The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

A simple synthesis of [11C]carfentanil using an extraction disk instead of HPLC.

[11C]Carfentanil was prepared without the need for purification by HPLC. The tetrabutylammonium salt of the precursor carboxylate was reacted with [11C]methyl triflate in DMSO. The resulting [11C]carfentanil was trapped on an Empore extraction disk and washed to remove precursor and most radioactive contaminants. The product was eluted by a small volume of ethanol, mixed with water and passed through a small column containing fibrous anion exchanger to remove remaining radioactive contaminants.

The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats.

Antagonists selective for either kappa- [e.g. nor-binaltorphimine (nor-BNI)] and mu- (e.g. beta-funaltrexamine) opioid receptors have previously been shown to reduce both kappa- and mu-opioid-induced feeding. In the present studies, the anorectic effects of GNTI, a newly synthesized antagonist selective for kappa-opioid receptors, were studied in rats. GNTI (0.032-0.32 nmol; i.c.v.), administered 15 min prior to food access, reduced feeding induced by the kappa-opioid agonist U50,488 (producing a 70% maximal decrease), the mu-opioid agonist DAMGO (90% maximal decrease), and 24 h acute food deprivation (60% maximal decrease). GNTI did not reduce the orexigenic effects of butorphanol, an agonist that binds to both kappa- and mu-opioid receptors, and neuropeptide Y (NPY). Taken together, these results suggest that GNTI is a potent anorectic agent and opioid antagonist in rats. Like nor-BNI, GNTI reduced feeding induced by both kappa- and mu-opioid agonists. However, unlike nor-BNI, GNTI did not alter the orexigenic effects of butorphanol or NPY. Given the selectivity of GNTI and its effectiveness in several of the present experiments, its potency, and its short duration of action compared to nor-BNI, GNTI may serve to be a useful tool to study behavioral effects mediated by kappa-opioid receptors.

Regional mu opioid receptor regulation of sensory and affective dimensions of pain.

The endogenous opioid system is involved in stress responses, in the regulation of the experience of pain, and in the action of analgesic opiate drugs. We examined the function of the opioid system and mu-opioid receptors in the brains of healthy human subjects undergoing sustained pain. Sustained pain induced the regional release of endogenous opioids interacting with mu-opioid receptors in a number of cortical and subcortical brain regions. The activation of the mu-opioid receptor system was associated with reductions in the sensory and affective ratings of the pain experience, with distinct neuroanatomical involvements. These data demonstrate the central role of the mu-opioid receptors and their endogenous ligands in the regulation of sensory and affective components of the pain experience.

Buprenorphine-induced changes in mu-opioid receptor availability in male heroin-dependent volunteers: a preliminary study.

A principle of opioid pharmacotherapy is that high medication doses should occupy fractionally more opioid receptors that mediate heroin effects. In this preliminary study we examined in vivo mu opioid receptor (muOR) binding in three healthy opioid-dependent volunteers during maintenance on 2 and 16 mg sublingual buprenorphine (BUP) liquid, and after detoxification (0 mg) under double-blind, placebo-controlled conditions, and once in matched controls. Binding measures were obtained with the muOR-selective radioligand [11C]carfentanil (CFN) and PET 4 hrs after BUP administration. BUP induced dose-dependent reductions in muOR availability, 36-50% at 2 mg and 79-95% at 16 mg relative to placebo. Heroin abusers also had greater muOR binding potential in the inferofrontal cortex and anterior cingulate regions during placebo, compared to matched controls. Further studies are warranted to examine the relationship of muOR availability with BUP therapeutic actions, and the clinical implications of increased muOR binding during withdrawal.

Sucrose consumption increases naloxone-induced c-Fos immunoreactivity in limbic forebrain.

Opioids have long been known to have an important role in feeding behavior, particularly related to the rewarding aspects of food. Considerable behavioral evidence suggests that sucrose consumption induces endogenous opioid release, affecting feeding behavior as well as other opioid-mediated behaviors, such as analgesia, dependence, and withdrawal. In the present study, rats were given access to a 10% sucrose solution or water for 3 wk, then they were injected with 10 mg/kg naloxone or saline. Brains were subsequently analyzed for c-Fos immunoreactivity (c-Fos-IR) in limbic and autonomic regions in the forebrain and hindbrain. Main effects of sucrose consumption or naloxone injection were seen in several areas, but a significant interaction was seen only in the central nucleus of the amygdala and in the lateral division of the periaqueductal gray. In the central nucleus of the amygdala, naloxone administration to those rats drinking water significantly increased c-Fos-IR, an effect that was significantly enhanced by sucrose consumption, suggesting an upregulation of endogenous opioid tone in this area. The data from this study indicate that the central nucleus of the amygdala has a key role in the integration of gustatory, hedonic, and autonomic signals as they relate to sucrose consumption, if not to food intake regulation in general. Furthermore, the data from this study lend further support to the hypothesis that sucrose consumption induces the release of endogenous opioids.

Synthesis of 1-[11C]methylpiperidin-4-yl propionate ([11C]PMP) for in vivo measurements of acetylcholinesterase activity.

Synthesis of 1-[11C]methylpiperidin-4-yl propionate ([11C]PMP), an in vivo substrate for acetylcholinesterase, is reported. An improved preparation of 4-piperidinyl propionate (PHP), the immediate precursor for radiolabeling, was accomplished in three steps from 4-hydroxypiperidine by (a) protection of the amine as the benzyl carbamate, (b) acylation with propionyl chloride, and (c) deprotection of the carbamate by catalytic hydrogenation. The final product was obtained in an overall 82% yield. Reaction of the free base form of PHP with [11C]methyl trifluoromethanesulfonate at room temperature in N,N-dimethylformamide, followed by high performance liquid chromatography (HPLC) purification, provided [11C]PMP in 57% radiochemical yield, > 99% radiochemical purity, and > 1500 Ci/mmol at the end of synthesis. The total synthesis time from end-of-bombardment was 35 min. [11C]PMP can thus be reliably prepared for routine clinical studies of acetylcholinesterase in human brain using positron emission tomography.

A simple synthesis of [11C]dihydrotetrabenazine (DTBZ).

[11C]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-[11C]methoxy-10 -methoxy-1,2,3,4,6,7,- hexahydro-11bH-bezo[alpha]-quinolizine) ([11C]DTBZ) was synthesized by reacting the 9-hydroxy precursor in DMSO with gas-phase [11C]methyl iodide on a column of alumina impregnated with KOH. The reaction was instantaneous at room temperature. This column was then connected to the inlet of a short column containing basic alumina. Elution with cyclohexane removed radioactive contaminants. The radioactive product was then eluted with a few milliliters ether containing 1% ethanol. The [11C]DTBZ was obtained in isolated yields of > 200 mCi and specific activities > 1600 Ci/mmol.

Absolute configuration of (+)-alpha-dihydrotetrabenazine, an active metabolite of tetrabenazine.

Chiral column liquid chromatography and enantiospecific enzymatic hydrolysis were utilized to separate the enantiomers of alpha- and beta-dihydrotetrabenazine and alpha-9-O-desmethyldihydrotetrabenazine, three benzo[a]quinolizines derived from the amine-depleting drug tetrabenazine. An X-ray crystal structure analysis of (-)-alpha-9-O-desmethyldihydrotetrabenazine gave an absolute structure of that compound as the 2S, 3S, 11bS isomer. Therefore, (-)-alpha-dihydrotetrabenazine also has the 2S, 3S, 11bS absolute configuration. (+)-alpha-Dihydrotetrabenazine, the single biologically active isomer from the metabolic reduction of tetrabenazine, thus has the absolute configuration of 2R, 3R, 11bR. For further in vitro and in vivo studies of the vesicular monoamine transporter, it is now possible to use the single enantiomer of radiolabeled alpha-dihydrotetrabenazine.

Decreased striatal monoaminergic terminals in olivopontocerebellar atrophy and multiple system atrophy demonstrated with positron emission tomography.

We used [11C]dihydrotetrabenazine, a new ligand for the type 2 vesicular monoamine transporter (VMAT2), with positron emission tomography to study striatal monoaminergic presynaptic terminals in 4 patients with multiple system atrophy, 8 with sporadic olivopontocerebellar atrophy, and 9 normal control subjects. Specific binding in the striatum was significantly reduced in the multiple system atrophy patients as compared with the normal control group, with average reductions of 61% in the caudate nucleus (p = 0.002) and 58% in the putamen (p = 0.009). Smaller reductions were found in the sporadic olivopontocerebellar atrophy group, averaging 26% in the caudate nucleus (p = 0.05) and 24% in the putamen (p = 0.11). Mean blood-to-brain [11C]dihydrotetrabenazine transport (K1) was significantly different between groups only in the cerebellum, with values for the sporadic olivopontocerebellar atrophy group diminished compared with the normal control group. Cerebellar K1 was not significantly decreased in the multiple system atrophy group. The finding of reduced striatal VMAT2 in sporadic olivopontocerebellar atrophy patients suggests nigrostriatal pathology, indicating that some may later develop symptomatic extrapyramidal disease.

Kinetic evaluation of [11C]dihydrotetrabenazine by dynamic PET: measurement of vesicular monoamine transporter.

(+)-alpha-[11C]Dihydrotetrabenazine (DTBZ) binds to the vesicular monoamine transporter (VMAT2) located in presynaptic vesicles. The purpose of this work was to evaluate various model configurations for analysis of [11C]DTBZ with the aim of providing the optimal measure of monoamine vesicular transporter density obtainable from a single dynamic PET study. PET studies on seven young normal volunteer subjects, ages 20-35, were performed following i.v. injection of 666 +/- 37 MBq (18 +/- 1 mCi) of (+)-alpha-[11C]DTBZ. Dynamic acquisition consisted of a 15-frame sequence over 1 h. Analysis methods included both creation of pixel-by-pixel functional images of transport (K1) and binding (DVtot) and nonlinear least-squares analysis of volume-of-interest data. Pixel-by-pixel calculations were performed for both two-compartment weighted integral calculations and slope-intercept estimations from Logan plots. Nonlinear least-squares analysis was performed applying model configurations with both two-compartments, estimating K1 and DVtot and three compartments, estimating K1-k4. For the more complex configuration, we examined the stability of various binding-related parameters including k3 (konBmax'), k3/k4 (Bmax'/Kd), DVsp[(K1/k2)(k3/k4)], and DVtot [K1/k2(1 + k3/k4)]. The three-compartment model provided significantly improved goodness-of-fit compared to the two-compartment model, yet did not increase the uncertainty in the estimate of the DVtot. Without constraining parameters in the three-compartment model fits, DVtot was found to provide a more stable estimate of binding density than either k3, k3/k4, or DVsp. The two-compartment least-squares analysis yielded approximately 10% underestimations of the total distribution. However, this bias was found to be very consistent from region to region as well as across subjects as indicated by the correlation between two- and three-compartment DVtot estimates of 0.997. We conclude that (+)-alpha-[11C]DTBZ and PET can provide excellent measures of VMAT2 density in the human brain.

Discriminative stimulus effects of a centrally administered, delta-opioid peptide (D-Pen2-D-Pen5-enkephalin) in pigeons.

The present study assessed the discriminative stimulus effects of the delta-opioid agonist [D-Pen2-D-Pen5]enkephalin (DPDPE) in pigeons. Food-restricted pigeons were trained to discriminate between i.c.v injections of 100 micrograms [D-Pen2-D-Pen5]enkephalin (DPDPE) and saline in a two-key operant procedure; acquisition of discriminative control was rapid (14-28 daily sessions). [D-Ser2, Leu5, Thr6]enkephalin (DSLET) and [D-Ala2]deltorphin II, peptides selective for delta-opioid receptors, produced discriminative stimulus effects similar to DPDPE, and were approximately equipotent to DPDPE. The non-peptidic, delta-opioid agonist BW373U86 (0.032-100 mg/kg, i.m.) partially generalized to DPDPE. The kappa-opioid agonist U69,593 (0.01-1 mg/kg, i.m.), and the mu-opioid agonists, DAMGO (0.1-3.2 micrograms, i.c.v) and morphine (1-10 mg/kg, i.m.), did not produce discriminative stimulus effects similar to DPDPE, up to doses that markedly decreased response rates. Naltrindole (0.1 mg/kg. i.m.), an antagonist selective for delta-opioid receptors, produced approximately a 30-fold reduction in the potency of DPDPE. DPDPE's discriminative stimulus effect in pigeons appears to be mediated through a delta-opioid receptor; this effect may provide a procedure for assessing delta-opioid receptor function in vivo.

Nitric oxide synthase inhibitors produce phencyclidine-like behavioral effects in pigeons.

The present study assessed the ability of nitric oxide synthase (NOS) inhibitors to produce PCP-like behavioral effects in pigeons. Food-restricted pigeons were trained to discriminate between PCP (1.0 mg/kg, i.m.) from saline in a two-key operant procedure. NOS inhibitors 7-nitroindazole (7-NI) and N omega-nitro-L-arginine methyl ester (L-NAME) produced PCP-like discriminative stimulus effects. 7-NI (17.8 mg/kg, i.m.) completely generalized to PCP. L-NAME (320-1000 mg/kg) produced partial generalization to PCP. D-NAME, the enantiomer of L-NAME, did not produce PCP-appropriate behavior. L-NAME was approximately 200-times more potent i.c.v., but did not fully generalize to PCP. Both NOS inhibitors were effective in producing catalepsy, which is an effect commonly produced by competitive and uncompetitive NMDA antagonists. 7-NI (32 mg/kg) produced catalepsy in all subjects, whereas L-NAME (3200 mg/kg) produced catalepsy in 50% of the subjects, D-NAME did not produce catalepsy. Pretreatment with L-arginine (32-3200 mg/kg) prevented the PCP-like discriminative stimulus and cataleptic effects of 7-NI (17.8-32 mg/kg), demonstrating that 7-NI produced PCP-like effects through blockade of NO synthesis. The current studies reveal that NOS inhibitors induced two behavioral actions, discriminative stimulus effects and catalepsy, that are very selective for NMDA antagonists in pigeons.

Dichloromethane attracts diabroticite larvae in a laboratory behavioral bioassay.

A two-choice laboratory behavioral bioassay was used to demonstrate that dichloromethane elicits the dose-dependent attraction of secondinstar western and southern corn rootworms. Preliminary data suggest that second-instar banded cucumber beetles are also attracted to dichloromethane. An eluotropic series of 10 materials, including distilled water, ethanol, methanol, acetone, ethyl dichloroacetate, dichloromethane, diethyl ether, benzene, hexadecane, and hexane, was tested for attraction of western corn rootworm larvae. Dichloromethane was the only one attractive at all doses tested, and orthogonal comparisons revealed a quadratic trend (convex) for responses of larvae to increasing dose. Benzene and hexadecane also attracted larvae, but significantly fewer than dichloromethane, and only at three doses and one dose, respectively. Orthogonal comparisons revealed no linear or quadratic trend for responses of larvae to increasing doses of either compound. Dichloromethane is the first organic compound demonstrated to attract western corn rootworm larvae in the absence of carbon dioxide. Carbon dioxide has previously been reported to attract western corn rootworm larvae either independently or when combined with other organic compounds, and the sensitivity of our bioassay was tested by demonstrating the dose-dependent attraction of western corn rootworm larvae to carbonated water as a carbon dioxide source. We have also demonstrated the attraction of southern corn rootworm larvae to carbon dioxide and propose that carbon dioxide and dichloromethane behave analogously when they interact with chemoreceptor sites on larvae.

Sex pheromone of caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae).

The main component of the sex pheromone of the caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae) was identified as 6-methylnonan-3-one (enantiomeric composition has not yet been determined). Extracts of adult females elicited strong electroantennogram (EAG) responses from adult male antennae, but elicited significantly smaller EAG responses from adult female antennae. Extracts of adult males did not elicit appreciable EAG responses from antennae of either sex. Racemic 6-methylnonan-3-one was synthesized and elicited EAG responses from male antennae as strong as those obtained with female extracts. In field tests with baited sticky traps near lakes and streams, traps baited with synthetic racemic 6-methylnonan-3-one caught significantly more males than control traps. Female adults contained approximately 1 µg of 6-methylnonan-3-one per individual. Related ketones and alcohols of other chain lengths were also tentatively identified, being present in tiny amounts in female extracts. Extraction of different body parts showed that 6-methylnonan-3-one occurs only in a region near the intersegmental membrane between the fourth and fifth abdominal sternites of the female (no discrete glands were observed). Extracts of males did not contain 6-methylnonan-3-one, nor did pupae of either sex.

Hesperophylax occidentalis (Trichoptera: Limnephilidae): Electroantennogram structure-activity study of sex pheromone component 6-methylnonan-3-one.

Electroantennogram (EAG) analyses ofHesperophylax occidentalis male and female antennae were used to generate dose-response curves for synthetic, racemic 6-methylnonan-3-one and to demonstrate the chemical specificity of male antennal olfactory cells. Male antennae responded to 6-methylnonan-3-one, the main female pheromone component. Females also demonstrated a response to 6-methylnonan-3-one, but a smaller one than males. The chemical specificity of male antennal receptors was determined by comparing EAG activity of synthetic, racemic 6-methylnonan-3-one and a series of structural analogs. The three structural parameters considered, including keto position, chain length, and methyl-group position, were demonstrated to contribute significantly to the reception of synthetic, racemic 6-methylnonan-3-one by male antennae. For straight-chain nonanones with keto groups at different positions, nonan-3-one elicited the greatest EAG responses from male antennae. For straight-chain alkan-3-ones with different chain lengths, the EAG responses of male antennae to nonan-3-one and decan-3-one were the same, but greater than those to other chain lengths. For methyl-branched nonan-3-ones, 6-methylnonan-3-one elicited greater EAG responses from antennae than analogs with the methyl group at another position.

Nor-binaltorphimine: an ultra-long acting kappa-opioid antagonist in pigeons.

This study assessed the ability of naltrexone and nor-binaltorphimine (NBNI) to antagonize the rate-decreasing effects of opioid agonists. Food-restricted pigeons were trained to peck a lit key under a fixed-ratio (FR) 20 schedule of food reinforcement. Bremazocine, a kappa-opioid agonist, decreased food-reinforced responding (ED(50) = 0.02mg/kg), and naltrexone (5.6mg/kg) reduced the potency of bremazocine six-fold. The effect of naltrexone lasted less than 24h. A single injection of NBNI (1mg/kg) was given to four pigeons, and the time course of antagonism of the rate-decreasing effects of bremazocine was measured. One hour after NBNI was given, it was ineffective. Eight days later, NBNI produced a five-fold reduction in the potency of bremazocine. Between 12 and 20 days after NBNI, it reduced the potency of bremazocine 14-fold. NBNI continued to antagonize bremazocine for 11 weeks. Smaller doses of NBNI (0.001-0.1mg/kg) were ineffective. The effect of NBNI was not due to tolerance to bremazocine, since tolerance failed to develop to bremazocine administered repeatedly. NBNI (1mg/kg) did not antagonize the response rate-decreasing effects of morphine, a mu-opioid agonist, or BW373U86, a delta-opioid agonist. NBNI was an effective and extremely long-lasting kappa-opioid antagonist in the pigeon. The duration of action of NBNI is among the longest yet described in any species.

Improved method for computation of potentials in a realistic head shape model.

The Lead Field Analysis (LFA) algorithm, a new computational technique for the calculation of potentials on the surface of a realistic head shaped volume conductor model based on the Boundary Element Method and the Reciprocity Theorem, is presented. The new algorithm, in comparison to the Standard Boundary Element Method, offers improved computational efficiency and lower storage requirements. It also yields more accurate surface potential results in the face of varying dipole source locations for a head shape Boundary Element model with a given number of nodes. Additionally, the algorithm results in quasi-analytic expressions of the derivatives of the surface potential with respect to the location of the sources, allowing the use of optimization techniques with better convergence properties. A set of simulations demonstrating the increased robustness of the LFA Algorithm in the face of varying dipole source parameters is also described.

Effects of neuropeptide Y, insulin, 2-deoxyglucose, and food deprivation on food-motivated behavior.

The current study demonstrates the ability of neuropeptide Y (NPY) to increase break points under a progressive ratio 1 (PR1) reinforcement schedule. An initial response resulted in delivery of a food reinforcer (45 mg pellet) under the PR1, and an additional response was required for each successive reinforcer. The break point, the number of responses emitted to obtain the last reinforcer, is considered a measure of reinforcing efficacy or motivational strength of the food reinforcer. NPY (0.3-10 micrograms) significantly increased break point to levels comparable to those produced by 36-48 h of food deprivation. Although insulin (3-8 U/kg) and 2-deoxyglucose (150-250 mg/kg) also increased food intake, neither increased break points to levels produced by NPY or food deprivation. These data suggest that NPY may change the value of food in ways that cannot be accounted for by changes in insulin, glucose levels or intracellular glucoprivation. These results emphasize that simply measuring the amount of freely available food eaten is not a fully adequate measure of the strength of the feeding behavior.

Multiple-generator errors are unavoidable under model misspecification.

Model misspecification poses a major problem for dipole source localization (DSL) because it causes insidious multiple-generator errors (MulGenErrs) to occur in the fitted dipole parameters. This paper describes how and why this occurs, based upon simple algebraic considerations. MulGenErrs must occur, to some degree, in any DSL analysis of real data because there is model misspecification and mathematically the equations used for the simultaneously active generators must be of a different form than the equations for each generator active alone.