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1.  NEUROPEPTIDE Y INHIBITS CAPSAICIN-SENSITIVE NOCICEPTORS VIA A Y1-RECEPTOR-MEDIATED MECHANISM 
Neuroscience  2004;125(3):703-709.
Neuropeptide Y (NPY) is expressed in certain primary afferent fibers, is up-regulated in response to tissue injury and is capable of inhibiting nociceptive behavior at the spinal level. However, the spinal mechanism(s) for NPY-evoked antinociception is unknown. In this study, we evaluated the hypothesis that agonists at the NPY Y1 receptor subtype (Y1-R) inhibit exocytosis from the capsaicin-sensitive class of nociceptors. Using in vitro superfusion of rat dorsal spinal cord slices, pre-treatment with the Y1-R agonist [Leu31Pro34]NPY significantly inhibited capsaicin-evoked release of immunoreactive calcitonin gene-related peptide with an EC50 value of 10.6 nM. This inhibitory effect was concentration dependent, significantly attenuated by pre-treatment with the Y1 receptor antagonist BIBP3226 and reproduced by synthetic NPY. Examination of adult rat dorsal root ganglia using double immunofluorescent labeling revealed frequent co-localization of Y1 receptor immunoreactivity in vanilloid receptor type 1-immunoreactive neurons, indicating that Y1 agonists may directly modulate the capsaicin-sensitive class of nociceptors. Collectively, these results indicate that NPY is capable of inhibiting capsaicin-sensitive neurons via a Y1 receptor mechanism, suggesting the mechanisms for spinal NPY-induced antinociception is due, at least in part, to inhibition of central terminals of capsaicin-sensitive nociceptors.
doi:10.1016/j.neuroscience.2004.01.044
PMCID: PMC4516042  PMID: 15099684
superfusion; pain; spinal cord; dorsal root ganglion; exocytosis
2.  Neuroanatomical distribution of oxytocin and vasopressin 1a receptors in the socially monogamous coppery titi monkey (Callicebus cupreus) 
Neuroscience  2014;273:12-23.
The coppery titi monkey (Callicebus cupreus) is a socially monogamous New World primate that has been studied in the field and the laboratory to investigate the behavioral neuroendocrinology of primate pair bonding and parental care. Arginine vasopressin has been shown to influence male titi monkey pair-bonding behavior, and studies are currently underway to examine the effects of oxytocin on titi monkey behavior and physiology. Here, we use receptor autoradiography to identify the distribution of arginine vasopressin 1a (AVPR1a) and oxytocin receptors (OXTR) in hemispheres of titi monkey brain (n=5). AVPR1a are diffuse and widespread throughout the brain, but the OXTR distribution is much more limited, with the densest binding being in the hippocampal formation (dentate gyrus, CA1 field) and the presubiculum (layers I and III). Moderate OXTR binding was detected in the nucleus basalis of Meynert, pulvinar, superior colliculus, layer 4C of primary visual cortex, periaqueductal gray, pontine gray, nucleus prepositus, and spinal trigeminal nucleus. OXTR mRNA overlapped with OXTR radioligand binding, confirming that the radioligand was detecting OXTR protein. AVPR1a binding is present throughout the cortex, especially in cingulate, insular, and occipital cortices, as well as in the caudate, putamen, nucleus accumbens, central amygdala, endopiriform nucleus, hippocampus (CA4 field), globus pallidus, lateral geniculate nucleus, infundibulum, habenula, periaqueductal gray, substantia nigra, olivary nucleus, hypoglossal nucleus, and cerebellum. Furthermore, we show that, in titi monkey brain, the OXTR antagonist ALS-II-69 is highly selective for OXTR and that the AVPR1a antagonist SR49059 is highly selective for AVPR1a. Based on these results and the fact that both ALS-II-69 and SR49059 are non-peptide, small-molecule antagonists that should be capable of crossing the blood brain barrier, these two compounds emerge as excellent candidates for the pharmacological manipulation of OXTR and AVPR1a in future behavioral experiments in titi monkeys and other primate species.
doi:10.1016/j.neuroscience.2014.04.055
PMCID: PMC4083847  PMID: 24814726
neuropeptides; receptor binding; nonhuman primate; neuroanatomy; monogamy; pair bonding
3.  ACCURACY OF REGENERATING MOTOR NEURONS: INFLUENCE OF DIFFUSION IN DENERVATED NERVE 
Neuroscience  2014;273:128-140.
Following injury to a peripheral nerve the denervated distal nerve segment undergoes remarkable changes including loss of the blood-nerve barrier, Schwann cell proliferation, macrophage invasion, and the production of many cytokines and neurotrophic factors. The aggregate consequence of such changes is that the denervated nerve becomes a permissive and even preferred target for regenerating axons from the proximal nerve segment. The possible role that an original end-organ target (e.g. muscle) may play in this phenomenon during the regeneration period is largely unexplored. We used the rat femoral nerve as an in vivo model to begin to address this question. We also examined the effects of disrupting communication with muscle in terms of the accuracy of regenerating motor neurons as judged by their ability to correctly project to their original terminal nerve branch. Our results demonstrate that the accuracy of regenerating motor neurons is dependent upon the denervated nerve segment remaining in uninterrupted continuity with muscle. We hypothesized that this influence of muscle on the denervated nerve might be via diffusion driven movement of biomolecules or the active axonal transport that continues in severed axons for several days in the rat, so we devised experiments to separate these two possibilities. Our data show that disrupting ongoing diffusion driven movement in a denervated nerve significantly reduces the accuracy of regenerating motor neurons.
doi:10.1016/j.neuroscience.2014.05.016
PMCID: PMC4096846  PMID: 24846614
PNS regeneration; denervated nerve; diffusion; Band of Bungner
4.  Reduced Chrna7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABAA receptor subunits 
Neuroscience  2014;273:52-64.
Decreased expression of CHRNA7, the gene encoding the α7* subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7* receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout mice using quantitative western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia.
doi:10.1016/j.neuroscience.2014.05.004
PMCID: PMC4122271  PMID: 24836856
Chrna7; λ-aminobutyric acid (GABA); GABAA receptors; parvalbumin; glutamate decarboxylase 67 (GAD67); schizophrenia
5.  Activation of the ACE2/Ang-(1-7)/Mas pathway reduces oxygen-glucose deprivation induced tissue swelling, ROS production, and cell death in mouse brain with angiotensin II overproduction 
Neuroscience  2014;273:39-51.
We previously demonstrated that mice which overexpress human renin and angiotensinogen (R+A+) show enhanced cerebral damage in both in vivo and in vitro experimental ischemia models. Angiotensin converting enzyme 2 (ACE2) counteracts the effects of angiotensin (Ang-II) by transforming it into Ang-(1-7), thus reducing the ligand for the AT1 receptor and increasing stimulation of the Mas receptor. Triple transgenic mice, SARA, which specifically overexpress ACE2 in neurons of R+A+ mice were used to study the role of ACE2 in ischemic stroke using oxygen and glucose deprivation (OGD) of brain slices as an in vitro model. We examined tissue swelling, the production of reactive oxygen species (ROS), and cell death in cerebral cortex (CX) and the hippocampal CA1 region during OGD. Expression levels of NADPH oxidase isoforms, Nox2 and Nox4 were measured using western blots. Results show that SARA mice and R+A+ mice treated with the Mas receptor agonist Ang-(1-7) had less swelling, cell death, and ROS production in CX and CA1 areas compared to those in R+A+ animals. Treatment of slices from SARA mice with the Mas antagonist A779 eliminated this protection. Finally, western blots revealed less Nox2 and Nox4 expression in SARA mice compared with R+A+ mice both before and after OGD. We suggest that reduced brain swelling and cell death observed in SARA animals exposed to OGD results from diminished ROS production coupled with lower expression of NADPH oxidases. Thus, the ACE2/Ang-(1-7)/Mas receptor pathway plays a protective role in brain ischemic damage by counteracting the detrimental effects of Ang-II-induced ROS production.
doi:10.1016/j.neuroscience.2014.04.060
PMCID: PMC4159741  PMID: 24814023
cerebral cortex; hippocampus; NADPH oxidase; brain edema; stroke; hypertension
6.  Reward Contingencies and the Recalibration of Task Monitoring and Reward Systems: A high-density electrical mapping study 
Neuroscience  2014;273:100-117.
Task execution almost always occurs in the context of reward-seeking or punishment-avoiding behavior. As such, ongoing task monitoring systems are influenced by reward anticipation systems. In turn, when a task has been executed either successfully or unsuccessfully, future iterations of that task will be re-titrated on the basis of the task outcome. Here, we examined the neural underpinnings of the task-monitoring and reward-evaluation systems to better understand how they govern reward seeking behavior. Twenty-three healthy adult participants performed a task where they accrued points that equated to real world value (gift cards) by responding as rapidly as possible within an allotted timeframe, while success rate was titrated online by changing the duration of the timeframe dependent on participant performance. Informative cues initiated each trial, indicating the probability of potential reward or loss (four levels from very low to very high). We manipulated feedback by first informing participants of task success/failure, after which a second feedback signal indicated actual magnitude of reward/loss. High-density EEG recordings allowed for examination of event-related potentials (ERPs) to the informative cues and in turn, to both feedback signals. Distinct ERP components associated with reward cues, task preparatory and task monitoring processes, and reward feedback processes were identified. Unsurprisingly, participants displayed increased ERP amplitudes associated with task preparatory processes following cues that predicted higher chances of reward. They also rapidly updated reward and loss prediction information dependent on task performance after the first feedback signal. Finally, upon reward receipt, initial reward probability was no longer taken into account. Rather, ERP measures suggested that only the magnitude of actual reward or loss was now processed. Reward and task monitoring processes are clearly dissociable, but interact across very fast timescales to update reward predictions as information about task success or failure is accrued. Careful delineation of these processes will be useful in future investigations in clinical groups where such processes are suspected of having gone awry.
doi:10.1016/j.neuroscience.2014.05.002
PMCID: PMC4209734  PMID: 24836852
Reward; Punishment; Task Monitoring; EEG; ERP; Motivation
7.  The Role of D2-Autoreceptors in Regulating Dopamine Neuron Activity and Transmission 
Neuroscience  2014;S0306-4522(14)00037-2 10.1016/j.neuroscience.2014.01.025.
Dopamine D2-autoreceptors play a key role in regulating the activity of dopamine neurons and control the synthesis, release and uptake of dopamine. These Gi/o-coupled inhibitory receptors play a major part in shaping dopamine transmission. Found at both somatodendritic and axonal sites, autoreceptors regulate the firing patterns of dopamine neurons and control the timing and amount of dopamine released from their terminals in target regions. Alterations in the expression and activity of autoreceptors are thought to contribute to Parkinson’s disease as well as schizophrenia, drug addiction and attention deficit hyperactivity disorder (ADHD), which emphasizes the importance of D2-autoreceptors in regulating the dopamine system. This review will summarize the cellular actions of dopamine autoreceptors and discuss recent advances that have furthered our understanding of the mechanisms by which D2-receptors control dopamine transmission.
doi:10.1016/j.neuroscience.2014.01.025
PMCID: PMC4108583  PMID: 24463000
Psychostimulants; cocaine; VTA; Substantia Nigra; GPCR
8.  Dendritic protein synthesis in the normal and diseased brain 
Neuroscience  2012;232:106-127.
Synaptic activity is a spatially-limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases.
doi:10.1016/j.neuroscience.2012.12.003
PMCID: PMC4502914  PMID: 23262237
dendritic protein synthesis; mRNA localization; synaptic plasticity; neurological disease
9.  Serotonin modulates electrosensory processing and behavior via 5-HT2-like receptors 
Neuroscience  2014;271:108-118.
Efficient sensory processing of the environment is a critical function for any organism to survive and is accomplished by having neurons adapt their responses to stimuli based on behavioral context in part through neuromodulators such as serotonin. We have recently shown that one critical function of the serotonergic system in weakly electric fish is to enhance sensory pyramidal neuron responses within the electrosensory lateral line lobe to stimuli caused by same sex conspecifics, thereby enhancing their perception. This enhancement is accomplished by making pyramidal neurons more excitable through downregulation of potassium channels. However, the nature of the 5-HT receptors that mediate this effect is not known. Here we show that the 5-HT2 receptor antagonist ketanserin can effectively block the effects of 5-HT on pyramidal neuron excitability in vitro. Indeed, serotonin application subsequent to ketanserin application did not cause any significant changes in neuron excitability and responses to current injection. We further show that ketanserin applied in vivo can block the effects of serotonin on behavioral responses. Thus, our results strongly suggest that the previously observed effects of serotonin on sensory processing within ELL and their consequences for behavior are mediated by 5-HT2 receptors.
doi:10.1016/j.neuroscience.2014.04.033
PMCID: PMC4503408  PMID: 24780766 CAMSID: cams4739
neural coding; weakly electric fish; neuromodulation; neuroethology
10.  Isoflurane unveils a critical role of glutamate transporter type 3 in 1 regulating hippocampal GluR1 trafficking and context-related learning and memory in mice 
Neuroscience  2014;272:58-64.
Glutamate transporter type 3 (EAAT3) may play a role in cognition. Isoflurane enhances EAAT3 trafficking to the plasma membrane. Thus, we used isoflurane to determine how EAAT3 might regulate learning and memory and the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, such as GluR1, to the plasma membrane, a fundamental biochemical process for learning and memory. Here, isoflurane increased EAAT3 but did not change GluR1 levels in the plasma membrane of wild-type mouse hippocampus. Isoflurane increased protein phosphatase activity in wild-type and EAAT3−/− mouse hippocampus. Also, isoflurane reduced GluR1 in the plasma membrane and decreased phospho-GluR1 in EAAT3−/− mice. The phosphatase inhibitor okadaic acid attenuated these effects. Finally, isoflurane inhibited context-related fear conditioning in EAAT3−/− mice but not in wild-type mice. Thus, isoflurane may increase GluR1 trafficking to the plasma membrane via EAAT3 and inhibit GluR1 trafficking via protein phosphatase. Lack of EAAT3 effects leads to decreased GluR1 trafficking and impaired cognition after isoflurane exposure in EAAT3−/− mice.
doi:10.1016/j.neuroscience.2014.04.049
PMCID: PMC4077290  PMID: 24797327
cognition; GluR1; glutamate transporter; hippocampus; isoflurane; trafficking
11.  Attenuated orexinergic signaling underlies depression-like responses induced by daytime light deficiency 
Neuroscience  2014;272:252-260.
Light has profound effects on mood, as exemplified by seasonal affective disorder (SAD) and the beneficial effects of bright light therapy. However, the underlying neural pathways through which light regulates mood are not well understood. Our previous work has developed the diurnal grass rat, Arvicanthis niloticus, as an animal model of SAD (Leach et al., 2013a, Leach et al., 2013b). By utilizing a 12:12hr Dim Light:Dark (DLD) paradigm that simulates the lower light intensity of winter, we showed that the animals housed in DLD exhibited increased depression-like behaviors in the forced swim test (FST) and sweet solution preference (SSP) compared to animals housed in bright light during the day (BLD). The objective of the present study was to test the hypothesis that light affects mood by acting on the brain orexinergic system in the diurnal grass rat model of SAD. First, orexinA immunoreactivity (OXA-ir) was examined in DLD and BLD grass rats. The results revealed a reduction in the number of OXA-ir neurons in the hypothalamus and attenuated OXA-ir fiber density in the dorsal raphe nucleus of animals in the DLD compared to those in the BLD group. Then, the animals in BLD were treated systemically with SB-334867, a selective orexin 1 receptor (OX1R) antagonist, which led to a depressive phenotype characterized by increased immobility in the FST and a decrease in SSP compared to vehicle-treated controls. The results suggest that attenuated orexinergic signaling is associated with increased depression-like behaviors in grass rats, and support the hypothesis that the orexinergic system mediates the effects of light on mood.
doi:10.1016/j.neuroscience.2014.04.069
PMCID: PMC4090246  PMID: 24813431
orexin; seasonal affective disorder; diurnal grass rats; SB-334867
12.  Functional distinction between NGF-mediated plasticity and regeneration of nociceptive axons within the spinal cord 
Neuroscience  2014;272:76-87.
Successful regeneration after injury requires either the direct reformation of the circuit or the formation of a bridge circuit to provide partial functional return through a more indirect route. Presently, little is known about the specificity of how regenerating axons reconnect or reconstruct functional circuits. We have established an in vivo dorsal root entry zone model, which in the presence of NGF, shows very robust regeneration of peptidergic nociceptive axons, but not other sensory axons. Expression of NGF in normal, non-injured animals leads to robust sprouting of only the peptidergic nociceptive axons. Interestingly, NGF-induced sprouting of these axons leads to severe chronic pain, whereas, regeneration leads to protective like pain without chronic pain. Using this model we set out to compare differences in behavioral outcomes and circuit features between these two groups. In this study, we examined pre-synaptic and post-synaptic markers to evaluate the relationship between synaptic connections and behavioral responses. NGF-induced sprouting of CGRP axons resulted in a significant redistribution of synapses and cFos expression into the deeper dorsal horn. Regeneration of only the CGRP axons showed a general reduction in synapses and cFos expression within laminae I and II; however, inflammation of the hindpaw induced peripheral sensitization. This data shows that although NGF-induced sprouting of peptidergic axons induces robust chronic pain and cFos expression throughout the entire dorsal horn, regeneration of the same axons resulted in normal protective pain with a synaptic and cFos distribution similar, albeit significantly less than that shown by the sprouting of CGRP axons.
doi:10.1016/j.neuroscience.2014.04.053
PMCID: PMC4103020  PMID: 24797326
Plasticity; Regeneration; Nerve growth factor; Hyperalgesia; Nociceptor
13.  DIFFERENCES IN THE REINSTATEMENT OF ETHANOL SEEKING WITH GANAXOLONE AND GABOXADOL 
Neuroscience  2014;272:180-187.
The endogenous neuroactive steroid allopregnanolone (ALLO) has previously been shown to induce reinstatement of ethanol seeking in rodents. ALLO is a positive allosteric modulator at both synaptic and extrasynaptic GABAA receptors. The contribution of each class of GABAA receptors in mediating reinstatement of ethanol seeking is unknown. The first aim of the present study was to determine whether ganaxolone (GAN), a longer-acting synthetic analog of ALLO, also promotes reinstatement of ethanol seeking. The second aim was to examine whether preferentially activating extrasynaptic GABAA receptors with the selective agonist gaboxadol (THIP) was sufficient to reinstate responding for ethanol in mice. Male C57BL/6J mice were trained to lever press for access to a 10% ethanol (v/v) solution (10E), using a sucrose fading procedure. Following extinction of the lever pressing behavior, systemic THIP (0, 4 and 6 mg/kg) and GAN (0, 10, and 15 mg/kg) were tested for their ability to reinstate ethanol-appropriate responding in the absence of 10E access. GAN significantly increased lever pressing on the previously active lever, while THIP did not alter lever pressing behavior. The results of this study suggest that direct activation of extrasynaptic GABAA receptors at the GABA site is not sufficient to induce ethanol seeking in the reinstatement procedure. Future studies are necessary to elucidate the mechanisms and brain areas by which differences in the pharmacological activity of GAN and THIP at the GABAA receptor contribute to the dissimilarity in their effect on the reinstatement of ethanol seeking. Nonetheless, based on the increased use of these drugs in clinical trials across multiple disease states, the effects of GAN or THIP on alcohol seeking may be an important consideration if these drugs are to be used clinically in a population with a co-occurring alcohol use disorder.
doi:10.1016/j.neuroscience.2014.04.065
PMCID: PMC4122668  PMID: 24814021
THIP; GABAA receptor; alcohol; relapse; allopregnanolone; extrasynaptic
14.  Sequence variations at I260 and A1731 contribute to persistent currents in Drosophila sodium channels 
Neuroscience  2014;268:297-308.
Tetrodotoxin-sensitive persistent sodium currents, INaP, that activate at subthreshold voltages, have been detected in numerous vertebrate and invertebrate neurons. These currents are believed to be critical for regulating neuronal excitability. However, the molecular mechanism underlying INaP is controversial. In this study, we identified an INaP with a broad range of voltage dependence, from −60 mV to 20 mV, in a Drosophila sodium channel variant expressed in Xenopus oocytes. Mutational analysis revealed that two variant-specific amino acid changes, I260T in the S4–S5 linker of domain I (ILS4–S5) and A1731V in the voltage sensor S4 of domain IV (IVS4), contribute to the INaP. I260T is critical for the portion of INaP at hyperpolarized potentials. The T260-mediated INaP is likely the result of window currents flowing in the voltage range where the activation and inactivation curves overlap. A1731V is responsible for impaired inactivation and contributes to the portion of INaP at depolarized potentials. Furthermore, A1731V causes enhanced activity of two site-3 toxins which induce persistent currents by inhibiting the outward movement of IVS4, suggesting that A1731V inhibits the outward movement of IVS4. These results provided molecular evidence for the involvement of distinct mechanisms in the generation of INaP: T260 contributes to INaP via enhancement of the window current, whereas V1731 impairs fast inactivation probably by inhibiting the outward movement of IVS4.
Graphical Abstract
doi:10.1016/j.neuroscience.2014.03.028
PMCID: PMC4482340  PMID: 24662849
persistent current; Drosophila sodium channel; window current
15.  Electrophysiological characterization of activation state-dependent Cav2 channel antagonist TROX-1 in spinal nerve injured rats 
Neuroscience  2015;297:47-57.
Highlights
•TROX-1 exhibits activation state-dependent inhibition of Cav2.2 in vitro.•TROX-1 selectively attenuates neuronal responses to mechanical stimulation.•Anti-nociceptive effect of TROX-1 dependent on pathophysiological state.
Prialt, a synthetic version of Cav2.2 antagonist ω-conotoxin MVIIA derived from Conus magus, is the first clinically approved voltage-gated calcium channel blocker for refractory chronic pain. However, due to the narrow therapeutic window and considerable side effects associated with systemic dosing, Prialt is only administered intrathecally. N-triazole oxindole (TROX-1) is a novel use-dependent and activation state-selective small-molecule inhibitor of Cav2.1, 2.2 and 2.3 calcium channels designed to overcome the limitations of Prialt. We have examined the neurophysiological and behavioral effects of blocking calcium channels with TROX-1. In vitro, TROX-1, in contrast to state-independent antagonist Prialt, preferentially inhibits Cav2.2 currents in rat dorsal root ganglia (DRG) neurons under depolarized conditions. In vivo electrophysiology was performed to record from deep dorsal horn lamina V/VI wide dynamic range neurons in non-sentient spinal nerve-ligated (SNL) and sham-operated rats. In SNL rats, spinal neurons exhibited reduced responses to innocuous and noxious punctate mechanical stimulation of the receptive field following subcutaneous administration of TROX-1, an effect that was absent in sham-operated animals. No effect was observed on neuronal responses evoked by dynamic brushing, heat or cold stimulation in SNL or sham rats. The wind-up response of spinal neurons following repeated electrical stimulation of the receptive field was also unaffected. Spinally applied TROX-1 dose dependently inhibited mechanically evoked neuronal responses in SNL but not sham-operated rats, consistent with behavioral observations. This study confirms the pathological state-dependent actions of TROX-1 through a likely spinal mechanism and reveals a modality selective change in calcium channel function following nerve injury.
doi:10.1016/j.neuroscience.2015.03.057
PMCID: PMC4436437  PMID: 25839150
ANOVA, analysis of variance; APs, action potentials; Ctrl, control; DMSO, dimethylsulfoxide; DRG, dorsal root ganglia; EGTA, ethylene glycol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; I, input; PD, post-discharge; RM, repeated measures; SNL, spinal nerve ligated; TROX-1, N-triazole oxindole; VGCCs, voltage-gated calcium channels; WDR, wide dynamic range; WU, wind-up; electrophysiology; dorsal horn; dorsal root ganglia; spinal nerve ligation; Cav2.2; N-type calcium channel
16.  DISTRIBUTION AND TUMOR NECROSIS FACTOR-ALPHA ISOFORM BINDING SPECIFICITY OF LOCALLY ADMINISTERED ETANERCEPT INTO INJURED AND UNINJURED RAT SCIATIC NERVE 
Neuroscience  2009;160(2):492-500.
Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine that is implicated in the initiation of neuropathic pain. Locally administered TNF antagonist etanercept offers a promising new treatment approach to target neuropathic pain. Here we evaluate the distribution and binding specificity for TNF isoforms of locally administered etanercept into injured and uninjured rat sciatic nerve. Distribution and co-localization of etanercept and TNF in the injured and uninjured nerve was evaluated at 1, 24, 48 and 96 h after etanercept local application using immunohistochemistry. In addition, binding specificity of etanercept for TNF isoforms was analyzed using immunoblot assay system in nerve lysates. A new observation was that locally administered etanercept reached the endoneurium of the injured but not the uninjured nerve 1 h after its application and mainly co-localized with TNF-positive structures, morphologically similar to Schwann cells and macrophages. We further noticed that immunoblot analyses for etanercept demonstrated its preferential binding to transmembrane and trimer TNF isoforms. Finally, locally administered etanercept inhibited pain-related behaviors in a rat sciatic nerve crush model. We conclude that locally administered etanercept reaches the endoneurial space in the injured nerve and preferentially binds to trans-membrane and bioactive trimer TNF isoforms to modulate neuropathic pain. Locally administered etanercept has potential as a targeted immunomodulating agent to treat local pathogenesis in neuropathic pain after peripheral nerve injury.
doi:10.1016/j.neuroscience.2009.02.038
PMCID: PMC4479269  PMID: 19250961
peripheral nerve injury; neuropathic pain; tumor necrosis factor; etanercept
17.  Neurochemical Changes Within Human Early Blind Occipital Cortex 
Neuroscience  2013;252:222-233.
Early blindness results in occipital cortex neurons responding to a wide range of auditory and tactile stimuli. These changes in tuning properties are accompanied by an extensive reorganization of occipital cortex that includes alterations in anatomical structure, neurochemical and metabolic pathways. Although it has been established in animal models that neurochemical pathways are heavily affected by early visual deprivation, the effects of blindness on these pathways in humans is still not well characterized. Here, using 1H magnetic resonance spectroscopy in nine early blind and normally sighted subjects, we find that early blindness is associated with higher levels of creatine, choline and myo-Inositol and indications of lower levels of GABA within occipital cortex. These results suggest that the cross-modal responses associated with early blindness may, at least in part, be driven by changes within occipital biochemical pathways.
doi:10.1016/j.neuroscience.2013.08.004
PMCID: PMC4476245  PMID: 23954804
18.  MRI evaluation of BBB disruption after adjuvant AcSDKP treatment of stroke with tPA in rat 
Neuroscience  2014;271:1-8.
The primary limitation of thrombolytic treatment of ischemic stroke with tPA is the hemorrhagic risk. We tested AcSDKP (N-acetyl-seryl-aspartyl-lysyl-proline), as an auxiliary therapeutic agent, to reduce blood-brain barrier (BBB) disruption in a combination tPA thrombolytic treatment of stroke. Wistar rats subjected to embolic stroke were randomly assigned to either the tPA monotherapy group (n=9) or combination of tPA and AcSDKP treatment group (n=9) initiated at 4h after ischemia. MRI measurements were performed before and after the treatments. Immunohistochemical staining and measurements were performed to confirm MRI findings. Longitudinal MRI permeability measurements with Gd-DTPA demonstrated that combination treatment of acute embolic stroke with AcSDKP and tPA significantly reduced BBB leakage, compared to tPA monotherapy, at 3 and 6 days (18.3±9.8mm3 vs 65.0±21.0mm3, p<0.001) after onset of stroke, although BBB leakage was comparable between the two groups prior to the treatments (6.8±4.4mm3 vs 4.3±3.3mm3, p>0.18). The substantial reduction of BBB leakage observed in the combination treatment group was closely associated with reduced ischemic lesions measured by T2 maps (113.6±24.9mm3 vs 188.1±60.8mm3, p<0.04 at 6d). Histopathological analysis of the same population rats showed that the combination treatment significantly reduced parenchymal fibrin deposition (0.063±0.059mm2 vs 0.172±0.103mm2, p<0.03) and infarct volume (146.7±35.9mm3 vs 199.3±60.4mm3, p<0.05) compared to the tPA monotherapy at 6 days after stroke. MRI provides biological insight into the therapeutic benefit of combination treatment of stroke with tPA and AcSDKP 4 hours after onset, and demonstrates significantly improved cerebrovascular integrity with neuroprotective effects compared with tPA monotherapy.
doi:10.1016/j.neuroscience.2014.04.025
PMCID: PMC4044825  PMID: 24769225
BBB; stroke; tPA; AcSDKP; MRI; rat
19.  Ultrastructural localization of tyrosine hydroxylase in tree shrew nucleus accumbens core and shell 
Neuroscience  2014;271:23-34.
Many behavioral, physiological, and anatomical studies utilize animal models to investigate human striatal pathologies. Although commonly used, rodent striatum may not present the optimal animal model for certain studies due to a lesser morphological complexity than that of non-human primates, which are increasingly restricted in research. As an alternative, the tree shrew could provide a beneficial animal model for studies of the striatum. The gross morphology of the tree shrew striatum resembles that of primates, with separation of the caudate and putamen by the internal capsule. The neurochemical anatomy of the ventral striatum, specifically the nucleus accumbens, has never been examined. This major region of the limbic system plays a role in normal physiological functioning and is also an area of interest for human striatal disorders. The current study uses immunohistochemistry of calbindin and tyrosine hydroxylase (TH) to determine the ultrastructural organization of the nucleus accumbens core and shell of the tree shrew (Tupaia glis belangeri). Stereology was used to quantify the ultrastructural localization of TH, which displays weaker immunoreactivity in the core and denser immunoreactivity in the shell. In both regions, synapses with TH-immunoreactive axon terminals were primarily symmetric and showed no preference for targeting dendrites versus dendritic spines. The results were compared to previous ultrastructural studies of TH and dopamine in rat and monkey nucleus accumbens. Tree shrew and monkey show no preference for the postsynaptic target in the shell, in contrast to rats which show a preference for synapsing with dendrites. Tree shrews have a ratio of asymmetric to symmetric synapses formed by TH-immunoreactive terminals that is intermediate between rats and monkey. The findings from this study support the tree shrew as an alternative model for studies of human striatal pathologies.
doi:10.1016/j.neuroscience.2014.04.024
PMCID: PMC4060433  PMID: 24769226
electron microscopy; dopamine; striatum; immunohistochemistry; ultrastructure
20.  Extracellular pH modulates GABAergic neurotransmission in rat hypothalamus 
Neuroscience  2014;271:64-76.
Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15–26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 µM) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1 mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant α1β2γ2 GABAA receptors stably expressed in HEK 293 cells, The pH influence on GABAA receptors was similar in HEPES- and MES-buffered medium, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission, which is mediated by both presynaptic and postsynaptic mechanisms.
doi:10.1016/j.neuroscience.2014.04.028
PMCID: PMC4067938  PMID: 24780768
protons; IPSC; acidification; hypothalamus; HEPES; buffer
21.  MICE LACKING THE TRANSCRIPTIONAL COACTIVATOR PGC-1α EXHIBIT ALTERATIONS IN INHIBITORY SYNAPTIC TRANSMISSION IN THE MOTOR CORTEX 
Neuroscience  2014;271:137-148.
Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator known to regulate gene programs in a cell-specific manner in energy-demanding tissues, and its dysfunction has been implicated in numerous neurological and psychiatric disorders. Previous work from the Cowell laboratory indicates that PGC-1α is concentrated in inhibitory interneurons and required for the expression of the calcium buffer parvalbumin (PV) in the cortex; however, the impact of PGC-1α deficiency on inhibitory neurotransmission in the motor cortex is not known. Here, we show that mice lacking PGC-1α exhibit increased amplitudes and decreased frequency of spontaneous inhibitory postsynaptic currents in layer V pyramidal neurons. Upon repetitive train stimulation at the gamma frequency, decreased GABA release is observed. Furthermore, PV-positive interneurons in PGC-1α −/− mice display reductions in intrinsic excitability and excitatory input without changes in gross interneuron morphology. Taken together, these data show that PGC-1α is required for normal inhibitory neurotransmission and cortical PV-positive interneuron function. Given the pronounced motor dysfunction in PGC-1α −/− mice and the essential role of PV-positive interneurons in maintenance of cortical excitatory:inhibitory balance, it is possible that deficiencies in PGC-1α expression could contribute to cortical hyperexcitability and motor abnormalities in multiple neurological disorders.
doi:10.1016/j.neuroscience.2014.04.023
PMCID: PMC4068733  PMID: 24769433
22.  Eugenol and carvacrol excite first- and second-order trigeminal neurons and enhance their heat-evoked responses 
Neuroscience  2014;271:45-55.
Eugenol and carvacrol from clove and oregano, respectively, are agonists of the warmth-sensitive transient receptor potential channel TRPV3 and the irritant-sensitive TRPA1. Eugenol and carvacrol induce oral irritation that rapidly desensitizes, accompanied by brief enhancement of innocuous warmth and heat pain in humans. We presently investigated if eugenol and carvacrol activate nociceptive primary afferent and higher-order trigeminal neurons and enhance their heat-evoked responses, using calcium imaging of cultured trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons, and in vivo single-unit recordings in trigeminal subnucleus caudalis (Vc) of rats. Eugenol and carvacrol activated 20-30% of TG and 7-20% of DRG cells, the majority of which additionally responded to menthol, mustard oil and/or capsaicin. TG cell responses to innocuous (39°) and noxious (42°C) heating were enhanced by eugenol and carvacrol. We identified dorsomedial Vc neurons responsive to noxious heating of the tongue in pentobarbital-anesthetized rats. Eugenol and carvacrol dose-dependently elicited desensitizing responses in 55% and 73% of heat-sensitive units, respectively. Responses to noxious heat were briefly enhanced by eugenol and carvacrol. Many eugenol- and carvacrol-responsive units also responded to menthol, cinnamaldehyde and capsaicin. These data support a peripheral site for eugenol and carvacrol to enhance warmth- and noxious heat-evoked responses of trigeminal neurons, and are consistent with the observation that these agonists briefly enhance warmth and heat pain on the human tongue.
doi:10.1016/j.neuroscience.2014.04.019
PMCID: PMC4070529  PMID: 24759772
23.  Neuropeptide Y in the rostral ventromedial medulla reverses inflammatory and nerve injury hyperalgesia in rats via non-selective excitation of local neurons 
Neuroscience  2014;271:149-159.
Chronic pain reflects not only sensitization of the ascending nociceptive pathways, but also changes in descending modulation. The rostral ventromedial medulla (RVM) is a key structure in a well-studied descending pathway, and contains two classes of modulatory neurons, the ON-cells and the OFF-cells. Disinhibition of OFF-cells depresses nociception; increased ON-cell activity facilitates nociception. Multiple lines of evidence show that sensitization of ON-cells contributes to chronic pain, and reversing or blocking this sensitization is of interest as a treatment of persistent pain. Neuropeptide Y (NPY) acting via the Y1 receptor has been shown to attenuate hypersensitivity in nerve-injured animals without affecting normal nociception when microinjected into the RVM, but the neural basis for this effect was unknown. We hypothesized that behavioral anti-hyperalgesia was due to selective inhibition of ON-cells by NPY at the Y1 receptor. To explore the possibility of Y1 selectivity on ON-cells, we stained for the NPY-Y1 receptor in the RVM, and found it broadly expressed on both serotonergic and non-serotonergic neurons. In subsequent behavioral experiments, NPY microinjected into the RVM in lightly anesthetized animals reversed signs of mechanical hyperalgesia following either nerve injury or chronic hindpaw inflammation. Unexpectedly, rather than decreasing ON-cell activity, NPY increased spontaneous activity of both ON- and OFF-cells without altering noxious-evoked changes in firing. Based on these results, we conclude that the anti-hyperalgesic effects of NPY in the RVM are not explained by selective inhibition of ON-cells, but rather by increased spontaneous activity of OFF-cells. Although ON-cells undoubtedly facilitate nociception and contribute to hypersensitivity, the present results highlight the importance of parallel OFF-cell mediated descending inhibition in limiting the expression of chronic pain.
doi:10.1016/j.neuroscience.2014.04.035
PMCID: PMC4071144  PMID: 24792711
descending pain modulation; nociception; ON-cells; OFF-cells; NPY; raphe magnus
24.  Estrogen Promotes Learning Related Plasticity by Modifying the Synaptic Cytoskeleton 
Neuroscience  2012;239:3-16.
Estrogen's acute, facilitatory effects on glutamatergic transmission and long-term potentiation (LTP) provide a potential explanation for the steroid's considerable influence on behavior. Recent work has identified mechanisms underlying these synaptic actions. Brief infusion of 17β-estradiol (E2) into adult male rat hippocampal slices triggers actin polymerization within dendritic spines via a signaling cascade beginning with the GTPase RhoA and ending with inactivation of the filament severing protein cofilin. Blocking this sequence, or actin polymerization itself, eliminates E2's effects on synaptic physiology. Notably, the theta burst stimulation used to induce LTP activates the same signaling pathway as E2 plus events that stabilize the reorganization of the sub-synaptic cytoskeleton. These observations suggest that E2 elicits a partial form of LTP, resulting in an increase of fast EPSP's and a reduction in the threshold for lasting synaptic changes. While E2's effects on the cytoskeleton could be direct, results described here indicate that the hormone activates synaptic TrkB receptors for Brain Derived Neurotrophic Factor, a releasable neurotrophin that stimulates the RhoA to cofilin pathway. It is therefore possible that E2 acts via transactivation of neighboring receptors to modify the composition and structure of excitatory contacts. Finally, there is the question of whether a loss of acute synaptic actions contributes to the memory problems associated with estrogen depletion. Initial tests found that ovariectomy in middle-aged rats disrupts RhoA signaling, actin polymerization, and LTP consolidation. Acute applications of E2 reversed these defects, a result consistent with the idea that disturbances to actin management are one cause of behavioral effects that emerge with reductions in steroid levels.
doi:10.1016/j.neuroscience.2012.10.038
PMCID: PMC4472431  PMID: 23103216
actin; cofilin; pTrkB; hippocampus; LTP; RhoA
25.  ACTIVATION AND INTERNALIZATION OF THE μ-OPIOID RECEPTOR BY THE NEWLY DISCOVERED ENDOGENOUS AGONISTS, ENDOMORPHIN-1 AND ENDOMORPHIN-2 
Neuroscience  1999;90(3):1051-1059.
The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal μ-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for μ-opioid receptors. Since the μ-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the μ-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged μ-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the μ-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced μ-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins’ and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle–myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 ~ 8.4) is consistent with antagonism at the μ-opioid receptor in myenteric neurons.
These results indicate that endomorphins directly activate the μ-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced μ-opioid receptor activation can be visualized by receptor endocytosis.
PMCID: PMC4472477  PMID: 10218804
myenteric neurons; receptor endocytosis; opioid peptides; opiate alkaloids; neurogenic cholinergic contractions; excitatory and inhibitory enteric neurons

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