MK-801 Binding Research Articles

Manganese inhibits NMDA receptor channel function: Implications to psychiatric and cognitive effects

Humans exposed to excess levels of manganese (Mn 2+) express psychiatric problems and deficits in attention and learning and memory. However, there is a paucity of knowledge on molecular mechanisms by which Mn 2+ produces such effects. We now report that Mn 2+ is a potent inhibitor of [ 3H]-MK-801 binding to the NMDA receptor channel in rat neuronal membrane preparations. The inhibition of [ 3H]-MK-801 to the NMDA receptor channel by Mn 2+ was activity-dependent since Mn 2+ was a more potent inhibitor in the presence of the NMDA receptor co-agonists glutamate and glycine ( K i = 35.9 ± 3.1 μM) than in their absence ( K i = 157.1 ± 6.5 μM). We also show that Mn 2+ is a NMDA receptor channel blocker since its inhibition of [ 3H]-MK-801 binding to the NMDA receptor channel is competitive in nature. That is, Mn 2+ significantly increased the affinity constant ( K d) with no significant effect on the maximal number of [ 3H]-MK-801 binding sites ( B max). Under stimulating conditions, Mn 2+ was equipotent in inhibiting [ 3H]-MK-801 binding to NMDA receptors expressed in neuronal membrane preparations from different brain regions. However, under basal, non-stimulated conditions, Mn 2+ was more potent in inhibiting NMDA receptors in the cerebellum than other brain regions. We have previously shown that chronic Mn 2+ exposure in non-human primates increases Cu 2+, but not zinc or iron concentrations in the basal ganglia [Guilarte TR, Chen M-K, McGlothan JL, Verina T, Wong DF, Zhou Y, Alexander M, Rohde CA, Syversen T, Decamp E, Koser AJ, Fritz S, Gonczi H, Anderson DW, Schneider JS. Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates. Exp Neurol 2006a;202:381–90]. Therefore, we also tested the inhibitory effects of Cu 2+ on [ 3H]-MK-801 binding to the NMDA receptor channel. The data shows that Cu 2+ in the presence of glutamate and glycine is a more potent inhibitor of the NMDA receptor than Mn 2+. Our findings suggest that the inhibitory effect of Mn 2+ and/or Cu 2+ on the NMDA receptor may produce a deficit in glutamatergic transmission in the brain of individuals exposed to excess levels of Mn 2+ and produce neurological dysfunction.

Subunit‐specific mechanisms and proton sensitivity of NMDA receptor channel block

We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

Methamphetamine exposure antagonizes N-methyl- d-aspartate receptor-mediated neurotoxicity in organotypic hippocampal slice cultures

Glutamatergic systems have been increasingly recognized as mediators of methamphetamine's (METH) pharmacological effects though little is known about the means by which METH interacts with glutamate receptors. The present studies examined effects of METH (0.1–100 μM) on [ 3H]MK-801 binding to membranes prepared from adult rat cortex, hippocampus and cerebellum, as well as the neurotoxicity produced by 24-h exposure to N-methyl- d-aspartate (5–10 μM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [ 3H]MK-801 with METH (0.1–100 μM) did not reduce dextromethorphan (1 mM)-displaceable ligand binding. Exposure of slice cultures to NMDA for 24-h produced increases in uptake of the non-vital fluorescent marker propidium iodide (PI) of 150–500% above control levels, most notably, in the CA1 region pyramidal cell layer. Co-exposure to METH (> 1.0 μM) with NMDA (5 μM) reduced PI uptake by approximately 50% in each subregion, though the CA1 pyramidal cell layer was markedly more sensitive to the protective effects of METH exposure. In contrast, METH exposure did not reduce PI uptake stimulated by 24-h exposure to 10 μM NMDA. Co-exposure to the NMDA receptor antagonist d-2-amino-5-phosphonovaleric acid (20 μM) prevented toxicity produced by exposure to 5 or 10 μM NMDA. These findings indicate that the pharmacological effects of short-term METH exposure involve inhibition of NMDA receptor-mediated neuronal signaling, not reflective of direct channel inhibition at an MK-801-sensitive site.

Short and long term changes in NMDA receptor binding in mouse brain following chronic phencyclidine treatment

Phencyclidine (PCP) is an antagonist of the NMDA subtype of glutamate receptor. PCP treatment induces psychosis in normal humans, which provides a valuable model of schizophrenia. PCP administration also models some of the symptoms of schizophrenia in experimental animals. NMDA hypofunction has been hypothesized to explain these schizophrenia-like symptoms. Acute or chronic administration of the NMDA receptor antagonist PCP has been shown to induce several short or long-term effects in both humans and experimental animals. In an attempt to clarify the neurochemical substrates of these effects in the present study, we used quantitative autoradiography to examine the effects of chronic (14 days) PCP treatment on NMDA receptor binding in mouse brain following both a short- (1 and 24 h) and long-term (14 days) delay after the last PCP treatment. NMDA receptors were targeted using [(3)H]MK801. Chronic PCP treatment increased [(3)H]MK801 binding consistently in the hippocampus in the short-term (p < 0.01). Conversely in the long-term, there were widespread reductions in NMDA receptor binding and this effect was most evident in the hippocampus where a 35% reduction of binding was found (p < 0.001). These results suggest that the hippocampus has a strong involvement in both the short and long-term effects of PCP treatment and that reduced NMDA receptor function might be one of the neurochemical substrates of the long lasting actions of PCP or PCP-induced psychosis. Importantly, this study shows that the long-term delay following chronic PCP treatment more accurately represents a state of NMDA hypofunction than the short-term PCP model.

Effect of Resuscitation with 21% Oxygen and 100% Oxygen on NMDA Receptor Binding Characteristics Following Asphyxia in Newborn Piglets

The present study investigated the effect of reventilation with 21% and 100% oxygen following asphyxia in newborn piglets on NMDA receptor binding characteristics, Na(+), K(+)-ATPase activity, and lipid peroxidation. After achieving a heart rate less than 60 beats per minute, asphyxiated piglets were reventilated with 21% oxygen or 100% oxygen. (3)[H]MK-801 binding showed the Bmax in the 21% and 100% groups to be 1.53 +/- 0.43 and 1.42 +/- 0.35 pmol/mg protein (p = ns). Values for Kd were 4.56 +/- 1.29 and 4.17 +/- 1.05 nM (p = ns). Na(+), K(+)-ATPase activity in the 21% and 100% groups were 23.5 +/- 0.9 and 24.4 +/- 3.9 micromol Pi/mg protein/h (p = ns). Conjugated dienes (0.05 +/- 0.02 vs. 0.07 +/- 0.03 micromol/g brain) and fluorescent compounds (0.54 +/- 0.05 vs. 0.78 +/- 0.19 microg quinine sulfate/g brain), were similar in both groups (p = ns). Though lipid peroxidation products trended higher in the 100% group, these data show that NMDA receptor binding and Na(+), K(+)-ATPase activity were similar following reventilation with 21% or 100% oxygen after a single episode of mild asphyxia.

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABAA receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.

Abnormal Glutamate Receptor Expression in the Medial Temporal Lobe in Schizophrenia and Mood Disorders

Pharmacological and anatomical evidence suggests that abnormal glutamate neurotransmission may be associated with the pathophysiology of schizophrenia and mood disorders. Medial temporal lobe structural alterations have been implicated in schizophrenia and to a lesser extent in mood disorders. To comprehensively examine the ionotropic glutamate receptors in these illnesses, we used in situ hybridization to determine transcript expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor subunits in the medial temporal lobe of subjects with schizophrenia, bipolar disorder (BD), or major depression (MDD). We used receptor autoradiography to assess changes in glutamate receptor binding in the same subjects. Our results indicate that there are region- and disorder-specific abnormalities in the expression of ionotropic glutamate receptor subunits in schizophrenia and mood disorders. We did not find any changes in transcript expression in the hippocampus. In the entorhinal cortex, most changes in glutamate receptor expression were associated with BD, with decreased GluR2, GluR3, and GluR6 mRNA expression. In the perirhinal cortex we detected decreased expression of GluR5 in all three diagnoses, of GluR1, GluR3, NR2B in both BD and MDD, and decreased NR1 and NR2A in BD and MDD, respectively. Receptor binding showed NMDA receptor subsites particularly affected in the hippocampus, where MK801 binding was reduced in schizophrenia and BD, and MDL105,519 and CGP39653 binding were increased in BD and MDD, respectively. In the hippocampus AMPA and kainate binding were not changed. We found no changes in the entorhinal and perirhinal cortices. These data suggest that glutamate receptor expression is altered in the medial temporal lobe in schizophrenia and the mood disorders. We propose that disturbances in glutamate-mediated synaptic transmission in the medial temporal lobe are important factors in the pathophysiology of these severe psychiatric illnesses.

Decreased N-methyl- d-aspartic acid (NMDA) receptor levels are associated with mercury exposure in wild and captive mink

Mercury (Hg) impairs glutamate homeostasis but little is known about its effects on the N-methyl- d-aspartic acid (NMDA) receptor. Here, we investigated NMDA receptor levels, as determined by [ 3H]-MK801 binding, in both wild and captive mink ( Mustela vison) that experienced different levels of methylmercury (MeHg) exposure. Competitive in vitro binding experiments showed that inorganic Hg (HgCl 2; IC 50 = 1.5–20.7 μM), but not MeHg (MeHgCl; IC 50 > 320 μM), inhibited binding to the NMDA receptor in several brain regions of mink. In a survey of trapped wild mink, NMDA receptor levels in the brain were negatively correlated ( p < 0.005) with concentrations of total Hg ( R = −0.618) and MeHg ( R = −0.714). These findings were supported by a laboratory feeding study in which captive mink were exposed to dietary MeHg (0–2 ppm) for 89 days. Concentration-dependent decreases in NMDA receptor levels were found in the basal ganglia, cerebellum, brain stem and occipital cortex. These findings are of physiological and ecological concern because they demonstrate that Hg, at dietary concentrations as low as 0.1 ppm, can significantly reduce NMDA receptor levels.

Decreased NR1, NR2A, and SAP102 transcript expression in the hippocampus in bipolar disorder

Objectives: Schizophrenia is associated with dysfunction of glutamatergic neurotransmission, and several studies have suggested glutamatergic abnormalities in bipolar disorder. Recent data suggest involvement of the NMDA receptor signaling complex, which includes NMDA receptor subunits as well as associated intracellular interacting proteins critical for NMDA receptor assembly, trafficking, and activation; the most well-characterized being PSD93, PSD95, SAP102, and NF-L. Previously, studies from our laboratories have described changes in glutamate receptor subunit transcript and binding site expression in schizophrenia and changes in NMDA receptor binding site expression in bipolar disorder in postmortem brain tissue. In the present work, we focus on the expression of these molecules in hippocampus in schizophrenia and bipolar affective disorder I. Methods: We performed in situ hybridization to assess hippocampal expression of the transcripts encoding NMDA receptor subunits NR1, 2A, 2B, 2C and 2D, and the transcripts for the NMDA receptor associated PSD proteins PSD95, PSD93, NF-L, and SAP102 in subjects with schizophrenia, bipolar affective disorder I, and a comparison group. We also measured [ 3H]CGP39653 and [ 3H]MK-801 binding site expression in the hippocampus in schizophrenia. Results: There was a significant decrease in the expression of transcripts for NR1 and NR2A subunits and SAP102 in bipolar disorder. We did not detect any changes in these transcripts or in binding site expression in the hippocampus in schizophrenia. Conclusions: We propose that the NMDA receptor signaling complex, including the intracellular machinery that is coupled to the NMDA receptor subunits, is abnormal in the hippocampus in bipolar disorder. These data suggest that bipolar disorder might be associated with abnormalities of glutamate-linked intracellular signaling and trafficking processes.

New polyamine-sensitive inhibitors of the NMDA receptor: Syntheses and pharmacological evaluation

Derivatives of 5-(4-aminobutyl)-2-thiophene-octylamine, a potent polyamine-sensitive inhibitor of the NMDA receptor, were synthesized and evaluated as inhibitors of [ 3H]MK-801 binding to rat brain membranes. Alkylations of the terminal amino groups reduced inhibitory potency; only incorporation of the amino group of the short 4-aminobutyl arm into a piperidine ring was tolerated. Substitution of the thiophene nucleus with methyl or ethyl, and its replacement by a benzene nucleus, was of minor influence. The corresponding diguanidines exhibited high potency independent of chain length, whereas their sensitivity to spermine was sharply dependent on chain length. Insertion of an amide bond into the long octylamine arm increased sensitivity to spermine and to Tris buffer. Our results indicate that spermine sensitivity of [ 3H]MK-801 binding inhibition is responsive to subtle changes in inhibitor structure and represents a promising target for pharmaceutical research.

Effect of δ-aminolevulinic acid and vitamin E treatments on the N-methyl- d-aspartate receptor at different ages in the striatum of rat brain

We report the effects of the chronic treatments with the oxidant agent δ-aminolevulinic acid (ALA) and with the antioxidant vitamin E on the N-methyl- d-aspartate (NMDA) receptors in the striatum of 4-, 12- and 24-month-old male Wistar rats. ALA and vitamin E were administered daily for 15 days (40 mg/kg i.p. and 20 mg/kg i.p. respectively). NMDA receptors were labeled by membrane homogenate binding, using tritiated dizocilpine ([ 3H]MK-801). [ 3H]MK-801 binding in the striatum was significantly decreased at all ages in ALA-treated rats with respect to their controls, and in contrast, was significantly increased at all ages when rats received the treatment with vitamin E. Western blot assays were performed using antibodies against the NR2A subunit, a NMDA receptor subunit widely distributed in the brain. We did not find significant differences in the amounts of NR2A in rats treated with either ALA or vitamin E with respect to those rats not treated. We conclude that the NMDA receptor densities in the rat striatum are modified by the chronic treatment with oxidants and antioxidants in an age-independent way, at least until 24 months. Also, our results support the notion that NR2A is not involved in these modifications.

Attenuation of morphine tolerance and withdrawal syndrome by coadministration of nalbuphine

Morphine has been used widely on the treatment of many types of chronic pain. However the development of tolerance to and dependence on morphine by repeat application is a major problem in pain therapy. The purpose of the present study was to investigate whether combined administration of nalbuphine with morphine affects the development of tolerance to and dependence on morphine. We hypothesize that the use of nalbuphine, kappa-agonist may prove to be useful adjunct therapy to prevent morphine-induced undesirable effects in the management of some forms of chronic pain. Morphine (10 mg/kg) was injected to rats intraperitoneally for 5 day. The variable dose of nalbuphine (0.1, 1.0 and 5.0 mg/kg) was administered (i.p.) in combination with morphine injection. The development of morphine tolerance was assessed by measuring the antinociceptive effect with the Randall-Selitto apparatus. The development of dependence on morphine was determined by the scoring the precipitated withdrawal signs for 30 min after injection of naloxone (10 mg/kg, i.p.). Nalbuphine did not attenuate antinociceptive effect of morphine in rats. Interestingly, combined administration of morphine with nalbuphine (10:1) significantly attenuated the development of dependence on morphine. The elevation of [3H]MK-801 binding in frontal cortex, dentate gyrus, and cerebellum after chronic morphine infusion was suppressed by the coadministration of nalbuphine. In addition, the elevation of NR1 expression by morphine was decreased by the coadministration of nalbuphine in rat cortex. These results suggest that the coadministration of nalbuphine with morphine in chronic pain treatment can be one of therapies to reduce the development of tolerance to and dependence on morphine.

Neuroprotective cannabinoid receptor antagonist SR141716A prevents downregulation of excitotoxic NMDA receptors in the ischemic penumbra

Whether cannabinoids act as neuroprotectants or, on the contrary, even worsen neuronal damage after cerebral ischemia is currently under discussion. We have previously shown that treatment with the cannabinoid (CB1) receptor antagonist SR141716A reduces infarct volume by approximately 40% after experimental stroke. Since it is suggested that SR141716A may exert neuroprotection besides its cannabinoid receptor-blocking effect, we addressed the question whether SR141716A may act via modulation of postischemic ligand binding to excitatory NMDA and/or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. For this purpose, rats (n = 12) were treated with either intravenous saline (control) or CB1 receptor antagonist SR141716A (1 mg/kg) 30 min after permanent middle cerebral artery occlusion. Five hours after ischemia, quantitative receptor autoradiography was performed using [(3)H]CP 55,940, [(3)H]MK-801, and [(3)H]AMPA for labeling of CB1, NMDA, and AMPA receptors, respectively. Ligand binding was analyzed within the infarct core, cortical penumbra, and corresponding areas of the contralateral hemisphere and compared to that of sham-operated rats (n = 5). Both in ischemic controls and SR141716A-treated rats [(3)H]CP 55,940 ligand binding was not specifically regulated in the cortical penumbra or contralateral cortex. Importantly, reduced infarct volumes in SR141716A-treated rats were associated with maintained [(3)H]MK-801 binding to excitotoxic NMDA receptors in the penumbra, compared to a decrease in the control group. In summary, our data suggest that SR141716A may possess additional intrinsic neuroprotective properties independent of receptor-coupled pathways or due to action as a partial agonist.

Effects of β-carboline alkaloids on the object recognition task in mice

β-carboline alkaloids are found in several medicinal plants and display a variety of actions on the central nervous, muscular and cardiovascular systems. The aim of the present study was to evaluate the effects of systemic administration of β-carboline alkaloids on object recognition in mice. Adult Swiss mice received an intra-peritoneal injection (i.p.) of alkaloids (1.0, 2.5 or 5.0 mg/kg) 30 min before training in an object recognition task. The fully aromatic β-carbolines, harmine and harmol, induced an enhancement of short-term memory (STM) at all doses tested when compared to controls. Harmaline, a dihydro β-carboline and inverse agonist of the MK-801 binding site on the N-methyl- d-aspartate (NMDA) receptor, also induced an enhancement of both short-term memory (STM) and long-term memory (LTM). These results demonstrate that systemic administration of β-carboline alkaloids can improve object recognition memory in mice.

Repeated ethanol treatment in adolescent rats alters cortical NMDA receptor

Earlier we have reported that repeated ethanol treatment during adolescence causes long-lasting impairments in spatial learning and memory. The present study was undertaken to determine the cellular mechanisms underlying the persistent ethanol-induced cognitive dysfunction in adolescent male rats. Since in adult animals ethanol is known to affect the N-methyl- d-aspartate (NMDA) receptor–gated ion channel, the hypothesis tested here was that adolescent ethanol exposure modulates NMDA receptor (NR) regulation in the brain. Adolescent male rats were injected daily with ethanol (2 g/kg intraperitoneally) for 5 consecutive days. Control rats received isovolumetric saline for the same number of days. Groups of control and experimental rats were sacrificed 7 days after the last ethanol/saline administration, and NR activity was measured in specific brain regions (frontal cortex, hippocampus) using the [ 3H]MK-801 binding assay. In addition, some rats were sacrificed and their brains were used to investigate changes in NR pharmacology by measuring specific NR2 subunits immunohistochemically. Compared to saline-treated controls, ethanol-treated rats showed significant increases in [ 3H]MK-801 maximal binding in the frontal cortex. This was associated with increased cortical NR2B subunit protein. [ 3H]MK-801 binding in the hippocampus was minimally affected. These results indicate that ethanol exposure during the adolescent period produces brain region–specific alterations in NR activity. These changes are different from those reported in literature for ethanol administration during the perinatal period or adulthood. Together, these data suggest that adolescence represents a unique stage in brain development in its long-term sensitivity to ethanol.

In vitro and antinociceptive profile of HON0001, an orally active NMDA receptor NR2B subunit antagonist

The analgesic activity and side effect liabilities of a novel NR2B antagonist, 7-hydroxy-6-methoxy-2-methyl-1-(2-(4-(trifluoromethyl)phenyl)ethyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride (HON0001) were investigated. HON0001 inhibited [ 3H]MK-801 binding to rat brain membranes in a biphasic manner, with IC 50 values of 54.68 ± 4.96 nM and 46.48 ± 5.85 μM for high- and low-affinity sites, respectively. HON0001 inhibited [ 3H]ifenprodil binding to membranes of rat cerebral cortex with an IC 50 value of 57.01 ± 3.4 nM, consistent with the results obtained for high-affinity sites of [ 3H]MK-801 binding. HON0001 exhibited no or negligible affinity for other receptors, transporters and ion channels, while HON0001 had a moderate agonistic activity at μ-opioid receptors and affinity for dopamine D 1 receptors. HON0001 exhibited an analgesic effect in carrageenan-induced mechanical hyperalgesia and in the Seltzer model of partial sciatic nerve ligation following oral administration. In contrast, unlike MK-801, HON0001 did not affect spontaneous locomotor activity, rotarod performance and step-through latency in a passive avoidance task even at doses much higher than antinociceptive doses. HON0001 exhibited excellent brain penetration with a brain-to-plasma ratio of 34.5. These findings show that HON0001 is an orally active NR2B antagonist and that it may be useful for treating patients with neuropathic and other conditions without causing the side effects often observed with currently available non-subtype selective NMDA receptor antagonists.

The mechanism of 1,2,3,4‐tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate‐induced excitotoxicity

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

Perinatal allopregnanolone influences prefrontal cortex structure, connectivity and behavior in adult rats

Cortical neurosteroid levels vary dramatically across development; during the second week of life elevated levels of allopregnanolone are associated with decreased GABA A receptor function. Since GABA A receptor modulation plays a role in proliferative regulation in developing neocortex, it is possible that endogenous neurosteroids such as allopregnanolone, acting through GABA A receptors, modulate cortical development. We augmented normally low levels with exogenous administration of allopregnanolone (10mg/kg) during the first week of rodent life. The localization of parvalbumin-labeled cells was markedly altered; the ratio of cell number in the deep (layers V–VI) vs. superficial (layers I–III) layers of adult prefrontal cortex increased two-fold in rats administered allopregnanolone on postnatal day 1 or 5. The mechanism underlying these anatomical changes likely involves GABA A receptors because similar changes in interneuron placement were observed after neonatal benzodiazepine administration. Measures of mature cortical function were also altered after neonatal neurosteroid administration, including [ 3H]MK-801 binding, prepulse inhibition and amphetamine-induced locomotor activity. Moreover, neonatal allopregnanolone administration increases the number of parvalbumin-expressing neurons in medial dorsal nucleus of the thalamus while the total neuron number is decreased. These findings suggest that connectivity between the medial dorsal nucleus of the thalamus and prefrontal cortex is likely altered by neonatal neurosteroid administration and may result in a disinhibited frontal cortex. Disinhibition in the prefrontal cortex is associated with behavioral changes relevant to human psychosis and developmental disorders. If neurosteroids play a role in normal development of prefrontal/medial dorsal patency as suggested by these studies, then alterations in neurosteroid levels may contribute to abnormal neurodevelopment.

Commercial valerian interactions with [3H]Flunitrazepam and [3H]MK-801 binding to rat synaptic membranes

Valeriana officinalis extracts are used in folkloric medicine for their sedative, hypnotic and tranquilizer effects. Using [3H]flunitrazepam binding as an indicator, the interactions of commercial Valerian extracts with GABA(A) receptors were examined. There was considerable fluctuation among the different extracts, some mildly enhanced [3H]flunitrazepam binding, others had no effect and others had inhibitory effects, independent of standardization by valerenic acid. Central depression can also be accomplished by a reduction of excitatory transmission. Valerian extracts had modest inhibitory effects on [3H]MK-801 binding, an indicator of NMDA-Valerian interactions. Spectral analyses (UV region) did not show marked differences among the different extracts. The inhibitory effects of one of the extracts on [3H]flunitrazepam binding was somewhat stable, while on [3H]MK-801 binding the inhibitory effects were lost within months. These results suggest that particular care should be taken in analysing and interpreting results from commercial Valerian preparations.

Lipopolysaccharide-induced microglial activation induces learning and memory deficits without neuronal cell deathin rats

We used lipopolysaccharide (LPS) to activate microglia that play an important role in the brain immune system. LPS injected into the rat hippocampus CA1 region activated microglial cells resulting in an increased production of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha in the hippocampus during the initial stage of treatment. Immunostaining for IL-1beta was increased at 6 hr after LPS injection. IL-1beta-immunopositive cells were co-localized with immunostaining for CD11b. Subacute treatment with LPS by the same route for 5 days caused long-term activation of microglia and induced learning and memory deficits in animals when examined with a step-through passive avoidance test, but histochemical analysis showed that neuronal cell death was not observed under these experimental conditions. The increased expression of the heme oxygenase-1 (HO-1) gene, an oxidative stress maker, was observed. However, the genetic expression of brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, decreased during the course of LPS treatment. We found decreases in [3H]MK801 binding in the hippocampus CA1 region by LPS-treatment for 5 days. The data shows that glutamatergic transmission was attenuated in the LPS-treated rats. These results suggest that long-term activation of microglia induced by LPS results in a decrease of glutamatergic transmission that leads to learning and memory deficits without neuronal cell death. The physiologic significance of these findings is discussed.