A potential role for somatostatin signaling in regulating retinal neurogenesis.

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.

Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1.

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

Conformational analysis of a cyclic AKH neuropeptide analog that elicits selective activity on locust versus honeybee receptor.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II ß-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI ß-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II ß turn exhibited by cycloAKH from the type VI ß turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations.

I8-arachnotocin-an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor.

The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V1aR, V1bR, and V2R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod (Metaseiulus occidentalis) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V2R (EC50 34 nM) and V1bR (EC50 1.2 µM), a partial agonist at OTR (EC50 790 nM), and a competitive antagonist at V1aR [pA2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gαs pathway of V2R without recruiting either ß-arrestin-1 or ß-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of ß-arrestin-1 or -2 recruitment to the V2R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.

Beneficial effect of adipokinetic hormone on neuromuscular paralysis in insect body elicited by braconid wasp venom.

The effect of Habrobracon hebetor venom and the role of the adipokinetic hormone (AKH) in poisoned adult females of the firebug Pyrrhocoris apterus were studied 24 and 48h after treatments. Venom application elicited total neuromuscular paralysis in firebugs, but the co-application of venom and Pyrap-AKH significantly reduced paralysis (up to 3.2 times) compared to the application of venom only. Although the mechanisms of their action are unknown, both agents might affect neuromuscular junctions. Venom application significantly increased the expression of both P. apterus Akh genes (Pyrap-Akh 5.4 times and Peram-Cah-II 3.6 times), as well as the level of AKHs in the central nervous system (2.5 times) and haemolymph (3.0 times). In the haemolymph, increased AKH levels might have led to the mobilization of stored lipids, which increased 1.9 times, while the level of free carbohydrates remained unchanged. Total metabolism, monitored by carbon dioxide production, significantly declined in paralysed P. apterus individuals (1.4 times and 1.9 times, 24 and 48h after the treatment, respectively), probably because of a malfunction of the muscular system. The results suggest an active role of AKH in the defence mechanism against the stress elicited by neuromuscular paralysis, and the possible involvement of this hormone in neuronal/neuromuscular signalling.

Classical neurotransmitters and neuropeptides involved in generalized epilepsy in a multi-neurotransmitter system: How to improve the antiepileptic effect?

Here, we describe in generalized epilepsies the alterations of classical neurotransmitters and neuropeptides acting at specific subreceptors. In order to consider a network context rather than one based on focal substrates and in order to make the interaction between neurotransmitters and neuropeptides and their specific subreceptors comprehensible, neural networks in the hippocampus, thalamus, and cerebral cortex are described. In this disease, a neurotransmitter imbalance between dopaminergic and serotonergic neurons and between presynaptic GABAergic neurons (hypoactivity) and glutaminergic neurons (hyperactivity) occurs. Consequently, combined GABAA agonists and NMDA antagonists could furthermore stabilize the neural networks in a multimodal pharmacotherapy. The antiepileptic effect and the mechanisms of action of conventional and recently developed antiepileptic drugs are reviewed. The GASH:Sal animal model can contribute to examine the efficacy of antiepileptic drugs. The issues of whether the interaction of classical neurotransmitters with other subreceptors (5-HT7, metabotropic 5 glutaminergic, A2A adenosine, and alpha nicotinic 7 cholinergic receptors) or whether the administration of agonists/antagonists of neuropeptides might improve the therapeutic effect of antiepileptic drugs should be addressed. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²âº mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²âº mobilization and arrestin recruitment.

Resveratrol increases cerebral glycogen synthase kinase phosphorylation as well as protein levels of drebrin and transthyretin in mice: an exploratory study.

Alzheimer's disease (AD) is characterized by intraneuronal ß-amyloid plaques and hyperphosphorylated tau, leading to neuronal cell death and progressive memory losses. This exploratory work investigates if dietary resveratrol, previously shown to have broad anti-aging effects and improve AD pathology in vivo, leads to neuroprotective changes in specific protein targets in the mouse brain. Both wild-type and APP/PS1 mice, a transgenic AD mouse model, received control AIN-93G diet or AIN-93G supplemented with resveratrol. Pathology parameters and AD risk were assessed via measurements on plaque burden, levels of phosphorylated glycogen synthase kinase 3-ß (GSK3-ß), tau, transthyretin and drebrin. Dietary resveratrol treatment did not decrease plaque burden in APP/PS1 mice. However, resveratrol-fed mice demonstrated increases in GSK3-ß phosphorylation, a 3.8-fold increase in protein levels of transthyretin, and a 2.2-fold increase in drebrin. This study broadens our understanding of specific mechanisms and targets whereby resveratrol provides neuroprotection.

[The discovery of neuromedin U and its pivotal role in the central regulation of energy homeostasis]. / Odkrycie neuromedyny U i jej rola w centralnej regulacji homeostazy energetycznej.

 Neuromedin U (NMU) is a structurally highly conserved neuropeptide and has been paired with the G-protein-coupled receptors (GPCRs) NMUR1 and NMUR2, which were formerly classified in the orphan receptor family. Activation of the G protein Gq/11 subunit causes a pertussis toxin (PTX)-insensitive activation of both phospholipase C and mitogen-activated protein kinase (MAP), and activation of the Go subunit causes a PTX-sensitive inhibition of adenyl cyclase. Additionally, NMU selectively inhibits L-type high-voltage-gated Ca2+ channels in mouse hippocampus, as well as low-voltage-activated T-type Ca2+ channels in mouse dorsal root ganglia (DRG). NMU peptide and its receptors are predominantly expressed in the gastrointestinal tract and specific structures within the brain, reflecting its major role in the regulation of energy homeostasis. A novel neuropeptide, neuromedin S (NMS), is structurally related to NMU. They share a C-terminal core structure and both have been implicated in the regulation of food intake, as well as the circadian rhythms. The acute anorectic and weight-reducing effects of NMU and NMS are mediated by NMUR2. This suggests that NMUR2-selective agonists may be useful for the treatment of obesity.

The influence of orexins on the firing rate and pattern of rat intergeniculate leaflet neurons--electrophysiological and immunohistological studies.

Orexins influence various physiological processes associated with feeding behaviour, endocrine functions and wakefulness. One component of mammalian circadian timing systems, intergeniculate leaflet (IGL) of the lateral geniculate nucleus, is thought to contribute to circadian entrainment by processing photic and non-photic/arousal-related signals. Because the IGL is possibly innervated by the orexinergic system, using in vitro extracellular recording techniques we evaluated the influence of orexin A (OXA) and orexin B (OXB) on the rate and pattern of neuronal firing in this structure. Significant increases in the activity of 33 and 28% of IGL cells were observed after locally applied OXA (1 µm) and OXB (1 µm), respectively. In the great majority of neurons responses to OXA were maintained in the presence of orexin-1 receptor OX1R antagonist, SB 334867 (10 µm). Additionally, 75% of the OXB-responsive neurons were also sensitive to an orexin-2 receptor (OX2R)-selective agonist, [Ala11, D-Leu15]-OXB (1 µm). Immunohistochemical stainings showed putative synaptic contacts between OXA- and OXB-immunoreactive fibres and neuropeptide Y, and enkephalin-positive neurons in the investigated area. The outcome of our experiments reinforces previous reports indicating the possible linkage between the orexinergic and circadian systems. To our knowledge the presented findings are the first showing the direct influence of orexins on the IGL activity, mostly through activation of OX2R.

Disruption of insect diapause using agonists and an antagonist of diapause hormone.

The dormant state known as diapause is widely exploited by insects to circumvent winter and other adverse seasons. For an insect to survive, feed, and reproduce at the appropriate time of year requires fine coordination of the timing of entry into and exit from diapause. One of the hormones that regulates diapause in moths is the 24-aa neuropeptide, diapause hormone (DH). Among members of the Helicoverpa/Heliothis complex of agricultural pests, DH prompts the termination of pupal diapause. Based on the structure of DH, we designed several agonists that are much more active than DH in breaking diapause. One such agonist that we describe also prevents the entry into pupal diapause when administered to larvae that are environmentally programmed for diapause. In addition, we used the unique antagonist development strategy of incorporating a dihydroimidazole ("Jones") trans-Proline mimetic motif into one of our DH agonists, thereby converting the agonist into a DH antagonist that blocks the termination of diapause. These results suggest potential for using such agents or next-generation derivatives for derailing the success of overwintering in pest species.

Novel peptide ligands with dual acting pharmacophores designed for the pathophysiology of neuropathic pain.

The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted. CCK has been shown to act as an endogenous "anti-analgesic" peptide and neuropathic pain conditions promote endogenous CCK release in CNS regions of pain modulation. Administration of CCK into nuclei of the rostral ventromedial medulla induces pronociceptive behaviors in rats. RSA 504 and RSA 601 are novel bifunctional compounds developed to target neuropathic pain by simultaneously acting as agonists at two distinct opioid receptors and antagonizing CCK receptors in the CNS. RSA 504 and RSA 601 demonstrate agonist activity in vitro and antihypersensitivity to mechanical and thermal stimuli in vivo using the spinal nerve ligation model of neuropathic pain. Intrathecal administration of RSA 504 and RSA 601 did not demonstrate antinociceptive tolerance over 7 days of administration and did not display motor impairment or sedation using a rotarod. These are the first behavioral studies that demonstrate how multi-targeted molecule design can address the pathology of neuropathic pain. These compounds with δ and µ opioid agonist activity and CCK antagonist activity within one molecule offer a novel approach with efficacy for neuropathic pain while lacking the side effects typically caused by conventional opioid therapies.

Pharmacotherapies for alcoholism: the old and the new.

Alcoholism and other alcohol use disorders are major public health problems, and the success rates of non-pharmacological treatment of these disorders such as psychotherapy, cognitive-behavioral therapy, group therapy, or residential treatment programs,remain only modest at best. High rates of recidivism (relapse) in alcoholics attempting to remain abstinent are prevalent worldwide. In recent years abundant evidence has accumulated demonstrating that alcoholism is a complex and multifaceted disease of the brain caused by numerous genetic, neurobiological, developmental, environmental, and socioeconomic factors that are still not yet fully understood.There is thus a great need to improve the success rates of all forms of treatment of alcoholism not only in preventing relapse, but curbing active alcohol consumption and craving. The development of improved pharmacotherapies that could be used as adjuncts to the aforementioned non-pharmacological treatment approaches is one avenue of great interest to the scientific community and the general public. Currently there are only three medications approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of alcohol abuse and alcoholism--disulfiram, naltrexone, and acamprosate. Yet medication compliance issues and the modest efficacy of these compounds leave substantial room for improvement. This special issue is devoted to reviewing the current status of these FDA approved medications in the treatment of alcoholism. In addition, preclinical and clinical evidence suggesting that other classes of medications might also be of potential use are reviewed, including anticonvulsants, GABAB receptor agonists, cholinergic receptor partial agonists, corticotropin-releasing factor and cannabinoid CB1 receptor antagonists, nociceptin receptor ligands, and the novel antipsychotic aripiprazole.

Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor.

Pheromone biosynthesis-activating neuropeptide (PBAN) and pyrokinins belong to a family of insect peptide hormones that have a common FXPRLamide C-terminal ending. The G-protein-coupled receptors (GPCRs) for this peptide family were first identified from a moth and Drosophila with sequence similarity to neuromedin U receptors from vertebrates. We have characterized the PBAN-receptor (PBAN-R or PR) active binding domains using chimeric GPCRs and proposed that extracellular loop 3 is critical for ligand selection. Here, we characterized the 3rd extracellular domain of PBAN-R through site-directed point mutations. Results are discussed in context of the structural features required for receptor activation using receptor activation experiments and in silico computational modeling. This research will help in characterizing these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors.

A kisspeptin-10 analog with greater in vivo bioactivity than kisspeptin-10.

The kisspeptins are neuropeptides that stimulate the hypothalamo-pituitary-gonadal (HPG) axis. The smallest endogenous kisspeptin, kisspeptin-10 (KP-10), binds to the receptor KISS1R with a similar affinity to the full-length peptide, kisspeptin-54 (KP-54), but is less effective in vivo, possibly because of increased enzymatic breakdown or clearance. The kisspeptin system may have therapeutic potential in the treatment of reproductive disorders and endocrine cancers. We have rationally modified the structure of KP-10 and tested the binding affinity of these analogs for the KISS1R. Those analogs that bound with relatively high affinity to KISS1R were tested for ability to stimulate ERK1/2 phosphorylation in vitro and for their ability to stimulate the HPG axis in vivo. One analog, [dY](1)KP-10, bound to KISS1R with lower affinity to KP-10 and exhibited similar bioactivity in vitro. However, in vivo peripheral administration of [dY](1)KP-10 increased plasma LH and testosterone more potently than KP-10 itself at 20 min postinjection in mice. In addition, 60 min postinjection, 0.15 nmol [dY](1)KP-10 significantly increased total testosterone levels in mice whereas the same dose of KP-10 had no significant effect. Should manipulation of the kisspeptin/KISS1R signaling system prove therapeutically useful, long-lasting analogs such as [dY](1)KP-10 may have greater therapeutic potential than endogenous forms of kisspeptin.

A novel dihydroimidazoline, trans-Pro mimetic analog is a selective PK/PBAN agonist.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in the regulation of reproductive and developmental processes in a variety of insects. A transPro, type I beta-turn has been previously identified as important for the activity of PK/PBAN peptides. A PK/PBAN analog (PPK-Jo) incorporating a novel dihydroimidazole transPro mimetic motif was evaluated in four PK/PBAN bioassays (pheromonotropic, melanotropic, pupariation and hindgut myotropic). PPK-Jo proved to be a pure, selective melanotropic agonist in S. littoralis. The melanotropic receptor in S. littoralis demonstrates more tolerance to deviations from the ideal transPro structure than those of other PK/PBAN assays. The selective PK/PBAN agonist represents a new tool to better understand the endogenous mechanisms of these peptides and serves as a probe of the plasticity of PK/PBAN regulated systems and receptors. The dihydroimidazoline moiety is shown to function as a surrogate for a transPro in certain circumstances, and provides a novel scaffold with which to construct mimetic PK/PBAN analogs with enhanced selectivity and the potential to disrupt critical physiological processes in insect pests.

A possible mechanism underlying an antidepressive-like effect of Kososan, a Kampo medicine, via the hypothalamic orexinergic system in the stress-induced depression-like model mice.

Kososan, a Kampo (Japanese herbal) medicine, has an antidepressive-like effect in behavioral animal models of depression and has been used clinically for the improvement of depressive mood. However, mechanism(s) underlying the antidepressive-like effect of kososan remain unknown. Previous studies showed that orexin-A (OX-A), a neuropeptide that is involved in feeding and arousal, exhibits an antidepressive-like property via hippocampal cell proliferation. Here, we used immunohistochemical analysis with bromodeoxyuridine (BrdU), a marker of proliferating cells, to investigate the effect of long-term treatment with kososan on the orexinergic system and on hippocampal cell proliferation. Oral administration of kososan (1.0 g/kg) or milnacipran (60 mg/kg), a serotonin and noradrenaline reuptake inhibitor, for 28 d led to an antidepressive-like effect in the stress-induced depression-like model mice and reversed the stress-induced decrease in the number of OX-A-positive cells in the lateral hypothalamic area. In addition, both kososan and milnacipran alleviated the stress-induced decrease in the number of BrdU-positive cells in the hippocampal dentate gyrus. Moreover, the antidepressive-like effect and the increase in cell proliferation and in the number of neuropeptide Y (NPY, which is closely associated with orexinergic system)-positive cells in the dentate gyrus induced by kososan were blocked by treatment with SB-334867, an orexin receptor 1 antagonist. These results suggest that kososan exerts an antidepressive-like effect via the improvement of the stress-induced decrease in hippocampal cell proliferation and that the mechanism underlying the antidepressive-like effect of kososan, but not of milnacipran, may be associated with the regulation of orexinergic and/or NPYergic transmission.

The neuroactive peptide N-acetylaspartylglutamate is not an agonist at the metabotropic glutamate receptor subtype 3 of metabotropic glutamate receptor.

The peptide N-acetylaspartylglutamate (NAAG) is present in high concentrations in the mammalian central nervous system. Various mechanisms have been proposed for its action, including selective activation of the metabotropic glutamate receptor (mGluR) subtype 3, its action at the N-methyl-D-aspartate receptor, or the production of glutamate by its hydrolysis catalyzed by an extracellular protease. To re-examine its agonist activity at mGluR3, we coexpressed human or rat mGluR3 with G protein inward rectifying channels in Xenopus laevis oocytes. High-performance liquid chromatography analysis of commercial sources of NAAG showed 0.38 to 0.48% glutamate contamination. Although both human and rat mGluR3 were highly sensitive to glutamate, with EC(50) values of 58 and 28 nM, respectively, purified NAAG (100 microM) had little activity (7.7% of full activation by glutamate). Only in the millimolar range did it show significant activity, possibly due to residual traces of glutamate remaining in the purified NAAG preparations. In contrast, the unpurified NAAG sample did produce a full agonist response with mGluR3 coexpressed with G alpha(15), with an EC(50) of 120 microM, as measured by a calcium release assay. This response can be explained by the 0.38 to 0.48% glutamate contamination. Our results suggest that NAAG may not have a direct agonist activity at the mGluR3 receptor. Thus, several in vivo and in vitro published results that did not address the issue of glutamate contamination of NAAG preparations may need to be re-evaluated.