4-DAMP, the GABAB receptor antagonist CGP 52432, inclusion of GDP-β-S, cesium, QX314 in the intracellular patch clamp solution, or extracellular barium. These observations suggest that ACh release decreased SC synaptic transmission through an M3 muscarinic receptor-mediated increase in inhibitory interneuron excitability, which activate GABAB receptors and inwardly rectifying potassium channels on CA1 pyramidal cells. In contrast, the ratio of synaptic amplitudes in response to paired-pulse stimulation in the SLM was increased by ACh release, consistent with presynaptic inhibition. ACh-mediated effects in SLM were blocked by the M2 receptor antagonist AF-DX 116, presumably located on presynaptic terminals. Therefore, our data indicate that ACh release differentially modulates excitatory inputs in SR and SLM of CA1 through different cellular and network mechanisms.

How Gastrin-Releasing Peptide Opens the Spinal Gate for Itch

SummarySpinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.

Facilitation of neuropathic pain by the NPY Y1 receptor-expressing subpopulation of excitatory interneurons in the dorsal horn

Endogenous neuropeptide Y (NPY) exerts long-lasting spinal inhibitory control of neuropathic pain, but its mechanism of action is complicated by the expression of its receptors at multiple sites in the dorsal horn: NPY Y1 receptors (Y1Rs) on post-synaptic neurons and both Y1Rs and Y2Rs at the central terminals of primary afferents. We found that Y1R-expressing spinal neurons contain multiple markers of excitatory but not inhibitory interneurons in the rat superficial dorsal horn. To test the relevance of this spinal population to the development and/or maintenance of acute and neuropathic pain, we selectively ablated Y1R-expressing interneurons with intrathecal administration of an NPY-conjugated saporin ribosomal neurotoxin that spares the central terminals of primary afferents. NPY-saporin decreased spinal Y1R immunoreactivity but did not change the primary afferent terminal markers isolectin B4 or calcitonin-gene-related peptide immunoreactivity. In the spared nerve injury (SNI) model of neuropathic pain, NPY-saporin decreased mechanical and cold hypersensitivity, but disrupted neither normal mechanical or thermal thresholds, motor coordination, nor locomotor activity. We conclude that Y1R-expressing excitatory dorsal horn interneurons facilitate neuropathic pain hypersensitivity. Furthermore, this neuronal population remains sensitive to intrathecal NPY after nerve injury. This neuroanatomical and behavioral characterization of Y1R-expressing excitatory interneurons provides compelling evidence for the development of spinally-directed Y1R agonists to reduce chronic neuropathic pain.

NETO1 Regulates Postsynaptic Kainate Receptors in CA3 Interneurons During Circuit Maturation

Kainate type ionotropic glutamate receptors (KARs) are expressed in hippocampal interneurons and regulate interneuron excitability and GABAergic transmission. Neuropilin tolloid-like proteins (NETO1 and NETO2) act as KAR auxiliary subunits; however, their significance for various functions of KARs in GABAergic interneurons is not fully understood. Here we show that NETO1, but not NETO2, is necessary for dendritic delivery of KAR subunits and, consequently, for formation of KAR-containing synapses in cultured GABAergic neurons. Accordingly, electrophysiological analysis of neonatal CA3 stratum radiatum interneurons revealed impaired postsynaptic and metabotropic KAR signaling in Neto1 knockouts, while a subpopulation of ionotropic KARs in the somatodendritic compartment remained functional. Loss of NETO1/KAR signaling had no significant effect on development of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-receptor-mediated glutamatergic transmission in CA3 interneurons, contrasting the synaptogenic role proposed for KARs in principal cells. Furthermore, loss of NETO1 had no effect on excitability and characteristic spontaneous network bursts in the immature CA3 circuitry. However, we find that NETO1 is critical for kainate-dependent modulation of network bursts and GABAergic transmission in the hippocampus already during the first week of life. Our results provide the first description of NETO1-dependent subcellular targeting of KAR subunits in GABAergic neurons and indicate that endogenous NETO1 is required for formation of KAR-containing synapses in interneurons. Since aberrant KAR-mediated excitability is implicated in certain forms of epilepsy, NETO1 represents a potential therapeutic target for treatment of both adult and early life seizures.

Temporal-spacial relationships between facial stimulation-evoked filed potential responses in mouse cerebellar granular layer and molecular layer

The cerebellum receives sensory inputs from mossy fiber-granule cell or climbing fiber pathways, and generates motor-related outputs. However, the temporal and special mechanism of the sensory information processing in cerebellar cortex is still unclear. Therefore, we here investigated the temporal-spacial mechanism between the facial stimulation-evoked field potential responses in granular layer (GL) and molecular layer (ML), by duo-electrophysiological recording technique and pharmacological methods in urethane-anesthetized mice. Our results showed that air-puff stimulation of ipsilateral whisker pad evoked successively field potential responses in GL and ML. The field potential response in GL exhibited a strong excitatory component (N1) followed by an inhibitory component (P1), while the field potential response in ML exhibited a tiny excitatory component (N1) followed by strong inhibitory component (P1). The latency of N1 was decreased with the increase of recording depth in ML, and it was the shortest in GL. Notably, the latencies of P1 in GL and ML were similar regardless the relative recording sites. Furthermore, blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated parallel fiber excitatory inputs by application of AMPA receptor antagonist, NBQX prevented P1 in both ML and GL. Moreover, application of GABAA receptors antagonist, gabazine simultaneously abolished P1 in both ML and GL. These results indicate that the facial stimulation evoked a simultaneous GABAergic inhibition in both ML and GL via mossy fiber-GC-parallel fiber pathway, suggesting that the sensory stimulation simultaneously evoked excitation of molecular layer interneurons (MLIs) and GL Golgi cells in cerebellar cortex.

Time course of functional changes in locomotor and sensory systems after spinal cord lesions in lamprey.

Changes in motor and sensory properties occur either side of spinal cord lesion sites from lower vertebrates to humans. We have previously examined these changes in the lamprey, a model system for studying recovery after spinal cord injury. These analyses were performed 8-12 wk after complete spinal cord lesions, a time when most animals have recovered good locomotor function. However, anatomical analyses have been performed at earlier and later times than this. Because there have been no functional studies at these times, in this study we have examined changes between 2 and 24+ wk after lesioning. Functional changes developed at different times in different regions of the spinal cord. Spinal cord excitability was significantly reduced above and below the lesion site less than 6 wk after lesioning but showed variable region-specific changes at later times. Excitatory synaptic inputs to motor neurons were increased above the lesion site during the recovery phase (2-8 wk after lesioning) but only increased below the lesion site once recovery had occurred (8 wk and later). These synaptic effects were associated with lesion-induced changes in connectivity between premotor excitatory interneurons. Sensory inputs were potentiated at 8 wk and later after lesioning but were markedly reduced at earlier times. There are thus time- and region-specific changes in motor and sensory properties above and below the lesion site. Although animals typically recover good locomotor function by 8 wk, there were further changes at 24+ wk. With the assumption that these changes can help to compensate for the reduced descending input to the spinal cord, effects at later times may reflect ongoing modifications as regeneration continues. NEW & NOTEWORTHY The lamprey is a model system for studying functional recovery and regeneration after spinal cord injury. We show that changes in spinal cord excitability and sensory inputs develop at different times above and below the lesion site during recovery. These changes may occur in response to the lesion-induced removal of descending inputs and may subsequently help to compensate for the reduction of the descending drive to allow locomotor recovery. Changes also continue once animals have recovered locomotor function, potentially reflecting adaptations to further regeneration at later recovery times.

Action of Norepinephrine on Lamina X of the Spinal Cord

Lamina X is localized in the spinal cord within the region surrounding the central canal and receives descending projections from the supraspinal nuclei. Norepinephrine (NE) is a neurotransmitter in descending pathways emanating from the brain stem; NE-containing fibers terminate in the spinal dorsal cord, particularly in the substantia gelatinosa (SG). NE enhances inhibitory synaptic transmission in SG neurons by activating presynaptic α1-receptors and hyperpolarizes the membranes of SG neurons by acting on α2-receptors; NE may thus act directly on SG neurons of the dorsal spinal cord and inhibit nociceptive transmission at the spinal level. NE-containing fibers also reportedly terminate in lamina X, suggesting that NE also modulates synaptic transmission in lamina X. However, the cellular mechanisms underlying such action have not been investigated. We hypothesized that NE might directly act on lamina X and enhance inhibitory synaptic transmission therein. Using rat spinal cord slices and in vitro whole-cell patch-clamps, we found that the bath-application of NE to lamina X does not affect the excitatory interneurons but enhances GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and induces an outward current. NE-induced enhancement of mIPSCs was blocked by α1A-receptor antagonists, and NE-induced outward current was blocked by α2-receptor antagonists. NE did not affect GABA- or glycine- induced outward currents. These findings are similar to those obtained from SG neurons: NE may act at presynaptic terminals of GABAergic and glycinergic interneurons on lamina X to facilitate inhibitory-transmitter release through α1A-receptor activation and directly induce inhibitory interneuron membrane hyperpolarization through α2-receptors activation.

(226) Gastrin-Releasing Peptide Defines an Itch-Specific Circuit in the Spinal Dorsal Horn

Recent studies have indicated that the spinal activity of gastrin-releasing peptide (GRP) and its receptor, GRPR, selectively modulate itching behaviors over other somatosensations including pain behaviors. Despite behavioral selectivity, how GRP acts within the spinal cord to selectively convey itch to the brain is not understood. Utilizing a novel ex vivo somatosensory preparation, retrograde labeling of spinal projection neurons, and multiphoton Ca2+ imaging, the activity of excitatory interneurons and projection neurons in the ex vivo spinal cord can be recorded in response to natural stimulation of the skin and pharmacologic modulation of GRP signaling. Using this technique to visualize activity, we found that GRP activates a primary population of neurons at the spinal dorsal horn which subsequently drives activity in a larger secondary population of interneurons. The activity of this circuit also drives output from the spinal dorsal horn through activation of local projection neurons. These experiments begin to define a network of neurons that may be involved in itch-selective signaling.

Intramuscular Injection of Adenoassociated Virus Encoding Human Neurotrophic Factor 3 and Exercise Intervention Contribute to Reduce Spasms after Spinal Cord Injury.

Limb spasms are phenomena of hyperreflexia that occur after spinal cord injury. Currently, the clinical treatment is less than ideal. Our goal is to develop a combination therapy based on individualized medicine to reduce spasticity after spinal cord injury. In this study, rats received a severe contusive injury at the T9 segment of the spinal cord, followed by gene therapy with adenoassociated virus encoding human neurotrophic factor 3 (AAV-NT3) and a 2-week exercise program starting at 4 weeks after injury. We quantified the frequency of spasms during a swimming test at 4 and 6 weeks after injury and confirmed the results of the swimming test by measuring the H-reflex of the plantar muscle. We obtained weekly hind limb exercise scores to assess the effect of the interventions in hind limb motor function improvement. Then, we used immunofluorescence to observe the immunoreactivity of spinal motor neurons, synaptophysin, cholinergic interneurons, and GABAergic interneurons. We also measured the expression of KCC2 in the spinal cord by western blot. We found that AAV-NT3 gene therapy, exercise, and combination therapy all attenuated the frequency of spasms in the swimming test conducted at 6 weeks after spinal cord injury and increased rate-dependent depression of H-reflex. Combination therapy was significantly superior to AAV-NT3 alone in protecting motor neurons. Recovery of KCC2 expression was significantly greater in rats treated with combination therapy than in the exercise group. Combination therapy was also significantly superior to individual therapies in remodeling spinal cord neurons. Our study shows that the combination of AAV-NT3 gene therapy and exercise can alleviate muscle spasm after spinal cord injury by altering the excitability of spinal interneurons and motor neurons. However, combination therapy did not show a significant additive effect, which needs to be improved by adjusting the combined strategy.