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.

Sex pheromone specificity in the pine sawflies: interchange of acid moieties in an ester.

3,7-Dimethylpentadecan-2-ol was identified as the free alcohol in three species from two genera of pine sawflies (Hymenoptera: Diprionidae). In Neodiprion lecontei and Neodiprion sertifer the acetate of this alcohol is the major component of their sex attractant; in Diprion similis it is the propionate. By examining the responses of the male antennae of several species of Neodiprion through the electroantennographic technique, it was determined that four species responded to the acetate and six to the propionate.

Human auditory evoked potentials: possible brain stem components detected on the scalp.

Auditory potentials recorded from the vertex of humans by a modified averaging technique have very short latencies and are probably generated by brain stem structures located at a considerable distance from the recording point. The evoked waves, which shOW considerable detail and consistency within and across subjects, may be clinically useful in evaluating subcortical function.

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.

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.

In vivo muscarinic cholinergic receptor imaging in human brain with [11C]scopolamine and positron emission tomography.

Cerebral muscarinic cholinergic receptors were imaged and regionally quantified in vivo in humans with the use of [11C]scopolamine and positron emission tomography. Previous studies in experimental animals have suggested the utility of radiolabeled scopolamine for in vivo measurements, on the bases of its maintained pharmacologic specificity following systemic administration and the exclusion of labeled metabolites from the brain. The present studies describe the cerebral distribution kinetics of [11C]scopolamine in normal subjects following intravenous injection. Scopolamine is initially delivered to brain in a perfusion-directed pattern. After 30 to 60 min, activity is lost preferentially from cerebral structures with low muscarinic receptor density including the cerebellum and thalamus. Activity continues to accumulate throughout a 2 h postinjection period in receptor-rich areas including cerebral cortex and the basal ganglia. The late regional concentration of [11C]scopolamine does not, however, accurately parallel known differences in muscarinic receptor numbers in these receptor-rich areas. Tracer kinetic analysis of the data, performed on the basis of a three-compartment model, provides receptor binding estimates in good agreement with prior in vitro measurements. Kinetic analysis confirms significant contributions of ligand delivery and extraction to the late distribution of [11C]scopolamine, reconciling the discrepancy between receptor levels and tracer concentration. Finally, a novel dual-isotope method for rapid chromatographic processing of arterial blood samples in radiotracer studies is presented. The combination of rapid chromatography and compartmental analysis of tracer distribution should have broad utility in future in vivo studies with short-lived radioligands.

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.

Evaluation of misonidazole peripheral neurotoxicity in rats by analysis of nerve trains evoked response.

The clinical use of misonidazole and other nitroimidazole radiosensitizing agents is limited by the peripheral and central neurotoxicity that is produced in animals and humans. In a blinded study, rats treated with misonidazole at either 100 mg/kg or 300 mg/kg, 5 days/week for 3 weeks, were evaluated for peripheral neurotoxicity using nerve trains evoked responses. Only one rat treated at a dose of 100 mg/kg developed symptoms and signs of neurotoxicity, while all rats treated at 300 mg/kg developed these signs and symptoms. Nerve trains analysis made possible a diagnosis of neurotoxicity before overt clinical signs appeared. This test is non-invasive and may be useful for evaluating patients receiving nitroimidazole radiosensitizers as part of a radiation therapy regimen.

Simple synthesis of F-18-labeled 2-fluoro-2-deoxy-D-glucose: concise communication.

Fluorine-18 acetyl hypofluorite, produced by a gas/solid-phase reaction, was reacted with D-glucal in water, followed by treatment with HCl and serial passage through columns of charcoal, ion-retardation resin, and alumina. The product was an injectable solution of 2-[18F]fluoro-2-deoxy-D-glucose. The radiochemical purity was greater than 95% and the radiochemical yield was about 40%. The synthesis was completed in about 15 min. Acetyl hypofluorite was shown to be a stable, highly stereoselective fluorinating agent in aqueous systems over a wide pH range.

Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging.

Carbon-11-meta-hydroxyephedrine is a new radiotracer developed for mapping the sympathetic nerves of the heart. Carbon-11-meta-hydroxyephedrine is synthesized by direct N-methylation of metaraminol with [11C]methyl iodide in dimethyl formamide/dimethyl sulfoxide and purified by semi-preparative reversed-phase HPLC. Total synthesis time is 45 min from end-of-bombardment. Carbon-11-meta-hydroxyephedrine is produced in 40%-50% corrected radiochemical yield with a specific activity of 900 Ci/mmol. Routine radiochemical and chemical purity are 95% and 98%, respectively. Biodistribution studies in rats show high myocardial uptake. Pretreatment with desipramine, a drug known to selectively block neuronal uptake, results in a 92% decrease in tracer accumulation in the myocardium. Metabolic studies in guinea pigs show less than 5% metabolites in heart tissue 30 min after intravenous injection suggesting that [11C]meta-hydroxyephedrine is suitable for kinetic modeling. These preliminary results support this new tracer as a clinical agent for neuronal imaging of the heart.

Synthesis, rodent biodistribution, dosimetry, metabolism, and monkey images of carbon-11-labeled (+)-2 alpha-tropanyl benzilate: a central muscarinic receptor imaging agent.

Muscarinic cholinergic receptors (mAChR) are abundant in the brain, and the mAChR system mediates many aspects of brain function. There is evidence of alterations in muscarinic binding in degenerative brain disorders. A muscarinic receptor radioligand, carbon-11-(+)-2 alpha-tropanyl benzilate ([11C]TRB), has been prepared through N-[11C]methylation of N-desmethyl TRB, and evaluated in rodents and primates. Full body biodistribution in rats has been determined and the expected human dosimetry calculated. Comparisons with [11C]scopolamine in rats showed 2-6 times greater brain uptake of [11C] TRB. Highly specific and saturable binding of [11C]TRB in the striatum and cortex was demonstrated by greater than 85% blockade of uptake following QNB or scopolamine pretreatment. Striatum/cerebellum ratios in mice at 60 min exceeded 12.6. TLC analysis of rat tissues showed the absence of 11C-metabolites in brain and heart, and a rapid solid phase C-18 Sep-Pak method found that unmetabolized plasma [11C]TRB in monkeys fell from 81% at 5 min to 48% at 80 min. Finally, brains of living primates have been imaged using PET and [11C]TRB; regional localization was consistent with muscarinic receptor distribution. These results represent intermediate steps in the development of [11C]TRB for quantification of central muscarinic receptors in man.

Autonomic components of the human pupillary light reflex.

To investigate the autonomic components of the pupillary light reflex in humans, we used infrared pupillometry combined with a partial local cholinergic (tropicamide) or alpha-adrenergic (thymoxamine) blockade. The pupillary response curve was analyzed using parameters identical or similar to those employed previously to study the autonomic components of the pupillary light reflex. Tropicamide increased baseline pupil area and affected five of the eight measured parameters. Thymoxamine lowered baseline pupil area but did not affect any of the parameters. We found the expected cholinergic contribution to the constrictive phase of the pupillary light reflex but no evidence for peripheral alpha-adrenergic activity during redilation. We propose that redilation primarily involves parasympathetic relaxation, modulated by cholinergic inhibition of the dilator muscle and central sympathetic inhibition of the Edinger-Westphal nucleus.

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.

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.

Spatio-temporal distribution of auditory-evoked far field potentials in rat and cat.

Short latency (less than 5 msec) auditory-evoked 'far field' potentials were mapped over the head of anesthetized rats and cats using a monaural 'click' stimulus. Significant activity was found over most of the head and, in the rat, on other parts of the body. Different components of the observed waveforms have different spatial distributions, supporting earlier evidence that they come from different generators. No area was found on the head which could be considered as an electrically neutral reference point for all the waves. There was considerable asymmetry in the spatial distribution of the early waves, with the area near the contralateral ear showing larger magnitudes than the area near the ipsilateral ear. It was found that most of the head of the rat is not in the far field, as previously defined. Significant activity was found on the tongue in the cat, which tended to increase the apparent magnitude of waves I and II at the vertex when the tongue was used as a reference. The area near the ear canal was also found to show significant activity, raising questions as to the use of the ear lobe as a reference point in human studies. We conclude that the placement of the electrodes can markedly influence the waveforms obtained, in some cases enhancing detection of early components.

Planar curve analysis of three-channel auditory brain stem response: a preliminary report.

Recording of auditory brain stem responses (ABRs) were made in guinea pigs by means of far-field computer averaging from three orthogonal electrode pairs. The 'Y' locations were mouth and nuchal ridge, 'X' locations left and right mastoids, and 'Z' locations vertex and throat. Three-dimensional models were constructed to plot simultaneous points from the three averages in a voltage-voltage-voltage space. In all five animals tested, at least three subsets of sequential data points lie in separate planes. The data points in each plane roughly correspond to waves I to III in the guinea pig ABR as recorded from the vertex-throat electrode pair. The planarity of data points is a new observation and is not predicted from the single-channel recordings. Three-dimensional planes are also found by means of vectorcardiography, which suggests some underlying principle, such as synchrony and homogeneity of anatomical position of the generators. Planar analysis of ABRs may be useful in analyzing and utilizing the additional information obtainable from 'non-standard' electrode pairs.

Effects of undernutrition on development of far-field auditory brain stem responses in rat pups.

Timed-pregnancy Sprague-Dawley rats were placed on special diets on day 15 of their pregnancy. One group received a normal, 24% protein diet, and the second a deficient, 8% protein diet. After birth the litters within each group were cross-fostered, thinned to 8 pups each, and maintained on their respective diets. Beginning with postnatal day 16 the ABR (auditory brain stem response) was repeatedly recorded from the pups until day 43. Undernourished rat pup body weights were less than one-third of those of the well-nourished litters at the conclusion of testing. ABRs taken from the undernourished group were significantly delayed in early development compared with the controls, but the differences decreased with age despite continuation of a deficient diet. A second experiment was run to rule out body temperature differences between the two groups and showed that the ABR effect was not solely due to this variable. These results indicate that undernourishment is one of the factors that can affect the rate of maturation of the ABR.

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.

The discriminative stimulus effects of neuropeptide Y.

Neuropeptide Y (NPY), an endogenous peptide which strongly induces food intake, is demonstrated to have discriminative stimulus properties when administered intracerebroventricularly. Rats rapidly learned to press the appropriate lever during training. NPY discrimination was dose-dependent. NPY's discriminative stimulus properties were compared to those of two doses of Peptide YY (PYY) and 24 and 48 h of food deprivation, conditions which also increase feeding. Both doses of PYY generalized to NPY, supporting previous findings that PYY has effects similar to NPY. Although food deprivation increases feeding in a manner similar to NPY, food deprivation did not result in NPY-appropriate responding.

[Leu31,Pro34]neuropeptide Y (NPY), but not NPY 20-36, produces discriminative stimulus effects similar to NPY and induces food intake.

Rats were trained to discriminate between an intracerebroventricular injection of 1.15 nmol of Neuropeptide Y (NPY) and a sham injection. Rats rapidly learned to press the appropriate lever during training. NPY's discriminative stimulus effects were compared to those of saline, and 1.15-3.45 nmol [Leu31,Pro34]NPY, a Y1 receptor agonist and NPY 20-36, Y2 receptor agonist. [Leu31,Pro34]NPY resulted in NPY-appropriate responding, whereas saline and NPY 20-36 did not. [Leu31,Pro34]NPY also increased food intake, but NPY 20-36 did not. This suggests that NPY's discriminative stimulus and orexigenic effects involve the Y1, but not the Y2, receptor.