Amino Acid Antagonists Research Articles

9228 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

8388 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

Interactions of the branched-chain amino acids. 1. Influence of dietary isoleucine and leucine on the valine requirement of male Cobb MV × 500 broilers for a 29- to 42-day finisher period

Branched-chain amino acid requirements have traditionally been determined using dose-response titrations without accounting for the influence of branched-chain amino acid antagonism. As recent data has indicated that inherent levels of the non-tested amino acid may influence response curves in diets mimicking industry, these methods may result in over or under estimation of requirement values. Therefore, an experiment was conducted to determine if dietary levels of isoleucine and leucine influence 29 to 42 day valine needs through the implementation of a 5 × 2 × 2 factorial design. Twenty diets were fed to 7 replicate pens of 12 broilers. Body weight gain, feed intake, and FCR were determined for the 29 to 42 day period, and carcass traits were assessed on day 44. Valine × isoleucine and valine × leucine interactions were observed for BW gain and total breast yield ( P < 0.10). Body weight gain requirements ranged from 73 to 81 Val/Lys, whereas responses for total breast yield were generally negative. A 3 way interaction was observed for FCR in which requirements varied from 75 to 82 Val/Lys. These interactions indicate that formulation strategies based on previous data concerning valine or isoleucine may not produce the desired response in Cobb MV × 500 broilers and that the branched-chain amino acids should be considered as a whole and not individually.

Failure of excess leucine to impact live performance and carcass traits in male and female Cobb MV × 500 broilers during a 15- to 32-day grower period

Modern broiler diets may contain excess levels of leucine due to the increased level of this amino acid in cereal grains. Excessive levels of leucine have previously been shown to negatively influence broiler performance due to branched-chain amino acid antagonism. Therefore, 2 leucine titrations were conducted in male and female Cobb 500 broilers. Low and high leucine diets were formulated to contain leucine to lysine ratios of 115 and 170, respectively. These diets were subsequently blended to produce 6 experimental diets containing leucine to lysine ratios of: 115, 126, 137, 148, 159, and 170. Pelleted diets were fed to 8 replicate pens of male and female broilers during a 15 to 32-day period. A subset of broilers was processed on day 33 for determination of carcass traits. Male broilers did not respond to excessive levels of dietary leucine. Increased leucine resulted in a positive quadratic response ( P = 0.020) in feed intake for female broilers. The overall lack of a response of increased dietary leucine on male and female broilers indicate that industry levels of isoleucine and valine are likely of a sufficient level to overcome the effects of leucine excess.

Interactions of the branched-chain amino acids. 2. Practical adjustments in valine and isoleucine

Reductions in dietary crude protein have potentially allowed for the occurrence of branched chain amino acid antagonism which can negatively influence live performance and carcass traits. Therefore, 2 factorial studies were conducted with the first experiment evaluating valine and leucine interactions during a 32 to 45 d finisher period, while the second experiment evaluated valine and isoleucine interactions during a 39 to 52 d withdrawal period. Valine, leucine, nor their subsequent interaction influenced broiler live performance or carcass traits in Experiment 1. However, a valine × leucine interaction was observed for average woody breast score and occurrence of scores of 1 and 2. This interaction demonstrated that leucine does not increase woody breast in birds fed diets with a valine to lysine ratio of 77, but interaction differences for woody breast severity varied among valine and leucine ratios to lysine. In the second experiment, increasing valine marginally increased feed intake, while isoleucine influenced tender yield. No valine × isoleucine interactions were observed. Within the confines of the levels used in the current study, manipulation of dietary branched-chain amino acid levels had little influence on live performance or carcass traits during the tested growout periods. The occurrence of a valine × leucine interaction on the incidence and severity of woody breast is perplexing and requires more investigation to determine if a successful mitigation strategy could be developed.

Features of development of winter wheat plants in the autumn-winter vegetation period as affected by pre-sowing seed treatment

Purpose. To identify the peculiarities of growth and development of winter wheat plants in the autumn-winter vegetation period as affected by pre-sowing seed treatment in the conditions of the Right-Bank Forest-Steppe of Ukraine.&#x0D; Methods. The experiments were carried out at the Agronomic Research Station of the National University of Life and Environmental Sciences of Ukraine (Pshenychne, Bila Tserkva district, Kyiv region) in the years 2019–2021: inoculant Binok Grain, complex micro fertilizer Harvest Start, organic biostimulant containing enzymes Rhizomax and bacterial formulations Trichodermin and Planoryz at the application rates recommended by their manufacturers. The background (control) for the study was a treatment where seedbed preparation included the application of nitroammophos N32P32K32 together with a complex granular fertilizer Actibion. The cultivation technology in the experiment is generally accepted for the research area.&#x0D; Results. In the control treatment, on average for the years of research, the field germination of bread winter wheat seeds was 82.7% with a plant density of 414 per 1 m2. The use of protective and stimulating formulations for seed treatment increased these indicators compared to the control by 1.9–4.8% and 9–26 plants/m2, respectively. The highest sugar content in the tillering node (14.2%) and, accordingly, winter hardiness of plants (96.8%) was obtained in the treatment with Binok Grain + Harvest Start. In other seed treatments, these indicators ranged between 12.6 and 14.0% and 93.4–96.0%, while in the control between 12.4% and 82.7%, respectively. In the control treatment, the incidence of Septoria in autumn was 1.6%, powdery mildew 5.6%, and Fusarium root rot 2.9%, while in the case of using biological formulations the values were 0.1–0.9%, 0.1–3.0 and 0.0–1.4%, respectively. Complete control of these diseases in sowings (plant damage was not detected) is provided by the combined use of Binok Grain + Harvest Start. Most likely, this is because Binok Grain contains antagonists of pathogens, phytohormones, antibiotics, vitamins, amino acids, and growth regulators. Harvest Start, in addition, to trace elements, contains auxins, amino acids, and B vitamins.&#x0D; Conclusions. The use of all studied protective and stimulating formulations for the winter wheat seed treatment ensured a significant increase in the field germination of seeds, plant density, sugar content in the tillering node, winter hardiness, and limit the development of dangerous pathogens in crops. Consequently, these biological formulations may be recommended for implementation in the current cultivation technologies practiced in the Right-Bank Forest-Steppe of Ukraine.

Sex- and sub region-dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin-releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS ) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+ ]i ) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+ ]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+ ]i in 95%-100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%-8% of kisspeptin neurones located in other ARN subregions responded in females and 0%-12% of cells in any subregion in males (P<0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro-caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

Possibilities of Pharmacological Preconditioning

The review is devoted to the experimental and clinical data analysis about the effectiveness of preconditioning as a method of organism’s tolerance development to ischemia/hypoxia. Characteristics of the trigger, signaling and effector stages of the preconditioning mechanism are described. Medicinal agents which can stimulate processes of endogenous metabolic adaptation are discussed. As is shown in the review the role of inducers of preconditioning can play drugs from different pharmacological groups: adenosine receptor agonists, some agents for inhalation anesthesia, potassium channel activators, opioid analgesics, antagonists of excitatory amino acids, inducers of transcription factors, drugs of erythropoietin, inhibitors of the mitochondrial pore, bioflavonoids, drugs with antihypoxic action. Examples of successful preconditioning with help of pharmacological agents are presented. The prospects of pharmacological preconditioning usage in cerebral ischemia and myocardial infarction are discussed in the article.

Glutamate receptors in the hypothalamic paraventricular nucleus contribute to insulin-induced sympathoexcitation.

The sympathoexcitatory response to insulin is mediated by neurons in the arcuate nucleus (ARC) and hypothalamic paraventricular nucleus (PVH). Previous studies have reported that stimulation of ARC neurons increases sympathetic nerve activity (SNA) and arterial blood pressure (ABP) through glutamate receptor activation in the PVH. Therefore, the purpose of the present study was to determine whether glutamatergic neurotransmission in the PVH contributes to insulin-induced sympathoexcitation. Male Sprague-Dawley rats (275-400 g) were infused with isotonic saline or insulin (3.75 mU · kg(-1) · min(-1)) plus 50% dextrose to maintain euglycemia. Intravenous infusion of insulin significantly increased lumbar SNA without a significant change in mean ABP, renal SNA, heart rate, or blood glucose. Bilateral PVH injection of the excitatory amino acid antagonist kynurenic acid (KYN) lowered lumbar SNA and ABP of animals infused with insulin. Similarly, a cocktail of the NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced lumbar SNA and mean ABP during infusion of insulin. In a final experiment, bilateral PVH injection of AP5 only, but not CNQX, lowered lumbar SNA and mean ABP of animals infused with insulin. The peak changes in lumbar SNA and mean ABP of insulin-treated animals were not different between KYN, AP5 plus CNQX, or AP5 alone. These drug treatments did not alter any variable in animals infused with saline. Altogether, these findings suggest that glutamatergic NMDA neurotransmission in the PVH contributes to insulin-induced sympathoexcitation.

The nucleus raphe magnus OFF-cells are involved in diffuse noxious inhibitory controls

Diffuse noxious inhibitory controls (DNIC) are very powerful long-lasting descending inhibitory controls which are pivotal in modulating the activity of spinal and trigeminal nociceptive neurons. DNIC are subserved by a loop involving supraspinal structures such as the lateral parabrachial nucleus and the subnucleus reticularis dorsalis. Surprisingly, though, whether the nucleus raphe magnus (NRM), another supraspinal area which is long known to be important in pain modulation, is involved in DNIC is still a matter of discussion. Here, we reassessed the role of the NRM neurons in DNIC by electrophysiologically recording from wide dynamic range (WDR) neurons in the trigeminal subnucleus oralis and pharmacologically manipulating the NRM OFF- and ON-cells. In control conditions, C-fiber-evoked responses in trigeminal WDR neurons are inhibited by a conditioning noxious heat stimulation applied to the hindpaw. We show that inactivating the NRM by microinjecting the GABAA receptor agonist, muscimol, both facilitates C-fiber-evoked responses of trigeminal WDR neurons and strongly attenuates their inhibition by heat applied to the hindpaw. Interestingly, selective blockade of ON-cells by microinjecting the broad-spectrum excitatory amino acid antagonist, kynurenate, into the NRM neither affects C-fiber-evoked responses nor attenuates DNIC of trigeminal WDR neurons. These results indicate that the NRM tonically inhibits trigeminal nociceptive inputs and is involved in the neuronal network underlying DNIC. Moreover, within NRM, OFF-cells might be more specifically involved in both the tonic and phasic descending inhibitory controls of trigeminal nociception.

Characterization of Renal Toxicity in Mice Administered the Marine Biotoxin Domoic Acid

Domoic acid (DA), an excitatory amino acid produced by diatoms belonging to the genus Pseudo-nitzschia, is a glutamate analog responsible for the neurologic condition referred to as amnesic shellfish poisoning. To date, the renal effects of DA have been underappreciated, although renal filtration is the primary route of systemic elimination and the kidney expresses ionotropic glutamate receptors. To characterize the renal effects of DA, we administered either a neurotoxic dose of DA or doses below the recognized limit of toxicity to adult Sv128/Black Swiss mice. DA preferentially accumulated in the kidney and elicited marked renal vascular and tubular damage consistent with acute tubular necrosis, apoptosis, and renal tubular cell desquamation, with toxic vacuolization and mitochondrial swelling as hallmarks of the cellular damage. Doses≥0.1 mg/kg DA elevated the renal injury biomarkers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, and doses≥0.005 mg/kg induced the early response genes c-fos and junb. Coadministration of DA with the broad spectrum excitatory amino acid antagonist kynurenic acid inhibited induction of c-fos, junb, and neutrophil gelatinase-associated lipocalin. These findings suggest that the kidney may be susceptible to excitotoxic agonists, and renal effects should be considered when examining glutamate receptor activation. Additionally, these results indicate that DA is a potent nephrotoxicant, and potential renal toxicity may require consideration when determining safe levels for human exposure.

Dopamine is differentially involved in the locomotor hyperactivity produced by manipulations of opioid, GABA and glutamate receptors in the median raphe nucleus

The median raphe nucleus (MR) has been shown to exert a powerful influence on behavioral arousal and marked locomotor hyperactivity can be produced by intra-MR injections of a variety of drugs including GABAA and GABAB agonists, excitatory amino acid antagonists, and μ- and δ-opioid agonists. Other studies have indicated that the MR exerts an inhibitory influence on ascending dopamine systems, suggesting that MR induced alterations in activity may be mediated through changes in dopaminergic transmission. In the present study, we explored this possibility by examining whether systemic administration of the preferential D2 dopamine antagonist haloperidol is able to antagonize the hyperactivity produced by intra-MR injections of various drugs. We found that haloperidol completely blocked the locomotor response to intra-MR injections of the μ-opioid receptor agonist DAMGO and the δ-opioid receptor agonist DPDPE. In marked contrast, at doses which abolished the locomotor response to systemic amphetamine, haloperidol had no effect on the hyperactivity induced by intra-MR injections of GABAA agonist muscimol, the GABAB agonist baclofen, or the kainate/quisqualate antagonist pBB-PZDA, even though it suppressed baseline activity in these same animals. These results indicate that there must be at least two mechanisms capable of influencing behavioral arousal within the MR region, one of which is dependent on D2 dopamine receptors and the other is not.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Neuropharmacological specificity of brain structures involved in soman-induced seizures

Pharmacological control of seizure activity following nerve agent exposure is critical in reducing neuropathology and improving survival in casualties. Three classes of drugs, anticholinergics, benzodiazepines and excitatory amino acid (EAA) antagonists, have been shown to be effective at moderating nerve agent-induced seizures. However, little is known about which brain structures are involved in producing the anticonvulsant response. This study evaluated drugs from each class, injected directly into one of three specific brain structures, the perirhinal cortex, the entorhinal cortex, or the mediodorsal thalamus, for their ability to modulate seizures induced by the nerve agent soman. The drugs evaluated were the anticholinergic scopolamine, the benzodiazepine midazolam, and the EAA antagonist MK-801. For each drug treatment in each brain area, anticonvulsant ED50 values were calculated using an up-down dosing procedure over successive animals. There was no statistical difference in the anticonvulsant ED50 values for scopolamine and MK-801 in the perirhinal and entorhinal cortices. MK-801 pretreatment in the mediodorsal thalamus had a significantly lower anticonvulsant ED50 value than any other treatment/injection site combination. Midazolam required significantly higher doses than scopolamine and MK-801 in the perirhinal and entorhinal cortices to produce an anticonvulsant response and was ineffective in the mediodorsal thalamus. These findings support the contention that specific neuroanatomical pathways are activated during nerve agent-induced seizures and that the discrete brain structures involved have unique pharmacological thresholds for producing an anticonvulsant response. This study is also the first to show the involvement of the mediodorsal thalamus in the control of nerve agent-induced seizures.

Identification of Minimal Neuronal Networks Involved in Flexor-Extensor Alternation in the Mammalian Spinal Cord

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated invitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.

β-Kainic acid is not an amino acid antagonist

beta-Kainic acid has been reported to have anticonvulsant properties. In view of its structural relationship to alpha-kainic acid it has therefore been tested for antagonist activity at excitatory receptors for N-methylaspartate, quisqualate and alpha-kainate using anaesthetized rats and brain slice preparations. No antagonism was found against these three agonists though beta-kainate itself caused excitation. If beta-kainate interferes with excitatory amino acid neurotransmission it probably acts presynaptically.

Descending Facilitatory Pathways from the Rostroventromedial Medulla Mediate Naloxone-Precipitated Withdrawal in Morphine-Dependent Rats

Opioids produce analgesic effects, and extended use can produce physical dependence in both humans and animals. Dependence to opiates can be demonstrated by either termination of drug administration or through precipitation of the withdrawal syndrome by opiate antagonists. Key features of the opiate withdrawal syndrome include hyperalgesia, anxiety, and autonomic signs such as diarrhea. The rostral ventromedial medulla (RVM) plays an important role in the modulation of pain and for this reason, may influence withdrawal-induced hyperalgesia. The mechanisms that drive opiate withdrawal-induced hyperalgesia have not been elucidated. Here, rats made dependent upon morphine received naloxone to precipitate withdrawal. RVM microinjection of lidocaine, kynurenic acid (excitatory amino acid antagonist) or YM022 (CCK2 receptor antagonist) blocked withdrawal-induced hyperalgesia. Additionally, these treatments reduced both somatic and autonomic signs of naloxone-induced withdrawal. Spinal application of ondansetron, a 5HT3 receptor antagonist thought to ultimately be engaged by descending pain facilitatory drive, also blocked hyperalgesia and somatic and autonomic features of the withdrawal syndrome. These results indicate that the RVM plays a critical role in mediating components of opioid withdrawal that may contribute to opioid dependence. Perspective Manipulations targeting these descending pathways from the RVM may diminish the consequences of prolonged opioid administration-induced dependence and be useful adjunct strategies in reducing the risk of opioid addiction.

Bidirectional Plasticity Gated by Hyperpolarization Controls the Gain of Postsynaptic Firing Responses at Central Vestibular Nerve Synapses

Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here, we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, whereas long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.

Introduction

HomeStrokeVol. 41, No. 10_suppl_1Introduction Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIntroduction Richard J. Traystman, PhD Richard J. TraystmanRichard J. Traystman From the University of Colorado Denver, Anschutz Medical Campus, Aurora, Colo. Search for more papers by this author Originally published1 Oct 2010https://doi.org/10.1161/STROKEAHA.110.598557Stroke. 2010;41:S63Brain protection from focal or global cerebral ischemia is not new, not innovative, and so far, mostly not successful. There have been many methods and agents tried in an attempt to produce neuroprotection from cerebral ischemia: hypothermia, anesthetics, free radical scavengers, excitatory amino acid antagonists, calcium channel blockers, ionic pump modulators, growth factors, heparinization, antineutrophil/platelet factors, sex steroids, and genes and gene products. There are hundreds, perhaps thousands of neuroprotective drugs that have been used in animal models. So, if you were a mouse or a rat, and experienced a stroke or cardiac arrest, we would know just what to do for you. But, essentially none of these pharmacological agents have demonstrated usefulness in humans even though they have been shown to be successful in preclinical animal trials. What could account for this and why do we not have a “magic bullet”?One issue is that there are several important mechanisms of injury from focal and global cerebral ischemia, and the agents mentioned above usually work only on 1 potential mechanism. For example, ischemia can produce free radicals, result in the release of excitotoxins, and release mediators of inflammation, and there are other mechanisms of injury as well. All of these mechanisms of injury can result in neuronal cell death. Because there are multiple mechanisms of injury, it is likely that there are multiple mechanisms of neuroprotection. None of the agents mentioned above can affect all mechanisms of injury at once or at the appropriate time after injury. Another issue is that there is a temporal sequence of injury after ischemia. For example, excitotoxicity occurs very quickly, in minutes or hours after the ischemia. Inflammatory injury or apoptotic injury may not occur until hours or even days after the injury. There is even a temporal sequence of gene expression such that heat shock proteins and early genes are expressed early after the ischemia, whereas growth factor genes are expressed days after the injury. Thus, the pharmacological agents must be administered at the appropriate time to result in neuroprotection.There are other issues concerning drug dose and time of administration of the drug which can determine neuroprotection efficacy. A dose of drug that is effective in a mouse may not be the correct effective dose in the human. The window of opportunity in a mouse also may not be the correct window in the human. Combination therapy also needs to be attempted in humans. In addition, in humans there may be a genetic predisposition for injury from ischemia, and gender differences. Issues related to trial design and analysis also must be considered. We must also consider that we work mostly with normal, young, healthy animals, whereas in the human population this is not true. Finally, we must also ask the question of whether the animal models of ischemia we use accurately reflect ischemic disease in humans.In this neuroprotection session, we will address several novel mechanisms of protection from ischemia. Dr Anne Comi will address ischemic injury and neuroprotection in immature brain and will discuss new approaches to reduce apoptosis (prevention of apoptosis and the erythropoietin trial in newborns). Dr Midori Yenari will speak about hypothermic neuroprotection for cerebral ischemia and will discuss the status of clinical hypothermia and challenges in its clinical application. Dr Kyra Becker will discuss immune tolerance therapy for stroke and attempt to answer the question whether modulation of the postischemic immune response can improve stroke outcome? Finally, Dr Daniel Laskowitz will address neuroprotection in subarachnoid hemorrhage and ask the question of whether vasospasm is a good therapeutic target in subarachnoid hemorrhage?DisclosuresNone.FootnotesCorrespondence to Richard J. Traystman, PhD, Professor, Vice Chancellor for Research, University of Colorado Denver, Anschutz Medical Campus, 13001 E. 17th Place, Mail Stop F520, Building 500, Room C1000, P.O. Box 6508, Aurora, CO 80045. E-mail [email protected] Previous Back to top Next FiguresReferencesRelatedDetailsCited By Yang L, Islam M, Karamyan V and Abbruscato T (2015) In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment, Brain Research, 10.1016/j.brainres.2015.03.022, 1609, (1-11), Online publication date: 1-Jun-2015. October 2010Vol 41, Issue 10_suppl_1 Advertisement Article InformationMetrics https://doi.org/10.1161/STROKEAHA.110.598557PMID: 20876508 Manuscript receivedJuly 29, 2010Manuscript acceptedAugust 10, 2010Originally publishedOctober 1, 2010 KeywordsneuroprotectionprincetonPDF download Advertisement

ChemInform Abstract: Protection by Pyroglutamic Acid and Some of Its Newly Synthesized Derivatives Against Glutamate-Induced Seizures in Mice.

The protection by pyroglutamic acid (CAS 98-79-3) and derivatives Ia-i (injected i.p.) against glutamate- and NMDA (N-methyl-D-aspartate) (i.c.v.) induced seizures in mice has been studied in comparison with known antiepileptics and antagonists of excitatory aminoacids. The potency of pyroglutamic acid and some derivatives (Id,f,g,h) against glutamate-induced convulsions was similar to that shown by glutamic acid diethylester and by valproic acid. Interestingly, pyroglutamic acid did not affect NMDA-induced convulsions which were well antagonized by both 2-amino-5-phosphono valeric acid and by diazepam. Thus, pyroglutamic acid may represent the starting for synthesis of excitatory aminoacid antagonists acting at non NMDA receptors.